Salmonella: From Pathogenesis to Therapeutics - asm.org · PDF file9:00 – 9:30 Population Structure of Salmonella enterica with Emphasis on Typhi Mark Achtmann, ... Defense Systems

Salmonella:FromPathogenesis toTherapeutics

© 2006, American Society for Microbiology1752 N Street, N.W.

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ISBN: 1-55581-412-3

TABLE OF CONTENTS

ASM Conferences Information

General Information

Scientific Program

Abstracts for Invited Speakers

Abstracts for Poster Sessions

Index

4

5

7

12

36

101

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Robert LaRossa, ChairDuPont de Nemours & Company

Fred C. Tenover, Vice ChairCenters for Disease Control and Prevention

Bonnie L. BasslerPrinceton University

William GoldmanWashington University School of Medicine

Jo HandelsmanUniversity of Wisconsin

Virginia MillerWashington University School of Medicine

Lance R. PetersonNorthwestern University

Darren HigginsHarvard Medical School

Scott WeeseUniversity of Guelph

ASM CONFERENCES COMMITTEE

CONFERENCE PROGRAM MISSION

To identify emerging or underrepresentedtopics of broad scientific significance.

To facilitate interactive exchange inmeetings of 100 to 700 people.

To encourage student and postdoctoralparticipation.

To recruit individuals in disciplines not already involved in ASM to ASM

membership.

To foster interdisciplinary andinternational exchange and collaborationwith other scientific organizations.

5Salmonella: From Pathogenesis to Therapeutics (2nd)

GENERAL

GENERAL INFORMATION

CONFERENCE ORGANIZERS

B. Brett FinlayUniversity of British Columbia

Gordon DouganThe Wellcome Trust Sanger Institute

ACKNOWLEDGMENTS

The Conference Organizers and the AmericanSociety for Microbiology would like toacknowledge the following for theircontributions to this conference:

InimexIntervetBionicheNobilon VaccinesThe Wellcome TrustEmergent BioSolutionsSchering Plough Animal HealthCanadian Institutes of Health Research

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Laura BetancorVishal ChananaElena CircellaVenessa EeckhautJeremy EllermeierSebastien FaucherGemma Foster

GENERAL INFORMATION

REGISTRATIONDuring the scientific sessions, the conference registration desk will be located in the foyer outside theLecture Theatre in the Victoria Conference Centre. ASM staff will be able to assist you during sessionhours.

GENERAL SESSIONSAll general sessions will be held in the Lecture Theatre at the Victoria Conference Centre. A name badgeis required for entry into all sessions. In consideration of other participants, no children are permitted inthe sessions.

POSTER SESSIONSPoster boards are located in Saanich Room, Oak Bay Room, Esquimalt Room, and the foyer outside theLecture Theatre in the Victoria Conference Centre. Presenters should mount posters on Sunday morningand should leave posters up until Tuesday evening. Each poster is allotted one board space. Please checkyour assigned number in the abstract index and mount your poster on the board space bearing thatnumber.

Official poster sessions will be held on Sunday, Monday and Tuesday as noted within the program. Pleasepresent your poster during the session (A, B, or C) noted next to your poster number.

SOCIAL EVENTSThe opening reception will be held on Saturday, September 9, in the Palm Court Ballroom in TheFairmont Empress. The Conference Dinner will be held in the Crystal Ballroom on Tuesday, September12, in The Fairmont Empress. Social events and refreshment breaks are included in the registration forconference participants. Tickets for guests that wish to attend the reception and dinner may be purchasedat the registration desk.

STUDENT TRAVEL GRANTSASM encourages the participation of graduate students and new postdocs at ASM Conferences. Tosupport the cost of attending the conference, ASM has awarded travel grants of $500 to each of thefollowing individuals:

GENERAL INFORMATION

Kaoru GeddesNicola GilberthorpeNguyen HueValerie Le SageOfir MenasheChristy OliverKai Papenfort

Krisztina Papp-WallaceYongKeun ParkMatthew RolfeMiryan Sanchez JimenezJenee SmithSophia Tan


SCIENTIFIC PROGRAM

SATURDAY, SEPTEMBER 9, 2006

18:30 Welcome Address

19:00 Keynote AddressMy Salmonella life (with interruptions): Early History, BacterialGenetics and Pathogenicity with Culminating Converging useof Genetics, Pathogenesis Knowledge and MolecularEngineering for Vaccine DevelopmentRoy Curtiss III, Arizona State University, Tempe, AZ, USA

20:00 - 21:30 Welcome ReceptionFairmont Empress Hotel, Palm Court Ballroom

SUNDAY, SEPTEMBER 10, 2006

9:00 – 12:15 Session 1: Salmonella enterica Population Biology

9:00 – 9:30 Population Structure of Salmonella enterica with Emphasis onTyphiMark Achtmann, Max-Planck-Institute for Infection Biology, Munich, Germany

9:30 – 10:00 Diversity in Salmonella GenomesMichael McClelland, Sidney Kimmel Cancer Center, San Diego, CA, USA

10:00 – 10:30 H-NS Mediates Global Gene Silencing in SalmonellaJay C. Hinton, Institute of Food Research, Norwich, United Kingdom

10:30 – 11:00 Break

11:00 – 11:30 Population Dynamics of Salmonella Typhimurium in the Organsof Infected MiceDuncan Maskell, University of Cambridge, Cambridge, United Kingdom

11:30 – 11:45 Horizontal Transfer of A+T-rich DNA and the Role of an H-NSParalogue in Maintaining Fitness and Virulence in SalmonellaCharles J. Dorman, Trinity College Dublin, Dublin, Ireland

11:45 – 12:00 Regulatory Insights from EnviCom, a Compendium Database ofSalmonella Gene Expression ProfilesMark Alston, Institute of Food Research, Norwich, United Kingdom

12:00 – 12:15 Detection of other Pathogens by SalmonellaJenee N. Smith, Ohio State University, Columbus, OH, USA

12:15 - 14:00 Lunch on your own

14:00 – 17:15 Session 2: Non-human Infections

14:00 – 14:30 Pathogenesis of Salmonella enterica Serotype Typhimurium andSerotype Typhi Infection in the Calf ModelAndreas J. Baumler, University of California, Davis, CA, USA

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14:30 – 15:00 Streptomycin-Pretreated Mice – Towards the Cellular Basis of S.Typhimurium-Induced EnterocolitisWolf-Dietrich Hardt, ETH Institute for Microbiology, Zürich, Switzerland

15:00 – 15:30 In vivo Pathogenesis of S. enterica Infections at the Single CellLevel and its Relevance for Understanding ImmunityPietro Mastroeni, University of Cambridge, Cambridge, United Kingdom

15:30 – 16:00 Break

16:00 – 16:30 A Murine Model of Salmonella enterica Serovar TyphimuriumPersistence: Identification of Salmonella Persistence andTransmission FactorsDenise Monack, Stanford University, Stanford, CA, USA

16:30 – 16:45 Host-Specific Systemic Translocation of S. enterica SerovarDublin in Cattle Occurs via Efferent Lymphatics in anExtracellular Niche and is Dependent on Type III SecretionSystem (T3SS)-1 but not T3SS-2Gillian D. Pullinger, Institute for Animal Health, Compton, Newbury, United Kingdom

16:45 – 17:00 The Role of SPI1 and SPI2 type III Secretion Systems inPathogenesis and Colonisation of Salmonella enterica SerovarTyphimurium in the ChickenPaul Wigley, University of Liverpool, Wirral, United Kingdom

17:00 – 17:15 Identification and Characterization of Salmonella TyphimuriumGenes Required for Persistence in the Gut of CaenorhabditisElegansRosanna A. Alegado, Stanford University, Stanford, CA, USA

17:15 – 19:15 Poster Session 1

MONDAY, SEPTEMBER 11, 2006

9:00 – 12:00 Session 3: Pathogenic Mechanisms I

9:00 – 9:30 PhoQ Recognition of Acidic pH and Antimicrobial Peptides toPromote Salmonellae Virulence for AnimalsSamuel I. Miller, University of Washington, Seattle, WA, USA

9:30 – 10:00 Modification of Cellular Transport and Immune Responses byIntracellular Salmonella entericaMichael Hensel, FAU Erlangen-Nürnberg , Erlangen, Germany

10:00 – 10:30 The Unique Lifestyle of Salmonella in Fibroblast CellsFrancisco Garcia-del Portillo, Centro Nacional de Biotecnologia-CSIC, Madrid, Spain

10:30 – 11:00 Break

11:00 – 11:15 SPIs 1 and 2 in Murine, Bovine and Human SalmonellaenterocolitiBryan Coburn, University of British Columbia, Vancouver, BC, Canada


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11:15 – 11:30 Small Regulatory RNAs and their Targets in SalmonellaTyphimuriumJoerg Vogel, Max Planck Institute for Infection Biology, Berlin, Germany

11:30 – 11:45 Transcriptome of Salmonella enterica Serovar Typhi insideHuman Peripheral Blood Monocytes CellsSebastien P. Faucher, University of Montreal, Montreal, PQ , Canada

11:45 – 12:00 Proteomic Analysis of Salmonella enterica Serovar TyphimuriumIsolated from RAW 264.7 Macrophages: Identification of aNovel Protein that contributes to the Survival of SerovarTyphimurium Inside MacrophagesLiang Shi, Pacific Northwest National Laboratory, Richmond, WA, USA


14:00 – 17:00 Session 4: Pathogenic Mechanisms II

14:00 – 14:30 The Abstract Title was not Available at the Time of PrintJohn Brumell, Hospital for Sick Children, Toronto, Ontario, Canada

14:30 – 15:00 Signal Transduction by the phop/phoq Regulatory SystemEduardo Groisman, Washington University, St. Louis, MO, USA

15:00 – 15:30 Interaction of Salmonella with its Mammalian Host CellJorge E. Galan, Yale School of Medicine, New Haven, CT, USA

15:30 – 16:00 Break

16:00 – 16:15 Identificatin of Genes and Pathways Required for SPI-1 Type IIISecretion System Activity Using a Contact-DependentHaemolysis AssayAbigail N. Layton, Institute for Animal Health, Berkshire, United Kingdom

16:15 – 16:30 Salmonella’s Type III Secretion System Targets PMNs in theMurine SpleenKaoru Geddes, Oregon Health and Sciences University, Portland, OR, USA

16:30 – 16:45 Salmonella enterica Serovar Typhimurium Effectors SopB, SopE,SopE2, and SipA Disrupt Tight Junction Structure and FunctionErin C. Boyle, University of British Columbia, Vancouver, BC, Canada

16:45 – 17:00 Analysing the Interplay between Salmonella Effector ProteinsCharlotte A. Perrett, University of Bristol, Bristol, United Kingdom


TUESDAY, SEPTEMBER 12, 2006

9:00 – 12:00 Session 5: Pathogenic Mechanisms III

9:00 – 9:30 The Abstract Title was not Available at the Time of PrintSteven Chatfield, Emergent Europe Ltd., Berks, United Kingdom

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9:30 – 10:00 Activation of the Pro-Survival Kinase Akt by Salmonella inInfected Epithelial CellsOlivia Steele-Mortimer, Rocky Mountain Laboratory, NIAID/NIH, Hamilton, MT, USA

10:00 – 10:30 H-NS and Ferritin B - The Novel Contributions of Two BacterialDefense Systems to Salmonella PathogenesisFerric. C. Fang , University of Washington, Seattle, WA, USA

10:30 – 11:00 Break

11:00 – 11:15 Pyroptosis: Caspase-1-Dependent Pore Formation Leads toOsmotic Lysis of Host Macrophages Infected with SalmonellaBrad T. Cookson, University of Washington, Seattle, WA, USA

11:15 – 11:30 Salmonella Bile Sensing and Bile-Mediated Biofilm FormationRobert W. Crawford, The Ohio State University, Columbus, OH, USA

11:30 – 11:45 SodCI Tethering within the Periplasm Contributes to SalmonellaVirulenceByoungkwan Kim, University of Illinois, Urbana, IL, USA

11:45 – 12:00 Metal Ion Sensing During Salmonella InfectionClare M. Taylor, University of Manchester, Manchester, United Kingdom


14:00 – 17:15 Session 6: Vaccination and Clinical Disease I

14:00 – 14:30 The Abstract Title was not Available at the Time of PrintLars Eckmann, University of California, San Diego, CA, USA

14:30 – 15:00 Multidrug Resistant Non-Typhoidal Salmonella Infections inChildren, KenyaSam Kariuki, KEMRI, Nairobi ,Kenya

15:00 – 15:30 The Abstract Title was not Available at the Time of PrintMike Levine, University of Maryland, College Park, MD, USA

15:30 – 16:00 Break

16:15 – 16:30 Salmonella Inhibit T Cell Proliferation by a Direct,Contact-Dependent Immunosuppressive EffectAdrianus W. Van der Velden, Harvard Medical School, Boston, MA, USA

16:30 – 16:45 An Improved Live Oral Salmonella enterica Vaccine withReduced Fecal SheddingRobert A. Kingsley, The Wellcome Trust Sanger Institute, Hinxton, United Kingdom

16:45 – 17:00 Vaccinal Protection in Broiler Chickens based on theColonization-Inhibition Mechanism: in Search of the IdealVaccine StrainVenessa Eeckhaut, Ghent University, Merelbeke, Belgium


17:00 – 17:15 Anti-Inflammatory Salmonella Vaccine Therapy ElicitsRegulatory T Cells and Protects Against AutoimmunityIndependent of AutoantigenDavid W. Pascual, Montana State University, Bozeman, MT, USA


19:30 – 23:00 BanquetFairmont Empress Hotel, Crystal Ballroom

WEDNESDAY, SEPTEMBER 13, 2006

9:00 – 11:45 Session 7: Vaccination and Clinical Disease II

9:00 – 9:30 Recent Advances in the Identification and Characterization ofthe Complex Effector Immune Responses Elicited by OralImmunization with Attenuated Salmonella enterica SerovarTyphi Strains in HumansMarcelo B. Sztein, Center for Vaccine Development, University of Maryland, Baltimore, MD, USA

9:30 – 10:00 Clinical Aspects of Enteric FeverChristiane Dolecek, University of Oxford, Oxford, United Kingdom

10:00 – 10:30 Vaccination and Clinical Disease: Lessons from the MurineModelRichard Strugnell, University of Melbourne, Melbourne, Australia

10:30 – 11:00 Break

11:00 – 11:15 Transcriptional Response to Salmonella enterica Serovar TyphiInfection Examined in Peripheral Blood of Patients in Viet NamLucinda J. Thompson, Stanford University, Stanford, CA, USA

11:15 – 11:30 Intracellular Persistence of NTS in HIV - the Role of IFNã in anEx-Vivo Human Tissue Macrophage ModelMelita A. Gordon, Liverpool University, Liverpool, United Kingdom

11:30 – 11:45 Prophylactic Anti-Tumor Immunity against a MurineFibrosarcoma Triggered by the Salmonella Type III SecretionSystemHolger Russmann, University of Munich, Munich, Germany

11:45 – 12:00 Closing Remarks

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K.A.MY SALMONELLA LIFE (WITHINTERRUPTIONS): EARLY HISTORY,BACTERIAL GENETICS ANDPATHOGENICITY WITH CULMINATINGCONVERGING USE OF GENETICS,PATHOGENESIS KNOWLEDGE ANDMOLECULAR ENGINEERING FORVACCINE DEVELOPMENTR. Curtiss III, The Biodesign Institute and School of Life Sciences,Arizona State University, Tempe, AZ

An early interest in poultry (chickens and ducks)provided an early exposure to Salmonella pullorumdisease control and immunological methods. Eightyears later, I divorced myself from chickens andcommenced studies of bacteriophage P22 and S.typhimurium. Two years later, after a brief flirtationwith chlamydial nucleic acid chemistry and nonex-istent potential (then) for genetic analysis, I em-braced the study of Escherichia coli with its phagesand plasmids. With the development of genecloning technologies and our own involvement inthe development of biological containment sys-tems, I turned my interests to the genetic analysisof bacterial pathogens. In the 1970’s, because of amost fortunate personal juxtaposition, I becameexposed to most of the key discoveries in theevolution and characterization of the mucosalimmune network. I then recognized the potential touse recombinant attenuated Salmonella strains as adelivery vehicle to target recombinant antigensspecified by cloned genes from diverse pathogensto host lymphoid tissues to stimulate mucosal,systemic and cellular immune responses. At thetime, introducing virulence or colonizing determi-nants from one pathogen into another was notpermissible, if not illegal. John Clements obtainedreversal of this policy in the early 1980’s. At thattime, I returned to a more comprehensive interestin Salmonella genetics and pathogenicity and com-menced again to work with chickens as one of ouranimal systems. During the past 25 years, my grouphas gradually shifted from a major emphasis onstudies of mechanisms of bacterial colonizationand pathogenesis to the use of our collective

knowledge and wisdom to design, construct andevaluate recombinant attenuated Salmonella vaccinesto control a diversity of diseases in poultry, swineand humans. I will describe several recent resultsand the development of a couple of new ap-proaches that should contribute to both understand-ing certain aspects of Salmonella pathogenesis andthe use of Salmonella as vaccine vectors.

S1:2DIVERSITY IN SALMONELLA GENOMESM. McClelland1, C. Santiviago1, F. Long1, N. Arrach1, B. Ahmer2, L.Florea3, S. Clifton4, H. Andrews-Polymenis5, S. Porwollik1; 1SKCC, SanDiego, CA, 2Ohio State University, Columbus, OH, 3George WashingtonUniversity, Washington D.C., DC, 4Washington University, Saint Louis,MO, 5Texas A&M, College Station, TX

DNA sequences from genomes of Salmonella andrelated species revealed a highly conserved overallgene order, with embedded genes and gene clustersintroduced by lateral transfer. Comparative genomichybridization (CGH) using microarrays of PCRproducts then allowed hundreds of Salmonellastrains to be compared and the pattern of lateraltransfer events during evolution to be determined.We have utilized this information in three ways: (1)The vast majority of laterally transferred genes haveno known function. We have begun to assemble acollection of lambda-red mediated in-frame knock-outs for putative protein coding genes that havebeen laterally transferred to S. enterica, excludingphage structural genes. So far, we have knocked outover 300 such genes in one virulent Typhimuriumstrain, 14028. A mixture of specific knockouts isthen subjected to selection in different environ-ments. Mutants in genes that increase fitness in thatenvironment become rare in the population of allmutants and this change is monitored on amicroarray. (2) CGH studies revealed that genomecontent does not necessarily correlate with Salmo-nella serovar; some strains in the same serovar maybe very different from each other, and are referredto as “genovars”. We have exploited the distributionof genes among genovars to develop PCR-basedclassification schemes for Salmonella. (3) Membersof Salmonella enterica subspecies I can differ by up to500 genes. Therefore, there are probably thousands


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GENERAL INFORMATION

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of unknown genes and gene clusters, of restricteddistribution, including prophage, that reside inhitherto unsequenced Salmonella genomes. Weshow that a recent innovation in shotgun sequenc-ing, when templated on a closely related genome,allows Salmonella genomes to be sampled to ~99%completion, and an accuracy of >99.9% in thecovered regions, for less than $25K per genome.Deep shotgun sequencing should allow a cost-effective investigation of this diversity. In addition,it can be expected that resequencing on high densityoligo arrays will then allow confirmation of putativeSNPs, facilitating analysis of their distribution andcontribution to evolution of Salmonella genomes.

S1:3H-NS MEDIATES GLOBAL GENESILENCING IN SALMONELLAJ. C. Hinton; Institute of Food Research, Norwich, UNITEDKINGDOM

We have discovered that global gene silencingoccurs in bacteria, and is mediated by the highly-abundant H-NS protein. H-NS is a modular proteinthat relies upon its oligomerisation properties tostructure the bacterial nucleoid. We used chromatinimmunoprecipitation to determine the binding sitesof H-NS and RNA polymerase on the S.Typhimurium chromosome. H-NS acts to block theexpression of 254 genes in wild-type S.Typhimurium LT2; these include a large number ofgenes acquired through horizontal gene transfer,including pathogenicity islands, which are silencedby the binding of H-NS to AT-rich chromosomalregions. The same phenomenon was independentlyreported by Navarre et al in the strain 14028, con-firming the role this type of gene silencing plays inSalmonella virulence. Our data suggest that H-NSprevents the uncontrolled transcription of geneswithin pathogenicity islands to ensure that bacterialfitness is maintained. This new process of AT-dependent bacterial gene silencing suggests that H-NS may play a role in evolution by influencing boththe acquisition and maintenance of foreign DNA.

S1:4POPULATION DYNAMICS OFSALMONELLA TYPHIMURIUM IN THEORGANS OF INFECTED MICED. Maskell, A. Grant, M. Sheppard, O. Restif, P. Mastroeni; Universityof Cambridge, Cambridge, UNITED KINGDOM

There are many ways to investigate host-pathogeninteractions. The simplest is to inject the pathogenand assay mortality. More sophisticated is to injectthe pathogen and remove organs in which it growsto follow the progression of the infection in termsof viable bacterial counts. This has become the goldstandard for whole animal infection studies withsalmonellae. More recently many groups haveinvestigated how salmonellae interact with infectedcells in culture, and therefore in isolation from theirnormal anatomical location and the influence of theimmune system. We report experiments investigat-ing the relationship between bacterial growth in ananimal host and the precise interactions betweenthe bacteria and the cells that they infect. We haveused the mouse typhoid model combined withfluorescence imaging techniques and mathematicalmodelling. Salmonella injected intravenously into amouse appears in its liver and spleen very rapidly.Resident liver macrophages are infected by onebacterium at one hour post-infection. If the infec-tion is left to grow in the organs, at three days postinfection we observe that infected cells still containa modal number of one bacterium. The next mostfrequent event is two bacteria per cell, then threebacteria per cell and so on, to generate a negativebinomial distribution. One day further into theinfection, when there has been a 10-fold increase inviable count per organ, we still see essentially thesame distribution of bacteria per cell. The increasednumber of viable bacteria per organ is due toincreased numbers of infected cells per focus andinfection foci per organ. This indicates that salmo-nellae escape from their original host cell and infectanother cell either in the same lesion, or elsewhereto generate a new lesion. These data also indicatethat models of infection predicated on in vitrotissue culture experiments where salmonellae growto high numbers per macrophage, and that there-

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fore suggest that salmonellae attain high numbers inmost infected cells in vivo, are possibly flawed. Inother experiments, if salmonellae that are almostidentical apart from the fact that they can be la-belled with “red” or “green” fluorescence, are usedto infect the animals, we observe that one hourpost-infection each macrophage contains either ared or a green bacterium. Intriguingly, after fourdays of infection, lesions observed in the organsstill contain either red or green bacteria but notboth. This indicates that each lesion originates froma single bacterium and is therefore clonal for thebacterial population therein, and that there is verylittle spread of bacteria between established lesions.We have developed mathematical models to help tounderstand these observations. Further experimentsusing these systems will be presented, as will theconsequences for our understanding of the finestructure dynamics of the infection process.

S1:5HORIZONTAL TRANSFER OF A+T-RICHDNA AND THE ROLE OF AN H-NSPARALOGUE IN MAINTAINING FITNESSAND VIRULENCE IN SALMONELLAC. J. Dorman1, M. Fookes2, A. Ivens2, J. Wain2, M. Doyle1; 1TrinityCollege Dublin, Dublin, IRELAND, 2Wellcome Trust Sanger Institute,Wellcome Trust Genome Centre, Cambridge, UNITED KINGDOM

The broad host range plasmid pSf-R27 was discov-ered in Shigella flexneri 2a strain 2457T. It is veryclosely related to the A+T-rich IncHI1 plasmid R27from Salmonella. These plasmids encode a protein,Sfh, that is a paralogue of the H-NS nucleoid-associated DNA binding protein and universaltranscriptional repressor. Sfh can formheterodimers with H-NS and with another chromo-some-encoded paralogue, StpA, leading to anhypothesis in which such interactions might modifythe activities of H-NS and StpA with widespreadeffects on gene expression in bacteria that receivepSf-R27. However, when pSf-R27 was introducedto naive Salmonella by conjugation, the impact onthe transcriptome was negligible. In stark contrast,the introduction of a derivative of pSf-R27 with aknockout mutation in the sfh gene resulted in wide-

ranging changes in gene expression and severealterations in several phenotypes, including fitnessand virulence. We will discuss the roles of genescoding for H-NS-like proteins in the successfultransfer and integration of A+T-rich DNA ele-ments in Gram-negative pathogens, includingSalmonella and the implications of this process forbacterial evolution.

S1:6REGULATORY INSIGHTS FROM ENVICOM,A COMPENDIUM DATABASE OFSALMONELLA GENE EXPRESSIONPROFILESM. Alston, S. Lucchini, A. Thompson, J. Hinton; Institute of FoodResearch, Norwich, UNITED KINGDOM

Salmonella enterica serovar Typhimurium (S.Typhimurium) is a major pathogen of animals andman causing more human deaths than any otherfood-borne pathogen, and may be responsible foran even higher mortality rate than previouslyrealised. To successfully infect its host, Salmonellamust survive a number of host defences such asgastric acid, bile, iron and oxidative stress. TheHinton group have used DNA microarrays todefine the Salmonella transcriptome in response to13 infection-relevant environmental stresses andconditions that include: acid, iron, anoxia, salt,butyrate, temperature, peroxide, nitric oxide,biofilm, and growth phase. The transcriptomic datahave been assembled into the Salmonella Environ-mental Compendium Database (EnviCom). Thebackbone of EnviCom is the open source databasesoftware BASE (BioArray Software Environment)and the expression profiles can be readily interro-gated with GeneSpring and R. Use of a genomicDNA common-reference array design has allowedcomparisons to be made between the variousstresses and conditions. Organising the array datain this way has allowed similar, or opposing, pat-terns of gene expression to be readily highlightedand linked to gene regulation, and has helped usdefine interactions between infection-relevantregulatory networks. For example, using the Salmo-nella Pathogenicity Island (SPI1) invasion genes as afocus we have discovered that the nap operon


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appears to be co-regulated with SPI1. The environ-mental compendium has utility for the identificationof new regulatory networks and will be queried toinvestigate the role played by particular regulons inthe response to environmental stresses. Furthemore,it offers an ideal operon discovery tool. We are inthe process of expanding EnviCom and are in theprocess of developing equivalent compendia forregulons and for gene expression during infectionof different host niches.

S1:7DETECTION OF OTHER PATHOGENS BYSALMONELLAJ. N. Smith1, C. D. Ellermeier2, J. L. Dyszel1, C. Altier3, G. M. Young4,S. D. Lawhon5, G. Adams5, V. Konjufca6, R. Curtiss, III6, J. M. Slauch7,B. M. Ahmer1; 1Ohio State University, Columbus, OH, 2Harvard University,Cambridge, MA, 3North Carolina State University, Raleigh, NC, 4Univer-sity of California, Davis, CA, 5Texas A&M University, College Station,TX, 6Arizona State University, Tempe, AZ, 7University of Illinois, Urbana,IL

The normal flora of the host play a very importantrole in preventing pathogen colonization. Themechanisms by which pathogens circumvent oroutcompete the normal flora are largely unknown.We hypothesize that one mechanism used by Salmo-nella is to take advantage of another pathogen’ssuccess. Salmonella encodes SdiA, a LuxR homologthat responds to the N-acylhomoserine lactones(AHLs) produced by other microbial species. UsingRIVET (Recombination-based In Vivo ExpressionTechnology) we have determined that Salmonelladoes not detect AHLs during transit through thegastrointestinal tract of healthy animals (mice,guinea pigs, rabbits, chickens, pigs, cows, or turtles).This suggests that AHLs are not a normal compo-nent of the intestinal milieu. However, SdiA activitywas recorded in turtles that carry the pathogenAeromonas hydrophila. SdiA was also active duringtransit through mice infected with Yersiniaenterocolitica but not in mice infected with anisogenic yenI mutant of Y. enterocolitica that doesnot synthesize AHLs. SdiA conferred a fitnessadvantage upon Salmonella in mice infected withwild-type Y. enterocolitica but not in mice mock-infected with LB or in mice infected with the yenI

mutant. Therefore, SdiA is detecting and respond-ing to the opportunities presented by coinfection.

S2:1PATHOGENESIS OF SALMONELLAENTERICA SEROTYPE TYPHIMURIUM ANDSEROTYPE TYPHI INFECTION IN THE CALFMODELA. J. Baumler; University of California, Davis, CA

Human infections with non-typhoidal Salmonellaserotypes, such as S. enterica serotypeTyphimurium, are characterized by a massive neu-trophil influx in the colon and terminal ileum. Incontrast, neutrophils are scarce in intestinal infil-trates of typhoid fever patients. We show that thescarcity of neutrophils in intestinal infiltrates oftyphoid fever patients is due to a capsule-mediatedreduction of IL-8 production in the intestinalmucosa. S. enterica serotype Typhi, the causativeagent of typhoid fever, expresses a capsularpolysaccharide, the Vi-antigen, which reduced IL-8expression in polarized human epithelial cells (T84)and human macrophage-like cells (THP-1) byinhibiting Toll-like receptor (TLR) signaling in hostcells. The relevance of these in vitro observationsfor the interaction of serotype Typhi with its humanhost was further studied ex vivo, using humancolonic tissue explants, and in vivo, using bovineligated ileal loops. These data showed that the Vi-capsular antigen enables serotype Typhi to evade astereotypic TLR-mediated innate immune responseleading to neutrophil infiltration.

S2:2STREPTOMYCIN-PRETREATEDMICE – TOWARDS THE CELLULAR BASISOF S. TYPHIMURIUM-INDUCEDENTEROCOLITISW. Hardt, Institut für Mikrobiologie, Departement Biologie, Zürich,Switzerland

S. Typhimurium employs a large set of virulencefactors to manipulate host cells and cause diarrhea.Intriguingly, type III effector proteins play a centralrole in this process. At the cellular level, the func-tions of these factors are quite well understood;

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now, animal models are required for elucidatinghow these factors trigger acute intestinal inflamma-tion in vivo. Calf infection models have beenemployed successfully. Recently, a mouse model wasidentified: in streptomycin-pretreated mice S.Typhimurium causes acute colitis. This mousemodel is employed to analyze the interaction of S.Typhimurium with the gut mucosa. Bacterial viru-lence factors (incl. TTSS-1, TTSS-2, flagella) re-quired for triggering acute colitis have been identi-fied. Also, the host’s innate immune system contrib-utes significantly. Now, we study the cell types ofthe murine mucosa (incl. PMN, dendritic cells, NKcells, macrophages, enterocytes) which are manipu-lated by S. Typhimurium to trigger disease. Cytokineanalysis, knockout animals, cell-type specific deple-tion strategies and microscopic techniques areemployed to dissect the complex network of inter-actions between the pathogen and the cells of themucosal tissue.

S2:3IN VIVO PATHOGENESIS OF S. ENTERICAINFECTIONS AT THE SINGLE CELL LEVELAND ITS RELEVANCE FORUNDERSTANDING IMMUNITYP. Mastroeni, G. Foster, A. Grant, H. Uppington, N. Menager, D.Maskell; Department of Veterinary Medicine, University of Cambridge,Cambridge, UNITED KINGDOM

During systemic infections, S. enterica reside withinphagocytic cells in the spleen and liver in welldefined infection foci that are surrounded by nor-mal tissue. In concomitant infections, foci contain-ing different S. enterica isolates are spatially sepa-rated and local events contribute to the control ofbacterial growth within each individual focus.However, systemic events determining functionalinteractions between bacterial populations withinthe same host can occur despite the geographicalseparation of the foci. For example, the net growthrate of a virulent S. enterica strain can be interferedwith (increased) by the administration of a differentstrain, despite the fact that the two strains occupydifferent locations in the tissues. This interferencerequires functional TLR4, is not due to the systemicinhibition of IFNgamma or TNFalpha production

and is mediated by IL-10. During their growth inthe tissues, S. enterica distribute to new phagocytesafter escaping from already-formed infection foci.The spatial separation of the foci implies that,during their cell-to-cell spread, the bacteria aretransiently present in the extracellular space. HereS. enterica are opsonized by anti-LPS antibodies andtargeted to Fc receptors present on the surface ofphagocytes. Using bone marrow derived macroph-ages from gene-targeted mice deficient in individualFc gamma receptors (I, II and III), we have shownthat antibodies enhance bacterial internalization viaFc gamma receptor I. This results in increasednumbers of infected macrophages and in changes inthe overall numerical distribution of S. entericawithin the phagocytes. Targeting to Fc gammareceptor I also results in increased bacterial killingvia reactive oxygen intermediates. Fc gamma recep-tor I, II, III-/- (triple KO) mice cannot survive aninfection with virulent S. enterica even after previousimmunization with a live attenuated S. entericavaccine which induces Th1 responses and anti-S.enterica antibody production. The requirement forFc gamma receptors in immunity and the under-standing of the patterns of S. enterica spread anddistribution, complement our previous work whichestablished the need for both T-cells and antibodies(B-cells) in vaccine-induced protection againstsalmonellae. We have also found that B-cells do notcontribute to immunity to S. enterica solely viaantibody production, but they are also key cells inthe initiation and amplification of Th1 immunity toS. enterica. We are currently investigating the pos-sible mechanisms used by B-cells to engender T-cellresponses to salmonellae.

S2:4A MURINE MODEL OF SALMONELLAENTERICA SEROVAR TYPHIMURIUMPERSISTENCE: IDENTIFICATION OFSALMONELLA PERSISTENCE ANDTRANSMISSION FACTORSD. Monack, Department of Microbiology and Immunology, StanfordUniversity, Stanford, CA

We recently described a mouse model of Salmonellapersistence in which Salmonella enterica serovar



Typhimurium (serovar Typhimurium) colonizes129X1/SvJ (Nramp+/+) mice for up to a year and isshed in feces for extended periods. A microarray-based negative selection screen was performed toidentify serovar Typhimurium genes which contrib-ute to long-term systemic infection in mice. A highcomplexity transposon-mutagenized library wasused to infect mice intraperitoneally and the selec-tive disappearance of mutants was monitored after7, 14, 21 and 28 days post-infection. One hundredand eighteen genes were identified to contribute toserovar Typhimurium infection of the spleens ofmice by 28 days post infection. The negatively-selected mutants represent many known aspects ofSalmonella physiology and pathogenesis, althoughthe majority of the identified genes are of putativeor unknown function. Interestingly, several SPI1encoded type III secretion system effectors/translocases are required by serovar Typhimuriumto establish and, unexpectedly, to persist systemi-cally, challenging the present description of Salmo-nella pathogenesis. Moreover, we observed a pro-gressive selection against serovar Typhimuriummutants based upon the duration of the infection,suggesting that different classes of genes may berequired at distinct stages of infection. Overall,these data indicate that Salmonella long-term sys-temic infection in the mouse requires a diverserepertoire of virulence factors. This diversity ofgenes presumably reflects the fact that bacteriasequentially encounter a variety of host environ-ments and that Salmonella has evolved to respond tothese selective forces in a way that permits both thebacteria and the host to survive. We also describethe use of this model to study horizontal transmis-sion via the natural fecal-oral route. SerovarTyphimurium was rapidly transmitted fromorogastrically infected mice to cohoused naïve mice.During transmission, the naïve mice are colonizedby serovar Typhimurium at both the mucosal andsystemic sites at levels comparable to theorogastrically infected mice. In addition, miceinfected by the fecal-oral route developed a serovarTyphimurium mucosal IgA response with similarkinetics to that of orogastrically inoculated mice.To test the possible role of Salmonella Pathogenicity

Island 1 (SPI-1) in transmission, which is requiredfor Peyer’s patch colonization, we infected micewith the effector/translocator mutant sipB. Thelevels of the sipB mutant that were shed in the feceswere similar to wild-type bacteria. However, themutant bacteria were not transmitted to naïve mice,even when cohoused for 45 days. Thus, we haveshown that SPI1 is required for long-term carriageand transmission via the fecal-oral route in thismouse model.

S2:5HOST-SPECIFIC SYSTEMICTRANSLOCATION OF S. ENTERICASEROVAR DUBLIN IN CATTLE OCCURS VIAEFFERENT LYMPHATICS IN ANEXTRACELLULAR NICHE AND ISDEPENDENT ON TYPE III SECRETIONSYSTEM (T3SS)-1 BUT NOT T3SS-2G. D. Pullinger, S. M. Paulin, P. R. Watson, B. Charleston, B.Villarreal-Ramos, T. S. Wallis, M. P. Stevens; Institute for Animal Health,Compton, Newbury, UNITED KINGDOM

Salmonella enterica serovar Dublin is primarily associ-ated with infections of cattle, inducing both sys-temic and enteric disease. We have used a cannula-tion model to investigate the mode and genetics ofits dissemination from the gut to systemic sites inits natural host. Serovar Dublin inoculated into aligated ileal loop translocated efficiently throughmesenteric lymph nodes into cannulated efferentlymphatics. In contrast, the fowl-associated serovar,Gallinarum, translocated poorly. Neither serovarwas recovered in significant numbers from cannu-lated venules. These results suggested that translo-cation occurred mainly via efferent lymphatics andcorrelated with host-specificity. Gentamicin protec-tion and microscopical analyses showed that serovarDublin was predominantly extracellular within theefferent lymph. Despite this, fluorescence micros-copy showed that S. enterica serotypes Dublin,Gallinarum and Typhimurium were intracellularwhilst in the intestinal mucosa, and were initiallyassociated with cells expressing CD68 and MHCII.Analysis of selected mutants in the cannulationmodel showed that early systemic translocation viaefferent lymphatics required a functional T3SS-1

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but was independent of T3SS-2. The same T3SS-2mutant has previously been shown to be attenuatedfollowing intravenous inoculation of calves. Theseexciting preliminary results contrast markedly withobservations in the murine typhoid model, whichhad indicated that S. Typhimurium is deposited inblood in a CD18-leukocyte dependent, T3SS-1independent manner. Ongoing studies will track thefate of a more comprehensive selection of mutantsin this model.

S2:6THE ROLE OF SPI1 AND SPI2 TYPE IIISECRETION SYSTEMS IN PATHOGENESISAND COLONISATION OF SALMONELLAENTERICA SEROVAR TYPHIMURIUM IN THECHICKENP. Wigley1, P. Barrow2, M. Jones2; 1University of Liverpool, Wirral,UNITED KINGDOM, 2University of Nottingham, Sutton Bonnington,UNITED KINGDOM

Background - The role of type III secretion systems(TTSS) in Salmonella enterica serovar Typhimuriuminfection of mammals has been well described. Incontrast, the role of the SPI1 and 2 systems in avianS. Typhimurium infections has been only partiallydescribed, though the importance of SPI2 has beenshown in avian specific serovars causing systemicdisease. Previously, using a signature-tagged mu-tagenesis approach (Morgan et al. Mol Microbiol 54994-1010 2004) it was found that neither SPI1 orSPI2 played a major role in colonisation of thechicken gastrointestinal tract. In newly hatchedchicks their role in the systemic disease that resultsfrom S. Typhimurium infection is not known. Inthis work we describe the role of both type IIIsecretion systems in systemic disease in chicks andcolonisation in older birds. Methods - Infection ofnewly hatched chicks 1-day old SPF Rhode Island RedChicks were infected orally with 108 cfu S.Typhimurium F98 or spaS (SPI1), or ssaU (SPI2)mutants. Chicks were killed at 24, 48 and 72h postinfection. Samples of liver and caecal content weretaken for bacteriological analysis Infection of one weekold chicks At one day of age SPF Rhode Island Redchicks were given an adult gut flora preparation tominimise variation in flora. At 7 days of age birds

were infected as described above. At 1, 3, 7 and 14days post infection birds were killed and sampled asabove. Results - Infection of newly hatched chicksAll strains resulted in systemic infection. Howeverin the ssaU infected group pathology was reducedand viable counts significantly reduced (P<0.005) ataround 103 cfu per g compared to over 105 cfu per gin the other groups by 72h post infection. Nodifference was found in bacterial numbers in thecaecal content (all in excess of 108 cfu per g at 72 hPI). One week old birds The ssaU mutant showedsignificantly lower numbers in caecal content thanthe parent strain at 1 and 3d PI, and was not recov-ered from the liver following infection. The ssaUmutant was cleared more rapidly, and was notdetected in caecal contents by 7 d PI. The spaSmutant was also detected at lower, though notstatitistically ignorant, levels in the faecal content,but was significantly lower in the liver at 3d PI(P=0.03) and was cleared more rapidly. Discussion -These findings indicate that the SPI 2 TTSS isrequired for both the systemic infection of thechicken an in the long-term colonisation of thechicken gastrointestinal tract. The role of the SPI1system is not essential but also appears to play arole in both stages of infection. Although theseresults contradict those described above, there maybe some strain-to strain variation and the use ofquantitative bacteriology at post mortem examina-tion may allow for the more sensitive detection ofdifferences in phenotype than cloacal swabbing.

S2:7IDENTIFICATION ANDCHARACTERIZATION OF SALMONELLATYPHIMURIUM GENES REQUIRED FORPERSISTENCE IN THE GUT OFCAENORHABDITIS ELEGANSR. A. Alegado, M. Tan; Stanford University, Stanford, CA

Caenorhabditis elegans has been shown to be a valu-able model for studying host-pathogen interactions.Several Salmonella typhimurium virulence factorsimportant in murine and tissue culture models alsohave conserved roles in worm mortality. Previouswork has shown that Salmonella can colonize, prolif-erate, and persist in the gut of C. elegans leading to



pronounced gut distention. However, the bacterialfactors required for a persistent infection of the gutare not known. To identify Salmonella genes impor-tant for persistence in the worm, we took a geneticapproach. We reasoned that bacterial mutants lesseffective in persistence would be eliminated fromthe worm gut over time. A small transposon librarywas generated from wild type Salmonella containinga chromosomal copy of GFP and visually screenedfor failure to remain in the nematode intestine. Weisolated eighteen bacterial genes important inbacterial persistence in the worm gut. Persistencedefects of each Salmonella mutant were confirmedby viable cell counts from worms. Four of ourSalmonella persistence genes (ssaL, hemA, cheM, andcheR) have known roles in the murine typhoid modelwhereas the rest have no documented role in Salmo-nella virulence. Using the information from thevarious functional groups isolated from our persis-tence screen, we have begun to dissect the mecha-nisms for persistence in the worm gut. Recently, weshowed that bacterial resistance to C. elegans antimi-crobials contributes to Salmonella persistence in theworm gut. One class of mutants displays sensitivityto membrane stress. Mutations in kdpA and hemAwere susceptible to both osmotic stresses and theantimicrobial peptide polymyxin B. Other genes inthis class are sensitive specifically with polymyxin B(ssaL, glnK, and STM1049) and some of these werealso required for macrophage survival. Importantly,the persistence defects of ssaL and STM1049 couldbe rescued in vivo when either of the C. eleganssaposin-like or defensin-like antimicrobial wasknocked down. While the role of ssaL has beendescribed, the involvement of glnK is novel andillustrates the utility of our screen in identifyingadditional virulence determinants in mammalianmodels. A second class of mutations shows defectsin biofilm growth, implying that biofilm formationis a critical component for establishing persistenceinfection. Assessment of adherence to wormenterocytes in vivo and polarized MDCK epitheliallines is in progress. Together, these results suggestthat multiple mechanisms contribute to establish-ment of Salmonella in the nematode gut. This studyprovides additional insight into bacterial-host

interactions and the extent to which Salmonellaexploits similar mechanisms for establishmentwithin disparate hosts.

S3:1PHOQ RECOGNITION OF ACIDIC PHAND ANTIMICROBIAL PEPTIDES TOPROMOTE SALMONELLAE VIRULENCEFOR ANIMALSS. Miller, University of Washington

Salmonellae are Gram-negative bacteria that causediverse diseases including diarrhea and the systemicdisease enteric or typhoid fever. Central to theability to cause disease is the bacterial ability tosurvive and replicate within professional phagocytessuch as macrophages. This is accomplished throughsensing of the intracellular environment whichsignals cell surface remodeling to resist innateimmune effectors such as cationic antimicrobialpeptides and the assembly of apparati, termed typeIII secretion systems which function as proteintransport devices to deliver bacterial proteins acrossthe phagocyte membrane. Key to this sensing isrecognition of mammalian signals by a bacterialsensor kinase PhoQ. PhoQ is a membrane boundsensor kinase important for the pathogenesis of anumber of Gram-negative bacterial species. PhoQand its cognate response regulator PhoP constitutea signal-transduction cascade that controls inducibleresistance to host antimicrobial peptides. Salmonellatyphimurium PhoQ is directly activated by antimi-crobial peptides and at low pH. A highly acidicsurface of the PhoQ sensor domain participates inboth divalent-cation and antimicrobial-peptidebinding as a first step in signal transduction acrossthe bacterial membrane. Identification of PhoQsignaling mutants, binding studies with the PhoQsensor domain, and structural analysis of thisdomain can be incorporated into a model in whichantimicrobial peptides displace divalent cationsfrom PhoQ metal binding sites to initiate signaltransduction. Sensing of acid pH appears to involvedifferent components of the PhoQ sensor domainand a model can be generated in which antimicro-bial peptides and pH act together as a signal trans-duction is initiated by alteration of the PhoQ sensor

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domain. In contrast to Salmonellae, Pseudomonasaruginosa PhoQ functions largely as a sensor ofdivalent cations. Structural studies and analysisindicates that acquisition of the ability of Salmonel-lae PhoQ to respond to low pH and antimicrobialpeptides could be part of the adaptation of Gram-negative bacteria such as Salmonellae to animals.Therefore bacteria sense small innate immunemolecules to initiate a transcriptional program thatpromotes bacterial virulence.

S3:2MODIFICATION OF CELLULARTRANSPORT AND IMMUNE RESPONSES BYINTRACELLULARSALMONELLA ENTERICAM. Hensel; FAU Erlangen-Nürnberg , Erlangen, GERMANY

Intracellular survival and replication within eukary-otic host cells is of central importance for thepathogenesis of infections caused by Salmonellaenterica. Intracellular Salmonella translocate a setof effector proteins by means of a type III secre-tion system (T3SS) encoded by Salmonella Pathoge-nicity Island 2 (SPI2) that manipulate normal hostcell functions. Intracellular survival and replicationis linked to the function of the SPI2-T3SS, butrecent observations show that many additionalcellular functions are targeted by this virulencesystem. We investigated the interaction of Salmo-nella with dendritic cells (DC), antigen-presentingcells that form an important link between innateand adaptive immunity. Salmonella is able to persis-tent in DC irrespective of the function of anyknown virulence factor. However, the SPI2-T3SS isexpressed and functional in translocation of effec-tor proteins. By means of the SPI2-T3SS, intracellu-lar Salmonella inhibit the antigen presentation byDC and antigen-dependent T-cell proliferation. Theresulting suppression of adaptive immunity may bea prerequisite for persistent Salmonella infections.A possible reason for this phenotype could be thealteration of cellular transport processes by intracel-lular Salmonella. To characterize the interaction ofintracellular Salmonella with host cell transportprocesses, we utilized various model systems tofollow microtubule-dependent transport. The

vesicular stomatitis virus glycoprotein (VSVG) is acommonly used marker to follow protein transportfrom the Golgi to the plasma membrane. Using aVSVG-GFP fusion protein, we observed thatvirulent intracellular Salmonella alter exocytotictransport and recruit exocytotic transport vesiclesto the Salmonella-containing vacuole (SCV). Thisvirulence function was dependent on SPI2-T3SSeffector proteins. Furthermore, the Golgi to plasmamembrane traffic of the sphingolipid C5-ceramidewas redirected to the SCV by virulent Salmonella.We propose that Salmonella modulates the biogen-esis of the SCV by deviating this compartmentfrom the default endocytic pathway to an organellethat interacts with the exocytic pathway.

S3:3THE UNIQUE LIFESTYLE OF SALMONELLAIN FIBROBLAST CELLSF. Garcia-del Portillo; Centro Nacional de Biotecnologia-CSIC, Madrid,SPAIN

Salmonella enterica targets diverse host cell typesalong the infection, including M cells, enterocytes,dendritic cells and macrophages. In systemic infec-tions, macrophages are the preferred cell niche usedby this pathogen to survive and proliferate inorgans. As shown by several groups, a hallmark ofthe macrophage-Salmonella interaction occurring invivo is that intracellular bacteria do not proliferateextensively in neither chronic nor acute infections.A rather low proliferation rate of Salmonella is alsoobserved in vitro upon infection of cultured den-dritic cells or fibroblasts. We have exploited thefibroblast infection model with the aim ofunravelling new host-pathogen interactions. In thismodel, we have characterized a Salmonella invasionpathway that does not rely in the type III secretionsystem encoded in SPI-1. Evidence for a fibroblastresponse to Salmonella infection involving thestimulation of the Raf/MEK/ERK and the Aktpathways has also been found. We are currentlycarrying out microarray studies in intracellularbacteria, which are shedding lights on the lifestyleof Salmonella inside fibroblasts. These experiments,performed in parallel with wild-type bacteria and



the overgrowing phoP mutant, reveal that theactivity of the SPI-2 system may substantially differto what it has been reported in bacteria residingwithin macrophage cells. Thus, a distinction notedis that the SPI-2 is activated at higher extent in thephoP mutant than in wild-type bacteria. Lastly,evidence for Salmonella infection of non-phago-cytic cells located in the lamina propria has beenobtained. Remarkably, the bacteria in these cellsundergo a program to limit intracellular growth thatmimics the response observed in cultured fibro-blasts. We conclude that fibroblasts could be a newcell type targeted by Salmonella during infection inwhich unique host-bacteria interactions non-ob-served in other cell types may occur.

S3:4SPIS 1 AND 2 IN MURINE, BOVINE ANDHUMAN SALMONELLA ENTEROCOLITISB. Coburn1, Q. Hu2, B. Coombes1, W. Deng1, Y. Li1, N. Brown1, A.Potter3, B. Finlay1; 1University of British Columbia, Vancouver, BC,CANADA, 2Shenzhen Centres for Disease Control and Prevention,Shenzhen, CHINA, 3Vaccine and Infectious Disease Organisation,Saskatoon, SK, CANADA

Non-typhoidal Salmonella species are a significantcause of human diarrheal disease, incurring world-wide morbidity and mortality. The prevailing dogmaarising from animal models of Salmonellaenteropathogenesis is that the virulence associatedgenomic regions, Salmonella pathogenicity island(SPI) -1 and SPI-2, are essential for intracellularinvasion/intestinal disease and intracellular sur-vival/systemic disease, respectively. New animalmodels of Salmonella enteropathogenesis haveallowed a re-examination of this theoretical di-chotomy. Using these models, SPI-2 has beenshown to be required for completeenteropathogenesis in Salmonella enterica serovarTyphimurium infection. In addition, murine andbovine intestinal inflammation was identified in theabsence of SPI-1, previously thought to be essentialfor intestinal disease. We have corroborated thesefindings in human disease by the identification of aSPI-1 deficient human clinical diarrheal Salmonellaenterica isolate. These strains were isolated frompatients affected with severe diarrheal disease in

Shenzhen, China. These observations indicate thatdisease models, diagnostic and therapeutic ap-proaches predicated on the requirement for SPI-1 inintestinal disease do not accurately describe intesti-nal salmonellosis.

S3:5SMALL REGULATORY RNAS AND THEIRTARGETS IN SALMONELLA TYPHIMURIUMK. Papenfort1, S. Lucchini2, F. Mika1, V. Pfeiffer1, J. C. Hinton2, J.Vogel1; 1Max Planck Institute for Infection Biology, Berlin, GERMANY,2Molecular Microbiology Group, Institute of Food Research, NorwichResearch Park, Norwich, UNITED KINGDOM

Small non-coding RNAs (sRNAs) are an emergingclass of post-transcriptional regulators of bacterialgene expression. Such RNAs are typically 50-250nucleotides long, and are expressed from free-standing genes in the intergenic regions of bacterialchromosomes. Most sRNAs studied to date havebeen found to act on trans-encoded mRNAs tomodulate their translation and stability. Recentsystematic searches indicate that non-pathogenic E.coli may express several hundred sRNAs. Theabundance and functional roles of these moleculesin bacterial pathogens, however, remain largelyunknown. We have identified and studied morethan 25 sRNAs in Salmonella enterica serovarTyphimurium. These sRNAs are expressed from theSalmonella core genome as well as from the Salmo-nella pathogenicity islands. Nearly all of them havehomologues in other Salmonella species. Northernhybridizations with RNA extracted from severalgrowth conditions showed that many of thesesRNAs are upregulated in stationary phase as wellas under conditions relevant to Salmonella viru-lence. We have taken a large-scale approach toidentify the cellular targets of more than 20 Salmo-nella sRNAs. To this end, sRNAs were pulse-expressed, and the resulting changes of globalmRNA levels were monitored using microarrays.Our results indicate that several hundred SalmonellamRNAs are directly regulated by sRNAs at thepost-transcriptional level. We have also identified aregulatory network in which small RNAs controlthe expression of major Salmonella outer mem-brane proteins (OMPs). Two of these OMP-regulat-

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SPEAKER ABSTRACTS

SPEAKER ABSTRACTS

ing sRNAs are induced by anti-microbial peptides,and their transcription is dependent on the alterna-tive stress sigma factor, RpoE. The results of thisfirst comprehensive study of small non-codingRNAs and their functions in a bacterial pathogenwill be presented.

S3:6TRANSCRIPTOME OF SALMONELLAENTERICA SEROVAR TYPHI INSIDE HUMANPERIPHERAL BLOOD MONOCYTES CELLSS. P. Faucher1, S. Porwollik2, M. McClelland2, F. Daigle1; 1University ofMontreal, Montreal, PQ , CANADA, 2Sidney Kimmel Cancer Center, SanDiego, CA

Introduction: Salmonella enterica serovar Typhi isthe etiological agent of typhoid fever, a systemicinfection specific to humans. Many known patho-genic determinants have been identified by usingserovar Typhimurium in the mouse model ofinfection. However, this serovar is usually unable tocause systemic infections in humans and it is likelythat Typhi has specific virulence factors that causedisease in humans. To understand the molecularmechanism of pathogenesis, it is necessary toidentify new and specific virulence factors thatallow Typhi to survive in human macrophages.Since there is no animal model to study this patho-gen, cultured cells lines, like THP-1 are routinelyused. However, these cells may not reflect the actualproperties of normal macrophages, because theyhave been transformed and cultured in vitro formany years. Hypothesis: The Typhi response insideperipheral blood monocytes cells (PBMC) may bemore representative than THP-1. The study ofexpression profiles of Typhi inside PBMC shouldallow for a better understanding of its pathogenesisand the identification of specific and new virulencefactors. Methods: We have developed a method toacquire the transcriptome of the intracellular patho-gen from a low number of bacteria by using selec-tive capture of transcribed sequences. We used thismethod to obtain the gene expression profile ofTyphi inside human PBMC at 0, 2, 8 and 24 h post-infection. The supernatant of the infection wasused as the control. Results: Comparison with thetranscriptome of Typhi inside human THP-1 mac-

rophages reveals many similarities. In both celltypes, Typhi induced the SPI-2 type three secretionsystem (TTSS) but repressed the SPI-1 TTSS.Genes involved in motility and in iron transportwere also repressed in both cell types. However,Typhi seems to experience more oxidative stressinside PBMC than inside THP-1. Antimicrobialpeptide resistance genes are not induced in PBMCbut they were in THP-1. Conversely, LPS synthesisgenes seem to be induced in PBMC but not inTHP-1. Many genes with unknown function arespecifically induced in PBMC and may be potentialnew virulence factors. These results have beenconfirmed by real-time qPCR. Conclusion: This isthe first time that the transcriptome of a human-specific intracellular pathogen has been obtainedfrom human primary cells. Differences in thetranscriptome of Typhi within these cells comparedto the one obtained from THP-1 cells may repre-sent new virulence factors that have been previouslyunderestimated. Further characterisation of thesegenes should provide a better understanding ofTyphi pathogenesis and open the way for the devel-opment of novel therapeutic targets.

S3:7PROTEOMIC ANALYSIS OF SALMONELLAENTERICA SEROVAR TYPHIMURIUMISOLATED FROM RAW 264.7MACROPHAGES: IDENTIFICATION OF ANOVEL PROTEIN THAT CONTRIBUTES TOTHE SURVIVAL OF SEROVARTYPHIMURIUM INSIDE MACROPHAGESL. Shi1, J. N. Adkins1, J. R. Coleman1, A. A. Schepmoes1, A. Dohnkova1,H. M. Mottaz1, A. D. Norbeck1, S. O. Purvine1, N. P. Manes1, H. S.Smallwood1, H. Wang1, J. Forbes2, P. Gros2, S. Uzzau3, K. D. Rodland1, F.Heffron4, R. D. Smith1, T. C. Squier1; 1Pacific Northwest NationalLaboratory, Richland, WA, 2McGill University, Montreal, PQ , CANADA,3University of Sassari, Sassari, ITALY, 4Oregon Health Sciences University,Portland, OR

To evade host resistant mechanisms, Salmonellaenterica serovar Typhimurium (STM), a facultativeintracellular pathogen, must alter its proteomefollowing macrophage infection. To identify newcolonization and virulence factors that mediateSTM pathogenesis, we have isolated STM cells fromRAW 264.7 macrophages at various time-points


SPEAKER ABSTRACTS

following infection and used a liquid chromatogra-phy-mass spectrometry (LC-MS)-based proteomicapproach to detect the changes in STM proteinabundances. Because host resistance to STM infec-tion is strongly modulated by the expression of afunctional host resistant regulator, i.e., naturalresistance associated macrophage protein 1(Nramp1, also called Slc11a1), we have also exam-ined the effects of Nrmap1 activity on the changesof STM protein abundances. Total 315 STM pro-teins have been identified from isolated STM cellsby LC-MS, whose abundances are largely unchangedduring the time-course of infection and most oftheir functions are house-keeping related. However,39 STM proteins are strongly induced after infec-tion, suggesting their involvement in modulating thecolonization and infection. Of the 39 inducedproteins, 6 proteins are specifically modulated byNramp1 activity, including STM3117, as well asSTM3118-3119 whose time-dependent abundancechanges were confirmed using Western blot analy-sis. Deletion of the gene encoding STM3117 re-sulted in a dramatic reduction in the ability of STMto colonize wild-type RAW 264.7 macrophages,demonstrating a critical involvement of STM3117in promoting the survival of STM inside macroph-ages. The predicted function common forSTM3117-3119 is biosynthesis and modification ofpeptidoglycan layer of STM cell wall, emphasizingtheir critical roles in the colonization of Salmonellain macrophages.

S4:2

SIGNAL TRANSDUCTION BY THE PHOP/PHOQ REGULATORY SYSTEM

E. A. Groisman, Howard Hughes Medical Institute, Department ofMolecular Microbiology, Washington University School of Medicine, St.Louis, Missouri

The PhoP/PhoQ two-component system is a majorregulator of Salmonella virulence, controlling theexpression of products that are necessary at differ-ent steps during the infection process. The DNA-binding protein PhoP accomplishes this task by

binding to the promoter regions of its regulatedgenes and thus exerting direct transcriptionalcontrol as well as indirectly, by controlling the levelsand/or the activity of regulatory proteins that aredirectly involved in regulating gene transcription.We have determined the mechanism by PhoPprotein can enhance the levels of RpoS, the alterna-tive sigma factor of RNA polymerase, which hadbeen previously implicated in Salmonella virulenceand in the adaptation to a variety of nutritionallimitation and other stressful conditions. We havealso established the critical role that the positivefeedback loop that controls expression of thephoPQ operon plays for Salmonellq’s ability to cause alethal infection in mice. These mechanisms ensurethat PhoP-regulated determinants are produced atthe right time and in the correct amounts.

S4:3INTERACTION OF SALMONELLA WITH ITSMAMMALIAN HOST CELLJ. E. Galan; Yale School of Medicine, New Haven, CT

The bacterial pathogen Salmonella enterica hasevolved a very complex functional interphase withits host, the product of the work of co-evolutionaryforces operating over millions of years. Central tothis interface is the work of a specialized bacterialorganelle, the type III secretion system, whichdelivers a battery of bacterial proteins into the hostcell. These proteins have the capacity to modulate avariety of cellular processes ranging from actincytoskeleton rearrangements and nuclear responsesto macropinocytosis and programmed cell death. Atheme emerging from structural and functionalstudies of the Salmonella/host interactions is oneof mimicry as a strategy to modulate cellular func-tions. These bacteria utilize proteins that faithfullymimic, structurally and functionally, the activities ofhost cell protein to modulate a variety of responses.Two bacterial proteins, SopE and SopE2, work asexchange factors for the Rho-family GTPases,Cdc42 and Rac. Remarkably, although throughdifferent chemistry, the conformational changesinduced by these bacterial proteins in the hostGTPases, which result in nucleotide exchange, are

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virtually identical to those induced by Dbl-likeGEFs. In a remarkable Ying and Yang, anotherbacterial protein, SptP, mimics host GTPase-activat-ing proteins (GAPs) to reverse the bacterial-inducedactivation of Cdc42 and Rac. In this case, theSalmonella protein uses the same chemistry as hostGAPs (i. e. insertion of a key arginine) but utilizingdifferent structural context. These and other ex-amples will be discussed to illustrate the power ofmimicry as a central strategy to modulate cellularfunctions by microbial pathogens.Selected ReferencesMarlovitz, T. C., Kubori, T., Lara-Tejero, M., Tho-mas, D. R., Unger, V. M., and J. E. GalÃ¡n. 2006.Nature 441(7093):637-40.Akeda, Y., and J. E. GalÃ¡n. 2005. Nature 437:911-5.Marlotivs, T., Kubori, T. Sukhan, A, GalÃ¡n, J. E.and V. Unger. 2004. Science 306:1040-2.Hernandez, L. D., K. Hueffer, M. R. Wenk, J. E.GalÃ¡n. 2004. Science 304:1805-1807.Kubori, T. and J. E. GalÃ¡n. 2003. Cell 115:333-342.Galkin, V. E., A. Orlova, M. S. VanLoock, D. Zhou,J. E. GalÃ¡n, and E. H. Egelman. 2002. NatureStruct. Biol. 9:518-21.Stebbins, C. E. and J. E. GalÃ¡n. 2001. Nature.412:701-5.Stebbins, C. E. and J. E. GalÃ¡n. 2000. Mol. Cell 6:1449-1460.Fu, Y., and J. E. GalÃ¡n. 1999. Nature 401:293-297.Zhou, D., M. Mooseker, and J. E. GalÃ¡n. 1999.Science 283:2092-2095.Hardt, W.-D., Chen, L.-M., Schuebel, K. E., Bustelo,X. R., and GalÃ¡n, J. E. 1998. Cell 93, 815-826.

S4:4IDENTIFICATION OF GENES ANDPATHWAYS REQUIRED FOR SPI-1 TYPE IIISECRETION SYSTEM ACTIVITY USING ACONTACT-DEPENDENT HAEMOLYSISASSAYA. N. Layton, T. R. Field, E. Morgan, E. E. Galyov; Institute forAnimal Health, Berkshire, UNITED KINGDOM

Salmonella can cause lysis of erythrocytes in a con-tact-dependent manner through a mechanisminvolving the formation of pores in the erythrocyte

membrane by the Salmonella Pathogenicity Island -1(SPI-1)-encoded type III secretion system (TTSS-1)translocator proteins, SipB and SipC. We adaptedthis haemolysis assay to a 96-well plate format andused it to screen a bank of Salmonella Typhimuriumsignature-tagged transposon insertion mutants withthe aim of identifying novel genes and pathwaysrequired for the normal functioning of TTSS-1.Such pathways may include those involved in generegulation, protein expression, and assembly orstability of the needle complex. Over 2000 mutantswere screened in the haemolysis assay, of which 42were unable to induce haemolysis or showed muchreduced haemolytic activity. The mutants wereidentified by mapping the transposon insertion sites.As expected a number of the mutants containedtransposon insertions in genes encoding the TTSS-1apparatus and translocator proteins. Mutants withinsertions in genes other than those encoding theTTSS-1 and its known regulators and secretedproteins were selected for further analysis. Ex-amples of such genes include fimW and rfbU. Someresults of the detailed analysis of these mutants arereported here and the data provide new insightsinto novel pathways involved in the normal func-tioning of TTSS-1.

S4:5SALMONELLA’S TYPE III SECRETIONSYSTEM TARGETS PMNS IN THE MURINESPLEENK. Geddes, F. Heffron; Oregon Health and Sciences University, portland,OR

We wished to investigate the in vivo secretion pat-terns of both SPI-1 and SPI-2 type three secretionsystems (TTSS). We hypothesized that in the mousespleen, SPI-2 secretion would predominate and thatthe cells targeted for secretion would primarilyconsist of macrophages. We did not rule out thepossibility of SPI-1 secretion in the spleen, how-ever, if such secretion was observed, we postulatedthat both phagocytic and non-phagocytic cellscould be targeted. In order to detect secretion inhost cells, we constructed chromosomal beta-lactamase fusions to several effectors dependent onSPI-1 or SPI-2 for their secretion. Using the fluo-

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rescent substrate CCF2, whose emission spectrashifts upon cleavage by beta-lactamase, we moni-tored the secretion of effectors both by microscopyand FACS analysis. Strains expressing effector-beta-lactamase fusions were used to infect C57BL/6mice via IP injection, and mice were sacrificed 2days post inoculation. FACS analysis of CCF2-stained spleen cells revealed that SPI-2 is respon-sible for the observed secretion. CCF2-stainedspleen cells were stained with antibodies to detectlymphocytes and granulocytes then FACS analysiswas performed to identify the cell types that weretargeted for secretion. Surprisingly, most of thesecretion was detected in GR-1+/CD11b+ cells.CD11b+/GR-1+ cells were FACS sorted andidentified as PMNs based on nuclear morphology.PMNs have previously been shown to be the pri-mary cell type harboring Salmonella 1-day post IVinfection (Dunlap et al. Microbial Pathogenesis 1992;13: 181-190). However, in their study they did notdemonstrate that the Salmonella remained viable.Therefore, we sorted GR-1+/Cd11B+ cells, platedthem for CFUs and found that these cells containedgreater than 25-fold more bacteria than the samenumber of unsorted spleen cells. Our results showthat only SPI-2 secretion is detected in the C57B/6mouse spleen, and that the majority of the secretionis a result of viable bacteria found in PMNs.

S4:6SALMONELLA ENTERICA SEROVARTYPHIMURIUM EFFECTORS SOPB, SOPE,SOPE2, AND SIPA DISRUPT TIGHTJUNCTION STRUCTURE AND FUNCTIONE. C. Boyle, N. F. Brown, B. B. Finlay; Univeristy of British Columbia,Vancouver, BC, CANADA

Salmonella enterica serovar Typhimurium is a majorcause of human gastroenteritis. Infection of epithe-lial monolayers by S. Typhimurium disrupts tightjunctions which normally maintain the intestinalbarrier and regulate cell polarity. Tight junctiondisruption is dependent upon the Salmonella Patho-genicity Island-1 (SPI-1) type III secretion system(T3SS) but the specific effectors involved have notbeen identified. In this study we demonstrate thatSopB, SopE, SopE2, and SipA are the SPI-1-se-

creted effectors responsible for disruption of tightjunction structure and function. Tight junctiondisruption by S. Typhimurium was prevented byinhibiting host protein geranylgeranylation but wasnot dependent on host protein synthesis or secre-tion of host-derived products. Unlike wildtype S.Typhimurium, DELTAsopB, DELTAsopE/E2,DELTAsipA, or DELTAsipA/sopB mutants,DELTAsopB/E/E2 and DELTAsipA/sopE/E2mutants were unable to increase the permeability ofpolarized epithelial monolayers, did not disrupt thedistribution or levels of ZO-1 and occludin, and didnot alter cell polarity. These data suggest that SPI-1-secreted effectors utilize their ability to stimulateRho family GTPases to disrupt tight junctionstructure and function.

S4:7ANALYSING THE INTERPLAY BETWEENSALMONELLA EFFECTOR PROTEINSC. A. Perrett, M. A. Jepson; University of Bristol, Bristol, UNITEDKINGDOM

The virulence of Salmonella enterica depends on theirability to enter and survive in host cells. The Salmo-nella Pathogenicity Island I (SPI-1) type threesecretion system translocates bacterial effectorproteins into host cells, where, by subverting thehost cellular machinery to remodel the actin cytosk-eleton and plasma membrane, they mediate theuptake of Salmonella into the cell. Salmonella invasionprotein A (SipA) and Salmonella outer proteins Eand E2 (SopE/E2) modulate cell actin dynamics:SipA binds F-actin, while SopE/E2 stimulate hostRho GTPases. Activation of Rho GTPases modu-lates the actin cytoskeleton resulting in the mem-brane ruffles that drive uptake of Salmonella. SipAdeletion was originally shown not to affect invasionover prolonged infection time courses but subse-quent examination of early time points revealedthat SipA does subtly enhance invasion at theseearly stages of infection. The exact role and impor-tance of SipA remains controversial, and contradic-tory data have been reported regarding the role ofSipA in membrane ruffle formation. We are exam-ining the hypothesis that the role of SipA in inva-

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sion may vary between Salmonella strains, and aretherefore examining the effect of SipA deletion inserovar Typhimurium strains, including those thatpossess or lack SopE. We have compared sipA nullmutants with respect to the number, morphologyand dynamics of the membrane ruffles they induce,and their ability to invade cultured epithelial cells,using a range of microscopy techniques, includingconfocal and electron microscopy and live cellimaging. These studies have confirmed that SipAhas no obvious effect on the number of rufflesformed during infection and reveal that it optimisesentry of all strains into MDCK cells, although theextent of the invasion defect associated with SipAdeletion varies dramatically between Typhimuriumstrains. The SipA- SopE- Salmonella strains examinedto date have been found to induce ruffles withmarkedly different morphology compared to thatpreviously described for wild type and sipA nullmutants, including the presence of multiple finger-like protrusions and numerous filopodia, bothstructures being highly dynamic when examined bylive cell imaging. In describing a novel role for SipAin membrane ruffling induced by certain strains, ourdata reveal that SipA may be more important inSalmonella invasion than previously thought; inparticular, SipA may play a key role in induction ofinvasion-competent membrane ruffles in strainswhere SopE is absent.

S5:2ACTIVATION OF THE PRO-SURVIVALKINASE AKT BY SALMONELLA IN INFECTEDEPITHELIAL CELLSO. Steele-Mortimer; Laboratory of Intracellular Parasites, NIAID,NIH, Hamilton, MT

Invasion of non-phagocytic epithelial cells bySalmonella enterica is mediated by a type III secre-tion system (T3SS1) that translocates several bacte-rial effector proteins into the host cell. Several ofthese effector proteins coordinately induce actinrearrangements leading to membrane ruffling andthe subsequent uptake of Salmonella. In addition,T3SS1 effectors can interdict host cell signal trans-duction pathways with significant ramifications far

beyond invasion. One such effector, SopB/SigD, isan inositol phosphatase that mediates activation ofthe serine-threonine kinase Akt (also known asPKB) a key mediator of metabolism, cell survival,motility, transcription and cell-cycle progression.Canonical Akt activation is a short-lived, highlyregulated process dependent on phosphoinositide(PI) 3 kinase. PI 3 kinase-mediated generation ofPI(3,4,5)P3 causes translocation of Akt to theplasma membrane where it is activated via a processthat involves phosphorylation at two distinct sites.Thr308 phosphorylation is mediated by PDK1,however, the identity of the second kinase respon-sible for Ser473 phosphorylation remains controver-sial. Recently the rapamycin-insensitive mTORcomplex, which contains rictor, has been implicatedin this step but other kinases may participate underdifferent conditions. Intriguingly SopB-dependentAkt activation diverges from this canonical pathwayin several respects. In particular, it is unaffected bythe PI 3 kinase-inhibitor wortmannin and exhibits asustained activation state. To better understand thisprocess, and the role of Akt activation in Salmo-nella pathogenesis, we are characterizing the rolesof the Akt isoforms, Akt1 and Akt2, and Aktregulators in HeLa cells infected with Salmonellaenterica serovar Typhimurium.

S5:3H-NS AND FERRITIN B - THE NOVELCONTRIBUTIONS OF TWO BACTERIALDEFENSE SYSTEMS TO SALMONELLAPATHOGENESISF. C. Fang; University of Washington, Seattle, WA

Bacteria must defend themselves from a wide rangeof external insults including foreign DNA andoxidative stress. This talk will examine the contribu-tions of two resistance proteins, H-NS and FerritinB, to Salmonella pathogenesis. H-NS is an abundantsmall nucleoid-associated protein that can act as atranscriptional repressor and multimerize intohigher-order bridging complexes following DNAbinding. H-NS selectively silences foreign DNA byrecognizing and targeting sequences with AT-content higher than the resident genome. Salmo-nella Typhimurium sequences bound by H-NS

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include all of the pathogenicity islands and the spvplasmid virulence genes. “Xenogeneic silencing” byH-NS protects bacteria from detrimental conse-quences of invading DNA and accounts for thestriking predilection of H-NS to repress horizon-tally-acquired virulence determinants. AdditionalDNA-binding proteins such as SlyA act as anti-silencers by displacing H-NS at specific promoters,thereby enabling expression of the foreign genesunder conditions advantageous to the bacteria.Ferritin B is one of four ferritins produced bySalmonella that play a fundamental role in ironhomeostasis by sequestering iron within the cell tolimit oxidative stress and providing a storage formof iron. Ferritin B possesses distinctive features: (1)Absence of a conserved ferroxidase domain; (2) Aprimary role in iron-sulfur cluster repair despitelittle contribution to total Fe storage capacity orfree iron levels; (3) Expression during iron-repleteconditions; (4) Exacerbation of oxidative stress inthe absence of other ferritins; and (5) An essentialrole in Salmonella virulence. These characteristicsare best rationalized by a role of Ferritin B as afacile reservoir of Fe(II) that can be readily mobi-lized for the repair of iron-sulfur clusters damagedby reactive oxygen species. Salmonella has evolvedinto an effective pathogen through both the hori-zontal acquisition of novel virulence determinantsand the adaptation of existing stress responses tocounter innate immune defenses. These observa-tions illustrate how H-NS has allowed Salmonella torecruit and integrate new genetic elements intoexisting regulatory networks, and how Ferritin B hasenabled Salmonella to counter reactive oxygenspecies generated by the host.

S5:4PYROPTOSIS: CASPASE-1-DEPENDENTPORE FORMATION LEADS TO OSMOTICLYSIS OF HOST MACROPHAGES INFECTEDWITH SALMONELLAB. T. Cookson, S. L. Fink; University of Washington, Seattle, WA

Salmonella enterica serovar Typhimurium invades hostmacrophages and induces caspase-1-dependent celldeath, which is called pyroptosis because of the

inflammatory consequences of the process. Thispathway likely plays an important role in a varietyof pathological events, yet the mechanism ofpyroptosis remains poorly defined. We show thatDNA cleavage during pyroptosis results fromcaspase-1-stimulated nuclease activity. FragmentedDNA activates poly(ADP-ribose) polymerase(PARP) and depletes cellular ATP to cause lysisduring oncosis, in contrast, the rapid lysis character-istic of Salmonella-infected macrophages does notrequire PARP activity or DNA fragmentation.Increased macrophage membrane permeability tosmall molecules appears to initiate the final steps ofcellular destruction: the formation of membranepores between 1.1 and 2.4 nm in diameter causehost cell swelling and osmotic lysis, resulting in therelease of intracellular contents. Pore formationrequires host caspase-1 activity and actin cytoskel-eton rearrangements. Induction of pyroptosis bySalmonella requires the bacterial Type III secretionsystem (TTSS), but inserting functional TTSStranslocons into the host membrane is not sufficientto directly evoke pore formation during infection atlow MOI. Concurrently with pore formation, andtemporally preceding lysis, inflammatory cytokinesare released from infected macrophages. These datafurther define the mechanism of caspase-1-medi-ated cell death and support the notion thatpyroptosis is a novel pathway of inflammatory celldeath.

S5:5SALMONELLA BILE SENSING ANDBILE-MEDIATED BIOFILM FORMATIONR. W. Crawford1, D. L. Gibson2, W. W. Kay2, J. S. Gunn1; 1The OhioState University, Columbus, OH, 2University of Victoria, Victoria, BC,CANADA

Salmonellae interact with bile in the intestine andgallbladder, and bile affects expression of Salmonellagenes important for virulence phenotypes that likelyenhance colonization and persistence in bothorgans. We have previously demonstrated thatsalmonellae form biofilms on the surface of humangallstones, which may contribute to the develop-ment of the carrier state. To eliminate the relianceon human gallstones, an in vitro assay of biofilm

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formation on cholesterol gallstones was developed.We show that cholesterol-coated Eppendorf tubessupport Salmonella biofilm formation (TBA, tubebiofilm assay), suggesting that salmonellae bind tocholesterol on human gallstones. The TBA, likeassays using human gallstones, has shown biofilmformation to be enhanced by bile. This enhance-ment was shown to be due to the effect of bile onSalmonella and its biofilm forming capacity. Al-though cellulose and colanic acid are the primaryextracellular matrix products of Salmonella biofilmson various surfaces, a double mutant lacking thesefactors formed mature biofilms on gallstones and inthe TBA, suggesting that a novel exopolysaccharide(EPS) was involved in gallstone biofilms. A newlyidentified capsular structure, SEPS, was shown tobe enhanced by the presence of bile. The presenceof SEPS in the TBA and on gallstone biofilms wasexamined by ELISA and immunofluorescent mi-croscopy, showing it to be highly expressed withincholesterol biofilms. The SEPS encoding operon(yih operon) was identified and shown to be tran-scriptionally activated in the presence of bile. Abetter understanding of the transcriptional responseto bile as well as constituents of Salmonella gallstonebiofilms, such as the EPS, promises to better char-acterize the asymptomatic carrier state and lead tonovel drug therapies.

S5:6SODCI TETHERING WITHIN THE PERIPLASMCONTRIBUTES TO SALMONELLAVIRULENCEB. Kim, R. Krishnakumar, J. M. Slauch; University of Illinois, Urbana, IL

Salmonella encounter a number of antimicrobialmolecules including superoxide and other reactiveoxygen species during replication in macrophages.Salmonella typhimurium encodes two periplasmicsuperoxide dismutases, SodCI and SodCII. OnlySodCI plays a major role in Salmonella virulence,although both proteins are expressed in vitro and invivo. Thus some difference inherent in the twoproteins determines effectiveness during infection.SodCI is a dimer, where SodCII is monomeric.Also, SodCII, but not SodCI, is released by osmotic

shock. We created a monomeric SodCI by sitedirected mutagenesis. This mutant protein was fullyenzymatically active, but was released by osmoticshock and could not contribute to Salmonella viru-lence. These data suggest that SodCI tetheringwithin the periplasm is critical for Salmonella viru-lence. We hypothesize that during infection cationicantimicrobial peptides (CAMPs) from macrophagedisrupt the outer membrane, releasing periplasmicproteins. SodCI would be retained, providing arationale for why tethering is important for viru-lence. We found that osmotic shock and polymyxinB treatment are analogous and SodCII and mono-meric SodCI are preferentially released by poly-myxin B, while dimeric SodCI is retained. Recently,it is shown that mouse macrophages produceCathelicidin related antimicrobial peptide(CRAMP). We show that SodCI is also preferen-tially tethered within the periplasm upon CRAMPtreatment, whereas SodCII and monomeric SodCIare released. Currently this hypothesis is beingtested in mouse and macrophage models of infec-tion.

S5:7METAL ION SENSING DURINGSALMONELLA INFECTIONC. M. Taylor, D. Osman, J. S. Cavet; University of Manchester,Manchester, UNITED KINGDOM

The ability of intracellular Salmonella to competefor essential metal ions is crucial for survival andgrowth. However, sudden fluctuations in redoxactive metals accompanied by a sudden change inthe nature of reactive oxygen species may alsocharacterize bacterial killing within phago-lysos-omes. We are determining the nature of the metalstresses encountered by Salmonella within mac-rophage phagosomes, our aims being to resolve (i)whether Salmonella is exposed to elevated or de-pleted metal levels in late phago-lysosomes, (ii)which are the predominant metals that fluctuate inconcentration (as sensed by Salmonella), and (iii)the principle substrates of Slc11a1 (Nramp1) anddirection of transport. Metal-responsive promoterscan report bacterial cytosolic metal levels. We have

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therefore precisely calibrated the metal specificitiesof various metal-responsive promoters in S.typhimurium to directly monitor changes in thelevels of iron and other metals â€~sensedâ€™ bythis pathogen during macrophage infection. Theseinclude the iron-responsive promoters for ftnA(encoding ferritin), sodB (for iron-superoxidedismutase) and iroBCDE (for salmochelinglycosylation) from S. typhimurium. Expressionfrom both PftnA and PsodB is low in iron-depletedmedia but substantially increases in response toelevated concentrations of iron, but no othermetals. In contrast, expression from PiroBCDE iselevated in iron depleted media but is low in mediasupplemented with iron, cobalt and, to a lesserextent, manganese, but no other metals. We havealso calibrated the responses of several promoterswith different metal specificities, including copper,zinc, manganese and cobalt. Various other environ-mental stresses (including oxidative stress) do notalter expression from these promoters confirmingtheir feasibility for reporting phagosomal metallevels. Progress in identifying the changes in metallevels that occur during infection of Slc11a1+/+and Slc11a1-/- macrophages, and the influence ofGp91Phox, will be presented.

S6:2MULTIDRUG RESISTANT NON-TYPHOIDALSALMONELLA INFECTIONS IN CHILDREN,KENYAS. Kariuki1, G. Revathi2, J. Kiiru1, J. Mwituria1, C. A. Hart3; 1KEMRI,NAIROBI, KENYA, 2Aga Khan University Hospital, NAIROBI,KENYA, 3University of Liverpool, Liverpool, UNITED KINGDOM

Background: In sub-Saharan Africa community-acquired non-typhoidal Salmonella (NTS) is a majorcause of high morbidity and death among childrenunder 5 years of age especially from resource poorsettings. Early diagnosis and appropriate treatmentof life threatening invasive NTS infections poses amajor challenge in these settings. Methods: Pa-tients were children under 5 years of age admittedwith NTS bacteremia and those that presented withgastroenteritis. We obtained data on clinical presen-tation and serotype distribution of NTS. Antimicro-bial susceptibility of NTS isolates was performed

by MIC method and genetic relatedness of isolateswas determined by pulsed field gel electrophoresis(PFGE). Results: Overall 170 (51.2%) of childrenpresented with bacteraemia alone, 28 (8.4%) withgastroenteritis and bacteraemia and 134 (40.4%)with gastroenteritis alone. NTS serotypes obtainedfrom all the cases included S. Typhimurium (196;59%), S. Enteritidis (94; 28.3%) and other serotypesin smaller numbers (42; 12.7%); distribution ofthese serotypes among cases with bacteremia orgastroenteritis was not significantly different.Significantly higher proportion of invasive NTSwere obtained from younger children (< 3 years ofage) and those from the slums compared to olderchildren and those from upper socio-economicgroups (p < 0.001). 147 (44.3%) NTS were resistantto 3 or more antimicrobial agents, and out of these59% were resistant to ampicillin, chloramphenicoland tetracycline. There was no significant differencein antibiotic resistance between the two serotypes,S. Typhimurium and S. Enteritidis. Ceftriaxone andciprofloxacin were the only antimicrobials tested towhich all the NTS were fully susceptible. UsingPFGE there were 3 main PFGE patterns for S.Typhimurium and 2 patterns for S. Enteritidisamong cases of bacteraemia and gastroenteritis.Conclusion: Serotype distribution, antibioticsusceptibility and genotypes of NTS causingbacteraemia and gastroenteritis did not differsignificantly. The high prevalence of NTS strainsresistant to most of the commonly used antimicro-bials raises major concern in management of theseinfections.

S6:4SALMONELLA INHIBIT T CELLPROLIFERATION BY A DIRECT,CONTACT-DEPENDENTIMMUNOSUPPRESSIVE EFFECTA. W. van der Velden, M. K. Copass, M. N. Starnbach; HarvardMedical School, Boston, MA

T lymphocytes play a pivotal role in controlling andclearing infection with intracellular pathogens.During infection, professional antigen presentingcells, including dendritic cells (DC), internalize,process, and present microbial antigens to T cells.

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Upon antigen recognition, T cells become activated,proliferate, and acquire effector functions thatinclude cytokine production and the ability todirectly lyse infected cells. We report that DC, whenco-cultured with Salmonella, fail to efficiently stimu-late T cells for proliferation and production ofIFNgamma. We further demonstrate that the failureof T cells to respond to Salmonella-infected DC isnot simply due to Salmonella-induced programmedDC death, suggesting that Salmonella may directlyinhibit T cell replication. To test this hypothesis, weinfected T cells in the absence of DC and demon-strate that Salmonella have a direct, contact-depen-dent inhibitory effect on the T cell resulting inreduced proliferation and cytokine production inresponse to T cell receptor cross-linking. We haveobtained evidence indicating that a soluble inhibi-tory factor is secreted into the culture supernatantupon contact between Salmonella and the T cell. Wehave used both genetic and biochemical approachesto characterize this direct, contact-dependentinhibitory phenotype. Cumulatively, our results areevidence that Salmonella may interfere with thedevelopment of acquired immunity, providing newinsights into the complex nature of this host-pathogen interaction.

S6:5AN IMPROVED LIVE ORAL SALMONELLAENTERICA VACCINE WITH REDUCEDFECAL SHEDDINGM. Abd E L Ghany, G. Dougan, R. A. Kingsley; The wellcome TrustSanger Institute, Hinxton, UNITED KINGDOM

The ability of Salmonella enterica serotypes totarget immune cells during natural infections hasresulted in considerable interest in their use as liveoral vaccines and antigen delivery systems. How-ever, shedding of genetically manipulatedmicroorgansisms into the environment is one of theissues confronting developments in the field ofnovel multivalent vaccines. A novel S. typhi vaccinecandidate with mutations in the aroC and ssaVgenes has been reported to be more immunogenicthan the currently licensed live oral vaccine, Ty21a,in volunteer studies. Furthermore, this vaccinecandidate is only transiently shed with the feces.

However, development of multivalent S. Typhivectors expressing heterologous antigen has beenunsuccessful to date. Impressive immune responseto antigen expressed in attenuated S. Typhimuriumstrains during experimental infections of mice in anumber of different studies has been reported. Incontrast little or no response was observed toheterologous antigens expressed in S. Typhi vaccinecandidates in volunteer studies. However, a S.Typhimurium phoP/Q vaccine candidate expressingHelicobacter pylori urease did generate an immuneresponse in human volunteer studies suggesting thatS. Typhimurium may be a better multivalent vaccinevector. However, unlike that observed with S. Typhivaccinees, the intestine of volunteers dosed with S.Typhimurium vaccine candidates become heavilycolonised, as indicated by fecal shedding. We haveinvestigated the immunogenicity of a S.Typhimurium vaccine strain (Î”aroA) expressing anheterologous antigen (TetC) that also containadditional mutations in genes required for persistentintestinal colonisation. Mutations in the shdA andmisL genes resulted in a marked decrease in num-bers of the vaccine strain shed in the feces. How-ever, similar humoral and cellular immune responseswere detected in all vaccinated mice regardless ofthe additional mutations in the shdA or misL genes.Immunisation with the modified vaccine strain(containing shdA and misL double mutation)protected against an otherwise lethal challenge witha virulent strain of S. Typhimurium.

S6:6VACCINAL PROTECTION IN BROILERCHICKENS BASED ON THECOLONIZATION-INHIBITION MECHANISM:IN SEARCH OF THE IDEAL VACCINESTRAINL. Bohez, V. Eeckhaut, F. Pasmans, F. Haesebrouck, R. Ducatelle, F.Van Immerseel; Department of Pathology, Bacteriology and Avian Diseases,Research Group Veterinary Public Health and Zoonoses, Faculty ofVeterinary Medicine, Ghent University, Merelbeke, BELGIUM

Vaccination is regarded as a protective measurewhose protective effect begins after a period ofmaturation of the B- and T- cell response. Thus,after vaccination of one-day old chicks, production

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of significant amounts of specific antibodiesagainst Salmonella takes more than 10 days. Forinfections which may occur before this time, such asthose arising from hatchery infection, the immunitygap is too long. However, oral administration of liveSalmonella organisms can induce a very rapid protec-tion against challenge early in the life of the bird asa result of their colonisation-inhibiting activity. Ithas been shown that oral administration at day ofhatch of wild-type Salmonella strains inhibit coloni-zation by virulent strains of the same serotype thatare inoculated one day later. These data suggest thatit might be possible to administer live Salmonellavaccine strains to newly-hatched chicks such thatthey would colonize the gut extensively and veryrapidly, and that this should induce a profoundresistance to colonization by virulent Salmonellastrains of epidemiological significance, which maybe present in the poultry house or may also havearisen from the hatchery. One important restrictionto this approach is that the strains used as livevaccines should be cleared within 6 weeks in broil-ers, to prevent transmission of vaccine strains intothe food chain. A hilA mutant vaccine strain ofSalmonella Enteritidis or its parent strain were orallyinoculated at day of hatch and their capacity toinhibit colonization of a virulent strain of the sameserotype, inoculated one day later, was evaluated.The vaccine parent strain colonized the caeca andstrongly inhibited colonization of the virulentchallenge strain. The hilA mutant vaccine strain alsocolonized the caeca the first days after inoculation,but the number of organisms in the caeca decreaseddramatically over time. The hilA mutant vaccinestrain was almost completely cleared at 28 days ofage. The colonization of the caeca by the virulentchallenge strain was initially strongly inhibited inthe hilA mutant vaccinated chicks, but increaseddramatically at the moment the hilA mutant vaccinestrain seriously decreased in number, thus at about7 days of age. These results suggest that longerpresence of the vaccine strain bacteria is required tomaintain protection, and it can be questionedwhether a strain can be found that colonizes longenough to acquire complete protection, but still iscleared rapidly thereafter from chicken gut and

tissues, and therefore can be regarded as safe forthe consumer.

S6:7ANTI-INFLAMMATORY SALMONELLAVACCINE THERAPY ELICITS REGULATORYT CELLS AND PROTECTS AGAINSTAUTOIMMUNITY INDEPENDENT OFAUTOANTIGEND. W. Pascual, I. Kochetkova, T. Trunkle, J. Ochoa-Repáraz; MontanaState University, Bozeman, MT

Regulatory T (Treg) cells show promise for treatingautoimmune diseases, but their induction to el-evated potency has been problematic when themost optimally derived cells are from diseasedanimals. To circumvent reliance on auto-antigenreactive Treg cells, stimulation to vaccine antigens(Ags) may offer a viable alternative while maintain-ing potency to protect against proinflammatorydiseases, experimental autoimmune encephalomyeli-tis (EAE) and collagen-induced arthritis (CIA). Ourattenuated Salmonella vaccine expressing coloniza-tion factor Ag I (CFA/I) possesses anti-inflamma-tory properties: elevated Th2 cell (IL-4-dependent)immune responses, delayed onset of Th1 cells,reduced inflammatory cell infiltrates in the PeyerB˚,)ü¨�s patches, and an absence of TNF-á, IL-1,and IL-6 production from infected macrophages.Given these findings, we hypothesized whether thisvaccine would be therapeutic for EAE. To test thishypothesis, SJL mice were challenged with proteo-lipid protein, PLP139-151, by standard convention andafter 6 days, mice were orally treated with Salmo-nella-CFA/I. The treated mice showed greatlyreduced clinical disease that completely resolvedwhen compared to PBS-treated controls. Histo-pathological studies showed reduced demyelinationand no inflammation of spinal cords when com-pared to PBS- or Salmonella vector-treated mice. Toascertain whether the observed immune deviationwas in part supported by Treg cells, FACS analysisrevealed a 4-fold increase in Foxp3+ CD25+ CD4+ Tcells in vaccine-treated mice. Adoptive transfer ofthese induced TGF-beta+ Treg cells from vaccinatedmice showed complete protection in PLP139-151-challenged mice, but not by naive Treg cells. More-

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over, partial protection to EAE was also conferredby adoptive transfer of CD25- CD4+ T cells sug-gesting that Th2 cells also contributed. Likewise,IL-4- and TGF-beta-producing Treg cells and IL-10-producing (CD25-) Tr1 cells contributed to protec-tion against CIA in DBA/1 mice. Thus, these datashow that Treg cells, both CD25+ and CD25-, areinduced by oral vaccination with Salmonella-CFA/Icontributing to the efficacious treatment of EAEand CIA. This work is supported by NIH AI-41123.

S7:1

RECENT ADVANCES IN THEIDENTIFICATION ANDCHARACTERIZATION OF THE COMPLEXEFFECTOR IMMUNE RESPONSES ELICITEDBY ORAL IMMUNIZATION WITHATTENUATED SALMONELLA ENTERICASEROVAR TYPHI STRAINS IN HUMANSMarcelo B. Sztein, Center for Vaccine Development, University ofMaryland, Baltimore, MD, USA

The development of improved typhoid vaccines toprevent antibiotic-resistant typhoid fever in devel-oping countries is a high global public health prior-ity. However, the development of improved liveoral typhoid vaccines has been hampered by a lackof information of the specific determinants ofprotective immunity to S. Typhi infection in humans.Moreover, the effector and memory immune re-sponses to S. Typhi in circulation and in the gutmicroenvironment have not been characterized.Since the gut is the site of entry of S. Typhi, thepresence of effective and sustained effector im-mune responses in the local microenvironment arelikely to be pivotal in protection from infection.The lack of a suitable small animal model to studytyphoid fever has made the study of the immuneresponses to S. Typhi during the course of infectionand vaccination in humans very difficult. Resultsfrom studies in typhoid patients and vaccine trialswith attenuated S. Typhi strains suggest that al-though antibodies (Ab) to common S. Typhi anti-gens, particularly to O, H and Vi, appear to play arole in protection against S. Typhi infection, it is notknown if such Ab actually mediate protection, act

in conjunction with other adaptive responses orserve as a surrogate for the presence of other moredominant protective immune responses (e.g., cell-mediated immunity, CMI) that will eventually leadto the elimination of this intracellular bacteria fromthe host. We hypothesized that the survival of S.Typhi in the host depends on its ability to persistintracellularly, thereby avoiding destruction by Aband C’. Thus, strong CMI will be essential in elimi-nating S. Typhi infection. During the past decadewe focused our efforts on characterizing the sys-temic and mucosal short and long-term Ab andCMI induced by oral immunization with attenuatedS. Typhi live oral vaccines in US subjects in an effortto begin the identification of the adaptive effectorresponses that are critical for the development ofeffective typhoid vaccines. Phase I and II studiesperformed at the Center for Vaccine Development(CVD) with new recombinant strains of attenuatedS. Typhi oral vaccine candidates showed that thesewell-tolerated oral vaccines can elicit vigorousimmunity following administration of just a singledose. We demonstrated that immunization ofsubjects with attenuated S. Typhi strains (e.g., CVD908, CVD 908htrA, CVD 909), elicits potent sys-temic CMI, including proliferation, predominantlytype-1 cytokine responses (e.g., IFN-g, TNF-a) andthe generation of classical class Ia-restricted andnovel, non-classical class-Ib HLA-E-restricted,CD8+ cytotoxic T lymphocytes (CTL). We havealso characterized the mechanisms of this effectorresponse and determined the frequency of respond-ing cells elicited by S. Typhi immunization. Theseresponses were similar in scope and magnitude tothose observed following administration of 4 dosesof the licensed attenuated Ty21a strain oral typhoidvaccine. We have recently initiated the in depthcharacterization of memory T cell populations,including T central memory [TCM], T effectormemory [TEM] and CD45RA+ TEM [TEMRA] and theirhoming potential by mutichromatic flow cytometry.We observed that oral immunization of subjectswith an attenuated S. Typhi elicits diverse specificIFN-g-secreting CD4+ and CD8+ TCM and TEMpopulations that express, or not, the gut homingmolecule integrin a4/b7 , i.e., have the potential to

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migrate to the gut and to secondary lymphoidtissues. Moreover, we have generated CD4+ andCD8+ clones to initiate studies on the fine antigenspecificity of S. Typhi-specific CMI. Finally, wehave also addressed a series of critical questionsregarding the induction of immunity following oraladministration of attenuated S. Typhi strains,including (a) is there a correlation between Ab andCMI elicited in individual vaccinees?, (b) can theresponses be enhanced by the administration of a 2vaccine dose?, (c) what are the kinetics of inductionof the various effector immune responses and howlong do they persist?, (d) what is the clonality of theresponses?, and (e) what are the characteristics ofthe Ab responses (e.g., avidity, induction and persis-tence of memory B cells)? The results from thesestudies will be presented. In sum, we have uncov-ered that immunity to S. Typhi in humans elicitedby immunization is unexpectedly broad and com-plex, involving local and systemic Ab and CMIcomponents. The implications of these results fortyphoid vaccine development and protection fromnatural infection will be discussed.

S7:3VACCINATION AND CLINICAL DISEASE:LESSONS FROM THE MURINE MODELR. Strugnell, J. Price, K. Simpfendorfer, M. Galic, O. Wijburg; Universityof Melbourne, PARKVILLE, AUSTRALIA

The S. typhimurium murine model of typhoiddisease has revealed the detail of numerous interac-tions between the pathogen, and the innate andacquired immune systems. While fundamentaldifferences in the pathogenesis of S. typhi and S.typhimurium are obvious, eg. the effect of the Vicapsule and hence the prophylactic activity ofcapsule-specific antibodies, the murine model hasprovided considerable insight into the role ofeffectors such as interferon gamma (IFN-g) inresistance to primary infection and to re-infectionfollowing vaccination. While the murine model hasbeen particularly useful in testing experimentalvaccines, our recent studies have focussed on thenature of the responses which control primaryinfection and barriers to efficient natural transmis-

sion of S. typhimurium. The pIgR-/- mouse lacksthe capacity to actively secrete sIgA and sIgM. Thisanimal is extremely sensitive to natural infection byS. typhimurium - moreover, the animal acts as a‘superspreader’ of infection. Studies in the pIgR-/-mouse indicate that natural antibodies raised againstthe microflora and other environmental antigensappear to have a basal capacity to limit low doseSalmonella infection. Studies using the streptomy-cin-treated mouse model indicate that the size, butnot individual constituents, of the natural gut floraare also important in resistance to murine Salmonel-losis. Once the epithelial barrier has been breached,effective control of attenuated variants of S.typhimurium appears to be dependent on theproduction of IFN-g however the source of thiskey cytokine is not limited to CD4+ or CD8+ Tcells. Studies in mice transgenic for the expressionof antibodies which delete CD4+ and/or CD8+ Tcells have revealed that a population of ‘doublenegative’ CD3+ cells is capable of producingbacteriostatic levels of IFN-g. A similar cell hasbeen observed in the peripheral blood of HIV+individuals attempting to control fulminant Salmo-nella infections. Taken together, these data arguethat the innate defences including natural antibod-ies, flora and non-classical T cells are likely toprovide some protection against Salmonella infec-tions and that strategies that increase the potencyof these barriers may provide another means ofcontrolling Salmonella infection and transmission.

S7:4TRANSCRIPTIONAL RESPONSE TOSALMONELLA ENTERICA SEROVAR TYPHIINFECTION EXAMINED IN PERIPHERALBLOOD OF PATIENTS IN VIET NAL. J. Thompson1, S. J. Dunstan2, C. Dolecek2, T. Perkins3, D. House3,N. Dung2, T. Hien2, J. J. Farrar2, D. Monack1, G. Dougan3, S. Falkow1;1Stanford University, Stanford, CA, 2Center for Tropical Diseases, Ho ChiMinh City, VIET NAM, 3The Sanger Center, Cambridge, UNITEDKINGDOM

Salmonella enterica serovar Typhi (S. Typhi) is thecausative agent of Typhoid Fever, a systemic diseaseof man in which the bacterium disseminates to thereticuloendothelial system. Although various factors

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involved in the innate and adaptive immune re-sponses to S. Typhi are known, it is still not under-stood why these immune responses do not confercomplete protection against relapse or re-infection.We examined transcriptional signatures in theperipheral blood of individuals with Typhoid Feverin Viet Nam as compared with malarial patients anduninfected healthy controls. Multiple peripheralblood samples were drawn from each patient duringthe course of infection and analyzed using HumancDNA microarrays. Hierarchical clustering of thedata showed disease and temporal specificity in thetranscriptional profiles. Statistical analysis revealedseveral classes of genes up-regulated in acuteTyphoid as compared with healthy controls includ-ing those involved in cell cycle, immune responseand signal-transduction activity. Correlation of cellcount data with expression profiles confirmed thatincreased neutrophil activity could account for themajority of the induced gene set. The expression ofa large number of genes was also down-regulated inacute Typhoid, particularly a number of cell surfacereceptors, transcription factors and anti-prolifera-tive responses. This acute transcriptional responsewas found to be almost identical in an independentset of samples from patients with acute TyphoidFever, indicating the robustness of the response.Changes in gene expression were evident over thetime course of S. Typhi infection and convales-cence. Many of the perturbed gene expressionpatterns evident in the acute samples had returnedto normal in the day 28 and 6 month convalescentsamples, however distinct differences remained inthe majority of individuals at day 28 as comparedwith healthy controls. By 6 months four patientsstill retained this signature indicating a long-lastingresidual response to Typhoid infection. In summary,we were able to detect distinct and robust differ-ences in the transcriptional response to infection inblood RNA samples and these profiles convergedover time during treatment towards uninfectedcontrols, leaving a significant signature of convales-cence. The results have also revealed changes in anumber of gene categories with previously un-known involvement in Typhoid Fever pathogenesis.

S7:5INTRACELLULAR PERSISTENCE OF NTS INHIV - THE ROLE OF IFNÃ IN AN EX-VIVOHUMAN TISSUE MACROPHAGE MODELM. A. Gordon1,2, L. Musaya2, M. E. Molyneux2,1, S. B. Gordon1,2, R. C.Read3; 1Liverpool University, Liverpool, UNITED KINGDOM, 2WellcomeTrust Clinical Research Programme, Blantyre, MALAWI, 3SheffieldUniversity, Sheffield, UNITED KINGDOM

Background: Non-typhi salmonella (NTS)bacteraemia is a common invasive disease with 25-50% mortality among HIV-infected adults inMalawi. Bacteraemic recrudescence occurs in 40%of survivors, probably arising from intracellularpersistence within tissue macrophages. Clearance ofintracellular salmonellae is critically dependent ontype-1 cytokines. This model of persistence of NTSwithin ex-vivo human tissue macrophages fromadults with HIV infection tests the hypothesis thatthere may be important HIV-related defects inphagocytosis, killing, IFNã-responsiveness, andtype-1 cytokine secretion in response to Salmonellatyphimurium. Methods: Primary human alveolarmacrophages (AM) were harvested bybronchoalveolar lavage from 11 HIV-infected and10 HIV-uninfected volunteer Malawian adults. AMwere mock-primed or primed for 24h or 72h withIFNã, and challenged with S. typhimurium at d4.Internalisation of bacteria was studied by fluores-cent microscopy. Intracellular killing was assessedby gentamicin protection assay. Cytokine responseswere measured at 2h and 24h by cytometric beadarray. Data were analysed using 2-group and pairedtests of hypothesis and by ANOVA regressionResults: Unprimed AM: Internalisation and killingwere the same in unprimed AM from HIV infectedvs uninfected subjects (p=0.99 and p=0.12). Therewas, however, a marked increase in type 1 cytokinesecretion following challenge with S. typhimurium inunprimed AM from HIV-infected vs HIV-uninfected subjects (TNF p=0.020, IL-10 p=0.015,IL-12 p=0.03), despite equal baseline production byunchallenged control cells. In contrast, there was noincreased production of the pro-inflammatorycytokines IL-1b or IL-6 by AM from HIV-infectedsubjects. IFNã-primed AM: The effect of IFNã

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on AM from healthy adults was to reduceinternalisation (p=0.005) and to increase earlykilling (p<0.00001) of S. typhimurium. The reductionin internalisation was seen up to saturation MOI,occurred under different bacterial growth condi-tions, and was not mediated by downregulation ofFc or mannose receptors. There was equal IFNã-responsiveness of AM from HIV infected vsuninfected subjects for internalisation (p=0.61) andkilling (p=0.56). Discussion: These are the firstreported data from human primary tissue macroph-ages infected with NTS, and they confirm thecritical role of IFNã in phagocytosis and earlykilling. Reduced IFNã production in advancing HIVdisease may permit macrophage invasion andintracellular persistence, leading to clinical relapses.Additionally, these data demonstrate marked over-production of type-1 cytokines by AM from HIV-infected adults following challenge with S.typhimurium. This may be due to an intrinsic effectof HIV on the macrophage, or to the effect of thelung tissue milieu in HIV. Overproduction anddysregulation of type 1 cytokines may lead toadverse clinical outcomes in HIV.

S7:6PROPHYLACTIC ANTI-TUMOR IMMUNITYAGAINST A MURINE FIBROSARCOMATRIGGERED BY THE SALMONELLA TYPE IIISECRETION SYSTEMH. Russmann, K. M. Meinel, K. Panthel; University of Munich, Max vonPettenkofer-Institute for Hygiene and Medical Microbiology, Munich,GERMANY

The potential of an attenuated Salmonella entericaserovar Typhimurium strain as a prophylactic anti-tumor vaccine against the murine fibrosarcomaWEHI 164 was evaluated. Tumor cells were trans-fected with the DNA sequence encoding the MHCclass I-restricted peptide p60 217-225 from Listeriamonocytogenes. BALB/c mice received a singleorogastric immunization with Salmonella thattranslocates a chimeric p60 protein via its type IIIsecretion system. Mice were subsequently chal-lenged subcutaneously with p60 217-225 -express-ing WEHI cells. In vivo protection studies revealed

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that 80% of these mice remained free of the fibro-sarcoma after challenge, whereas all animals of thenon-vaccinated control group did develop tumorgrowth. In further experiments, the distribution oftetramer-positive p60 217-225 -specific effector andmemory CD8 T cells after Salmonella-based immu-nization and tumor application was analyzed.Costaining with CD62L and CD127 revealed apredominance of p60-specific central memory andeffector memory CD8 T cells in spleens, whereas inblood samples the majority of p60-specific lympho-cytes belonged to effector and effector memoryCD8 T cell subsets. This is the first report demon-strating that a bacterial type III secretion system canbe used for heterologous antigen delivery to inducecytotoxic effector and memory CD8 T cell re-sponses resulting in an efficient prevention oftumor growth.

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A1CHEMICAL-GENETICS IDENTIFIES HOSTKINASES AS TARGETS FOR ANTIBIOTICSGEARED TOWARDS INTRACELLULARBACTERIA SUCH AS SALMONELLA ANDMYCOBACTERIUM TUBERCULOSISN. D. Savage1, C. Kuijl2, M. Marsman2, E. Tuin3, L. Janssen2, D. Egan4,S. J. van den Eeden1, A. Geluk1, G. van Marcel3, R. Beijersbergen4, H.Overkleeft3, T. H. Ottenhoff1, J. J. Neefjes2; 1Department ofImmunohaematology and Bloodtransfusion, Leiden University Medical School(LUMC), 2 Albinusdreef, 2333ZA, Leiden, THE NETHERLANDS,2Division of Tumour Biology, Nederlandse Kanker Instituut (NKI),Plesmanlaan 121, 1066CX, Amsterdam, THE NETHERLANDS,3Department of Bioorganic Chemistry, Leiden Institute of Chemistry,2300RA, Leiden, THE NETHERLANDS, 4Division of ChemicalCarcinogenesis, Nederlandse Kanker Instituut (NKI), Plesmanlaan 121,Amsterdam, THE NETHERLANDS

With the increasing emergence of multidrug-resistant (MDR) bacteria, it is imperative to developnovel intervention strategies. Current antibioticstrategies focus on targeting pathogen-specificbiochemical pathways, providing a high degree ofprecision, but in the same instance allowing rapiddevelopment of drug resistance in cases of antibi-otic misuse. By assimilating forward and reversechemical-genetics we have identified and subse-quently developed chemical inhibitors specific forhost protein kinases which very rapidly inhibitgrowth in the intracellular infection phase ofvarious micro-organisms, ranging from Salmonellatyphimurium to Mycobacterium tuberculosis. Importantly,the kinase inhibitors also repressed intracellulargrowth of MDR M. tuberculosis. Identification of theprotein kinase(s) was performed by performing ashRNAi screen of the human kinome followed byinfection of GFP-expressing Salmonella and a highthroughput read out by flow cytometry. Using thismethod, we isolated eight host kinases capable ofinhibiting intracellular growth of salmonellae. Thekinases isolated are known to be involved in hostcytoskeletal modifications. Cloning the kinases andperforming in vitro kinase assays revealed that twoof the identified kinases were indeed inhibited bythe chemical compounds used in the initial chemi-cal-genetics screen. Extension of our studies in vivodemonstrated that, despite the lack of preciseknowledge on pharmaco-kinetics of these com-

POSTER ABSTRACTSPOSTER ABSTRACTS

pounds, administration of functional chemicalinhibitors to susceptible mice delayed Salmonellainduced lethality when compared to vehicle controlor non-inhibitory (but structurally similar) com-pounds. From these observations we propose thatmomentarily targeting host protein kinases with theuse of chemical compounds, is a novel strategy tocombat intracellular infection. This novel approachto developing antibiotics may be more difficult forthe pathogen to build resistance to. Furthermore,we established that different species of bacteriautilize similar pathways and mechanisms to evadeelimination from the host by phagosome matura-tion.

B2THE EFFECT OF THE SALMONELLAGENOMIC ISLAND 1 ON IN VITROGLOBAL GENE EXPRESSION INSALMONELLA ENTERICA SEROVARTYPHIMURIUM LT2G. R. Golding1, A. B. Olson1, B. Doublet2, A. Cloeckaert2, S.Christianson1, M. R. Graham1, M. R. Mulvey1; 1National MicrobiologyLaboratory, Winnipeg , MB, CANADA, 2Institut National de la RechercheAgronomique, Nouzilly, FRANCE

The 43 Kb Salmonella Genomic Island 1 (SGI1)harbours numerous antimicrobial resistance genesand has been associated with the emergence of theepidemic Salmonella enterica serotype Typhimurium(ST) DT104. In addition to the multi-drug resis-tance genes, the genes encoded within SGI1 mightalso play a role in hypervirulence. In this study weconstructed an SGI1 isogenic strain pair usingSalmonella enterica serovar Typhimurium LT2 (STLT2) for the purpose of examining the effect of theSGI1 on global gene expression. Expression studieswere conducted for ST LT2 +/- SGI1 using DNAexpression microarrays and reverse transcriptasequantitative real-time PCR (RT-PCR). DNAmicroarray analysis revealed twenty-two chromo-somal genes significantly upregulated in ST LT2harbouring SGI1. This included increased expres-sion of iron and sialic acid utilization genes, en-coded within the ccm and nan operons, respectively.Decreased expression was noted for 15 genes in STLT2 harbouring SGI1, including genes involved in


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bacterial culture. Bacterial strains were grown underSPI-1 conditions. Bacterial culture supernatants andpellets were used to investigate secreted-proteinsand cell-associated proteins, respectively. Expres-sion and secretion were analyzed by SDS-page andimmunoblotting with anti-FLAG antibodies. Invitro experiments showed that D220 mutant Salmo-nella Typhimurium secretes nearly half the amountof SPI-1 effector proteins (with the exception ofSopA) that are secreted by the wild type strain.These differences were statistically significant (p <0.05). We found that the secretion of SipA andSopE2 in D220 dam mutant is reduced by 40 %compared to the virulent strain, whereas the secre-tion of SopB and SopD is diminished by 38% and50%, respectively. The amount of SopA detected inthe supernatant of D220 culture was only 5% ofthat seen for the wild type strain (p < 0.01). Thisfact is likely to be related to the short half-life ofSopA rather than a diminished expression of theprotein. Indeed, all the effector proteins analyzed(including SopA) were expressed in D220 mutant atcomparable levels, around 50% lower than the wildtype. Complementation of the dam mutation fullyrestored the proteins profile observed in the wildtype strain. Taken together, our results demonstratethat Dam methylation modulates the SPI-1 encodedtype III secretion system.

A4PRESENCE OF SPVR GENE IN CLINICALISOLATES OF SALMONELLA ENTERICAFROM COLOMBIA PATIENTSM. M. Sanchez Jimenez1, N. M. Cardona Castro1, N. A. Cannu2, S.Uzzau2, S. Rubino2; 1Instituto Colombiano de Medicina Tropical-CES,Medellin, COLOMBIA, 2Sezione di Microbiologia Clinica e Sperimentale.University of Sassari, Sassari, ITALY

Salmonella enterica comprises many serovars, with avariety of clinical syndromes. Show 97,5% homol-ogy in their genome. The non homologies genesmay comprise the genes that explain host specific-ity; and clinical syndromes that produce. The re-search about pathogenic mechanisms of Salmonellaenterica in animal, environmental and some humanisolates, have determined the presence, function andlocation, in chromosomal and extra chromosomal

chemotaxis encoded within the che operon andmotility encoded by fliCD. Iron and sialic acidutilization, chemotaxis, and motility are knownfactors to contribute to virulence in many patho-gens. This study, which is the first report examininggene expression within the SGI1 and its effect onglobal gene expression in Salmonella, provides thefirst experimental evidence for factors contributingto increased virulence and fitness of Salmonellaisolates possessing SGI1.

C3EXPRESSION AND SECRETION OF SPI-1EFFECTOR PROTEINS IN DNA ADENINEMETHYLASE (DAM) MUTANTS OFSALMONELLA TYPHIMURIUMM. N. Giacomodonato1, S. Uzzau2, A. García Cattaneo1, S. H.Sarnacki1, S. Rubino2, M. C. Cerquetti1; 1CEFyBO-CONICET andSchool of Medicine, University of Buenos Aires, Buenos Aires, ARGEN-TINA, 2University of Sassari, Sassari, ITALY

DNA adenine methylase (Dam) protein is a globalregulator of bacterial gene expression.Dysregulation of Dam activity is potentially ageneral strategy for the generation of vaccinesagainst bacterial pathogens. We obtained a dammutant of Salmonella serovar Typhimurium, namedD220, which bears a defective Dam (ten amino-acidshorter). D220 mutant is highly attenuated andconfers a protective immune response. However weobserved that D220 mutant has a reduced capacityto invade the murine intestinal epithelium in theileal loop assay (40 % compared to wild type strain).This partial inability to invade epithelial cells couldbe related to a reduced secretion of invasion pro-teins encoded in the Salmonella pathogenicity island1 (SPI-1). Then, we investigated the involvement ofDam protein on the expression and secretion ofSPI-1 proteins essential for invasion, such as SipA,SopA, SopB, SopD and SopE2. SalmonellaTyphimurium SSM3213 sopA::3xFLAGsopE2::3xFLAG cat::FLAG, SSM3214 sopD::3xFLAGsipA::3xFLAG cat::FLAG, SSM3215 sopB::3xFLAGcat::FLAG and their derivative dam-231::Tn10dTetwere used throughout the experiments. The expres-sion and secretion of these effector proteins weredetermined in vitro, analyzing different fractions of

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regions, of specific genes involved in the virulenceprocess. There aren’t many studies that have usedisolates from specific geographic regions whereSalmonella enterica frequently circulates. The objec-tive of the present work was to determine in 125Salmonella enterica clinical isolates from Colombianpatients, the presence of the spvR gene of SalmonellaVirulence Plasmid. The gene detection was achievedusing polymerase chain reaction (PCR) amplifica-tion, to detect the presence of this gene involved invirulence processes. The clinical isolates wereclassified by serotype in Salmonella Typhimurium (48from stool and 5 from other samples), S. Enteritidis(21 from stool and 6 from other samples), S. Typhi(16 from blood and 4 from other samples), S.Muenchen (5 from stool), S. Choleraesuis (4 fromstool), S. Dublin (2 from stool and 1 from articularliquid), S. Paratyphi C (3 from stool), S. Panama (2from stool), S. Paratyphi B (2 from blood), S.Agona (1 from stool), S. Derby (1 from stool), S.Javiana (1 from stool), S. Braenderup (1 from stool),S. Virginia (1 from stool) and S. Weltevrenden (1from stool). The spvR gene was present in 22isolates (41,5%) of Salmonella Typhimurium and in 7isolates (35%) of S. Typhi. This finding in S. Typhihas not been reported previously in literature. InSalmonella Enteritidis, the spvR gene was present in20 isolates (74,1%). In the other serotypes (25isolates), the spvR gene was absent in 18 isolates(72%). The results of this analysis do not show astatistically significant relation between these char-acteristics and the clinical syndrome. The searchfor specific genes that are responsible for thevariability of clinical syndromes produced bySalmonella enterica in humans is being done by severalresearch groups around the world; so far the answerthat clarifies the differences among each serovar hasnot been discovered.

B5ATTENUATED DAM MUTANTS OFSALMONELLA ENTERICA HAS A DEFECT INTHE O ANTIGEN LIPOPOLYSACCHARIDE(LPS) SYNTHESISS. H. Sarnacki1, M. N. Giacomodonato1, M. Noto Llana1, M. A.Valvano2, M. C. Cerquetti1; 1CEFyBO-CONICET and School ofMedicine, University of Buenos Aires, Buenos Aires, ARGENTINA,2Department of Microbiology and Immunology, University of WesternOntario, London, ON, CANADA

The DNA adenine methylase (Dam) enzyme modu-lates a variety of processes such as DNA replica-tion, mismatch repair and gene transcription. Salmo-nella enterica dam mutants have decreased infectivityin the murine model and have been recently sug-gested as promising vaccine candidates. We ob-tained Salmonella enterica serovar Enteritidis dammutants by targeted and transposition mutagenesis.Using a murine model of salmonellosis, we ob-served that the S. Enteritidis dam mutants wereattenuated when inoculated by intraperitoneal, oral,and intragastrical routes. Protection rates conferredby these mutants in immunized mice challengedwith the parental strain of S. Enteritidis, variedbetween 40 to 60 %. We also found that dam mu-tants induced lower levels (p < 0.05) of intestinalIFN-ã than the parental strain (509 + 62 vs 798 +82 pg/ ìg of protein, respectively), measured byELISA. Western blot analysis showed that dammutants induced a delayed activation of NF-kB. Wealso observed by silver-stained SDS-PAGE that dammutants of S. Enteritidis produce a defective LPS,which was characterized by a reduced length in theO antigen polysaccharide. This LPS pattern wasalso observed in our laboratory in S. Typhimuriumdam mutant, a strain proposed as a vaccine candi-date, but no evident defect was observed in E. colidam mutant. These observations suggest that theDam protein in S. enterica may be involved in modu-lating LPS synthesis, more specifically the lengthdistribution of O antigen polysaccharide, a functionencoded by the wzz gene. In order to test whetherthe methylation by Dam might be involved in wzzregulation the transcriptional expression of the wzzgene, was studied with a wzz::lacZ fusion containedin a low copy number plasmid, pFZY1. The activity



A7INHIBITORY ACTION OF BITTER LEAF(VERNONIA AMYGDALIRA) ONSALMONELLA TYPHI: THE IN VITROEVALUATIONO. I. Olawuyi, A. Falegan; UNIVERSITY COLLEGE HOSPITAL,IBADAN, NIGERIA.

Introduction: Bitter Leaf (Vernonia amygdalina) is asmall teee that grows up to 23 feet tall in most partof Nigeria. The plant has been noted for its medici-nal qualities. This abstract showed the evaluation ofthe Inhibitory action of the bitter leaf extract onSamonella typhi in vitro.Both Minimum InhibitoryConcentration (MIC) and Minimal bactericidalConcentration (MBC) were also evaluated.Method: S. typhi colonies were isolated from feacalsamples using Leifson‘s Deoxycholate Citrate Agar(DCA) and Strontium chloride broth forselectivity.Three plates were prepared and thefollowing were incorporated therein: Plate A wasincoporated with Bitter leaf ethanol extract, Plate Bwith Bitter leaf water extract , and Plate C with noextract, serving as control plate. The three plateswere then dried and inoculated with the isolated S.typhi colonies, and cultured for 24 hours and 48hours at 37 degree Celsius. The MIC and MBC wereevaluated respectively. Result: Plate A and Bshowed no growth of the organism while Plate Cshowed considerate and significant growth of S.typhi. MIC and MBC evaluated and calculated are 1and 2 mg/ml respectively, and the ratio of MIC toMBC is less than 4 (MIC/MBC < 4).Conclusions: The In vitro and microbial analysisshowed that Bitter Leaf extract has a strong inhibi-tory substance that inhibit the growth of S. typhi invitro.This holds a promising therapeutic alternativeagainst Salmonella pathogenesis. However, there isneed for further exploration of the plant againstSalmonella pathogenesis and diseases,especially asthe plant is generally regarded as safe for humanconsumption.

of Beta-galactosidase was assessed in wild type anddam mutant strains of S. Enteritidis. Analysis ofBeta-galactosidase production by cells carryingplasmids with the fusion indicated that lack of Damenzyme, increased transcription of the wzz pro-moter. Therefore, our results demonstrate that thedam gene modulates LPS synthesis and this defec-tive LPS could explain, at least in part, the avirulentphenotype of S. Enteritidis dam mutants.

C6

PREVALENCE AND DRUG SENSITIVITY OFSALMONELLA SEROGROUPS ISOLATES INCHICKEN MEAT OF CHAIN STORES OFIRANS. Dordari; Iranian aghricultural Research & Education organization,Tehran, IRAN (ISLAMIC REPUBLIC OF)

Bacteriological examination was curried out on 600broiler samples from packaged chicks sold in chainstores of Tehran. During 2 successive years. Sero-type and antibiotic sensitivity of salmonellae iso-lated from these broilers were carried out. Eighteensalmonella samples were isolated from the packagedchickens by using standard methods. Eighteensalmonellae were serogrouped and serotyped withspecific antisera. Salmonella serotypes isolated fromchickens were: tompson (1.67%), typhimurium(1%), eastborne (0.17%) and muenchen (0.17%).The isolates were tested for drug sensitivity with 20different antimicobials. All of the tested salmonellawere resistant to penicillin, erythromycin, tylosin,streptomycin, bacitracin and tiamulin and all of thetested salmonella were susceptiple to difloxacin,enrofloxacin and ciprofloxacin. The percentage ofsalmonella susceptible to chloramphenicol furazoli-done, flumequine, cefixime, soltrim, linco-spectinand gentamicin were 83.4%, 33.3%, 27.8%, 22.2%,22.2%, 11.1%, 11.1%. As the results shows theprocess of packaging and flaying of broilers andwashing it decreasses the contamination of salmo-nella on the broiler meat. Because of the increasedacqaired resistance against the regular antibioticsmedication alone is not enough to control thesalmonella infections in poultry and even can causesproblems in public health.

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B8BIOFILM FORM OF SALMONELLAPARATYPHI A: THE POTENTIALTHERAPEUTIC AGENT AGAINSTSALMONELLA PATHOGENESISE. Egbejule, O. Ogunlowo; Lagos State University, Lagos, NIGERIA

Background: This work revealed the inhibitoryproperties of biofilm form Salmonella paratyphi Alipopolysaccharide substance from the extract ofbiofilm of S. paratyphi A and Staphylococcus aureus.This substance inhibit the wild types of Salmonellaorganism in vitro. Method: The S. paratyphi A wasisolated from the extract of biofilm of S.paratyphi Aand S. aureus, and was cultured on DeoxycholateCitrate Agar (DCA) along with wild types ofSamonella species. The control plate was set up con-taining only the wild type isolate colonies. The twoplates were cultured for 24 hours at 37 degreeCelsius. Result: After 24 hours incubation, plate Ashowed only the significant growth of only biofilmform of S. paratyphi A while plate B showed thesignificant growth of all the wild type species(S.typhi and S.enteritidis) cultured. Conclusions: Themicobial analysis of Biofilm of S. paratyphi Ashowed that it possesses an enhanced lipopolysac-charide substances which was very bactericidalagainst the wild type species, and this was thoughtto be got from the cell-cell exchange and signalingin the biofilm.

C9MAJOR PULSED FIELD GELELECTROPHORESIS PATTERNS FORPHAGE-TYPES (PT) 4, 8, AND 13ASALMONELLA ENTERICA SEROVARENTERITIDIS AND INVASIVENESS ASSAYSFOR SUBTYPES IN PT4 AND 8H. Tsen1, J. C. Pang1, J. S. Lin2, C. C. Tsai1; 1Hung-Kuang University,Department of Food Science and Nutrition, Taichung , TAIWAN, 2NationalYang-Ming University, Department of Biochemistry and Molecular Biology,Taipei, TAIWAN

This study was attempted to elucidate the majorglobal subtypes of Salmonella Enteritidis with phagetype (PT) 4, 8 and evaluate their invasiveness.Seventy seven animal isolates of Salmonella Enteriti-

dis obtained from United States were analyzed byphage typing and pulsed field gel electrophoresis(PFGE). Thirty nine strains were found with themajor phage types of 4, 8, and 13a. PFGE patternsof the S. Enteritidis strains with PT4 and PT8 werethen compared with those of the S. Enteritidisstrains with the same phage types obtained fromGermany and Taiwan. Results showed that for these39 US isolates, when XbaI, SpeI and NotI were usedfor chromosomal DNA digestion followed byPFGE analysis, 28 strains were found with a pat-terns combination of X4S4N4, which was themajor subtype. When PFGE patterns of the USisolates with PT4 and 8 were compared with thoseof the Taiwan and German isolates previouslyreported, patterns X4S4N4 and X3S3N3 werefound as the most common subtypes shared by PT8and PT4 isolates, respectively. Since strains used insuch comparison were unrelated, this study revealsthe major world-wide clones of PT8 and PT4Salmonella Enteritidis. The presence of such majorworld-wide clones, however, may not associate withtheir virulence in terms of invasiveness to culturedhuman intestinal epithelium cell line Int-407 andBALB/ mice.

A10ADMINISTRATION OF AN ATTENUATED S.ENTERICA STRAIN CAN INCREASE THEGROWTH RATE OF A VIRULENT STRAINWITHIN THE HOST VIA THE PRODUCTIONOF IL10G. L. Foster, A. Grant, C. Bryant, D. Maskell, P. Mastroeni; Universityof Cambridge, Cambridge, UNITED KINGDOM

During systemic disease, Salmonella enterica growintracellularly within discrete infectious foci in thespleen and liver. In concomitant infections, focicontaining different S. enterica isolates are spatiallyseparated. We have investigated whether functionalinteractions between bacterial populations withinthe same murine host can occur despite the knowngeographical separation of the foci. In this studywe have demonstrated that the net growth rate of avirulent S. enterica serovar Typhimurium C5 straincan be increased by the presence of the attenuatedaroA- S. Typhimurium SL3261 vaccine strain in the



RtsA, InvF and global regulators, including nucle-oid-associated proteins Hha and H-NS. A singlecopy transcription fusions were used to assesseffects of nucleoid-associated proteins Hha and H-NS on hilA, rtsA, and hilC expression under differ-ent osmolarity conditions. Single mutants in hha andhns moderately increased expression of all threegenes. In the double hha hns mutant derepression ofhilA, rtsA, and hilC genes in low osmolarity condi-tions was significantly greater, 26-fold, 11-fold, and4-fold, respectively. Electronic mobility shift assaysrevealed the presence of binding sites for the Hhaand H-NS proteins in the A+T reach sequences ofhilA, rtsA, and hilC promoters, which overlap theDNA-binding sites for activators of invasion HilD,HilC, and RtsA. Proposed model of osmotic regula-tion of Salmonella invasion genes through competi-tion between activators and the Hha/H-NS repress-ing complex for overlapping regulatory sites.

C12FACTORS INFLUENCING INVASION ANDSURVIVAL OF SALMONELLA ENTERICASEROVAR TYPHIMURIUM INACANTHAMOEBA POLYPHAGAP. Wigley, R. Birtles, P. Lott, C. Shepherd; University of Liverpool,Wirral, UNITED KINGDOM

Background The persistence of food-borne bacte-rial pathogens in the environment is an essential, yetpoorly understood part of their transmission.Protozoa, particularly free-living amoebae, may beimportant reservoirs for environmental persistenceand transmission of Salmonella enterica. Salmonellaenterica serovars Typhimurium and Dublin have bothbeen shown to survive within Acanthamoeba.Acanthamoeba exploitation by Legionella pneumophila isthe model system from which much of the knowl-edge of bacteria-protozoal interactions has beenderived. Various virulence factors have been impli-cated in exploitation of both free-living amoebaand host macrophages during disease, includingtype II and type IV secretion systems and flagella.Salmonella too survive and replicate within macroph-ages with virulence factors, notably the SPI2 typeIII secretion system, the PhoP/Q two-component

same tissue. Acceleration of the growth of the C5strain does not require simultaneous co-injection ofthe attenuated bacteria. SL3261 can be administeredup to 48 hours after or 24 hours before the C5strain and still cause an increase in the growth rateof the virulent bacteria. This indicates that intrave-nous administration of an S. enterica vaccine straincould potentially exacerbate an established infectionwith wild type bacteria. This data also suggests thatthe severity of an infection with a virulent S. entericastrain can potentially be increased by the prioradministration of a live attenuated vaccine strain.Systemically, administration of SL3261 induces hosthypo-responsiveness to LPS challenge by 24 hoursfollowed by hyper-responsiveness at 48 hours asmeasured by altered TNFá and IFNã production.SL3261 administration also triggers the release ofserum IL10 and corticosterone. Exacerbation of C5growth requires functional TLR4 and can be repro-duced by administration of TLR4 ligands such ashigh doses of heat killed S. enterica or LPS. Further,we have found that the SL3261 strain fails to exac-erbate the growth of the C5 strain in IL10-/- mice.Together, these results suggest that IL10, which isknown to suppress macrophage function, is re-quired for the increased C5 growth rate. Our workhas shown that the injection of a vaccinating doseof a live attenuated S. enterica strain increases thegrowth rate of virulent S. enterica organisms via amechanism that requires the activation of TLR4and the presence of IL10. Better understanding ofthe potential interactions between bacterial livevaccine strains and environmentally acquired wildtype strains is advisable to inform safer vaccinationstrategies in areas of the world where typhoid feveris endemic.

B11OSMOREGULATION OF SALMONELLAINVASION BY HHA/H-NS REPRESSINGCOMPLEX AND ARAC/XYLS FAMILYACTIVATORS HILD, HILC, AND RTSAI. N. Olekhnovich; University of Virginia, Charlottesville, VA

Transcription control of invasion genes located onthe Salmonella pathogenicity island-1 is mediated bySalmonella-specific activators HilA, HilD, HilC,

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regulatory system implicated in this process. Arange of stable mutants of S. Typhimurium F98have been used in our studies including a SPI2-deficient DELTAssaU strain, a DELTAPhoP strain,a SPI1-deficient DELTAspaS strain and aDELTAfliM strain, lacking flagella. These mutantshave been characterised for their ability to invadeand survive within A. polyphaga.Methods Invasionand persistence of Salmonella was determined bygentamicin protection assay. Briefly, A. polyphagawas grown axenically in PYG media to a level of5×106 cells ml-1. Salmonella enterica Typhimurium F98parent and spaS, ssaU, fliM and phoP mutant strainswere grown to late log phase and added at a MOI10. After 1h extracellular bacteria were killed by theaddition of gentamicin. At this point and at 4, 8,24,48 and 72 h post challenge, the amoeba wereremoved, lysed and plated to determine numbers ofinternalised Salmonella Results Counts in excess of107cfu ml-1 of S. Typhimurium F98 were recoveredat 1h post infection, rising to over 108 cfu by 4hpost infection indicating rapid invasion and replica-tion. Visualisation using GFP-expressing strainssuggested Salmonella were mainly within vacuoles.Bacterial numbers remained high at 24h post infec-tion, then declined to 104 cfu ml-1 at 72 h postinfection. The spaS (SPI1) mutant was reduced byaround 95% in its ability to invade A. polyphaga,though survived at comparable levels to the parentstrain. In contrast the ssaU (SPI2) and phoP mutantsshowed no difference in invasion, but both survivedless well, the SPI2 mutant was not recovered be-yond 8h post infection. The absence of flagella hadlittle effect on phenotype. Discussion As in mac-rophages both the SPI2 type III secretion systemand PhoP/PhoQ two compenent regulatory systemsplay a role in persistence within protozoal cells. TheSPI1 system appears to be involved in initial inva-sion, but is not required for intra-amoebal survival.As in Legionella it appears that there are a number ofsystems common to intra-amoebal and intra-mac-rophage survival.

A13EFFICACY OF PLANT ESSENTIAL OILS ASANTIMICROBIAL AGENTS AGAINSTSALMONELLA ENTERITIDIS IN POTATOSALADM. Jalali, IV, D. Abedi, IV, G. Asghari, IV, M. Taghipor, III, K.Ghokasian, III; Isfahan University of Medical science, Isfahan, IRAN(ISLAMIC REPUBLIC OF)

The purpose of present study was to evaluate theantimicrobial activity of three plant essential oils(OE) against S. enteritidis in potato salad at 10 °Cand 5 °C. EO of Zataria, Satureja and Pelargoniumwere applied to artificially contaminated potatosalad and stored for up to 14 days to determinetheir anti-Salmonella activity. Zataria and SaturejaEO were applied to potato salad to reach the finalconcentration of 6 μl/g (high), 3 μl/g (medium)and 0.6 μl/g (low). Pelargonium EO was applied atthe level of 0.3 μl/g (high), 0.15 μl/g (medium) and0.03μl/g (low). The addition of Zataeia EO at highand medium level effectively reduced Salmonellapopulation to d”1.0 log cfu ml-1 at second day ofstorage at 10 °C. Zataria EO at low concentrationshowed no inhibitory effect on growth/ survival ofSalmonella in potato salad at 10 °C. Zataria EOapplied at high, medium and low level and stored at5 °C reduced the population of Salmonella to d”1.0log cfu ml-1 at second, sixth and twelfth day ofstorage respectively. The addition of Satureja EO atlow level and stored at both 10 °C and 5 °C showedno inhibitory effect on population of Salmonella.Satureja EO at medium level indicate 2 log10 reduc-tion of Salmonella population during storage at 10°C. In contrast medium level of Satureja EO inpotato salad stored at 5 °C reduced Salmonellapopulation to d”1.0 log cfu ml-1 at day twelfth ofstorage. The high level of Satureja EO reducedSalmonella population to d”1.0 log cfu ml-1 at fourthand eighth day of storage at 10 °C and 5 °C respec-tively. The Pelargonium EO at the high, medium andlow level showed no inhibitory effect on Salmonellapopulation at both 10 °C and 5 °C. These resultssuggest that plant EO might be useful as antimicro-bial in potato salad



additive during the first week posthatch couldprovide increased protection against a variety ofbacterial pathogens.

C15TEMPERATE BACTERIOPHAGE AND THEMOLECULAR EPIDEMIOLOGY OFANTIBIOTIC RESISTANCE IN SALMONELLAS. Tan1, M. D. Barton2, M. W. Heuzenroeder3; 1School of Molecular andBiomedical Science, The University of Adelaide, Adelaide, South Australia,AUSTRALIA, 2The University of South Australia, Adelaide, SouthAustralia, AUSTRALIA, 3The Institute of Medical and VeterinaryScience, Adelaide, South Australia, AUSTRALIA

Antibiotic resistance in bacterial pathogens presentsa challenge to public health by limiting the thera-peutic options available for treatment of infectiousdiseases. Bacteria have various genetic transfersystems which facilitate the acquisition and ex-change of antibiotic resistance genes. Salmonella is acommon cause of foodborne disease worldwide andis known to harbour multiple resistance factors toantimicrobial agents. Genetic transfer of multipledrug resistance has been demonstrated in SalmonellaTyphimurium DT104 (H. Schmieger and P.Schicklmaier, FEMS Microbiol. Lett. 170: 251-256,1999). The aim of the current study was to detecttemperate phages in Salmonella serovars commonlyisolated from animals and humans in Australia, andto determine if these phages were capable oftransferring antibiotic resistance genes to antibiotic-sensitive Salmonella strains, that have caused diseasein humans, by generalised transduction. Temperatephage were induced with mitomycin C from antibi-otic-resistant S. Typhimurium, S. subsp. 2 Sofia, S.Kiambu, S. Derby, S. Bovismorbificans from chick-ens and S. Anatum from pigs. Experiments demon-strated that these phages plaque on antibiotic-sensitive S. Typhimurium DT1, 2, 41, 108, 135, 12and 4 as well as S. Enteritidis PT1. These phageswere also shown to plaque on Shigella sonnei and S.flexneri, but did not plaque on the E. coli indicatorstrains. Furthermore, it was demonstrated thatfunctional ampicillin and streptomycin resistancewas transferred by temperate bacteriophages in-duced from antibiotic-resistant Salmonella isolates toantibiotic-sensitive S. Enteritidis and S.

B14TAMUS CATIONIC PEPTIDES DECREASESUSCEPTIBILITY OF YOUNG CHICKENS TOSALMONELLA ENTERICA SEROVARENTERITDIS INFECTION BYUP-REGULATION OF THE INNATE IMMUNERESPONSEM. H. Kogut1, K. J. Genovese1, H. He1, Y. Jiang2; 1USDA-ARS, CollegeStation, TX, 22Department of Medical Biochemistry & Genetics, TexasA&M University System Health Science Center, College Station, TX

The TAMUS cationic peptides are a group ofrelated cationic peptides produced by a Gram-positive bacterium. Cationic amphiphilic peptideshave been found to stimulate or prime the innateimmune responses in mammals. The innate immunesystem of poultry is functionally inefficient duringthe first week posthatch enabling pathogens such asSalmonella enterica serovar Enteritidis (SE) to invadeand colonize the visceral organs of these immaturebirds. The objective of the present study was toevaluate the effect of TAMUS as animmunostimulator of the innate immune responseof young chickens. TAMUS was provided as a feedadditive at three different concentrations (12, 24, or48 ppm) for 4 days post-hatch significantly in-creased protection against SE organ invasion in aconcentration-dependent manner. The functionalefficiency of heterophils isolated from chickens fedthe TAMUS rations at the three concentrations wassignificantly up-regulated when compared toheterophils isolated from chickens fed a controlstarter ration as determined with an array of func-tional assays. Phagocytosis, oxidative burst, anddegranulation were all significantly increased in aconcentration-dependent manner in heterophilsisolated from chickens fed the TAMUS diets. This isthe first report of bacterial cationic peptides induc-ing the up-regulation of the avian innate immuneresponse that provides protection againstextraintestinal Salmonella infections. The significanceof these data is that the orally delivered cationicpeptides stimulate the innate response at a time ofimmunologic inefficiency and increased susceptibil-ity to bacterial infections (first week posthatch).Because of the non-specific nature of the innateresponse, we speculate that TAMUS given as a feed

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Typhimurium. These results show that phages fromantibiotic-resistant Salmonella are capable of infect-ing antibiotic-sensitive strains and therefore may beable to transfer antibiotic resistance genes. Resultsindicate that generalised transduction is a mecha-nism for antibiotic resistance gene transfer inSalmonella.

A16FLUOROQUINOLONE RESISTANCE ANDGENETIC RELATEDNESS OF SALMONELLAISOLATED FROM SALMONELLOSIS PIGS INTAIWANY. Wang1, K. Yeh2, T. Chen3; 1Department of Veterinary Medicine,National Chung Hsing University, Taichung , TAIWAN, 2Department ofMicrobiology and Immunology, School of Medicine, Taipei Medical University,Taipei, TAIWAN, 3The Graduate Institute of Veterinary Public Health,National Chung Hsing University, Taichung , TAIWAN

Background: The emergence offluoroquinolone(FQ) resistant SalmonellaCholeraesuis has become an important public healthissue in Taiwan. The objectives of this study wereto investigate the potential risk of salmonellosis pigsin transmitting FQ-resistant Salmonella to humans inTaiwan, and to characterize epidemiologic relation-ship and antimicrobial resistance among the isolates.Methods: Animal Disease Diagnostic Center con-ducted a surveillance of Salmonella from salmonello-sis pigs in the central Taiwan from 2004-2005. TheSalmonella isolates were characterized usingserotyping, pulsed-field gel electrophoresis (PFGE)and antimicrobial susceptibility testing. DNAsequences of the quinolone resistance-determiningregions were determined. Results: During the two-years study, fourty-nine Salmonella isolates wascollected. Most of them were S. Choleraesuis(40/49) and S. Typhimurium(5/49). S. Choleraesuisisolates were showed the high frequency of resis-tance to the tetracycline(92.5%), ampicillin(95%),chloramphenicol(80%), trimethoprim-sulfamethoxazole(82.5%), nalidixic acid(100%) andfluoroquinolone(77.5%). All of S. Choleraesuisstrains have the closely related PFGE patterns, andthe major genotype was the same as the gt-1a.Mutations of gyrA and parC were also conserved inall of the FQ-resistant isolates. Conclusions: In

conclusion, our study indicated that the isolates ofS. Choleraesuis from diseased pigs in Taiwan from2004 to 2005 were caused by gt-1a clone that hadhigh prevalence of resistance to FQ and otherantimicrobial agents. Increasing prevalence of FQ-resistant S. Choleraesuis indicate that a more effi-cient veterinary policy is essential to control it.

B17ROLE OF ZINC IN THE HOST-SALMONELLAINTERACTIONS. Ammendola1, P. Pasquali2, A. Battistoni1; 1Dipartimento di Biologia,Università di Roma Tor Vergata, Rome, ITALY, 2Dipartimento di SanitàAlimentare e Animale, Istituto Superiore di Sanità, Rome, ITALY

Bacterial growth is critically dependent on anadequate supply of different transition metals, asthey play crucial catalytic and/or structural role in awide number of macromolecules. The problem ofmetal recruitment is particularly acute for infectiousbacteria, as they colonize environments wheremetals may be scarcely available in accessible forms.For example, it is well established that within thehost iron is sequestered in forms which preventmetal reactivity and limit its availability for invasivemicroorganisms. Although iron is usually consid-ered the most important transition metal involvedin the host-pathogen interaction, recent studiesindicate that active mechanisms ensuring efficientuptake of other transition metals play an importantrole in bacterial pathogenicity. In particular, there isgrowing attention about the relevance of themechanisms of zinc uptake in bacterial infections.Under conditions of metal starvation, adequate zincrecruitment in Gram-negative bacteria is mediatedby the high affinity Zn2+ ABC transporter encodedby the znuABC genes. To analyze the relevance ofthe ZnuABC transporter in Salmonella virulence, wehave constructed S. Typhimurium ATCC 14028 andS. Enteritidis LK5 deletion mutants, lacking thegene encoding the periplasmic subunit (ZnuA) ofthe transporter. This mutation does not alter theability of Salmonella to grow in rich media, butdrastically reduces bacterial multiplication in pres-ence of EDTA or in defined media containing onlytrace amounts of zinc. Moreover, even if the znuAmutants are not impaired in their ability to pen-



deletion of fur (ferric uptake regulator) causes a 6 to7 fold decrease in hilA expression. Fur regulation ofSPI1 is dependent on HilD and controls hilD at thepost-transcriptional level. Fur “activation” of hilD ispresumably via repression of a negative regulatorof hilD translation and/or mRNA stability. Thisregulation is not mediated through the Fur regu-lated small RNAs FrrA (RyhB) and FrrB, whichredundantly control expression of sodB, or theknown SPI1 repressor HilE. Thus, iron is poten-tially an important trigger in the regulation of SPI1.

B20EVOLUTION AND GENOME VARIATIONOF THE MULTIDRUG RESISTANTSALMONELLA ENTERICA SEROVARTYPHIMURIUMI. Rychlik, H. Hradecka, J. Matiasovicova, M. Malcova; VeterinaryResearch Institute, Brno, CZECH REPUBLIC

Multidrug resistant Salmonella enterica serovarTyphimurium (S. Typhimurium) harboring theSalmonella Genomic Island 1 (SGI-1) was detectedfor the first time in the UK in mid 1980’s and sincethat time this clone has reached a worldwide distri-bution. Due to its multidrug resistance, it is epide-miologically highly important. In this study weperformed microarray genomotyping of 4multidrug resistant SGI-1 positive strains andcompared their genomes with the genome of theLT2 strain. All the multidrug resistant strains werefree of Fels-1 and Fels-2 prophages. Two of themultidrug resistant strains lacked the rrtT retronreverse transcriptase locus and one strain was alsofree of Gifsy-2 prophage. All multidrug resistantstrains also lacked genes STM0517-STM0529allowing utilization of allantoin as a nitrogensource. The rrtT retron reverse transcriptase locusis localized immediately downstream of the SGI-1and its absence in S. Typhimurium genome wasobserved only among SGI-1 positive strains. Whencharacterizing the deletion we found that altogether8164 bp was missing in the chromosome of the rrtTnegative strains. Since the deletion happened exactlybetween the right inverted repeat of IS6100 andinside the yieE gene, we propose that intramoleculartransposition of IS6100, a part of SGI-1, followed

etrate and survive in cultured macrophages, theirpathogenicity is dramatically reduced in orally orintraperitoneally infected BALB/c and DBA2 mice.ZnuA accumulation in Salmonella was studied intro-ducing a DNA sequence encoding the 3xFLAGepitope at the 32 end of the znuA gene. We havefound that znuA is expressed in bacteria growing inmedia containing less 10-7M zinc, but is repressed incultured macrophages or in bacteria recovered fromthe spleens of infected animals, thus suggesting thatthere is not severe zinc shortage in intracellularenvironments. Our results indicate that the bacterialmechanisms of zinc uptake play a very importantrole in the regulation of the host-Salmonella interac-tion and that Salmonella requires a functionalZnuABC transporter to obtain an adequate zincsupply within its host.

C18ENVIRONMENTAL REGULATION OF THESPI1 TYPE THREE SECRETION SYSTEM INSALMONELLA ENTERICA SEROVARTYPHIMURIUMJ. R. Ellermeier, J. M. Slauch; University of Illinois at Urbana/Champaign, Urbana, IL

Salmonella enterica serovar Typhimurium invades non-phagocytic intestinal epithelial cells using a TypeThree Secretion System (T3SS) encoded on Salmo-nella Pathogenicity Island 1 (SPI1). HilC, HilD, andRtsA are members of the AraC family of transcrip-tional activators, each of which can independentlyactivate expression of SPI1 genes by binding up-stream of the master regulatory gene hilA to induceits expression. HilA, in turn, activates the SPI1T3SS structural genes. We recently proposed amodel in which HilC, HilD, and RtsA form a feedforward regulatory loop controlling expression ofhilC, hilD, and rtsA. Thus, production of the SPI1T3SS is controlled by the combined action of HilC,HilD and RtsA. Our goals are to characterize therole of HilC, HilD, and RtsA in SPI1 regulation andto understand the overall regulatory circuit thatcontrols this important virulence function. Currentfocus is on understanding how environmentalsignals feed into the system. We show that highlevels of iron induce expression of hilA and a

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POSTER ABSTRACTS

by homologous recombination was responsible forthe excision. Excision of retron together with theright end of SGI-1 may also result in its stabiliza-tion in the Salmonella typhimurium genome.The absence of genes allowing utilization ofallantoin as a nitrogen source in all 4 multidrugresistant strains was also unexpected. We extendedthis observation by PCR screening of an additional120 S. Typhimurium field strains and found that thislocus was absent in all SGI-1 encoded multidrugresistant strains and also in 24 % of SGI-1 negativestrains. Such strains did not grow in minimal me-dium with allantoin as a sole nitrogen source, wereof highly similar PFGE pattern and prophagecontent as proved by PCR. Based on these findingswe propose that a lineage of the original S.Typhimurium sensitive to antibiotics lost the abilityto metabolize allantoin, then S. Typhimurium ofthis lineage acquired SGI-1 and finally, some ofthese strains lost the retron reverse transcriptaselocus. Finally, all the strains of this lineage areprototrophic, grow to high cell densities in variousculture media and are capable of efficient biofilmformation what may explain their efficient spreadthroughout the world.

C21IDENTIFICATION OF A SUBCELLULARLOCALIZATION DOMAIN IN SALMONELLATYPHIMURIUM SOPD2 AND THE ROLE OFTHE EFFECTOR IN SALMONELLA INDUCEDFILAMENT DYNAMICSJ. Szeto, J. H. Brumell; Hospital for Sick Children, Program in CellBiology, Toronto, ON, CANADA

Salmonella enterica serovar Typhimurium (S.Typhimurium) causes gastroenteritis in humans.Using two type III secretion systems, S.Typhimurium injects bacterial effector proteins intothe host cell to modulate several activities. Internal-ized bacteria reside in the Salmonella-containingvacuole (SCV) from which membranous tubulestermed Salmonella-induced filaments (Sifs) arise.While their function is unknown, Sif formationrequires the effector SifA and is aided by a secondeffector, SopD2. SopD2 contributes to S.Typhimurium virulence in mice and localizes to late

endocytic (LE) compartments and to Sifs.The first 150 amino acids (aa) of SopD2 can bindLE compartments, hence these studies were per-formed to identify SopD2 N-terminal residues thatmediate its intracellular targeting. SopD2 is a Sal-monella-Translocated Effector (STE) family mem-ber with a conserved N-terminal WEK(I/M)xxFFmotif previously implicated in translocation intohost cells. We hypothesized that this motif inSopD2 (WDRFKDCF; aa 37-44) may also directsubcellular localization of the effector. To examinethis, site-directed mutations or deletions in SopD2were generated, and their effects on SopD2-GFPlocalization were examined. Substitution or deletionof conserved residues within the SopD2WDRFKDCF motif disrupted the targeting ofSopD2-GFP to LE vesicles. Interestingly, shortSopD2 peptides containing the WDRFKDCF motiffused to GFP localized exclusively to the Golgi andnot to LE vesicles. We show that the SopD2WDRFKDCF sequence itself has striking similarityto a eukaryotic Golgi targeting motif. These studiesindicate the N-terminal WDRFKDCF motif ofSopD2 may have roles in both translocation and indirecting proper subcellular localization of theeffector protein. Sif formation requires intactmicrotubule networks and associated host motorproteins, suggesting Sifs might display motility.However, conventional fixed cell immunofluores-cence cannot show this. By employing Lamp1-GFPto label Sifs in infected HeLa cells, we used live cellimaging to show that Sifs produced by either wild-type or sopD2 mutant S. Typhimurium are verymotile. Lamp1-GFP-positive Sifs were observed tomove with rapid extension and retraction events,and also formed de novo from the sides of pre-existing Sifs by an active branching mechanism.Closer inspection of individual elongating Sifsrevealed their end tips consisted of a thin Lamp1-GFP-positive extension that acted as a leader underwhich the remaining Sif structure, characterized bya thicker Lamp1-GFP-positive filament, wouldfollow. These studies show that Sifs are highlydynamic structures; however, SopD2 is not requiredfor Sif motility nor directionality.


POSTER ABSTRACTS

POSTER ABSTRACTS

mutant in the global regulator, FNR, was examined.The fnr mutant was unable to produce NO suggest-ing that the protein responsible for NO productionis positively regulated by FNR.It is clear that the relationship between Salmonellaand NO is complex. NO is used by mammaliansystems to limit the survival of the pathogen, butconversely NO can be produced by Salmonella undercertain conditions.

B23IN VIVO EFFECTS OF SALMONELLAFLAGELLIN ADMINISTRATION TO DAY-OLD CHICKENSK. J. Genovese, H. He, M. H. Kogut; FFSRU, USDA ARS SPARC,College Station, TX

Flagellin is a highly evolutionarily conserved bacte-rial product that has been shown to be recognizedby the innate immune system through toll-likereceptor (TLR) 5. Previous work has shown thatflagellin is a potent stimulator of in vitro phagocyticcell functions of chickens. The purpose of thepresent studies was to define the effects of flagellinadministration to chickens in vivo. Day-old chickswere administered, by intra-abdominal injection,either 2.0 μg flagellin in sterile saline/bird or sterilesaline alone. 1 hr after flagellin administration,chicks were challenged by intra-abdominal injectionwith Salmonella enteritidis [5 x 103 colony formingunits (CFUs)/bird]. Peripheral blood samples andabdominal leukocyte infiltrates were monitored at 0,4, 8, and 24 hr post-infection. Chick mortality wasrecorded over a 72 hour period. Flagellin adminis-tration was found to protect chicks from SE-associated mortality, reducing mortality by 50% ascompared to the SE infected control group. At 4hours post-infection, total white blood cells (WBCs)in the peripheral blood showed a significant in-crease in flagellin, flagellin + SE, and SE alonegroups as compared to control birds. At 4, 8, and 24hr post-infection, chicks in both the flagellin aloneand the SE + flagellin groups had significantlyhigher total WBCs than the SE alone control group.The largest portion of the increase in peripheralblood WBCs was due to a significant increase in

A22THE NITRIC OXIDE METABOLISM INSALMONELLA ENTERICA SEROVARTYPHIMURIUM AND ITS ROLE WITHINMACROPHAGESN. J. Gilberthorpe, R. C. Read, R. K. Poole; University of Sheffield,Sheffield, UNITED KINGDOM

One of the most potent anti-microbial defencemechanisms used by macrophages is the productionof nitric oxide (NO). The ability of Salmonella tosurvive within macrophages is central to its capacityto cause disease. The survival of hmp and fur mutantstrains of Salmonella in the murine macrophage cellline, J774.2 was investigated. Survival assays werecarried out in J774.2 cells that had been cultured inthe absence and presence of the immuno-stimula-tory cytokine, IFN-ã. Intracellular survival of thefur mutant was attenuated compared to an isogenicwild-type control strain in infection of both non-stimulated and stimulated macrophages. We suggestthat this is a consequence of exacerbated oxidativestress sustained by the mutant, caused by elevatedlevels of free iron. In contrast, theflavohaemoglobin, hmp mutant, was attenuatedcompared to the wild-type only in stimulated mac-rophages. These results indicate that the levels ofNO produced in non-stimulated J774.2 can beavoided or detoxified by the hmp mutant to thesame extent as the wild-type. However, in infectionof IFN-ã-stimulated J774.2 cells, the hmp mutantsurvived relatively poorly, illustrating enhancediNOS activation and the requirement of Hmp to aidmicrobial defence. Ability of the hmp mutant tosurvive intracellularly was negatively correlated withaccumulation of nitrite and nitrate in the tissueculture media. Paradoxically, in vitro studies haveshown that wild-type Salmonella that has beencultured anaerobically using nitrate as an electronacceptor, will produce NO from nitrite anaerobi-cally. Interestingly, the hmp mutant of Salmonellaproduces NO under aerobic as well as anaerobicconditions. It is suggested that this is due to theinability of the hmp mutant to detoxify NO aerobi-cally. To aid determination of the nitric oxide-producing factor, the NO-producing capacity of a

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heterophils in the SE, SE + flagellin, and flagellingroups, with the flagellin and SE + flagellin groupshaving a significantly greater increase in the num-bers of heterophils in the peripheral blood. Totalleukocyte numbers in the abdominal cavity of birdsin the flagellin alone group were significanltygreater than in the SE alone and SE + flagellingroups at 4 and 8 hours post-injection, then wereon the decline by 24 hours post-injection. In the SE+ flagellin group, abdominal total leukocytes in-creased overall, with a slight decrease in numbers at8 hours post-injection, but were significantly greaterthan the SE alone group at 4 and 24 hr post-injec-tion. Leukocytes in the abdomen of birds in the SEalone group steadily increased over the 24 hourperiod. The predominant cell type associated withincreasing numbers of leukocytes in the abdominalcavity was the heterophil, with the flagellin and SE+ flagellin groups having significantly greaternumbers of heterophils in the abdominal cavitythan did birds in the SE alone group. The datasuggest that the TLR 5 agonist, flagellin, is also apotent stimulator of a heterophil-mediated innateimmune response in vivo, with subsequent protectiveeffects on bacterial infections in the chicken andmay have applications as an adjuvant in vaccines oras a general prophylactic innate immune enhancingagent in poultry during the first week post-hatch.

C24STRUCTURAL AND FUNCTIONAL STUDIESOF THE SHDA AUTOTRANSPORTER OF S.TYPHIMURIUMJ. D. Taylor1, R. A. Kingsley2, G. Dougan2, K. A. Brown1; 1ImperialCollege, London, UNITED KINGDOM, 2The Wellcome Trust SangerInstitute, Hinxton, UNITED KINGDOM

Non-typhoidal serotypes of Salmonella entericasubspecies I to VII and Salmonella bongori generallycause gastroenteritis in man. However, whilst thereare no significant differences in morbidity or mor-tality rates within this pathogenic group, the vastmajority of human clinical cases involve subspeciesI. The shdA gene, found exclusively within particu-lar serovars of subspecies I (e.g. Typhimurium), maycontribute to this epidemiology. The shdA geneencodes a large (200 kDa) outer membrane

autotransporter (AT) protein, which binds to the13th type III repeat (Hep-2 domain) of fibronectin(Fn) during in vitro and in vivo studies. We hypoth-esize that during infection the interaction betweenShdA and host intestinal Fn contributes towards theenhanced persistence displayed by subspecies I. Insupport of this, an S. typhimurium shdA knockoutstrain was recovered from murine fecal pellets inlower numbers and over a shorter duration com-pared to wild type strains. Our research aims toprovide structural characterisation of ShdA andbiophysical analysis of its physiologicalbiomolecular complexes. Such work serves as amodel for understanding bacterial ATs in generaland assists in the development of therapeuticstargeted against S. enterica subspecies I.Bioinformatic analysis of the ShdA amino acidresidue sequence revealed the presence of an N-terminal signal peptide, followed by a ~400 residuenon-repeat region, a ~1300 residue repeat region,consisting of three repeat types, termed A, B and C,and a C-terminal Beta-barrel domain. The repeatregion of ShdA is sufficient for interaction with Fn,with a low micromolar dissociation constant (Kd).Since a monoclonal antibody recognising the A3repeat abrogates binding to Fn, we are alsoanalysing the binding capacity of the isolated A3repeat, and other truncated forms of ShdA. Muchof the passenger domain is hypothesised to adopt aright-handed parallel Beta-helix structure on thebasis of ab initio secondary structure predictions,homology to other bacterial ATs, and in vitro circu-lar dichroism (CD) studies. Using this information,we have constructed a tertiary structural molecularmodel of ShdA. We are currently undertakingcrystallisation trials using truncated forms of ShdAwith the view of obtaining atomic-resolution struc-tures.



The annotation component is also accessed via theportal. For more information about the databaseand tools, contact [email protected].

B26PULSED FIELD GEL ELECTROPHORESISPATTERNS AND INVASIVENESS ASSAYSFOR SALMONELLA SCHWARZENGRUNDSTRAINS ISOLATED FROM RAW CHICKENMEAT IN TAIWANM. Chen1, Y. C. Hu2, T. C. Chen1, T. H. Chiu3, H. Y. Tsen2; 1Depart-ment of Food Science and Biotechnology, National Chung-Hsing University,Taichung , TAIWAN, 2Department of Food Science and Nutrition, Hung-Kuang University, Taichung County, TAIWAN, 3Department of FoodScience, National Penghu University, Penghu Hsien, TAIWAN

Salmonella Schwarzengrund has been in recent yearsrecognized as one of the most common Salmonellaserotypes contaminated in chicken meat in Asiaarea. In this study, 144 raw chicken meat isolates ofSalmonella Schwarzengrund obtained in differentyears from 2000 to 2003 from conventional marketsin Taiwan area were analyzed by pulsed field gelelectrophoresis (PFGE). Results showed that forthese 144 raw chicken meat isolates, when XbaI andAvrII were used for chromosomal DNA digestionfollowed by PFGE analysis, a total of 20 and 12PFGE patterns, respectively, were identified. Ofthem, 25 (17.4%) and 60 (41.7%) strains belong to asingle pattern of X1 and A2, respectively, and 28strains belong to a pattern combination of X1A2,27 strains belong to a pattern combination ofX3A2. They were clonally highly related and wereboth the major subtypes. However, neither thestrains in major subtypes nor the strains in minorsubtypes were not associated with their virulence interms of invasiveness to cultured human intestinalepithelium cell line Int-407, colon epithelium cellline Caco-2, and liver epithelium cell line HEP G2.In conclusion, this study revealed the subtypingdata and the related invasiveness capability forSalmonella Schwarzengrund strains collected inTaiwan area. Reports regarding the subtyping andvirulence study for S. Schwarzengrund were rare, ifany.

A25SALMONELLA GENOME ANNOTATION INTHE ERIC-BRC DATABASEV. Burland1, G. Plunkett, III1, J. D. Glasner1, E. Cabot1, B. Anderson1,E. Neeno-Eckwall1, Y. Qiu1, M. Rusch1, P. Liss1, T. Hampton2, R.Martell2, D. Pot2, R. Sfeir2, M. Shaker2, L. Schaull2, P. Shetty2, M. Wong2,F. R. Blattner1, J. M. Greene2, N. T. Perna1;1University of Wisconsin,Madison, WI, 2SRA International, Rockville, MDERIC (Enteropathogen Resource InformationCenter) is an NIAID Bioinformatics ResourceCenter for Biodefense and Emerging/Re-emergingInfectious Disease, formed by a partnership be-tween the University of Wisconsin-Madison andSRA International. This Center provides an infor-mation resource via a Web-based bioinformaticsportal, for five related enteropathogens: Salmonella,E. coli, Shigella, Yersinia pestis and Y. enterocolitica.ERIC contains 36 genomes including the fivecompleted genomes of Salmonella strains that arehuman pathogens, with more in the pipeline. Thedatabase integrates information on the genomicsequence, genome annotations, and other types ofrelated biological data, to facilitate research anddesign of therapeutics, diagnostics, and vaccines.Detailed annotations are being generated with eachentry supported by evidence with links to thesources of information, such as experimentalevidence from papers in PubMed. Ortholog infor-mation across all the enterobacteria is provided forevery gene. All entries will be curated. The projectprovides a forum for any researcher to share dataimmediately in a standardized format. Communitycontributions are strongly encouraged, using theASAP component (A Systematic Annotation Pack-age) developed at the University of Wisconsin-Madison expressly for distributed user annotations.ERIC/ASAP provides researchers with a way tolend their expertise in updating and correctinggenome annotations with new information via auser-friendly interface, to create a resource that isuseful for all Enterobacterial researchers. TheERIC system is also evolving to incorporate dataand provide analysis tools for comparativegenomics, microarrays and proteomics and supportscross-database queries. All tools and data developedin this project are freely accessed at the Web portalhttp://www.ericbrc.org, and for available download.

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C27ROLE OF PURINE BIOSYNTHESIS ININTRACELLULAR SURVIVAL ANDREPLICATION OF SALMONELLA ENTERICASEROVAR TYPHIMURIUMR. R. Mantena, O. L. Wijburg, R. A. Strugnell; Department ofMicrobiology and Immunology, The University of Melbourne, Melbourne,AUSTRALIA

Pathogenic Salmonellae are considered facultativeintracellular pathogens. While some species ofSalmonella exhibit a narrow host range (eg. S. Typhifor humans) and other species are much less hostrestricted (eg. S. Typhimurium), intra-macrophagesurvival appears to be a common pathogenic trait.Transposon mutagenesis was used to define genesthat were necessary for S. Typhimurium replicationwithin RAW264.7 cells. These studies revealed thatpurine biosynthesis was essential to macrophagesurvival and/or replication. Since earlier studies hadidentified purine metabolism as a key metabolic traitin Salmonella virulence, we selected the purinebiosynthetic pathway for detailed investigation. S.Typhimurium carrying a mutation in purG wascompared with bacteria deficient in purA, guaA andguaB. A purG deficiency resulted in reduced growthin RAW264.7 cells that was reflected by reduced netreplication in mice. This deficiency was comple-mented by phagosome escape, suggesting that thephagosome, but not the cytoplasm, is restricted forpurine availability. purA mutants were less capableof intracellular replication than purG mutants,indicating that both de novo and salvage purinepathways were important to net bacterial replicationin macrophage-like cells. A variety of inhibitors ofmacrophage killing were used to determine whetherpurine deficiency resulted in increased killing orreduced bacterial growth within macrophages - theresults from these studies suggest that mutantsdeficient in purine metabolism were highly sensitiveto killing by the fused phagolysosome. Thesestudies reinforce previous observations that thepurine biosynthetic pathway provides a target forthe development of novel anti-microbials, and thatbacterial survival and replication within macroph-ages is dependent on a complex bacterial metabo-lism.

A28ENVIRONMENTAL REGULATION OFSALMONELLA INVASION: USINGSHORT-CHAIN FATTY ACIDS TOMODULATE INVASIVENESS IN THECHICKEN GUTF. Van Immerseel1, V. Eeckhaut1, I. Gantois1, F. Pasmans1, I. Hautefort2,A. Thompson2, F. Haesebrouck1, J. C. Hinton2, R. Ducatelle1; 1Departmentof Pathology, Bacteriology and Avian Diseases, Research Group VeterinaryPublic Health and Zoonoses, Faculty of Veterinary Medicine, GhentUniversity, Merelbeke, BELGIUM, 2Molecular Microbiology Group,Institute of Food Research, Norwich, NR4 7UA, Norwich, UNITEDKINGDOM

During infection of a host, Salmonella bacteria areexposed to several environmental factors includingthe acidity of the stomach and the anoxia of thegut. In the intestine, the anaerobic environment isdominated by fermentative bacteria, which mainlyaccumulate three organic acids: acetate, propionateand butyrate. We assessed the role of short-chainfatty acids as environmental modulators of Salmo-nella virulence, using invasion assays of epithelialcells, DNA microarray studies and in vivo trials. Wehave shown that exposure of Salmonella to lowconcentrations of acetic acid increases invasion ofSalmonella in intestinal epithelial cells, while propi-onic and butyric acids decrease invasion, belowgrowth inhibitory concentrations. A comparativetranscriptomic analysis of Salmonella Enteritidis andSalmonella Typhimurium grown in medium supple-mented with butyrate was performed. We foundthat butyrate down-regulated the expression of 19genes common to both serovars by more than 2-fold, and that 17 of these genes were localised onthe Salmonella Pathogenicity Island I (SPI1), includ-ing the SPI1 regulatory hilD and invF. Expression ofgenes involved in central metabolism was notaltered, highlighting the specificity of action ofbutyric acid. We have also shown that supplementa-tion of the feed with acetic acid-coated productsincreases the ability of Salmonella Enteritidis tocolonise a chicken caecum model by 100-fold aftera 3 day-treatment. In contrast, propionic and bu-tyric acid-coated products decrease colonization ofthe chicken intestinal tract by 100-fold in the sameperiod of time. Moreover, we showed that butyricacid-coated products decreased faecal shedding of



of significant mutagenic effect. However, twofractions showed weak mutagenic effect, thereforefurther experiments, including animal test, shouldbe conducted.

C30SALMONELLA ENTERICA SEROVARCHOLERAESUIS REPLICATES WITHINMACROPHAGES AND INDUCESAPOPTOSIS THROUGH THE ACTIVATIONOF CASPASE-9-DEPENDENT INTRINSICPATHWAYC. Chiu1, W. Tang2, H. Chen1, S. Liu2; 1Chang Gung Children’s Hospitaland Chang Gung University College of Medicine, Kweishan, Taoyuan,TAIWAN, 2Department of Molecular Biotechnology, Da-Yeh University,Changhua, TAIWAN

There are more than 2,500 serovars among Salmo-nella. S. Typhi and S. Paratyphi cause typhoid andparatyphoid fevers in humans, respectively, whileother non-typhoid Salmonella, such as S.Typhimurium, S. Enteritidis, and S. Choleraesuis,usually cause gastrointestinal infections and bacter-emia. Survival within macrophages and induction ofapoptosis appear to be an important virulencemechanism that is used by Salmonella to suppress thehost immune response and to persist at sites ofinfection. In this study, we used human macrophagecell line, THP-1, and different serovars of Salmonellato study the Salmonella-host cell interaction. Allserovars of Salmonella examined appeared to survivewithin THP-1 cells following the ingestion. Never-theless, S. Choleraesuis, compared to S.Typhimurium, S. Typhi, and S. Enteritidis, ex-pressed higher ability to survive and replicate withinmacrophages. Flow cytometry with Annexin V-FITC/PI double staining was used to evaluate thecell death of macrophages caused by Salmonella. At12 and 14 hr post-infection, S. Choleraesuis pro-voked 16% and 18% of THP-1 macrophages to celldeath. Only the PMA-activated THP-1 cells exhib-ited significant level of apoptosis, compared tonon-activated THP-1 cells, following Salmonellainfection. Transmission electron microscopy dem-onstrated typical morphological changes ofapoptosis in THP-1 cells, including nuclear conden-sation, membrane blebbing, and cytoplasmic vacu-olization. We also used Oligo GEArray (Superarray)

S. Enteritidis from a seeder model throughout therearing period up to slaughter age. Taken together,all our data suggest that Salmonella modulates itsinvasiveness according to the short-chain fatty acidpattern it encounters. This is the first proposedexplanation for why Salmonella preferentially invadesand colonises the chicken caecum, where it encoun-ters the appropriate level of acetic acid to triggerinvasion. We suggest that the modification offermentation pattern in the gut can be used tolower levels of Salmonella colonization of chickensby elevating levels of butyric acid and reducinglevels of acetic acid.

B29SALMONELLA TYPHIMURIUM AS A TOOLTO DETERMINE LACTA DROPMUTAGENICITYB. Fazly Bazzaz, M. Homauni; School of Pharmacy &BiotechnologyResearch Center, Mashhad, IRAN (ISLAMIC REPUBLIC OF)

Objective: Salmonella typhimurium, genetically ma-nipulated tester strains, are used in Ames test todetermine the mutagenicity of compounds. In thisinvestigation we report the results of Ames test onmixture of some plant extracts, namely” Lactadrop”. This has been claimed to be useful forimproving lactation. Materials and Methods:Lacta drop, as a mixture of plant extracts, wasreceived from Dr. F. Moatar, after confirming thegenotype of standard tester strains (TA97, TA98,TA100 and TA102 of S. typhimurium), the Amesmutagenicity test was performed on differentfractions of Lacta drop (5 fractions) in the presenceand absence of S9 mix. Results: From five frac-tions of “Lacta drop” , fractions 2 and 5 (aqueousfractions) showed weak mutagenic activity, espe-cially in the presence of S9 mix (number of rever-tant colonies were: 470, 495 (TA97), 420, 480(TA100), 500, 497 (TA102) for fractions 2 and 5respectively). While Lacta drop as a whole was notmutagenic (number of revertant colonies were: 85(TA97), 60 (TA100) and 73 (TA102). Conclusion:Analysis of Ames test using S. typhimuriun strains asa tool to measure be conducted.mutagenicity ofLacta drop in the presence and absence of S9 mixin comparison with positive controls, indicated lack

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to assess the gene expression profiles of macroph-ages that underwent apoptosis following ingestionof Salmonella. S. Choleraesuis apparently inducedthe expression of genes associated with apoptosis inTHP-1, including caspase-3, caspase-9, Bax andBad. Our data suggest that Salmonella induced thelate-phase apoptosis of macrophages mainlythrough the activation of intrinsic pathway. Theability to modulate the activity of effector caspasesmay represent an unexploited avenue for therapeu-tic intervention in systemic Salmonella infections.

A31INVESTIGATION INTO THE SEASONALSALMONELLOSIS IN LACTATING DAIRYCATTLET. S. Edrington1, T. T. Ross2, T. R. Callaway1, C. H. Martinez2, M. E.Hume1, K. J. Genovese1, R. C. Anderson1, D. J. Nisbet1; 1USDA-ARS-FFSRU, College Station, TX, 2New Mexico State University, Las Cruces,NM

Sporadic salmonellosis has been reported in maturelactating dairy cattle in the southwestern UnitedStates and is responsible for substantial loss ofincome for the farmers. This problem is intriguingin that Salmonella can be cultured from the feces ofthese cattle throughout the year, however it ispathogenic only during the late summer/early falland in certain years. In attempting to elucidatepossible explanations, we sampled apparentlyhealthy and diarrhetic cattle during an outbreak ona 2000 head dairy in 2003. The following year, 30fresh cows were identified and monthly fecalsamples (February - October) collected. Samples ofthe total mixed ration (TMR), trough water, and pensoil were also sampled monthly for Salmonellaculture. No appreciable differences in serogroup orserotype were observed in comparison of isolatesfrom healthy and sick cattle. No genotypic differ-ences were noted when comparing sick versushealthy isolates using pulsed-field gel electrophore-sis, although multiple genotypes within serotypewere observed for a number of the isolates exam-ined. Antimicrobial susceptibility patterns weresimilar among Salmonella isolates cultured from sickand healthy cattle. During year two of the study,Salmonella was routinely cultured from the dairy

cattle and the environment each month of thesurveillance period, however no outbreak of salmo-nellosis was observed. Monthly fecal Salmonellaprevalence ranged from 19 to 96%, averaging 54%over the 9-month period. The prevalence of Salmo-nella isolated from total mixed ration (TMR), waterand pen soil samples was also highly variable frommonth to month. Serogroup prevalence varied bymonth and by sample type with multiple serogroupsidentified in feces, TMR, water and soil. Multipleserogroups and serotypes were identified in fecalsamples collected from individual cattle. Fiveisolates randomly selected from one cow’s positivefecal sample were identified as five different sero-types. The results of this research highlight thecomplexity of monitoring Salmonella at the farmlevel. Not only is the prevalence of Salmonellaconstantly changing in cattle and the environment,but also the predominant serogroup and serotype.As the salmonellosis outbreaks typically occur inlate August, we hypothesize that they are a result ofchronic heat stress, weakening the immune system,and enabling the already present Salmonella tobecome problematic.

B32PREVALENCE OF MULTI-DRUG RESISTANTSALMONELLA IN DAIRY CATTLET. S. Edrington, T. R. Callaway, R. C. Anderson, D. J. Nisbet; USDA-ARS-FFSRU, College Station, TX

To determine the prevalence of multi-drug resistant(MDR) Salmonella in dairy cattle and which groupsof cattle may be more likely to harbor MDR Salmo-nella, we sampled animals on four commercial dairyfarms that all utilized a central calf/heifer raisingfacility. Fecal samples were obtained from hutchcalves, 12 and 24 month old heifers, lactating cows,dry cows, and cows in the sick/fresh pen in Octo-ber 2005 and again in March 2006. Fecal Salmonellaprevalence varied (0 to 96% positive) among groupsof cattle and between the two collection periods.Hutch calves and cattle in the sick/fresh pen weremore likely to harbor MDR Salmonella than heifersor lactating and dry cows. However, a significantnumber of MDR Salmonella were detected in lactat-ing cattle on one farm during the October collec-


POSTER ABSTRACTS

POSTER ABSTRACTS

a bimodal O-antigen chain length distribution: an L-LPS determined by wzzST and a very long (VL-LPS)controlled by wzzfepE. In S. typhi wzzfepE is apseudogene We previously demonstrated that Oantigen production by S. Typhi increases at theonset of stationary phase and correlates with agrowth-regulated expression of the transcriptionelongation factor RfaH. This study shows thatexpression of both L-LPS and VL-LPS by S.Typhimurium also increases during stationary phase,compared to exponential phase. In this work weshow that expression of both L-LPS and VL-LPSOAg by serovar Typhimurium also increases duringstationary phase compared to exponential phase.We constructed non-polar deletion mutants in wzzSTand wzzfepE genes. S. Typhimurium DELTAwzzST didnot produce L-LPS while expression of VL-LPSremained relatively constant during growth;DELTAwzzfepE was devoid of VL-LPS but synthe-sized L-LPS and, as in S. Typhi, the expression ofL-LPS was growth phase regulated. Serum sensitiv-ity assays showed that wild type S. Typhimurium isquite resistant to serum regardless of the growthphase. Mutant DELTAwzzST was as resistant as thewt; DELTAwzzfepE was significantly more serumsensitive but only in exponential phase. Theseresults indicate that, VL-LPS is required for survivalin serum; but in the absence of VL-LPS, as in S.Typhi and S. Typhimurium DELTAwzzfepE, L-LPSplays a relevant role in serum resistance, conferringprotection to exponentially-grown bacteria. We alsoinvestigated the role of RfaH in LPS synthesis andserum resistance. Non-polar deletions of the rfaHgene in S. Typhimurium did not produce O antigenbut was able to synthesize a complete core region.This mutant was did not survive in human serum atall times of incubation. When complemented withthe wild type rfaH gene, both LPS profiles andserum resistance were restored, confirming the roleof the transcriptional antiterminator in LPS biosyn-thesis. Supported by grants 1040562 from Fondecyt(to IC) and MOP- 2110206 from the CanadianInstitutes of Health Research (to MAV)

tion. Even so, as the 24 month old heifers andmature cows were largely negative for MDR Salmo-nella, the use of a central calf/heifer facility doesnot appear to facilitate the spread of MDR Salmo-nella among dairy herds. Eleven differentserogroups were identified with C1 being the pre-dominant serogroup. The MDR Salmonella belongedto only one serogroup (B) and were predominantlyof the serotypes Reading and Typhimurium.Twenty-eight different serotypes were identifiedwith more types identified in the March collection(27), when overall Salmonella prevalence was sub-stantially lower, compared to the October collection(17). The five most common serotypes in all classesof cattle in descending order of frequency were:Reading, Senftenberg, Montevideo, Fresno, andCerro. No Salmonella Newport was identified. Only asmall proportion of the cultured Salmonella isolatesexamined for antimicrobial resistance were MDR,although 30 isolates were resistant to 9 or 10 antibi-otics on the NARM’s panel. While the presence ofMDR Salmonella is a cause for concern, all isolatesexamined were susceptible to ciprofloxacin andceftriaxone, two antibiotics used in the treatment ofsevere cases of human salmonellosis.

C33GROWTH-PHASE REGULATION OFLIPOPOLYSACCHARIDE O-ANTIGENCHAIN LENGTH AND MODALDISTRIBUTION INFLUENCE SERUMRESISTANCE IN SALMONELLAI. Contreras1, D. Bravo1, J. Carter1, A. Hoare1, S. Alvarez1, C. Blondel1,M. Zaldivar1, M. A. Valvano2; 1University of Chile, Santiago, CHILE,2University of Werstern Ontario, London, ON, CANADA

Lipopolysaccharide (LPS) plays a critical role in thepathogenesis of infections by Salmonella. The LPSmolecule is composed of three structural domains:the lipid A region which is embedded in the outermembrane, the core oligosaccharide, and the O-specific polysaccharide chain (or O antigen), that isexposed on the bacterial surface. The number of Ounits attached to the lipid-A core is regulated by thechain-length regulator, Wzz. S enterica serovar Typhihas a long chain modal length (L-LPS) controlledby the wzz gene, while serovar Typhimurium shows

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A34IDENTIFICATION OF DIFFERENCES INGENE CONTENT BETWEEN SALMONELLAENTERICA SEROVARS FROM SUBSPECIESII, IIIA, IIIB AND IVM. A. Ponder1, M. F. McClelland2, S. Clifton3, K. Delehaunty4, J.Godfrey3, R. Wilson3, P. I. Fields1; 1Center for Disease Control, Atlanta,GA, 2Sidney Kimmel Cancer Center, San Deigo, CA, 3WashingtonUniversity Genome Sequencing Center, St. Louis, MO, 4WashingtonUniversity Genome Sequencing Center, St.Louis, MO

Background: Salmonella is a routine food-bornegastrointestinal pathogen. “Salmonella” consists of 2species, 7 subspecies, and >2500 different sero-types. The majority of Salmonella enterica isolatesfrom humans belong to subspecies (ssp) I, althoughhuman infections due to the other subspecies occur.Current knowledge on the genetics and pathogen-esis of Salmonella is limited to a few ssp I serotypes.Methods and Results: The partial genome sequenceof S. enterica subspecies arizonae serotype 62:z4,z23:-(ssp IIIa), and S.enterica subspecies diarizonae61:1,v:1,5,(7) (ssp IIIb) were annotated using RPS-BLAST. Putative genes of <95% identity in awindow of 100bp with any Salmonella sequence wereconsidered novel. The predicted gene content ofthe ssp IIIa genome revealed 251 (~5.3%) novelgenes, 16.9% of these putative genes have nopredicted function. . The ssp IIIb genome con-tained 361 novel genes (~9.5%), 46% of the novelpredicted genes have no known function or arehypothetical. Among the genes with homology toknown virulence factors are 28 genes, including thehigh pathogenicity island of uropathogenic E.coliand Yersinia, and putative toxins. Both genomespossess homologues to virulence factors describedin other pathogenic bacteria and found in any ssp Igenome sequences including pili, fimbrae,aerobactin and putative toxins. Additional genesthat may be involved in virulence and/or host rangein serovars of ssp II and IV were detected bysubtractive hybridization experiments between sspII or IV serovars and Salmonella enterica serotypeTyphimurium LT2. Carbon source utilization variedbetween selected serotypes of subspecies IV andsubspecies II which may allow exploitation ofdifferent niches. Conclusions: The availability of

ssp IIIa and ssp IIIb genomic sequence offers theopportunity to identify potential virulence and/orhost range-associated genes not present in ssp Iserovars. The presence of pseudogenes and ~40mobile element associated genes may reflect adapta-tion to a reptilian host of IIIa 62:z4,z23:-. Thepresence of potential virulence genes, metabolismgenes and mobile element associated genes mayallow for the expansion of its host range anddisease etiology in IIIb 61:l,v:1,5,7. Future com-parative hybridization of genomic DNA to Salmo-nella serovar microarrays, including novel IIIa andIIIb genes, will explore the genetic diversity withinSalmonella and improve our understanding of thespectrum of Salmonella diseases.

B35EFFECT OF SALMONELLA ENTERICASEROVAR TYPHI VI-ANTIGEN ONTOLL-LIKE RECEPTOR SIGNALLINGPATHWAYS IN HUMAN EPITHELIAL CELLLINESS. E. Winter1, M. Raffatellu1, D. Chessa1, B. A. Cobb2, C. J. Lewis2, S.C. Szu3, J. B. Robbins3, H. Rüssmann4, A. J. Baumler1; 1UC Davis, Davis,CA, 2Case Western Reserve University, Cleveland, OH, 3NIH/NICHD,Bethesda, MD, 4Max von Pettenkofer-Institut LMU, Munich, GERMANY

S. enterica Serovar Typhimurium causes a localisedinfection resulting in inflammatory diarrhea that ischaracterised by neutrophil influx in the intestinalmucosa. In contrast, infection with S. entericaSerovar Typhi leads to a systemic infection (typhoidfever) and about one third of patients developdiarrhea witin 5-9 days of ingesting the organism.However, no neutrophil influx in the intestinallumen can be observed. We have previously shown,that a non-capsulated S. Typhi viaB mutant (Vi-)was able to induce the expression of the CXC-chemokine IL-8 in polarized human epithelial cells,macrophage-like cells and human colonic explants,while this response was significantly downregulatedin S. Typhi wild type (Vi+) infected cells. In humanepithelial cells, the secretion of IL-8 was at leastpartially mediated by TLR4 and TLR5, indicating,that the Vi-antigen interferes with TLR signaling.Our central hypothesis states that the Vi-antigeninterferes with Toll-like receptor signaling pathways,


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creating strains of Salmonella and other pathogenswith resistance to several drugs. Such resistantchromosomal genes and plasmids are faithfullyreplicated and inherited by all subsequent progenythat ultimately makes its way into humans andrapidly transmit around the world. In current study,we investigated antimicrobial resistant profile of111 Salmonella enterica strains from poultry tissuesand table eggs. These strains were isolated from1747 epidemiologicaly unrelated random samples,collected from twelve different sources and 56locations in the Northern Punjab area of Pakistan.The Salmonella entercia pool comprised of 63(56.76%) S. Albany and 48 (43.24%) S. Enteritidisisolates. All these strains were screened for resis-tance to a panel of 14 antimicrobial agents by discdiffusion method. This included amoxicillin (20μg/disc), ampicillin (10μg/disc), cefamizin (30μg/disc),cefoperazone (75μg/disc), chloramphenicol (30μg/disc), erythromycin (15μg/disc), gentamycin (10μg/disc), kanamycin (20μg/disc), nalidixic acid (30μg/disc), neomycin (30μg/disc), plasmacolin (10μg/disc), quinalcen (10μg/disc), sulfamethoxazole(23.75 μg/disc), and tetracycline (30μg/disc).Multiple drug resistance was detected in all 63isolates of S. Albany, which were resistant to kana-mycin, tetracycline and sulsfamethoxazole. In S.Enteritits sulfamethoxazole resistance was detectedin 100% of the isolates followed by kanamycin 29(60.42%) and tetracycline 27 (65.25%). All isolatesof S. Albany and S. Enteritis were highly sensitiveto plasmacoloin, quinolcen, cefoperazone and restof the antimicrobial agents included in the assay.

A37THE PRIMORDIAL FUNCTION OF AVIRULENCE GENE REGULATOR: SLYACONTROLS A FUSARIC ACID EFFLUXPUMPW. W. Navarre, K. L. Main-Hester, T. E. Bumgarner, F. C. Fang , S. J.Libby; University of Washington, Seattle, WA

SlyA is a MarR family transcriptional regulator thatis widely distributed in the gamma-proteobacteria.SlyA is critical for virulence in both Salmonella andYersinia sp., but appears to be cryptic in other

resulting in downregulation of mitogen-activatedprotein (MAP) kinase cascades and the NFêBpathway, thereby inhibiting the secretion of CXC-chemokines. As a model, we used T84 cells, a TLR5expressing epithelial cell line. The activation ofcertain MAP kinases and NFêB upon infection wasmeasured by Western Blot using phosphorylationstate specific antibodies. Significant upregulation ofERK1/2, JNK, p38 MAPK and NFêB was ob-served in T84 cells infected with S. Typhi viaBmutant in comparsion to cells infected with the wildtype strain, a viaB fliC mutant or uninfected cells. Asexpected, IRF-3 (MyD88-independent pathway) wasnot activated. Since masking of the flagella by theexopolysaccharide cannot be ruled out in thisexperiment, the activity of purified componentswas tested. T84 cells incubated with flagellin puri-fied from S. Typhimurium showed upregulation ofERK1/2, JNK, p38 MAPK and NFêB, which couldbe restored to levels comparable to untreated cellsby addition of purified Vi-antigen. By using trans-fected HEK293 cells expressing different TLRs, wewill further investigate, whether the Vi-antigen isable to block signaling through different TLRsignaling pathways.

C36ANTIMICROBIAL RESISTANCE INSALMONELLA ENTERICA SUBSP.ENTERICASEROVARS ALBANY AND ENTERITIDISISOLATES FROM PAKISTANS. U. Sajid; Foundation University Medical College, Rawalpindi,PAKISTAN

The widespread drug resistance in bacteria is be-lieved to be an inevitable adaptive or genetic re-sponse to the selective pressure associated withextensive oral use of antibiotics. An unfortunatebut common practice that has also selected forresistant strains, especially intestinal pathogens suchas Salmonella, is spiking poultry feed with antibioticsto increase growth rate and help prevent intestinalinfections. It has now become evident that normalintestinal flora maintain a gene pool of resistantplasmids and transposable drug resistant sequencesthat are readily shared with other intestinal bacteria,

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species including Escherichia coli. We have previ-ously found that Salmonella SlyA-repressed genesare highly conserved among enteric species. How-ever, all SlyA-activated genes were acquired afterSalmonella diverged from E. coli, suggesting thatSlyA evolved to be an activator after Salmonellabecame a distinct species. In parallel studies we andothers have found that SlyA (called RovA inYersinia) can activate virulence gene expression byantagonizing silencing by the nucleoid protein H-NS. We have recently found that H-NS selectivelysilences horizontally-acquired DNA by recognizingsequences with low-AT content. In this manner, H-NS can protect bacteria from unregulated expres-sion of foreign DNA while simultaneously facilitat-ing the acquisition of such sequences. We haveundertaken studies to determine the evolutionarypathway of SlyA as a virulence regulator in Salmo-nella. We report here that SlyA is divergently tran-scribed from a putative efflux pump (ydhIJK) in E.coli, Klebsiella, and Salmonella. Expression of theydhIJK locus is repressed by SlyA. One substrate ofthe YdhIJK pump is shown to be the antibioticfusaric acid. We have identified an ~80 bp region ofthe SlyA promoter that is radically divergent inSalmonella sp. and E. coli, and we have obtainedevidence that this region may help to explain differ-ences in slyA expression between the two species.We also find that the region upstream of theYersinia rovA gene differs from slyA in eitherSalmonella or E. coli, suggesting a distinct evolu-tionary pathway for rovA regulation.We suggest that SlyA, an endogenous regulator of aminor efflux pump, has evolved through indepen-dent events in Salmonella and Yersinia to become avirulence regulator that unlocks expression of asubset of H-NS silenced genes critical for survivalwithin an animal host. The unique characteristics ofSlyA that allow it to serve this role remain to bedetermined.

B38TOLL-LIKE RECEPTOR 4 (TLR4) ANDTYPHOID FEVER IN VIETNAMN. T. Hue1,2, M. N. Lanh3, L. T. Phuong3, H. Vinh2, N. T. Chinh2, T.T. Hien2, J. J. Farrar1,4, S. J. Dunstan1,4; 1Oxford University ClinicalResearch Unit, Hospital for Tropical Diseases, Ho Chi Minh, VIET NAM,2Hospital for Tropical Diseases, Ho Chi Minh, VIET NAM, 3Dong ThapProvincial Hospital, Dong Thap, VIET NAM, 4Centre for TropicalMedicine, Nuffield Department of Clinical Medicine, Oxford University,UNITED KINGDOM

Understanding host genetics may yield answers thatlead to the development of new therapeutics forinfectious disease such as Typhoid fever. Using agenetic approach we aim to investigate a number ofimmune response genes that may be important inthe defense against Typhoid fever. Here we describestudies investigating the genetic variation within thehuman TLR4 gene encoding the principal receptorfor bacterial endotoxin recognition, an element ofinnate immunity that contributes to the first line ofdefense against infectious disease. Any geneticchange in TLR4 could detrimentally affect the firstline of defense against S. Typhi infection. Mutationdetection and genotyping of the TLR4 gene wasperformed using DNA isolated from 372 Vietnam-ese kinh typhoid fever patients and 372 populationcontrols. dHPLC was used to detect mutationswithin the upstream and exonic regions of TLR4. Atotal of 10 mutations were identified in the Viet-namese kinh, of which 9 are novel. Two low fre-quency SNPs (T4025A, 0.04 and C4215G, 0.01) hadallele frequencies that were higher in cases thancontrols however they only showed borderlinesignificance (T4025A: OR 1.9, 95%CI 0.9-4.3, P0.07 and C4215G: OR 6.7, 95%CI 0.8-307, P 0.04).Another interesting finding from this study is thatthe majority of non-synonymous exonic SNPs andone promoter SNP (A-271G) were only present intyphoid cases, however these SNPs had low allelefrequencies. In conclusion we determined theextent of genetic variation within TLR4 in a Viet-namese kinh population. It appears that this genemay be involved in defense against Typhoid fever,as evidenced by weak associations with two SNPsand the presence of low frequency non-synony-mous SNPs in only Typhoid fever cases.


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effect), or by induction of changes in the host thatreflect the intracellular Salmonella (indirect effect).

A40MOTILITY OF SALMONELLA-CONTAININGVACUOLES IS RESTRICTED BY SSEG IN ASUBCELLULAR-SPECIFIC MANNERL. J. Mota, A. E. Ramsden, D. W. Holden; Imperial College London,London, UNITED KINGDOM

Intracellular replication of Salmonella enterica occursin membrane-bound compartments, called Salmo-nella-containing vacuoles (SCVs). Following invasionof epithelial cells, most SCVs migrate to a peri-nuclear region and become surrounded by Golgimembranes, whereupon bacterial replication occurs.The association of SCVs with the Golgi is depen-dent on the Salmonella-pathogenicity island-2 (SPI-2)type III secretion system (T3SS) effector SseG.However, little is known about SCV movement andhow SseG works. Here we show that in epithelialcells, 2 hours were required for accumulation ofSCVs within 5 ìm from the microtubule (MT)organizing center (MTOC), located in the samesubcellular region as the Golgi network. This initialSCV movement was saltatory, bidirectional and MT-dependent. An intact Golgi, SseG and SPI-2 T3SSwere dispensable for SCV localization to theMTOC/Golgi, but were essential for retention ofSCVs in that region. Between 6-8 h post-invasion,we observed an MT-dependent redistribution ofsseG::aphT SCVs away from the MTOC/Golgiregion. Live-cell imaging during this period revealedthat wild-type SCVs were relatively non-motilewhereas most sseG::aphT SCVs were highly motile.Further live-cell experiments indicated that SseGcontrols SCV motility by restricting vacuole speedwithin the MTOC/Golgi region, suggesting a modelin which SseG mediates tethering between the SCVand Golgi-related molecules.

C39GENTAMICIN HAS A PHYSIOLOGICALEFFECT ON INTRACELLULAR SALMONELLAENTERICA SEROTYPES TYPHIMURIUM ANDVIRCHOWO. Menashe, T. Baazov, S. Yaron; Technion – Israel Institute ofTechnology, Haifa, ISRAEL

Background: The ability of Salmonella enterica tosurvive and even to replicate within the pagosomeis an effective strategy of avoiding the defensemechanism of the host. Gentamicin (Gn) is com-monly used to eliminate the extracellular Salmonellain tissue culture studies. The goal of this study wasto determine the influence of aminoglycosides,particularly Gn on two intracellular Salmonellaenterica serotypes: Virchow 101591 (Gn MIC=8μgml-1) and Typhimurium ATCC 14028 (Gn MIC=4μg ml-1). Methods: In the present study we devel-oped a method to monitor the effectiveness ofantimicrobial agents in real time. By using thebacterial luciferase system as a reporter to physi-ological changes, we were able to estimate theantibiotic permeability and activity in the broth andalso within the host cells. Results: Only 1-logreduction of intracellular Salmonella within mac-rophages (J774A.1) was observed after treatmentwith 100-150μg ml-1 Gn for 1 hour. However,significant physiological changes were observed byusing even lower doses of Gn as indicated by theluciferase system. When we treated the intracellularbacteria with Gn (15μg ml-1 for S. Typhimurium and100μg ml-1 for S. Virchow) the effect of Gn on lightemission was significant within 30±5 minutes.When higher doses of Gn were used (over 50μg ml-1 in S. Typhimurium and 150μg ml-1 in S. Virchow), thiseffect was observed within less than 5 minutes. Incontradiction, Gn influence on the light emissionof planktonic Salmonella in broth was observedwithin less than 5 minutes with all concentrations.Similar results were obtained when we used othertypes of aminoglycosides such as tobramycin andkanamycin. Conclusion: Our findings indicate thatexpression of proteins in intracellular Salmonella isaffected by Gn. This effect can be achieved byeither Gn penetration into the bacterial cells (direct

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B41INVOLVEMENT OF CASPASE-3, OXIDANTSAND TNF-ALPHA IN CAUSING APOPTOSISOF MACROPHAGES BY COORDINATELYEXPRESSED IMMUNODOMINANTSALMONELLA 69 KDA PHENOTYPE UNDERSTRESS CONDITIONSV. Chanana1, S. Majumdar2, P. Rishi1; 1Panjab University, Chandigarh,India., Chandigarh, INDIA, 2Post Graduate Institute of Medical Educationand Research (PGIMER), Chandigarh, INDIA

Invasive Salmonella has been reported to inducemacrophage apoptosis as a part of its infectionprocess, which may allow it to avoid detection bythe innate immune system. However, the inductionof apoptosis remains to be looked into under thedifferent host environments experienced by patho-gen. Successful pathogens overcome the hostenvironmental stresses by the coordinated expres-sion of various genes and eventually proteins. Since,the surface of the microbe is likely to come incontact with the host initially, an attempt was madeto identify the Salmonella outer membrane proteins(OMPs) which may get expressed under more thanone environmental conditions simulating the in vivoones and to assess their apoptotic potential. In thepresent study, S. typhi was grown under iron, oxida-tive and anaerobic stress conditions and their OMPprofiles were compared. The phenotypic similarityamong the stresses-induced proteins was assessedon the basis of their homogeneity, cross reactivityand HPLC. For apoptosis; assessment of nucleoso-mal DNA, nuclear staining with Acridine orangeand Hoechst33342 as well as flow cytometry wasdone. Estimations of caspase-3 activity, cytokines(TNF-á, Il-6, IL-1á), oxidants (ROS and RNIs),levels of antioxidants (SOD and catalase) were donefor their possible role in causing the macrophageapoptosis. A 69 kDa OMP was found to expresswith enhanced intensity under the selected stressconditions. The protein expressed under oxidativestress and anaerobic conditions reacted with theantibodies raised against iron regulated outer mem-brane protein, indicating the sharing of at leastsome of the epitopes. A single peak observed aftersubjecting the pooled 69 kDa protein sample toHPLC confirmed the purity and phenotypic similar-

ity of this protein. Reactivity of pooled 69 kDaprotein with 85% of widal positive sera fromtyphoid patients revealed its in vivo expression.Analysis of data revealed that stress-induced 69kDa OMP caused apoptotic cell death in 51% ofmacrophages whereas OMPs of S. typhi grownunder normal conditions accounted for only 30%apoptosis. A significantly enhanced activity ofcaspase-3 and cytokines (TNF-á, IL-1á, IL-6) wereobserved during macrophage apoptosis induced bythis protein. A significant increase in the levels ofoxidants and decrease in antioxidants was alsoobserved. These results suggest that caspase-3 andTNF-á in conjunction with other cytokines mayinduce apoptotic cell death by coordinately ex-pressed 69 kDa OMP of S. typhi under iron, oxida-tive and anaerobic stress conditions, through theup-regulation of oxidants and down-regulation ofantioxidants. These findings may be relevant for thebetter understanding of the disease pathophysiol-ogy and for the future developments of diagnosticand preventive strategies during the host-pathogeninteractions.

C42VARIANT SALMONELLA GENOMICISLAND 1 ANTIBIOTIC RESISTANCE GENECLUSTERS IN SALMONELLA ENTERICASEROVAR DERBY ISOLATES FROMHUMANS IN TAIWANC. Hsiu-Ling1, K. Lin-Xian1, D. Benoit2, P. Karine2, C. Axel2, C. Chi-Shih3, C. Cheng-Hsun1; 1Department of Pediatrics, Chang Gung Children’sHospital, Chang Gung University College of Medicine, Taoyuan, TAIWAN,2Plasticité Génomique, Biodiversité, Antibiorésistance, Infectiologie Animale,Santé Publique, Centre de Tours, Nouzilly, FRANCE, 3Department ofApplied Microbiology, National Chiayi University, Chiayi, TAIWAN

Salmonella genomic island 1 (SGI1) is a genomicisland containing an antibiotic resistance genecluster, located in a complex class 1 integron. SGI1was initially described in multidrug-resistant (MDR)Salmonella enterica serovar Typhimurium DT104.Such strains containing SGI1 are resistant to ampi-cillin, chloramphenicol/florenicol, streptomycin/apectinomycin, sulfonamides, and tetracycline, andthus display an ACSSuT-resistance phenotype. Notonly confined to DT104, SGI1 has also been found



secreted by a Type I secretion system also encodedwithin SPI4. An investigation of the regulation ofSPI4 expression has identified an operon polaritysuppressor (ops) sequence within the 5’ untranslatedregion. The ops sequence is predicted to bind RfaH,a factor that promotes RNA polymerase bindingand facilitates transcription through large operons.Using quantitative RT-PCR and mutational analysis,we have shown that the transcript level ofSTM4261 is decreased in an rfaH mutant or a straincarrying a scrambled ops sequence. Using quantita-tive RTPCR and a number of isogenic mutants, wehave also found that SPI4 shares a number ofregulatory characteristics with SPI1, includingactivation by SirA, HilA, HilC, and HilD, andnegative regulation by PhoP and HilE. However,SPI4 is not highly expressed under conditions ofhigh osmolarity and low aeration that result inmaximal SPI1 induction, suggesting that SPI1 andSPI4 do not respond identically to environmentalsignals. The genetic elements responsible for differ-ences in SPI1 and SPI4 expression are currentlybeing defined. Given the similarities in SPI1 andSPI4 regulation, we have investigated the role ofSPI4 in an in vitro model of Salmonella invasionusing human epithelial cells. Preliminary observa-tions indicate that a SPI4 mutant is efficientlyinternalized by epithelial cells but is impaired in itsability to replicate intracellularly, and this phenotypeis SPI1-independent. Collectively, our observationssuggest a role of SPI4 in Salmonella interactionswith host epithelial cells.

B44POULTRY AS SALMONELLA INFECTIONSOURCE FOR WORKERS IN LAYING HENSFARMS AND EGG PACKAGING PLANTSE. Circella1, G. Bruni1, P. Battista1, D. Pennelli2, A. Camarda1;1University of Bari, Valenzano, ITALY, 2Istituto ZooprofilatticoSperimentale, Brescia, ITALY

Salmonella (S.) enteritidis and S. typhimurium are themain responsible of human food poisoning. Poultryis the principal reservoir of Salmonella. Thesebacteria are transmitted by infected meat and oftenby eggs and eggs products. Workers employed infarms and in selection and packaging eggs plants are

in other serovars of Salmonella, including Agona,Paratyphi B, Newport, and Albany. SGI1 has beenrecently demonstrated to be an integrativemobilizable element. Here we have screened a setof MDR S. Derby human isolates from Taiwan forthe presence of SGI1. Characterization of the SGI1variants in these isolates was based on PCR map-ping and sequence analysis. Out of the 23 isolates,13 isolates were found to harbor variant SGI1-likeelements and could be defined into three types asfollows, (i) typical SGI1, which was encountered inmost of the isolates (7/13); (ii) SGI1-A, whichcontained an additional region carrying orf513, aputative transposase gene, and dfrA10 locatedbetween duplicated qacEá1/sulI genes (1/13); and(iii) SGI1-I, which included the dfrA1-orfC cassettearray instead of the blaPSE-1 (5/13). S. Derby is acommon serovar of non-typhoid Salmonella thatcauses infection in humans in Taiwan. SGI1 mayplay an important role in the dissemination ofmultidrug-resistance among S. Derby and otherserovars of Salmonella.

A43THE ROLE OF SALMONELLAPATHOGENICITY ISLAND-4 (SPI4) INBACTERIAL INTERACTIONS WITHEPITHELIAL CELLSK. L. Main-Hester1, G. A. Thomas2, S. J. Libby1, F. C. Fang1;1University of Washington, Seattle, WA, 2VWR, Inc., Bethesda, MD

Salmonella enterica serovar Typhimurium harbors fivepathogenicity islands (SPI) encoding functionsrequired for infection in a variety of vertebratehosts. SPI4 is a 24kb pathogenicity island contain-ing six ORFs numbered STM4257 to STM4262(siiA-F). The contribution of SPI4 to Salmonellavirulence is incompletely characterized. A recentstudy by Morgan et al (Mol. Micro. 54:994, 2005)demonstrated that SPI4 is required for intestinalinfection in calves and systemic infection in micefollowing oral but not intraperitoneal administra-tion, suggesting that SPI4, like SPI1, plays animportant role in the intestinal phase of infection.Here we demonstrate that ORF STM4261 (siiE)encodes a large protein approximately 600kD that is

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particularly exposed to the infection. In order tovalue the contamination risk in these employed, amonitoring for Salmonella enterica has been carriedout from 2002 to 2005 in 189 laying hens flocks, in13 selection and packaging eggs plants and inworkers in Apulia (South Italy). In each flock, wecollected at least 30 eggs, faeces and feed samples.Swabs from the floor and working desks werecollected in egg selection plants. Furthermore,swabs were collected from egg selection machinesand from workers. The bacteriological assays wereundertaken according to the International standardOrganization (ISO). Salmonella strains wereserotyped. Salmonella spp. was found in 15,87% oftested flocks. In 3,7% flocks, Salmonella was foundboth in faeces and on egg shells. Besides, the pres-ence of Salmonella was sporadically found in feedsamples. In selection and packaging plants, floorand working desks, egg graders and egg conveyorbelts contamination rates was 33,57% and 29,02%respectively. S. enteritidis and S. typhimurium wasprevalent serovars. In many cases, different serovarsfollowed one another at different times in the samefarm. No specific symptoms was observed ininfected animals. The same serovar tended tobecome endemic in a certain farm. The contamina-tion of the workers employed in selection plants isworth mentioning: 11,43% of isolated Salmonellastrains was from samples collected from workers’shoes. S. typhimurium was isolated by workers,besides in flocks and selection and packaging plants.Such result is particularly worrying if we considerthat often the same staff is employed both in flocksand in selection and packaging plants. As a conse-quence, they cannot only represent an importantsource of bacterial spreading, but they are alsoparticularly exposed to the infection.

C45SALMONELLA ENTERICA SEROVARTYPHIMURIUM PERIPLASMIC SUPEROXIDEDISMUTASE, SODCI, IS A MEMBER OF THEPHOPQ REGULON AND IS INDUCED INMACROPHAGESY. A. Golubeva, J. M. Slauch; University of Illinois at Urbana-Champaign, Urbana, IL.

Salmonella enterica serovar Typhimurium replicateswithin host macrophages during the systemic stageof infection. In the macrophage, the bacteria mustsurvive the respiratory burst that produces superox-ide. Serovar Typhimurium strain 14028 producestwo periplasmic superoxide dismutases, SodCI andSodCII, but only SodCI contributes to virulence.Although we have shown that this is primarily dueto differences in the two proteins, evidence suggestsdifferential regulation of the two genes. Usingtranscriptional sodCI- and sodCII-lac fusions, weshow that sodCII is under the control of the RpoSsigma factor, as was known for the E. coli ortholog,sodC. In contrast, we show that sodCI is transcrip-tionally controlled by the PhoPQ two-componentregulatory system, which regulates an array ofvirulence genes required for macrophage survival.Introduction of a phoP null mutation into the sodCIfusion strain resulted in a decrease in transcriptionand loss of regulation. The sodCI-lac fusion showedhigh level expression in a background containing aphoQ constitutive allele. We provide evidence thatPhoP binds and directly activates the sodCI pro-moter. The sodCI gene is induced 15 fold in bacteriarecovered from either tissue culture macrophages orspleens of infected mice. Induction in macrophagesis dependent on PhoP. The sodCII fusion was in-duced 3-4 fold in macrophages and animals; thisinduction was unaffected by loss of PhoP. Thus,sodCI, which is horizontally transferred by theGifsy-2 phage, is regulated by PhoPQ such that it isinduced at the appropriate time and place to com-bat phagocytic superoxide.



to connect the clinical manifestation of infectionwith phenotypical and plasmid characteristics ofpathogen and to draw a conclusion on the impor-tance of virulence plasmids in clinics of Salmonellainfection at the human.The aim of this work was studying of the microbio-logical characteristic of Salmonella enteritidis strainsnot containing virulence plasmid with typicalmolecular weight 57 kb and also epidemiologicalfeatures and clinical manifestation of Salmonellainfection caused by them. S. enteritidis strains notcontaining virulence plasmid with weight 57 kbisolated in Primorye Region from 160 patients andfrom 11 specimens of food were investigated. Theplasmid analysis was carried out on method de-scribed by Kado (1981). The presence in strainsvirulence plasmids was investigated by PCR on thespvC gene located on virulence plasmid (Chiu,1996). S. enteritidis strains not containing plasmidswith weight 57 kb have made 3.3% in 1995-2005from investigated ones. The spvC gene was notrevealed in one of these strains. Hence, the strainsnot containing plasmid 57 kb are simultaneously thestrains not containing serovar specific virulenceplasmid. The presence of other low-molecular andhigh-molecular plasmids was not reflected in bio-chemical properties, antigenic structure and antibi-otic resistance of S. enteritidis strains not contain-ing virulence plasmid. The clinical manifestation ofan infection at group of 41 patients isolated S.enteritidis without virulence plasmid (group I) wascompared to those at group of 131 patients isolatedthe pathogen with plasmid 57 kb (group II). At allpatients the disease proceeded as gastroenteritis.Distinctions in a degree of dehydration and fre-quency of an infectious-toxic shock have beenrevealed. At 9.8% of group I patients was dehydra-tion shown the expressed vomiting and diarrhea.On the contrary, the dehydration with such symp-toms was observed at 29.0% of group II patients.Besides essential distinctions were revealed at fecesanalysis at both groups of patients. So, at patientsof group II mucus and blood were detected muchmore often, that indicates heaviergastroenterocolitis. Carriers in the group I havemade 12.2±5.1%, whereas in the group II only

A46INDUCTION OF PROTECTIVE EFFECTS INMICE WITH SALMONELLA TYPHIMURIUMHARBORING INTERLEUKIN-12 SECRETINGPLASMIDW. Yoon, Y. Park; Korea University,Seoul, Korea, Seoul, REPUBLIC OFKOREA

IL-12 is known to be an essential cytokine whichappears to provide protective immunity againstintracellular bacteria, such as Salmonella. In thisstudy, we investigated the possibility of developinga vaccine adjuvant using IL-12 secreting plasmidagainst virulent Salmonella. The highly virulentSalmonella strain (Salmonella typhimurium UK-1) wastransformed with IL-12 expressing plasmids. Theselive, wild-type pathogens were used as vaccinestrains without undergoing any other biological orgenetic attenuating processes. The newly developedstrains induced partial protection from infections(30-40%). Of note, the interleukin-12 transformedpathogen was safe upon immunization with lowdoses (103 CFU), induced IgG responses, andstimulated protective immune responses againstSalmonella typhimurium in mice (80-100%). Theseresults suggest that IL-12 induces attenuation ofwild-type Salmonella in the host infection stage andvaccine adjuvant development using the wild-typestrain harboring IL-12 secreting plasmids may beeffective in developing the intracellular bacterialvaccine adjuvants.

B47SALMONELLA ENTERITIDIS INFECTIONCAUSED BY STRAINS NOT CONTAININGVIRULENCE PLASMIDA. V. Rakov, F. N. Shubin; Research Institute of Epidemiology andMicrobiology, Vladivostok, RUSSIAN FEDERATION

Some Salmonella serovars which are adapted toanimals have virulence plasmids which allow strainsto cause system infections in mice. There is rathersteady opinion that the presence of virulenceplasmid in strains of pathogen is not necessary fordevelopment of Salmonella gastroenteritis (Fierer,2001). However, the insufficiency of clinical andepidemiological data in these works does not allow

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4.6±1.8%. Hence, S. enteritidis infection caused bystrains not containing virulence plasmid is charac-terized by easier gastroenteritis that is shown bysignificant decrease at patients of dehydration andfrequency of an infectious-toxic shock and alsohigh frequency of carrier state.

C48THE EVOLUTION OF VIRULENCE PLASMIDSOF S. ENTERICA SEROVAR DUBLIN ANDITS INFLUENCE IN THE INFECTION OFTHP-1 CELLSC. Chu1, Y. Chen1, N. Wang1, S. Chen1, C. Yeh1, C. Chiou2, C. Chiu3;1National Chiayi University, Chiayi, TAIWAN, 2Center for Disease control,Taichung , TAIWAN, 3Chang-Gung Memorial Hospital, Taoyuan,TAIWAN

Based on the incompatibility, two forms of viru-lence plasmids (pSDV) with similar size of 80 kbare found in S. enterica serovar Dublin. Previouslywe reported that pSDVr (pOU1115) of S. DublinLane (OU7409) is possibly derived from a recombi-nation event between pSDVu (pOU1113) and a36.6-kb non-virulence plasmid (pOU1114), whichreside simultaneously in strain OU7025. InpOU1115, most part of pOU1114 replaces the F-like transfer region of pOU1113. Recently thesethree plasmids have been sequenced. The compari-son of recombination sites between the two pSDVsdemonstrated a more divergent change than asimple recombination event. A major differencebetween these two pSDVs is that pOU1115 consistsof a 9-kb type IV pilus operon, which is also lo-cated on pOU1114 and may play a role in enhance-ment of the bacteria to adhere to the target cells.Two independent experiments were performed todetermine the effect of this type IV pilus. First,clinical S. Dublin strains were collected from ChangGung Memorial Hospital and CDC of Taiwan inthe period of 1997-2004. Primers specific for eachplasmid were designed to determine their pSDVpatterns. The result showed that most clinicalDublin strains harbors pOU1115, rather thanpOU113. We are currently doing an in vitro studyby using OU7025 (pOU1113 and pOU1114),OU7029 (pOU1114), OU7052 (pOU1113),OU7409 (pOU1115), and two clinical S. Dublins

strains (pOU1115) to infect THP-1 cells, a human-derived macrophage cell line. The invasion andsurvival of these bacteria within the cells andcytokine expression are examined following theinfection.

A49DSBA AFFECTS TRANSCRIPTION OF THESPI1 TYPE THREE SECRETION SYSTEM INSALMONELLA TYPHIMURIUMD. Lin, J. M. Slauch; University of Illinois at Urbana-Champaign,Urbana, IL

Type III secretion systems (TTSS) are importantvirulence factors that Gram-negative bacteria use totranslocate proteins into the cytoplasm of eukary-otic host cells. Upon contact with intestinal epithe-lial cells, Salmonella typhimurium inject a set of effec-tor proteins into the host cell cytoplasm via theSalmonella Pathogenicity Island 1 (SPI-1) TTSS toinduce bacterial uptake. The master SPI1 regulatorygene hilA, is controlled directly by three AraC-likeregulators: HilD, HilC and RtsA. Previous worksuggested a role for DsbA, a periplasmic disulfidebond oxidase, in SPI1 TTSS assembly or function.RtsA directly activates dsbA. Deletion of dsbA leadsto loss of SPI1 function. Transcription of effectorproteins was also significantly decreased in the dsbAmutant background. These results led us to proposea model in which RtsA coordinates expression ofDsbA with SPI1 and that DsbA is required forfunctional assembly of the SPI1 machinery. Weproposed that loss of the machine caused feedbackinhibition of effector gene expression. We havestudied dsbA mediated feedback regulation bymonitoring expression of SPI1 structural genes,regulatory genes, and the effector gene sopB. In thiswork, we present evidence that dsbA-mediatedfeedback inhibition is not simply through the SPI1TTS apparatus. Deletion of SPI1 structural genesdoes not cause feedback inhibition and the dsbAeffect is evident in these deletion backgrounds.Rather, we show that DsbA feeds into the SPI1regulatory network by posttranscriptionally control-ling HilD production. HilD activates both rtsA andhilA. Presumably, the regulatory network is sensing



of the cellulose-synthesis coding genes bcsA andbcsE. Cellulose is one of the known components ofthe biofilm matrix. Therefore, Salmonella in a biofilmnot only survives after exposure to triclosan andchlorine, but also stimulates mechanisms that mightconfer further resistance to a wide variety of anti-microbial. This link between the usage of biocidslike triclosan and chlorine and development ofresistance to antibiotics is of a great concern be-cause triclosan and chlorine are widely used.

C51HHA AND YDGT SHOW FUNCTIONALOVERLAP IN THE REGULATION OF S.TYPHIMURIUM VIRULENCE GENEEXPRESSIONG. Rowley, S. Lucchini, J. C. Hinton; Institute of Food Research,Norwich, UNITED KINGDOM

S. Typhimurium can survive within a number ofdifferent environmental niches. The ability to dothis is governed by transcriptional responses toexternal stimuli such as host defence factors. Theseresponses involve a number of regulators one suchgroup being the nucleoid associated proteins. Thenucleoid of Gram-negative bacteria comprises acircular chromosome and at least 12 distinct typesof associated proteins that direct DNA folding andpackaging. They also modulate global gene expres-sion in response to environmental and physiologicalstimuli. In this study we have focused on twonucleoid associated proteins, Hha and YdgT. We seerelatively limited phenotypic changes in singlemutants that lack hha or ydgT but severe phenotypeswhen these deletion mutations are combined.Phenotypic perturbations include slower growthrate, reduced motility, poor survival in macrophagesand reduced virulence in a mouse model. We werekeen to investigate the transcriptional profiles ofthese mutants, throughout different growth phasesand conditions. Our data demonstrate that Hha andYdgT work together to regulate expression ofseveral virulence-associated genes, including thoseinvolved in motility and chemotaxis. We also discov-ered that lrhA (a NADH dehyrogenase transcrip-tional repressor) known to affect RpoS translation

some property of the periplasm that is affected byDsbA. Current experiments are focused on deter-mining if DsbA is required for SPI1 functionindependent of its effects on transcription.

B50THE EFFECT OF TRICLOSAN ANDCHLORINE ON SALMONELLATYPHIMURIUM BIOFILMK. Scher1, M. Tabak1, E. Hartog1, K. R. Matthews2, M. L. Tchikindas2,S. Yaron1; 1Technion, Haifa, ISRAEL, 2Rutgers, The State University ofNew Jersey, New Brunswick, NJ

Bacterial biofilms have great significance for publichealth, since biofilm-associated bacteria exhibitdramatically decreased susceptibility to antibioticsand disinfection treatments. In this work we investi-gated the susceptibility of the foodborne pathogenSalmonella enterica serovar Typhimurium to chlorineand triclosan and identified potential strategies ofthe pathogen’s adaptation and survival.Biofilm cells were significantly more resistant tochlorination and triclosan compared to planktoniccells from the logarithmic and stationary phases ofgrowth. There was only a log reduction in viabilityof the cells in the intact biofilm compared to 8 logsreduction in viability of the exponentially-growingcells when treated with 1 mg/ml triclosan. Theplanktonic cells were killed by chlorination (50ppm) within 15 min. In contrast, about half of thecells in the biofilm survived exposure to the sameconcentration. We developed an accurate system forquantification of fabI, micF, marA, acrA, bcsA andbcsE mRNAs using Real-time RT-PCR. Whentreated with sub-inhibitory concentrations oftriclosan both cultures, planktonic and biofilmassociated cells overproduced FabI, the main targetof triclosan, and micF, an antisense RNA thatregulates the expression of OmpF, through whichhydrophilic substances enter the cell. But only thebiofilm-associated bacteria over-expressed theAcrAB efflux pumps and their regulator, MarA.When treated with chlorine, biofilm-associatedbacteria also over-expressed micF, acrAB and marA.Upon exposure of biofilms to triclosan or chlorinewe observed a significant increase in transcription

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in E. coli, was up-regulated in theDELTAhhaDELTAydgT double mutant. Wehypothesise that some of the phenotypes we ob-served reflect RpoS instability.

A52THE BACTERIAL SIGNAL MOLECULEPPGPP MEDIATES THE ENVIRONMENTALREGULATION OF BOTH THE INVASIONAND INTRACELLULAR VIRULENCE GENEPROGRAMS OF SALMONELLAA. Thompson1, M. Rolfe1, S. Lucchini1, P. Schwerk2, J. C. Hinton1, K.Tedin2; 1Inst. of Food Research, Norwich, UNITED KINGDOM, 2Institutfür Mikrobiologie und Tierseuchen, Freie Universität, Berlin, GERMANY

During infection of mammalian hosts, facultativeintracellular pathogens must adjust rapidly todifferent environmental conditions encounteredduring passage through the gastrointestinal tractand following uptake into epithelial cells and mac-rophages. Successful establishment within the hosttherefore requires the co-ordinated expression of alarge number of virulence genes necessary for theadaptation between the extracellular and intracellu-lar phases of infection. In this study we show thatthe bacterial signal molecule, ppGpp, plays a majorrole in mediating the environmental signals involvedin the regulation of both the extracellular andintracellular virulence gene programs. Underoxygen-limiting conditions, we observed a strongppGpp-dependence for invasion gene expression,reflecting severe reductions in expression of theSalmonella pathogenicity island 1 (SPI1) transcrip-tional regulator genes hilA, C, D and invF in mu-tants lacking ppGpp. Over-expression of the non-SPI1-encoded regulator, RtsA, restored hilA expres-sion in the absence of ppGpp. SPI2-encoded genes,required for intracellular proliferation in macroph-ages, were activated in the wildtype strain underaerobic, late-log phase growth conditions. Theexpression of SPI2 genes was also shown to beppGpp-dependent under these conditions. Theresults from this study suggest a mechanism for thealternate regulation of the opposing extracellularand intracellular virulence gene programs, andindicates that ppGpp shows some specificity in theregulation of genes involved in virulence compared

to the rest of the genome. This is the first demon-stration that the ppGpp alarmone is responsible fora highly conserved regulatory system that controlsbacterial virulence gene expression on a globalscale.

B53TRANSCRIPTION OF ACRA, MICF ANDTHEIR REGULATORS ENCODING GENES,MARA, SOXS AND ROB IN SALMONELLAENTERICA SEROVAR TYPHIMURIUME. Hartog1, L. Ben-Shalom1, D. Shachar1, K. R. Matthews2, S. Yaron1;1Technion, Haifa, ISRAEL, 2Rutgers, The State University of New Jersey,New Brunswick, NJ

Background: MarA, SoxS and Rob are closelyrelated regulatory proteins that either activate orrepress the expression of a large set of genes (themar/sox/rob regulon) and confer a low level ofresistance to superoxides, antibiotics, bile salts andorganic solvents (mar phenotype). Resistance toantimicrobials is mediated mainly through a boostof efflux by induction of the AcrAB efflux pumpsand a reduction of influx by repression of theOmpF porin. The main goal of the research was tostudy the transcriptional regulation of the marRAB,rob and soxS regulatory encoding genes as well asacrAB and micF genes from Salmonella enterica serovarTyphimurium. Methods: To investigate the tran-scriptional activation of these genes in planktonicEscherichia coli and Salmonella, we developed anaccurate system for real time monitoring. Thissystem relies on a panel of reporter plasmids, inwhich GFP is under the control of one of thepromoters. RT-PCR was also used to confirm theresults. The transcriptional activation of marA andsoxS has been examined also in Salmonella infectedmacrophages, using FACS analysis. Results: Basaltranscription of marA, acrA, micF and rob in plank-tonic Salmonella increased when temperature wasraised from 30°C to 37°C, while transcription ofmarA, acrA and rob declined in E. coli. The tran-scription of marA was strongly up-regulated bysalicylate and decanoate, soxS by paraquat, and robtranscription was down-regulated by all threeagents. Both, micF, and acrAB were up-regulated bythe inducers, but the effect on micF was noteworthy



Isolates were clustered into 14 closely related PFGEpattern groups, and two main phage types PT26 andPT31. There were no significant differences inresistance profiles, phage types or PFGE profile ofstrains originated from human or poultry.The rapid emergence of S. Virchow is of greatconcern, since it has a higher tendency to causeinvasive disease, particularly in very young children.The invasion of serotype Virchow to J77.4A1macrophages and its survival after 24 h was com-pared to other serotypes by Fluorescence ActivatedCell (FACS) analysis. S. Virchow isolates succeededto survive and to proliferate 10 to 100 fold higherthan S. Enteritidis and S. Typhimurium. Results ofPCR and Southern blot confirmed that the Salmo-nella virulent plasmid (spv-plasmid) is absent inserotype Virchow. Thus, the ability of S. Virchow toinvade the host cells can not be explained by thepresence of known virulence plasmid. Coupledwith its invasive propensity, the increasing incidenceof highly resistant S. Virchow in Israel is of realconcern.

A55INNATE SECRETORY ANTIBODIES PROTECTAGAINST NATURAL SALMONELLATYPHIMURIUM INFECTIONO. Wijburg1, T. Uren1, K. Simpfendorfer1, F. Johansen2, P. Brandtzaeg2, R.Strugnell1; 1The University of Melbourne, Parkville, Melbourne,AUSTRALIA, 2LIIPAT Institute of Pathology, Oslo, NORWAY

The predominance of secretory antibodies (SAbs),particularly secretory IgA (SIgA), at mucosal sur-faces has led to the hypothesis that a major functionof the secretory immune system is the productionof antigen/pathogen-specific SAbs which protectthe mucosal surfaces. However, upon first encoun-ter with an antigen/pathogen, such a specific SAbsresponse would be absent. In addition, numerousstudies in mice lacking various elements of the SAbresponse strongly argue against a major role forantigen-specific mucosal antibodies in protectionagainst many infections. The production of largeamounts of IgA is, in normal individuals, induced inan antigen-unspecific manner by commensal flora.These ‘innate’ SAbs may bind multiple antigens and

higher. Kinetic studies showed that genes re-sponded immediately to salicylate and decanoateand slowly to paraquat. The results of the FACSanalysis indicated that in macrophages infected withSalmonella, marA was also up-regulated by salicylateand soxS by paraquat.Conclusion: Results indicate that this regulatorynetwork is controlled similarly in planktonic andintra-macrophages Salmonella. Furthermore, resultsshow that in the planktonic E. coli and Salmonella,although the sequences and functions are similar,the regulation of the genes differs in the responseto environmental signals such as temperature.

C54CHARACTERIZATION OFMULTIPLE ANTIBIOTIC RESISTANTSALMONELLA ENTERICA SEROTYPEVIRCHOW IN ISRAELH. Solnik-Isaac1, M. Weinberger2, S. Yaron1; 1Technion, Haifa,ISRAEL, 2Assaf Harofeh Medical Center, Zerifin, ISRAEL

Recently we have been facing the emergence andspread of Salmonella enterica serotype Virchow inhumans and farm animals in Israel. While theoverall incidence of Salmonella infections decreased(from 69 to 53 per 100,000) from 1997-2002, theincidence of S. Virchow increased (from 7 to 9).The present research was undertaken to study theresistance and virulence properties of serotypeVirchow in Israel from 1997-2004. Looking atantimicrobial resistance of 243 and 93 S. Virchowisolates obtained from human and poultry sourcesrespectively, a high incidence of resistance tomultiple antibiotic agents was detected. Fifth of theisolates from clinical and veterinary sources werefound to be resistant to specific five drugs includingnalidixic acid, streptomycin, tetracycline,trimethoprim-sulfamethoxazole and chlorampheni-col. About half of all isolates harbored class-1integrons. Extremely high rates of the isolates(about 90%) showed resistance to nalidixic acidwith a slight decreased susceptibility tociprofloxacin (MIC: 0.125 - 0.25 mg/L). In allnalidixic acid resistant isolates we found mutation inthe GyrA protein: the switch of Asp87 to Tyr.

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are thought to eliminate commensal bacteria andself-antigens to avoid systemic recognition. It couldtherefore be hypothesized that the continuoussecretion of innate SAbs provides a first-line barrierto microorganisms present at mucosal surfaces.However, a role for innate SAbs in protection ofthe gut against pathogenic organisms is less clear. Inthis study, we addressed the role of innate SAbs inprotection against infection of the gastrointestinaltract. We used polymeric immunoglobulin receptor(pIgR-/-) knock-out mice, which are unable to bindand actively transport dimeric IgA and pentamericIgM to the mucosae, and examined the role ofinnate SAbs in protection against the invasivepathogen Salmonella serotype Typhimurium. Invitro experiments suggested that innate IgA inpIgR-/- serum bound S.Typhimurium in a cross-reactive manner that inhibited epithelial cell inva-sion. Using a ‘natural’ infection model, we demon-strated that pIgR-/- mice are profoundly sensitiveto infection with S.Typhimurium via the fecal-oralroute and, moreover, shed more bacteria that readilyinfected other animals. These results imply animportant evolutionary role for innate SAbs inprotecting both the individual and the herd againstinfections, and suggest that the major role of SAbsmay be to prevent spread of microbial pathogensthroughout the population, rather than protectionof local mucosal surfaces.

B56THE H-NS-LIKE PROTEIN STPASPECIFICALLY REPRESSES EXPRESSION OFRPOS-DEPENDENT GENES OFSALMONELLA ENTERICA SEROVARTYPHIMURIUMS. Lucchini, P. McDermott, A. Thompson, J. C. Hinton; Institute of FoodResearch, Norwich, UNITED KINGDOM

Pathogenic bacteria have a variety of systems tointegrate environmental signals to ensure the cor-rect timing of gene expression during the infectiousprocess. Nucleoid associated proteins such as H-NSare key to this process. In Salmonella Typhimurium,H-NS has been shown to play an important role inthe regulation of virulence genes in response to

changes in temperature. Interestingly, many Entero-bacteriaceae code for additional H-NS-like proteins,which may interact with H-NS to regulate geneexpression. A widespread paralogue is StpA. Inter-estingly, no clear phenotypes have been reported formutants lacking StpA, while hns mutants of E. coliand S. Typhimurium have pleiotropic effects. Wehave investigated the physiological role of StpA inS. Typhimurium SL1344. We determined the effectof deleting and over-expressing stpA on the Salmo-nella transcriptome during early exponential growth,at a time-point corresponding to the peak of StpAexpression. The results showed that like H-NS,StpA mostly acts as a repressor of gene expression.Deletion of stpA caused de-repression of 89 genesand repression of only 11 genes. Over-expressionof stpA had the opposite effect, with more genesshowing repression than an increase in gene expres-sion. We observed that the majority of StpA-regulated genes were growth-phase dependent.These StpA-dependent changes in gene expressionwere no longer observed in a rpoS- background.Accordingly, strains over-expressing StpA showphenotypes associated with rpoS mutants such asincreased sensitivity to hydrogen peroxide or acid.We propose that a major function of StpA is toprevent premature expression of stationary-phasegenes during exponential growth.

C57CONTROL OF SALMONELLA INFECTIONSIN THE ABSENCE OF CD4+ AND CD8+ TCELLSJ. Price1, O. Wijburg1, M. Kaparakis1, Y. Zhan2, J. Brady3, W. Heath2, A.Lew2, R. Strugnell1; 1The University of Melbourne, Parkville, Melbourne,AUSTRALIA, 2Walter and Eliza Hall Institute for Medical Research,Parkville, Melbourne, AUSTRALIA, 3Walter and Eliza Hall Institute ofMedical Research, Parkville, Melbourne, AUSTRALIA

Previous studies in immunodeficient mice suggestthat CD4+ T cells are essential in the control of S.typhimurium infections. Here, S. typhimuriuminfection was examined in GK mice, a mouse strainthat lacks CD4+ T cells through the constitutivetransgenic expression of the anti-CD4 antibodyGK1.5. The GK mouse more closely mimics CD4+



effects of various CpG ODNs on the innate im-mune functions of turkey immune cells, includingdegranulation and respiratory burst in theheterophils and nitric oxide (NO) production in themonocytes. Our results indicate that CpG-ODNsdo not stimulate significant degranulation andrespiratory burst activities in the turkey heterophils.However, many CpG-ODNs have shown greatstimulatory effects (more potent than bacteriallipopolysaccharide) on NO production in the turkeymonocytes. The optimal CpG-ODN for inducingNO production in the monocytes is the one con-taining GTCGTT motif. Based on these results, weconducted additional experiments to test the hy-pothesis that immune stimulatory CpG-ODN mayincrease resistance to bacterial infection in theyoung turkeys by stimulating their innate immunity.In this study, the newly hatched turkeys, obtainedfrom a commercial source, were given CpG-ODNsor a control ODN that does not contain CpG motifat 50 μg/bird via intra-peritoneal (i.p.) injection.Twenty-four hours after the CpG-ODN treatments,turkeys were challenged with live Salmonella entericasubsp. Arizonae (SEA) (suspended in PBS). For theorgan colonization experiment, 0.5-1x107 cfu/birdwas given orally to the turkeys. For the mortalityexperiment, 0.5-1x107 cfu/bird was given to theturkey via i.p. injection. Twenty-four hours after theoral SEA challenge, birds were euthanized with CO2and liver and spleen were aseptically removed fromeach bird and cultured for the organ colonizationof SEA. The mortality was monitored for a periodof 7 days after i.p. SEA challenge. A significantreduction (p<0.05) of organ invasion by SEA wasobserved in turkeys pretreated with CpG-ODNscontaining the immunostimulatory GTCGTT motif.These CpG-ODNs also significantly reduced mor-tality of turkeys with acute peritoneal infection ofSEA. Our study provides evidence thatimmunostimulatory CpG-ODN stimulated innateimmune activities and enhanced the resistance toinfectious pathogens in neonatal turkeys.

T cell deficiency in AIDS compared with the MajorHistocompatibility Complex (MHC) Class II-/-mice. In contrast to MHC Class II-/- mice, GKmice controlled infection with aroAD- S.typhimurium, albeit with delayed kinetics comparedwith C57BL/6 mice. Production of interferongamma (IFN-gamma) by CD4-deficient mice wasdelayed and prolonged when compared to C57BL/6mice, and subsets of IFN-gamma -producing Tcells were identified as CD3+CD4-CD8+ andCD3+CD4-CD8- by flow cytometry. Experimentsin mice lacking CD8+ cells and double transgenicmice lacking both CD4+ and CD8+ cells showedthat further depletion of CD8+ cells did not delaybacterial clearance. Further, control of aroAD-Salmonella infections correlated with a populationof ‘double negative’ T cells that produced IFN-gamma in the spleen and liver of all mouse strains.This is the first study where protection against S.typhimurium has been demonstrated in the absenceof CD4+ T cells, and suggests that replication ofthe bacterium can be controlled by double negativeT cells.

A58PROPHYLACTIC APPLICATION OFIMMUNE STIMULATORY CPG-ODNREDUCES SALMONELLA ENTERICA SUBSP.ARIZONAE ORGAN COLONIZATION ANDMORTALITY IN YOUNG TURKEYSH. He, K. J. Genovese, C. L. Swaggerty, M. H. Kogut; Food and FeedSafety Research Unit, Southern Plains Agricultural Research Center, USDA-ARS, College Station, TX

Synthetic oligodeoxynucleotides (ODN) containingCpG dinucleotides (CpG-ODN) mimics bacterialDNA and are stimulatory to innate immune systemin most vertebrate species through Toll-like recep-tor 9. The immunostimulatory activities of CpG-ODN have been studied extensively and well char-acterized in human and murine immune cells.However, information on immune responses ofavian species to CpG-ODN is limited. The immunestimulatory activity of CpG-ODN has not beenstudied in turkeys, an important species in thepoultry industry. Here, we have evaluated the

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B59THE EXTRACYTOPLASMIC STRESSRESPONSE (ESR) AND THE RESISTANCE TOB-LACTAM ANTIBIOTICS IN FIELD ANDCLINICAL ISOLATES OF SALMONELLAENTERITIDISA. R. Ureta1, L. Betancor1, R. Vignoli2, L. Yim1, A. Martinez1, J. A.Chabalgoity1; 1Laboratory for Vaccine Research, Department of Biotechnol-ogy, Instituto de Higiene, Facultad de Medicina, Montevideo, URUGUAY,2Department of Bacteriology and Virology, Instituto de Higiene, Facultad deMedicina, Montevideo, URUGUAY

In the last decade the enteric pathogen Salmonellaenterica serovar Enteritidis was the main cause ofhuman gastroenteritis of bacterial origin in Uru-guay. In each step of the infection process (en-trance to the organism, adhesion to the intestinalepithelial cells, cell invasion and survival inside hostcells) Salmonella must sense the changes associatedto its interaction with the host in order to appropri-ately modulate the production of virulence factors.In Salmonella as in other gram-negative pathogensthis is in great part controlled by the ESR systemscomposed of the alternative sigma factor SigmaEand the CpxAR two-component system (TCS) (1).DegP is a periplasmic protease that also has chaper-one activity and whose expression is induced eitherby SigmaE or the CpxAR TCS. It is clear the impor-tance of DegP in particular and the ESR in generalin bacterial pathogenesis (2,3). Another aspect ofthe bacterial physiology with important clinicalimplications is the resistance to b-lactam antibiotics.Because these antibiotic target molecules are lo-cated in the bacterial envelope and the mechanismsthat lead to b-lactams resistance profoundly modifythis compartment (4,5) it is likely that the ESRsystems play a critical role in the resistance pheno-type. By growing field and clinical isolates of S.Enteritidis in increasing concentrations of cephal-othin (a cephalosporin) we were able to induceresistance to this and other b-lactam antibiotics.However, while resistant to b-lactams these cellsremain sensitive to cloramphenicol or nalidixic acid.Western blots and serological analysis show that theresistance phenotype is associated with the induc-tion and deregulation of DegP and possibly withthe alteration of the lipopolisaccharides (LPS) at the

bacterial surface. Interestingly the CpxAR TCS butnot SigmaE is responsible for the induction andderegulation of DegP expression. To understandthe physiological role of the ESR in this phenotype,fractionation analysis are being performed in orderto characterize the protein content of each enve-lope compartment and the outer membrane LPS.Given the importance of ESR in bacterial patho-genesis we are evaluating the virulence potential ofthese antibiotic resistant bacteria on human intesti-nal epithelial cell cultures. This work could lead tothe identification of the signal(s) that regulate theactivity of the ESR systems, and to the develop-ment of additional treatments to Salmonella infec-tions. The results of these analyses will be dis-cussed.References1- Raivio,T and Silhavy,T. 2000. Bacterial StressResponses, ASM Press p19-32.2- Rowley,G et al. 2006. Nat. Rev. Microbiol. 4:p383-394.3- Raivio,T. 2005. Mol. Microbiol. 56: p1119-1128.4- Alekshun,M and Levy,S. 2000. Bacterial StressResponses, ASM Press p 323-366.5- Poole,K. 2004. Cell.Mol. Life Sci. 61: p2200-2223.

C60POST-TRANSCRIPTIONAL REGULATION OFTHE AVRA EFFECTOR PROTEIN

Z. Ben-Barak Zelas1, W. Streckel2, T. Kerrines2, U. Strutz2, R. Prager 2,H. Tschäpe2, S. Yaron11Department of Biotechnology and Food Engineering ,Technion Institute, Haifa, Israel 2National Reference Centre for Salmonellaeand other enterics, Robert Koch Institute, Wernigerode, Germany

The effector protein AvrA of Salmonella entericacontributes to the complexity of the virulencenetwork of Salmonella. AvrA is a cysteine protease,which turns the inflammatory response of the hostby inhibition of the production of IL8 and helps toinduce apoptosis. The avrA gene is absent in severalserovars such as Typhi, which usually cause severesystemic disease. The aim of this research was tostudy the expression of avrA in Salmonella serovars.Using Western blot technique we found that thetranslation of avrA in S. enterica serovars took place


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either constitutively (class 1), or after acid inductiononly (class 2) or not at all (class 3 if they carry theavrA gene or class 0 if avrA is absent). The differ-ent expression profiles of AvrA were independenton any DNA-sequence variations of the promoterand /or structural part of the avrA. Cloning ofavrA gene and promoter on low copy plasmids gaverise to a strong AvrA production in all tested S.enterica strains. This was found to be in contrast toEscherichia coli which failed to produce AvrA by therespective plasmids. The transcription levels wereexamined in two different ways: by using GFPmarker it was concluded that avrA cannot be tran-scribed in E. coli but easily in S. enterica belonging toclass 1, 2, or 3 and also in class 0. By using real timeRT-PCR method we showed that E. coli strainscarrying the plasmids have the same level of tran-scription as S. enterica. Results demonstrated thatthe control of expression of the AvrA protein isdue to post-transcriptional regulation, probably apost-transcriptional control factor, which might beabsent in E. coli.

A61ANALYSIS OF THE MECHANISMSRESPONSIBLE FOR THE INITIATION OFGROWTH IN SALMONELLA ENTERICASEROVAR TYPHIMURIUM USING DNAMICROARRAYSM. D. Rolfe1, C. Pin1, M. W. Peck1, A. Thompson1, R. P. Betts2, M. F.Stringer2, J. C. Hinton1; 1Institute of Food Research, Norwich, UNITEDKINGDOM, 2Campden and Chorleywood Food Research Association,Chipping Campden, UNITED KINGDOM

Lag phase is an important stage of bacterial growthwith particular relevance for human food-bornedisease and food spoilage. The physiology of lagphase remains poorly understood; before this study,it was not known whether the initiation of bacterialgrowth would involve large-scale changes at thetranscriptional level or not. We aimed to provideinsight into the physiology of growth initiation.We developed a protocol which allowed isolation ofRNA from lag phase bacteria in sufficient quantitiesfor microarray studies, and performedtranscriptomic analyses. We discovered that theexpression of half the S. Typhimurium genes

changed during lag phase, and identified key genesand pathways which are activated during the earlieststages of growth. Notably, genes involved in inor-ganic phosphate uptake, iron uptake and oxidativestress responses/repair showed characteristicinduction patterns.We have also looked at the role of the Fis protein, aglobal regulator known to be rapidly induced fol-lowing nutrient upshift. Careful experimentsshowed that a fis deletion mutant did not have anincreased lag time compared to wild-type in oursystem. Furthermore, the characteristic pattern oflag-phase-regulated gene expression was not con-trolled by Fis, suggesting that this regulator doesnot play a central role during initiation of growth.This work has suggested the physiological processesthat are involved in the resumption of growth of abacterial cell after nutrient upshift.

B62VARIANT SALMONELLA GENOMIC IS-LAND 1-L IN SALMONELLA ENTERICASEROVAR NEWPORTB. Doublet1, K. Praud1, M. Demartin2, F. Weill3, A. Cloeckaert1;1INRA, Nouzilly, FRANCE, 2Pasteur, Paris, FRANCE, 3pasteur, Paris,FRANCE

The 43 Kb Salmonella Genomic Island 1 (SGI1)harbours several antimicrobial resistance genes andhas been associated with the emergence of theepidemic multidrug-resistant Salmonella entericaserovar Typhimurium DT104 clone. Since then,SGI1 has become widespread in several Salmonellaenterica serovars, including Agona, Albany, Cerro,Derby, Dusseldorf, Emek, Infantis, Kentucky,Kiambu, Newport, and Paratyphi B. SGI1 containsa complex class 1 integron that most often confersresistance to ampicillin (Ap), chloramphenicol(Cm)/florfenicol (Ff), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracy-clines (Tc). The SGI1 complex integron harbourstwo attI1 sites with classically the gene cassettesaadA2 and blaPSE-1, conferring resistance to Sm/Sp and Ap respectively. However, variant SGI1complex integrons conferring various multidrugresistance phenotypes have also been identified in

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different S. enterica serovars and were classified asSGI1-A to -K. In this study we identified a newvariant SGI1 complex integron in a S. entericaserovar Newport strain isolated from a humanreturning from Egypt to France. This isolate dis-played the multidrug resistance profileApCmFfSmSpSuTc typical of the SGI1 antibioticresistance gene cluster but with the additionalresistances to gentamicin (Gm) and trimethoprim(Tm). SGI1 was detected in this strain and charac-terization of its complex integron showed that theaadA2 gene cassette was replaced by a dfrA15 genecassette conferring resistance to Tm. The isolatecontained another class 1 integron probably inte-grated elsewhere in the chromosome containing theaac(3)-Id and aadA7 gene cassettes, conferringresistance to Gm and Sm, respectively. This genecassette replacement in SGI1 of serovar Newportconstitutes a new variant SGI1 complex integronnamed SGI1-L.

C63MECHANISMS FOR IRON UPTAKE INRESPONSE TO OXIDATIVE STRESS VARYBETWEEN SALMONELLA TYPHIMURIUMAND SALMONELLA ENTERITIDISV. E. Danino1, L. Sait2, T. Humphrey2, M. Bailey2, J. Hinton1;1Institute of Food Research, Norwich, UNITED KINGDOM, 2Universityof Bristol Veterinary School, Bristol, UNITED KINGDOM

Salmonella Typhimurium and Salmonella Enteritidisvary in their ability to infect and survive in theiranimal hosts. Crucially, S. Enteritidis has a greaterability to infect chicken eggs than S. Typhimurium.During the process of host infection these bacteriaencounter a number of different environmentalconditions moving from the gut to the inside ofanimal cells, where they encounter oxidative stress.We have used a transcriptomic approach to deter-mine the effect of oxidative stress on gene expres-sion of S. Typhimurium and S. Enteritidis in re-sponse to the addition of hydrogen peroxide. It isknown that under conditions of oxidative stressbacteria sequester free iron, possibly to prevent ironacting as a catalyst for the generation of oxygenradicals by the Fenton reaction. As might be pre-

dicted, most genes required for iron scavengingwere up regulated in both Salmonella serovars.However a significant difference was observed; inresponse to oxidative stress, the ent genes are up-regulated in S. Typhimurium but show no change inexpression in S. Enteritidis. The implications of thedifferential response to oxidative stress and ironuptake in these two serovars will be presented.

A64A CENTRAL DOMAIN OF THESALMONELLA EFFECTOR SIPACONTRIBUTES TO INTRACELLULARLOCALIZATION AND VIRULENCEM. C. Schlumberger1, R. Kaeppeli1, M. Wetter1, A. J. Mueller1, M.Kremer2, W. Hardt1; 1Institute of Microbiology, ETH Zürich, Zurich,SWITZERLAND, 2Institute of Pathology, Technische UniversitätMünchen, Munich, GERMANY

Salmonella enterica is a prime cause of bacterialenterocolitis in humans. Injection of effectorproteins into intestinal cells by the type III secretionsystem (TTSS) encoded on Salmonella pathogenicityisland 1 is a key virulence mechanism for bacterialinvasion into epithelial cells and for the inductionof acute intestinal inflammation. Among theseeffectors, SipA plays a major role in Salmonella-induced enterocolitis. In cell culture experiments,SipA has been shown to accelerate bacterial inva-sion. The N-terminal region of SipA (amino acids1-105) encodes the TTSS transport signals and theC-terminal domain of SipA (amino acids 446-685)binds actin and promotes polymerization of F-actinfilaments. Little is known about the internal regionsof SipA. In the host cell cytosol, SipA specificallyaccumulates in small foci located in close proximityto the bacteria - host cell interface. Thus, specificinteractions may restrict the localization of SipAinside the host cell. In this study, we performed adeletion analysis to identify SipA domains involvedin intracellular focus formation. We found thatefficient formation of SipA foci required a regioncomprising amino acids 170-271 of SipA which wetermed accumulation-enhancing domain (AED). Incontrast, the actin-binding domain of SipA (ABD,amino acids 446-685) is dispensable for SipA focusformation. We demonstrate that both the ABD and



the AED of SipA are essential for SipA-mediatedinduction of intestinal inflammation and for effi-cient host cell invasion. Thus, while distinct func-tions can be assigned to the AED (enhanced focusformation) and the ABD (actin binding and poly-merization) of SipA, the activities of both domainsare required for virulence. This suggests that theAED functions to increase local SipA concentra-tions within foci, which may play a role in enhanc-ing host cell manipulation.

B65TYPE III SECRETION: IS THERE AHIERARCHY OF EFFECTOR PROTEINTRANSPORT?B. Winnen, M. Schlumberger, W. Hardt; ETH,Zürich, SWITZERLAND

Type III secretion systems (TTSS) are utilised bymany Gram-negative pathogens. They mediatecontact-dependent translocation of effector pro-teins directly into the host cell cytosol. The translo-cated effector proteins trigger host-signaling cas-cades which mediate a variety of responses, includ-ing pathogen uptake. Efficient delivery of effectorsand rapid manipulation of host cell signaling iscrucial for the outcome of the bacteria-host cellinteraction. Recently, by using time lapse micros-copy, the delivery of the Salmonella type III effectorprotein SipA into animal cells was imaged in realtime. However, very little is known about thedelivery kinetics of other type III effector proteins.Specifically, it has remained unclear whether all typeIII effector proteins are translocated at the sametime. Alternatively, a “transport”-hierarchy mayexist among the > 10 different TTSS-1 effectoreffector proteins and some groups of effectorproteins may be translocated earlier than others. Toaddress this question, we are currently developingsuitable assay systems. The results of these studieswill be presented.

C66COPPER HOMEOSTASIS IN SALMONELLASP.: SENSING, TRANSPORT ANDTRAFFICKINGD. Osman, C. M. Taylor, J. S. Cavet; University of Manchester,Manchester, UNITED KINGDOM

The ability to sense and respond rapidly to fluctuat-ing metal levels is crucial for bacterial pathogenicity.Copper is required by bacteria as a cofactor forseveral fundamental enzymes, but is highly toxic inexcess. Hence, stress can be caused by surplus ordeficiency of this metal and may arise in responseto environmental transients. Intracellular concentra-tions of copper must therefore be precisely con-trolled and it is apparent that, at least some, bacteriapossess elaborate copper homeostatic mechanismsthat maintain essentially no free cytosolic copperwhilst allocating copper to copper-requiring pro-teins. We are examining the mechanisms used bySalmonella typhimurium to sense and respond tochanging copper levels and their contributions tosurvival within a host. Analysis of the S. typhimuriumgenome has revealed a number of genes encodingdeduced copper homeostatic proteins, severallacking homologues in closely related organisms.Expression from the promoters of cuiD (encoding amulti-copper oxidase), copA (for a deduced copper-transporting P1-type ATPase) and copB/copR/copZ(for a deduced copper-transporting P1-type ATPase,copper-responsive transcriptional regulator &copper metallochaperone, respectively) is substan-tially induced in cells exposed to elevated concen-trations of copper, and to a lesser extent silver. Inaddition, copper-induced transcription from PcopB/

copR/copZ is enhanced at low pH. No other environmen-tal stresses affect expression from these promoters.S. typhimurium possesses two deduced MerR-familytranscriptional regulators, SctR (Kim et al., 2002;FEMS Microbiol Lett. 210) and CopR, with se-quence features of copper sensors. The contribu-tions of these proteins to the copper-responsive-ness of PcopA, PcopB/copR/copZ and PcuiD are being exam-ined. Similar levels of copper induction from PcopAand PcuiD are observed in wild-type and DELTAcopRcells, but copper-responsiveness is abolished in

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DELTAsctR, consistent with SctR acting as a cop-per-sensing regulator of expression from bothpromoters. In contrast, PcopB/copR/copZ retains somecopper-responsiveness in DELTAsctR andDELTAcopR single mutants, but not aDELTAcopR/DELTAsctR double mutant, consis-tent with copB being under the control of both SctRand CopR. It is tempting to speculate that thecytosolic copper sensors detect available copperions, bound or free, and mediate rapid transportinto the cell envelope for incorporation into cop-per-requiring proteins, or for ejection when inexcess. Deduced copper-requiring proteins in S.typhimurium include cytochrome oxidase and twoperiplasmic copper/zinc superoxide dismutaseswhich represent key virulence factors. Work iscontinuing to establish the roles of the coppersensors and target gene products in copper trans-port and trafficking during infection.

A67INFECTION OF HUMAN EPITHELIAL CELLSCAUSES NOVEL TIME-DEPENDENTTRANSCRIPTIONAL REMODELLING OFSALMONELLA ENTERICA SV.TYPHIMURIUM SL1344I. Hautefort1, A. Thompson1, S. Ygberg-Eriksson2, M. L. Parker1, S.Lucchini1, N. Ahmad1, M. Rhen2, J. C. Hinton1; 1Institute of FoodResearch, Norwich, UNITED KINGDOM, 2Microbiology and TumorBiology Center, Karolinska Institute, Stockholm, SWEDEN

The last ten years have seen impressive develop-ments in our understanding of the genesis andevolution of the Salmonella-containing vacuole(SCV) inside mammalian cells; nevertheless, thecapacity of Salmonella to modulate its gene expres-sion pattern in response to changes occurring insidethe host cell remains poorly described. We reporthere the complete transcriptomic profile of S.Typhimurium inside epithelial cells and highlight itssimilarities and the differences with our previouslystudy in macrophage cells [Eriksson et al., 2003,Mol. Microbiol. 47: 103-18]. The expression profilessuggest differences in metabolic activity betweenSalmonella isolated from epithelial cells and mac-rophages. Surprising expression patterns of viru-lence genes were observed inside epithelial cells;

SPI1 effectors and structural genes were up-regu-lated inside epithelial cells. We discovered thatflagella biosynthetic genes were induced in a time-dependent manner inside epithelial cells, but are notexpressed inside macrophages. This de novo produc-tion of flagellin and up-regulation of SPI1 expres-sion coincides with intriguing changes in the SCVmicroenvironment.

B68CORA, A MAGNESIUM TRANSPORTER, ISREQUIRED FOR VIRULENCE INSALMONELLA ENTERICA SEROVARTYPHIMURIUMK. M. Papp-Wallace, M. E. Maguire; Case Western Reserve University,Cleveland, OH

CorA, the primary Mg2+ transporter in Salmonellaenterica serovar Typhimurium, is required for fullvirulence in the mouse. A corA mutant has analtered ability to invade Caco-2 epithelial cells;however entry of J774A.1 macrophage-like cells isnot changed. Some components of Salmonellapathogenicity island 1 (SPI-1) are downregulated ina corA mutant. Whereas, mgtC, mgtB, and mgtA,components of the PhoPQ regulon, are upregulatedin a corA mutant. Lack of Mg2+ does not appear tobe the cause of these phenotypes, because totalintracellular Mg2+ content in a corA mutant is notdifferent from wild type. Thus, the CorA proteinalone may be a factor in these phenotypes. Since,CorA from Methanococcus jannaschii fails to comple-ment the SPI-1 defects of a S. Typhimurium corAmutant.

C69PRE-HARVEST CONTROL OF SALMONELLATHROUGH VACCINATION OF FOODANIMALSD. A. Emery1, D. E. Straub1, J. D. Sandstrom1, L. M. Slinden1, D. T.Burkhardt1, L. D. Wonderling2; 1Epitopix, Willmar, MN, 2Syntiron, St.Paul, MN

Salmonella enterica is a ubiquitous bacterium thatis able to infect and colonize the intestinal mucosaof all animals in the human food chain and isconsidered to be amongst the most prevalent food



borne pathogens on the planet. The ability ofSalmonella to evade host defenses and the emergenceof antibiotic-resistant strains highlight the urgentneed for developing improved strategies for theprevention and control of this pathogen. Ourinvestigations show that a composition of outermembrane proteins expressed by Salmonella inresponse to iron restriction constitutes an effective,safe and broad spectrum vaccine. The studiesdemonstrate the efficacy of the vaccine in multiplefarm animal species both under controlled labora-tory conditions as well as in large commercial fieldtrials with both systemic and oral homologous andheterologous challenge models. Field and experi-mental challenge studies in both domestic poultryand cattle show the vaccine to be highly efficaciousin reducing morbidity/mortality and decreasingfecal shedding of numerous serotypes of Salmonella.Taken together, the results of our studies suggestthat administration of the vaccine to farm animalswill not only reduce morbidity and mortality inlivestock populations, but may also serve to reducethe transmission of Salmonella as a food bornepathogen to human populations.

A70OCCURRENCE AND CHARACTERIZATIONOF EXTENDED-SPECTRUMCEPHALOSPORIN RESISTANCE IN SALMONELLA DURING 1999-2004L. Martin, C. Poppe, E. Weir, P. Boerlin, A. Muckle; Laboratory forFoodborne Zoonoses, Guelph, ON, CANADA

Background: The increase of drug resistance inSalmonella in recent years has been well docu-mented. Of particular concern is the developmentof resistance to antimicrobials that are of impor-tance in human medicine. We sought to determinethe frequency of extended-spectrum cephalosporin(ESC) resistance and to characterize this resistancein Salmonella isolated from animals and food ofanimal origin in Canada submitted to our laboratoryfor serotyping during the years 1999 - 2004.Methods: Antimicrobial susceptibility (AMS)testing was performed on 18,605 isolates between1999 and 2004 using the agar dilution methodaccording to Clinical and Laboratory Standards

Institute guidelines. ESC resistant isolates wereexamined for the presence of the blaCMY-2, blaTEM andblaSHV genes by PCR. Products were sequenced toconfirm gene identity. Southern blots were preparedfrom plasmid DNA of selected strains (n = 241)and the blots were hybridized with a DIG-labeledblaCMY-2 probe. Results: A total of 652 ESC resis-tant isolates were identified during the 6-yearperiod. The annual percentage of resistant isolatesincreased over the study period, from 1.1% of AMStested isolates in 1999 to 7.3% of isolates in 2004.These isolates belonged to an increasing number ofserovars. The ESC-resistant isolates from 1999belonged to seven serovars, while in 2004 they weremembers of 20 serovars. More than 90% of theexamined isolates contained the blaCMY-2 gene. Thisgene was often located on large plasmids that were60-145 kb in size. Resistance to other antimicrobialsincluded resistance to the aminoglycosides, tetracy-cline, sulfonamides and trimethoprim. Conclusion:Resistance of Salmonella isolated from animals, theanimal environment, and foods of animal origin toESCs increased during the 6-year study period.Humans may become infected with ESC-resistantSalmonella via the food-borne route or by directcontact with infected animals. Options to treathumans or animals infected with ESC-resistantSalmonella are limited.

B71CATIONIC ANTIMICROBIAL PEPTIDESMODULATE SOLELY THE KINASE ACTIVITYOF THE SALMONELLA ENTERICA PHOQSENSORV. Le Sage, H. Le Moual; Department of Microbiology and Immunology,McGill University, Montreal, PQ , CANADA

In Salmonella enterica, the PhoP/PhoQ two-compo-nent regulatory system plays a central role in con-trolling virulence. PhoQ is the transmembranekinase sensor that detects specific environmentalsignals. PhoP is the cognate response regulator thatelicits the cellular response. PhoQ has been shownto respond to the external divalent cations Mg2+,Ca2+ or Mn2+ and to some cationic antimicrobialpeptides of host origin. Like most kinase sensors,PhoQ possesses both kinase and phosphatase

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activities that play opposite roles in controlling thelevel of phospho-PhoP. Environmental signalsrecognized by PhoQ are thought to affect thekinase and/or the phosphatase activities of thekinase sensor. Previous work has shown that highconcentrations of external Mg2+ stimulate specifi-cally the PhoQ phosphatase activity and, in turn,result in PhoP dephosphorylation. In contrast toMg2+, cationic antimicrobial peptides were shown tostimulate PhoP phosphorylation. In this study, weused purified PhoQ reconstituted in E. coli lipo-somes to determine whether the kinase and/or thephosphatase activities are targets for regulation bycationic antimicrobial peptides. Reconstituted PhoQinserts into liposomes with the periplasmic domainfacing the vesicle lumen and the catalytic domainfacing the extraluminal environment. To assesswhether the PhoQ kinase activity was affected bythe antimicrobial peptide C18G, we measured thekinetics of phosphorylation of PhoQ reconstitutedin the presence of increasing concentrations ofintraluminal C18G. Initial rates of PhoQ phospho-rylation increased by 2- to 5-fold when the concen-tration of C18G varied from 0.1 to 5 μg/ml, indi-cating that C18G enhances the PhoQ kinase activityin a concentration-dependent manner. In contrast,similar concentrations of intraluminal C18G did notaffect the PhoQ phosphatase activity both in theabsence and presence of ADP, which has beenshown to stimulate the PhoQ phosphatase activity.We conclude that cationic antimicrobial peptidesstimulate the PhoP/PhoQ system by acting solelyon the PhoQ kinase activity, as opposed to magne-sium that inhibits the phosphorylation cascade byenhancing solely the phospho-PhoP phosphataseactivity of PhoQ.

C72INTESTINAL SHORT CHAIN FATTY ACIDSCONTROL SALMONELLA INVASIONTHROUGH BARA/SIRA AND THE CSRREGULATORY SYSTEMD. R. Fortune, M. Suyemoto, C. Altier; Cornell University, Ithaca, NY

As pathogenic bacteria move through the intestinaltract of an animal host, they encounter changes in

environmental conditions that provide potentialcues for the control of virulence. The penetrationof the intestinal epithelium, a required step inSalmonella pathogenesis, occurs preferentially in theileum, and requires the invasion genes of Salmonellapathogenicity island 1 (SPI1). We have shown thatinvasion is controlled by the two-component regula-tor BarA/SirA, which itself controls the csr post-transcriptional regulatory system. The csr systemconsists of CsrA, which binds to target messagesand alters their half-lives, and two functional RNAs,CsrB and CsrC, that bind to CsrA and titrate it fromits targets. BarA/SirA induces both CsrB and CsrC,thus reducing the effective concentration of CsrA.We show here that specific types of short chainfatty acids (SCFAs) present in the intestine cansignal Salmonella to either induce or repress invasionthrough these integrated regulatory systems. For-mate, found in high concentration in the ileum,induces invasion through BarA/SirA. An ackA-ptamutant was deficient in the expression of SPI1genes and invasion of cultured epithelial cells. Thismutant excreted 68-fold less formate into culturemedium, but the addition of formate to the me-dium completely restored SPI1 expression andinvasion. Formate, however, had no effect in mu-tants that lacked barA or sirA, indicating that it actsthrough BarA/SirA. The metabolism of formatewas also not required for its activity, as disruptionof all known routes of formate metabolism, aloneand in combination, failed to replicate the defect ofthe ackA-pta mutant. Thus, it is likely that formateitself is the signal for induction of invasion. Incontrast, propionate and butyrate repress invasionby inducing the expression of csrA. These twoSCFAs at physiologically relevant concentrationsand pH induced csrA by 3-fold and induced produc-tion of a second csrA-specific transcript. They alsofailed to repress invasion in a csrA mutant. csrA-mediated repression of invasion by propionaterequired the structural elements of the prp operon,but repression by butyrate required only prpR, thepositive regulator of that operon. Protein productsof the prp operon metabolize propionate, creatingthe intermediate 2-methylcitrate, which is an acti-vating co-factor for PrpR. These findings thus


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indicate that PrpR activates csrA and further sug-gests that 2-methlycitrate is the required activatingco-factor for PrpR produced by the metabolism ofpropionate though prp, but that butyrate producesan analogous activating co-factor by a pathwayindependent of prp. As these two SCFA are in lowconcentration in the ileum, but high concentrationin the large intestine, they provide likely signals forthe repression of invasion in the area of the intes-tine where penetration of the epithelium by Salmo-nella is less productive.

A73MOLECULAR EPIDEMILOGY OFSALMONELLA ENTERITIDIS INFECTION INSIBERIA AND FAR EAST OF RUSSIAF. N. Shubin, A. V. Rakov, N. A. Kuznetsova; Research Institute ofEpidemiology and Microbiology, Vladivostok, RUSSIAN FEDERATION

Monitoring for Salmonella enteritidis as the mostsignificant pathogen, based on a plasmid analysis ofstrains, has allowed to carry out genetic marking ofthe pathogen population in Primorye Region and tofind out a degree of its heterogeneity. The aim ofthe study consists in the analysis of the S. enteriti-dis population structure in a number of regions ofSiberia and Far East of Russia and mechanisms offormation of genetically marked S. enteritidispopulations. During the centralized microbiologicalmonitoring were investigated S. enteritidis strainsfrom 1652 patients in Primorye Region (1432patients), Magadan Region (103 patients),Krasnoyarsk Region (30 patients), Irkutsk Region(27 patients), Kamchatka Region (54 patients),Chita and Sakhalin Region (6 patients) and 60specimens of food isolated in 2003-2005. Microbio-logical monitoring for S. enteritidis based on theplasmid analysis data in complex with the analysisof the population morbidity is used. It is estab-lished that S. enteritidis population in the investi-gated regions of Siberia and Far East is presentedby ten plasmid variants (plasmidovars) of pathogen.In all regions plasmid profile of S. enteritidis strainsisolated from food corresponded those for strainsisolated from patients. The comparative analysis ofS. enteritidis strains isolated on various administra-

tive territories has allowed to differentiate amongthem plasmidovars of pathogen circulating onwhole territory of Siberia and Far East (strains of38 : 1.4 MDa plasmidovar), on the majority ofadministrative territories (38 : 26 : 1.4 MDa, 38 : 2.6: 1.4 MDa, 38 : 3.2 : 2.9 : 1.4 MDa) and specific toseparate territories (38 : 4.2 MDa, 38 : 2.3 MDa, 38: 35 : 1.4 MDa). The analysis of the Salmonellainfection morbidity in Primorye Region has shownthat S. enteritidis plasmidovars have various etho-logical values. Most frequently S. enteritidis infec-tion is caused by widely distributed 38 : 1.4 MDaplasmidovar and 38 : 4.2 MDa, 38 : 2.3 MDaplasmidovars specific to Primorye Region. Themechanism of the formation of the S. enteritidispopulation in Siberia and Far East is connected totransport of animal production contaminated by thepathogen and as result of deliveries of the breedinglivestock infected by salmonellas from the uniformcenters of poultry cultivation. Wide application ofthe centralized microbiological monitoring behindsalmonellas in Siberia and Far East is a basis forunderstanding of formation mechanisms of Salmo-nella populations in concrete territories and realiza-tion of purposeful preventive actions.

B74

SALMONELLA ENTERICA SODC GENESENCODE TWO PERIPLASMIC CU,ZNSUPEROXIDE DISMUTASES WITHNON-EQUIVALENT STRUCTURAL ANDFUNCTIONAL PROPERTIESS. Ammendola, F. Pacello, M. D’Orazio, G. Rotilio, A. Battistoni;Dipartimento di Biologia, Università di Roma Tor Vergata, Rome, ITALY

Several Salmonella enterica strains possess two genesencoding periplasmic Cu,Zn superoxide dismutase,sodC1 and sodC2, located on a lambdoid prophageand on the chromosome, respectively. While bothCu,ZnSODs can protect bacteria from damage dueto extracellular reactive oxygen species generated invitro, studies carried out with Salmonella entericaserovars Typhimurium and Enteritidis have estab-lished that only disruption of sodC1 significantlyimpairs the ability of Salmonella to systemically

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infect mice. These investigations have suggestedthat the different contribution of SodC1 and SodC2to Salmonella virulence can be explained by differ-ences in gene expression levels and/or in stillunidentified enzyme properties. In fact, it has beenshown that SodC1 is the only one of the twoproteins synthesized to high levels in intracellularbacteria and that the two coding sequences are notfunctionally interchangeable, SodC1 performingbetter than SodC2. To identify the structural/functional properties differentiating SodC1 andSodC2, we have carried out an extensive compara-tive analysis on the two purified proteins. The twoenzymes display nearly identical resistance to hydro-gen peroxide inactivation, but clearly differ forseveral other properties, including superoxidedismutation rates, peroxidative activity, thermalstability, resistance to proteolytic attack, pH sensi-tivity and affinity for the active site metals. Most ofthese differences can be explained by the differentquaternary structure of the two enzymes, as SodC1is a dimeric protein while SodC2 is monomeric.However, when compared to other bacterial dimericCu,ZnSODs, SodC1 displays much greater struc-tural stability and higher catalytic activity. Similarexceptional properties can be observed also in thebacteriophage-encoded Cu,ZnSOD present inenterohemorrhagic Escherichia coli O157:H7. Theseresults increase the interest for the functionalimplications of the huge structural variabilityobserved in bacterial Cu,ZnSODs and suggest thatthe phage-associated sodC genes encode enzymevariants optimized to resist the harsh environmentalconditions encountered by bacteria during infec-tion.

C75SALMONELLA TRAFFICKING IS DEFINEDBY DYNAMIC INTERACTIONS WITH THEENDOLYSOSOMAL SYSTEMD. Drecktrah, L. A. Knodler, O. Steele-Mortimer ; Rocky Mountain Lab,NIAID, NIH, Hamilton, MT

Biogenesis of the Salmonella containing vacuole(SCV) involves sequential interactions with theendocytic pathway. However, Salmonella is believed

to limit these interactions and, in particular, toavoid fusion of terminal lysosomes with the SCV.Here we have reexamined SCV biogenesis using anextremely sensitive live-cell imaging approach. Wediscovered an unanticipated level of interactionbetween the SCV and the endocytic pathway,including pre-formed lysosomes. Direct interac-tions, in which lysosomal content was transferred toSCVs, could be detected within 30 min followinginvasion and continued for at least 8 h. Mechanisti-cally lysosome-SCV fusion appeared similar tophagosome-lysosome fusion since it was character-ized by rapid acidification of the SCV, was blockedby chemical perturbation of microtubules or vacu-olar acidification and involved the small GTPaseRab7. Despite the dynamic nature of these interac-tions, Salmonella do appear to delay lysosome-SCVfusion compared to vacuoles containing Escherichiacoli or heat-killed Salmonella, although we wereunable to detect a requirement for either type IIIsecretion system in this delay. Altogether theseresults provide compelling evidence that inhibitionof SCV-lysosome fusion is not a major determinantin establishment of the Salmonella replicative nichein epithelial cells.

A76THE VI ANTIGEN OF SALMONELLAENTERICA SEROTYPE TYPHI: A SWEETCURE OF DIARRHEAM. Raffatellu1, R. L. Santos1, D. Chessa1, R. P. Wilson1, S. E. Winter1,S. D. Lawhon2, J. F. Figueiredo2, L. G. Adams2, A. J. Baumler1; 1Universityof California at Davis, Davis, CA, 2Texas A&M University, CollegeStation, TX

Human infections with non-typhoidal Salmonellaserotypes, such as S. enterica serotype Typhimurium,are characterized by a massive neutrophilic infiltratein the colon and terminal ileum. In contrast, neutro-phils are scarce in intestinal infiltrate of typhoidfever patients. In light of our recent finding that aS. Typhi-specific virulence factor, the Vi capsularantigen, blocks intestinal inflammation by interfer-ing with TLR signaling in human intestinal epithelialcells, human macrophage-like cells and humanintestinal explants, we have begun investigatingwhether the Vi antigen also modulates host re-



sponses elicited in in vivo models of gastroenteritis.Bovine ligated ileal loops were infected with acapsulated and a non-capsulated strain of S. Typhi(wild-type and viaB mutant) in comparison with S.Typhimurium wild-type. The non-capsulated S.Typhi mutant triggered more fluid accumulation (asurrogate of diarrhea), more neutrophil influx andmore GRO-alpha expression than the isogenic S.Typhi wild-type. In addition, introducing a plasmidencoding the viaB operon into S. Typhimurium (i.e.a S. Typhimurium strain expressing the Vi antigen)reduced the inflammatory response elicited inbovine ileal loops. We are currently investigatingwhether the Vi antigen attenuates the host inflam-matory response in other animal models of infec-tion.

B77TRANSPOSON BASED SCREEN FORREGULATORY GENES CONTROLLINGEXPRESSION OF THE STDA FIMBRIALANTIGEND. Chessa, S. Nuccio, M. Winter, A. J. Bäumler; University of Californiaat Davis, Davis, CA

Fimbriae are surface structures important forbacterial colonization of mucosal surfaces. Theprecise roles during host pathogen interaction ofmost fimbrial operons present in the genomes ofSalmonella serotypes are poorly understood, mainlybecause conditions for their expression have notbeen identified, thereby preventing functionalanalysis in vitro. Only two genetic adherence deter-minants, encoded by tcfABCD and stdABCD are notaffected by genome degradation (i.e. pseudogeneformation) in genomes of human adapted Salmonellaserotypes (Salmonella enterica serotypes Typhi andParatyphi A). To identify regulatory signals control-ling std expression, we used a transposon library toenrich for mutants expressing StdA, the majorfimbrial subunit, on their cell surface by using para-magnetic beads coated with anti-StdA serum. Weidentified 7 mutants expressing Std fimbriae ontheir surface as determined by Western blot, elec-tron microscopy and immuno gold labeling. Threestrains had insertions in or upstream of dam (3),

setB, lrhA, csgC and STM4463. STM4463 is the lastof five open reading frames that form a small islandpresent in all sequenced Salmonella genomes butabsent from all sequenced Escherichia coli genomesexcept those from strains associated with urinarytract infections. The four open reading framesupstream of STM4463 encode proteins with homol-ogy to enzymes of the urea cycle. Subsequentanalysis of STM4463 showed that it represses stdexpression in the absence of urea. These data raisethe interesting possibility that Std fimbriae functionduring colonization of the urinary tract, a site thatwas found to be colonized by the human adapted S.Typhi in 25% of patients in the pre-antibiotic era.

C78UNCOUPLING PHENOTYPES OFSALMONELLA ENTERICA SEROVARTYPHIMURIUM SWARM CELLSA. L. Turnbull, M. G. Surette, University of Calgary, Calgary, Alberta,Canada

Swarming is a form of flagella-driven surfacemotility occurring in diverse groups of bacteria.Swarm cells are morphologically distinct fromvegetative cells; typically elongated andhyperflagellated. Swarming is usually consideredsimply as a motility phenotype, however in Salmo-nella enterica serovar Typhimurium, migrating swarmcells have been shown to undergo further differen-tiation. In nutrient rich conditions on semi-solidagar, actively migrating swarm cells have beenshown to undergo further differentiation into aunique physiological state characterized by: 1.adaptive antibiotic resistance (elevated resistanceto many classes of antibiotics), 2. metabolic differ-entiation (a shift in central metabolism fromcatabolic to anabolic growth) and 3. communityadaptations (enhanced production and detectionof cell-cell signaling molecules). The aforemen-tioned adaptations aren’t necessarily coupled toswarm motility but are phenotypes of activelymigrating swarm cells The adaptive antibioticresistance exhibited by swarm cells is unlike otherexamples of non-inherited antibiotic resistance suchas persistence or tolerance exhibited by non-grow-

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ing cells: swarm cells show elevated resistance to awide variety of antibiotics in exponentially growingcells. This study aims to elucidate physiologicalchanges involved in swarm cell differentiation andadaptive antibiotic resistance. Is adaptive antibioticresistance coupled to metabolic differentiation? Toaddress this, auxotrophs were isolated that werepredicted to block metabolic differentiation. Allauxotrophs examined were capable of activelyswarming, however metabolic differentiation wasimpaired to varying degrees with different mutants.Cysteine auxotroph swarm cells were more sensitiveto antibiotics than the wild type, while other aminoacid auxotrophs retained the adaptive antibioticresistance. Using reporters for glycolysis and theTCA cycle, up-regulation of TCA cycle enzymes incysteine auxotrophs was not observed to the sameextent as in wild type S. typhimurium swarm cells.These results indicate that cysteine auxotrophs wereable to swarm but had lost both metabolic differen-tiation and adaptive antibiotic resistance suggestingthese two processes are coupled. Additionally,cysteine auxotrophs had altered swarm colonymorphology, depending on the mutated gene. Achemical genetics approach was used to comple-ment these genetic studies. Inhibitors of cysteinebiosynthesis did not inhibit swarm cell motility butblocked metabolic differentiation and adaptiveantibiotic resistance. It thus seems that adaptiveantibiotic resistance is coupled to metabolic differ-entiation in swarm cells and that cysteine biosynthe-sis is central to swarm cell physiology. Furthermorethese results suggest that inhibitors of metabolicpathways not usually considered antibacterial mayact synergistically with antibiotics by blockingadaptive resistance pathways.

A79ROLE OF SALMONELLA PATHOGENICITYISLAND 4 (SPI-4) IN COLONISATION OFCATTLE BY SALMONELLA ENTERICASEROVAR TYPHIMURIUME. Morgan1, A. J. Bowen1, T. S. Wallis2, M. P. Stevens1; 1Institute forAnimal Health, Compton, Nr Newbury, Berkshire, UNITED KING-DOM, 2Ridgeway Biologicals Ltd, Compton, Nr Newbury, Berkshire,UNITED KINGDOM

Salmonella enterica serovar Typhimurium infects abroad range of animal species and it is becomingincreasingly apparent that Salmonella uses differentrepertoires of genes to infect different hosts. Werecently demonstrated through signature-taggedmutagenesis that genes encoded on SalmonellaPathogenicity Island 4 (SPI-4) are required forintestinal colonisation of cattle, but not chickens orpigs and thus may contribute to the host-specificityof Salmonella. The purpose of this study was todefine the role of SPI-4 in intestinal colonisation ofcattle. SPI-4 is a 24 kb pathogenicity island whichcarries six open reading frames named siiA-F. Wehave constructed mutants with defined non-polardeletions of siiE and siiF and characterised thesemutants in vitro and in vivo. Here we demonstratethat siiE encodes a large protein of c. 600 kDawhich is secreted into the culture supernatantduring growth in LB broth. Secretion of SiiErequires siiF which is predicted to encode part of aType 1 secretion system which is also carried onSPI-4. We have also shown that siiE and siiF influ-ence intestinal colonisation of cattle by S.Typhimurium as quantified by pyrexial responses,faecal shedding and colonisation of sites along thegastro-intestinal tract. Both siiE and siiF contributeto invasion of bovine enterocytes as determined ina bovine ileal loop invasion model, but are notrequired for survival or persistence in bovine pri-mary macrophage cells.


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B80DOWN-REGULATION OF KEY VIRULENCEFACTORS MAKES THE SALMONELLA RFAHMUTANT A PROMISING LIVE-ATTENUATEDVACCINE CANDIDATEG. Nagy1, V. Danino2, B. Kocsis1, U. Dobrindt3, L. Emody1, J. C.Hinton2, J. Hacker3; 1University of Pecs, Pecs, HUNGARY, 2Institute ofFood Research, Norwich, UNITED KINGDOM, 3University ofWurzburg , Wurzburg, GERMANY

RfaH is a transcriptional anti-terminator whichregulates the expression of long transcripts inenteric bacteria, such as those required for produc-tion of lipopolysaccharide (LPS). A mutant ofSalmonella enterica sv. Typhimurium that lacks RfaH isno longer virulent in the murine model of typhoidand elicits protection against a subsequent lethalchallenge by the wild-type strain. We have deter-mined which genes are regulated by RfaH in Salmo-nella enterica to understand the mechanism of attenu-ation. Whole genome Salmonella microarrays wereused to identify RfaH-dependent genes in S.Typhimurium and Enteritidis. These data showeddown-regulation of genes responsible for LPS-synthesis and other phenotypes. To differentiatebetween genes directly regulated by RfaH and thosewhich differ in expression due to the resulting deep-rough phenotype, a transcriptomic analysis of astructural deep-rough LPS mutant (waaG) was done.This showed the only genes directly regulated byRfaH are those involved in LPS-core and O-antigensynthesis. The deep-rough phenotype of rfaHmutants resulted in increased invasiveness of bothepithelial and macrophage cells. However, intracel-lular growth was severely impaired, possibly as aconsequence of increased susceptibility to antimi-crobial peptides. Down-regulation of major surfaceimmunogenic molecules (e.g., LPS and flagella)allows the rfaH mutant to provoke a cross-reactiveimmune response. Sera of mice vaccinated with therfaH mutant showed reactivity to not only theparental S. Typhimurium strain, but also to heter-ologous serovariants and even other members ofEnterobacteriaceae. This cross-reactivity was caused byantibodies reacting to several conserved outermembrane proteins. Vaccination of mice with the S.Typhimurium rfaH mutant elicited protection

against a heterologous challenge by S. Enteritidis.The ability of the Salmonella rfaH mutant to showincreased invasiveness into antigen-presenting cells,combined with impaired intracellular growth andthe potential of raising a cross-protective immuneresponse, makes it a promising vaccine candidate.

C81VISUALIZING BACTERIAL PROTEOMESJ. Huang1, M. Leong1, R. M. Watt1, J. Wang1, A. Danchin2, D. Liu3;1The University of Hong Kong, Pokfulam, HONG KONG, 2InstitutPasteur, Paris, FRANCE, 3PUMC & CAMS, Beijing, CHINA

Traditional methods to tag bacterial proteins withepitope sequences are time consuming and effi-ciency of the tagging process is low. Thereforedevelopment of an efficient tagging method isnecessary for the increasing demand of highthroughput proteomics in bacteria as well as eukary-otic organisms. We constructed 9 fluorescent andtandem affinity purification (TAP) plasmid tem-plates suitable for tagging all the open readingframes (ORFs) in bacterial genome. These fluores-cent tags can be used to localize proteins in bacte-ria, while the TAP tag can be used to purify proteincomplexes, providing valuable resources for ge-nomic and proteomic studies. These tag templateplasmids are designed to combine recent advancesin recombineering and site-specific recombinationtechnologies to tag E. coli chromosomal genes.Recombineering mediated by the ë red prophagesystem allows insertion, deletion or replacement ofDNA fragment at a precise location on the chromo-some or plasmid without the needs for restrictionenzymes. Sequence homology 36 -50 nucleotidelong is sufficient for high efficiency homologousrecombination in E. coli. Therefore tagging vectorswith short sequence homology to target site can beprepared by PCR or synthetic oligonucleotides.Using gel purified tagging vector, 78 - 100 % ofcorrectly tagged recombinant clones was obtainedby PCR confirmations. Success rates from 20 - 75% were achievable using PCR fragments purified byQiagen spin column kit, reducing the time in tag-ging procedure further. We were able to purifyprotein complexes containing the tagged proteins.

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Although this technology was developed in E. coli, itcan be applied to Salmonella.

A82EFFECT OF CHLORATE, MOLYBDATE, ANDSHIKIMIC ACID ON SALMONELLATYPHIMURIUM IN AEROBIC ANDANAEROBIC CULTURESC. E. Oliver1, D. J. Smith2, T. A. Casey3, S. M. Horrocks4, R. C.Anderson4, D. J. Nisbet4; 1North Dakota State University, Fargo, ND,2USDA/ARS Biosciences Research Laboratory, Fargo, ND, 3Pre-harvestFood Safety & Enteric Diseases Research Unit, National Animal DiseaseCenter, Ames, IA, 4USDA/ARS, Food & Feed Safety Research Unit,Southern Plains Agricultural Research Center, College Station, TX

Two studies were conducted to examine the effectsof shikimic acid (60 ìg/mL) and(or) molybdate (1mM) on the sensitivity of Salmonella enterica serovarTyphimurium to sodium chlorate (5 mM) duringanaerobic (90% N2:5% CO2:5% H2) or aerobicgrowth in brain heart infusion broth supplementedwith 5 mM NaNO3. In the first study, aerobiccontrol cultures without additions reached theirmaximum absorbance (A600) of 1.99 by 4 h whereasthe A600 of aerobic cultures incubated with addedchlorate peaked at 0.62 at 5 h. Anaerobic controlsincubated without additions reached maximum A600of 1.60 by 8 h. Growth of anaerobic culturesincubated with added chlorate was practically nil(A600 < 0.12) until 10 h, whereupon cultures grewrapidly, attaining an A600 of 1.46 at 24 h, which wascomparable to that measured in controls incubatedwithout chlorate. In the second study, chloratemarkedly affected growth of Salmonella, with growthof all cultures containing added chlorate by itselfpeaking at A600 of 0.43 compared to 1.14 measuredin non-chlorate containing counterparts. In bothstudies, cultures incubated without chlorate did notdevelop chlorate resistance, as assessed by an agaroverlay method, at concentrations above 103 CFUml-1, which was our lower limit of detection. In thefirst study, two of the triplicate anaerobic culturesincubated with chlorate had detectible chlorateresistance by h 6 and had acquired or propagated to100% resistance (>109 CFU ml-1) in all three by 24h. In the aerobic chlorate-containing cultures, twoof the triplicates had detectible chlorate resistance

by 6 h, but only one retained detectible resistance at24 h. In the second study, chlorate resistance inchlorate-containing anaerobic cultures ranged from86 to 95% of total viable colonies at 8 h and 72 to96% at 24 h. Anaerobic incubation of chlorate-containing cultures with molybdate and shikimicacid had no effect on the quantitative developmentof chlorate resistance. In aerobic cultures, only oneof the chlorate-containing triplicates had resistanceat 8 or 24 h (72 and 83% of total viable colonies,respectively). When aerobic cultures were incubatedwith chlorate and either shikimic acid or molybdate,chlorate resistance was observed at 8 h (62 to 78%of total viable colonies in two of three and three ofthree cultures, respectively) but sensitivity was fullyrecovered in all tubes by 24 h. When shikimic acidand molybdate were included together in the pres-ence of chlorate, they did not mitigate the effectsof chlorate as chlorate resistance was observed inall triplicates at 8 h (64 to 72%) and 24 h (67 to80%). Results illustrate the differential effects ofchlorate against anaerobic- or aerobically grownSalmonella Typhimurium and demonstrate thatshikimic acid or molybdate added individually, butnot in combination, were able to reverse chlorateresistance in aerobically, but not anaerobicallygrown cultures.

B83MICROARRAY-BASED COMPARATIVEGENOMIC ANALYSIS OF SALMONELLAENTERICA SEROVAR ENTERITIDISISOLATED IN URUGUAYL. Betancor1, M. Fookes2, A. Martinez1, L. Yim1, D. Pickard2, F.Schelotto1, A. Ivens2, D. Maskell3, G. Dougan2, J. A. Chabalgoity1; 1Institutode Higiene, Facultad de Medicina, Universidad de la Republica, Montevideo,URUGUAY, 2The Wellcome Trust Sanger Institute, Cambridge, UNITEDKINGDOM, 3University of Cambridge,Cambridge, UNITED KINGDOM

Salmonella enterica serovar Enteritidis (S. Enteriti-dis) is a major cause of food-borne disease. Before1994 S. Enteritidis was sporadically isolated inUruguay, but since 1995 yearly outbreaks have beenfrequent and from 1997 onwards it became themost frequently agent of food borne disease in thecountry. This situation persisted for 10 years, but



within the last twelve months the number of iso-lates has markedly diminished, suggesting a newchange in the epidemiology of salmonellosis. Wepreviously conducted a genotyping study of fieldisolates circulating in Uruguay since 1995. UsingRAPD-PCR we demonstrated the existence of apredominant genetic type that is a significant causeof human disease. It also revealed the existence ofseveral other different genotypes circulating inpoultry. Only some of these were occasionallyrecovered from humans, suggesting the existence ofdifferent pathogenic traits among them. To test thishypothesis, we defined a project aimed at decipher-ing the genomic diversity between these isolates andto investigate whether these differences are associ-ated with different pathogenic capacities. We havestarted by selecting 10 isolates with higher chancesof being different, covering varied genetic types,origin (animals, food and human infection cases)and time of isolation (pre epidemic, epidemic andpost- epidemic), and subjected them to DNAmicroarray-based comparative genomic analysisusing the sequenced strain of S. Enteritidis PT4NCTC 13349 as reference. We used the S. TyphiCT18-based Gen-III Pan-Salmonella array availableat Sanger Institute which covers genomes from 3different S. enterica serovars as well as from S.bongori. Emerging data show that some of the fieldisolates have insertions of genomic regions contain-ing virulence-associated genes described in otherserotypes but absent at PT4. At least 2 strains fromhuman origin (one from the epidemic and the otherfrom the post-epidemic period) contain genes so farconsidered as specific of S. Typhi, including geneswithin SPI6. Conversely, some PT4 specific regionsare absent in several of our field isolates, includinggenes within SPI10 also absent in DT104. Further,some isolates show absence of relevant mobileelements as phages and plasmids usually found inSalmonella, including sopE phage and pSLT. Asexpected, several strains are almost identical to PT4strain. The extent of genomic diversity found sofar, diverge from previous reports showing lowextent of genomic divergence within a single sero-type. Noteworthy, condition trees constructed basedon microarray results are similar to those con-

structed using RAPD results suggesting that RAPDis consistent enough to out light relevant differ-ences. Three of the most diverse isolates are nowbeing used for in vitro assays of infection of humanepithelial cell lines.Results from all these analysis will be presented anddiscussed.

C84THE YFGL ORF IS ESSENTIAL FORSALMONELLA ENTERITIDIS VIRULENCE INMICEY. Fardini, Sr., J. Trotereau, Sr., E. Bottreau, Sr., P. Velge, Sr., I.Virlogeux Payant, Sr.; INRA, Nouzilly, FRANCE

Salmonellosis are an important worldwide healthproblem. Type III secretion systems (TTSS) andtheir effector proteins play a major role in Salmonellainfections. The TTSS-1 and flagella are involved inthe Salmonella-host cell interaction with the intestineand the TTSS-2 is required for the intracellularsurvival and multiplication. Previously, we identi-fied, by transposon mutagenesis, a new locus in-volved in Salmonella Enteritidis virulence. This locusis composed of 4 open reading frames (ORF) :yfgM, yfgL, engA and yfgJ and was shown to beinvolved in host-cell invasion and in motility. RT-PCR analyses revealed that the yfgL and yfgM ORFsbut also yfgL and engA are co-transcribed. In orderto determine the precise role of each ORF, deletionmutants were constructed and characterized. A S.Enteritidis DELTAyfgM mutant was as virulent asthe wild type strain in a murine model of systemicinfection. We didn’t manage to construct aDELTAengA mutant, suggesting that engA is anessential gene for Salmonella as is the case for its E.coli der homolog. A DELTAyfgL mutant showed areduced ability to invade enterocytes and macroph-ages cell lines. In line with this phenotype, themutant secreted smaller amounts of TTSS-1 effec-tor proteins SipA and SipC and flagella proteinsFliC and FliD. Transcriptome analyses revealed aglobal down-regulation of the transcription ofTTSS-1 and flagella encoding genes. Taken to-gether, these results and the fact that a DELTAyfgLmutant is completely avirulent in the mouse model

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of infection highlight a critical role for the yfgLORF in S. Enteritidis virulence.

A85MORE ROBUST DETECTION OF MOTIFS INSALMONELLA TYPHIMURIUMCOEXPRESSED GENES BY USINGPHYLOGENETIC INFORMATIONP. Monsieurs1, A. Fadda2, G. Thijs1, B. De Moor1, J. Vanderleyden2, S. C.De Keersmaecker2, K. Marchal2,1; 1ESAT-SCD, K.U.Leuven,BELGIUM, 2Dep Microbial and Molecular Systems, K.U.Leuven,BELGIUM

Several motif detection algorithms have beendeveloped to discover overrepresented motifs insets of coexpressed genes. However, usually theirperformance rapidly drops as the signal to noiseratio (defined as the number of sequences thatactually contain the motif versus the number ofsequences lacking the motif) in the data set de-creases. However, coexpressed sequence sets de-rived from microarray studies are usually noisy. Thehigh noise level in such sequence sets comes as aresult of the processing of the microarray data, aswell as of the nature of the biological processes inthe cell itself. For instance, when comparing mRNAexpression between a wild type and a regulatorknock out strain, besides the direct targets of theregulator, indirect targets are also affected in theirmRNA expression in the knock out. In such mu-tants the complete regulatory network acting down-stream of the mutated regulator is disturbed. Listsof genes derived from these experiments containtargets of more than one regulatory protein lower-ing the relative overrepresentation of a particularmotif. To still recover motifs which are not signifi-cantly enriched but still present, we developed aprocedure in which we use phylogeneticfootprinting to first delineate all potential motifs ineach gene. Then we mutually compare all detectedmotifs and identify the ones that are shared by atleast a few genes in the data set as potential candi-dates. We applied our methodology to a compiledtest data set containing known regulatory motifsand to biological data sets derived from genomewide expression studies on Salmonella typhimurium.By executing four consecutive steps of 1) identify-

ing conserved regions in orthologous intergenicregions, 2) aligning these conserved regions, 3)clustering the conserved regions containing similarregulatory regions followed by extraction of theregulatory motifs and 4) screening the inputintergenic sequences with detected regulatory motifmodels, our methodology proves to be a powerfultool for detecting regulatory motifs when a lowsignal to noise ratio is present in the input data set.Comparing our results with two other motif detec-tion algorithms (AlignACE and PhyloCon) pointsout the robustness of our methodology.Conclusively, we developed an approach that canreliably identify multiple regulatory motifs lacking ahigh degree of overrepresentation in a set ofcoexpressed genes (motifs belonging to sparselyconnected hubs in the regulatory network) byexploiting the advantages of using bothcoexpression and phylogenetic information.

B86A NOVEL SMALL NON-CODING RNA,INVR, OF THE SALMONELLA INVASIONGENE ISLAND REGULATES OUTERMEMBRANE PROTEIN EXPRESSIONV. Pfeiffer1, A. Sittka1, K. Papenfort1, A. Müller2, W. D. Hardt2, J.Vogel1; 1Max Planck Institute for Infection Biology, Berlin, GERMANY,2Institute of Microbiology, ETH Zurich, SWITZERLAND

The Salmonella chromosome possesses large inser-tions, the Salmonella pathogenicity islands (SPIs),which encode the proteins that are the major deter-minants of Salmonella virulence. Of these islands,SPI1 encodes the virulence factors that are requiredfor invasion of epithelial cells. We show that asidefrom invasion proteins, SPI1 expresses an abundantnon-coding RNA, InvR. Transcription of this 80 ntRNA peaks under conditions that favour cell inva-sion, and is controlled by HilD, a key transcriptionfactor of Samonella virulence genes. The invR geneis highly conserved among known Salmonellaspecies, including the early-branching S. bongori.Global transcriptome and proteome analysis ofinvR deletion and overexpression strains revealedthat InvR RNA regulates the expression of mem-brane and surface proteins. Using a mouse model inwhich Salmonella causes acute colitis, we obtained



evidence that InvR RNA contributes to Salmonellavirulence in vivo. The invR gene is the first smallnon-coding RNA gene identified in the Salmonellapathogenicity islands, and the first post-transcrip-tional regulator of gene expression encoded bySPI1.

C87IN SEARCH FOR THE ROLE OF LUXS INSALMONELLA ENTERICA SV.TYPHIMURIUM BIOFILM FORMATIONS. C. De Keersmaecker1, D. De Coster1, N. van Boxel1, K. Sonck1, I.M. Thijs1, H. Zhao1, K. Engelen1, P. Monsieurs2, K. Marchal1,2, J.Vanderleyden1; 1Centre of Microbial and Plant Genetics, K.U. Leuven,Leuven, BELGIUM, 2ESAT-SCD/SISTA, K.U. Leuven, Leuven,BELGIUM

The term “quorum sensing” describes bacterial cell-to-cell communication, using small diffusible mol-ecules to cooperate in diverse behaviors. Amongdifferent quorum-sensing systems, the only oneshared by Gram-positive and Gram-negative bacte-ria involves the production of autoinducer-2 (AI-2).LuxS, the AI-2 synthase, is widely spread, suggest-ing that AI-2 is a common language for interspeciescommunication. However, LuxS is also an integralcomponent of the activated methyl cycle in bacte-ria. In Salmonella enterica serovar Typhimurium (S.typhimurium), AI-2 is produced and released duringexponential growth. It subsequently isreinternalized by the bacteria via the Lsr (LuxS-regulated) ABC transporter. AI-2 induces transcrip-tion of the lsrACDBFGE operon, of which the firstfour genes encode the Lsr transport apparatus. InSalmonella, no AI-2-regulated genes other than thosein the lsr operon have yet been identified. Neverthe-less, a Salmonella luxS deletion mutant has beenreported to be defective in biofilm formation.However, biofilm formation of the luxS mutantcannot be restored by addition of AI-2, althoughaddition of AI-2 induced lsr expression. In contrast,introduction of luxS under control of its ownpromoter complemented biofilm formation. Fur-ther results demonstrated that biofilm formation ofthe luxS mutant cannot be restored with luxS undercontrol of the strong nptII promoter. This suggeststhat regulatory elements in the promoter region of

luxS affect Salmonella biofilm formation. Thishypothesis was further supported by the observa-tion that a luxS insertion mutant (luxS::Km) is stillable to form biofilms. In Escherichia coli a smallregulatory RNA (sRNA), SraD, is located upstreamof luxS, with its promoter region overlapping withthe luxS coding sequence and this sRNA is con-served in other species, such as S. typhimurium. InVibrio spp. multiple sRNAs have been reported tocontrol quorum sensing. sRNAs can act to regulateboth the synthesis of proteins, by affecting mRNAtranscription, translation and stability, and theactivity of specific proteins by binding to them.These sRNAs regulate bacterial genes involved inenvironmental adaptation, which makes themputative interesting members of the LuxS/AI-2quorum sensing system. Efforts are ongoing todisentangle to role of luxS and its upstream ele-ments in Salmonella biofilm formation by mutagen-esis and high-throughput expression profilingexperiments.

A88GENOME-WIDE LOCATION ANALYSIS INSALMONELLA ENTERICA SV.TYPHIMURIUM: EXPANDING THE HILAREGULONI. M. Thijs1, S. C. De Keersmaecker1, K. Marchal1,2, K. Engelen1, M.McClelland3, S. Porwollik3, J. Vanderleyden1; 1Centre of Microbial and PlantGenetics, KULeuven, Leuven, BELGIUM, 2ESAT-SCD/SISTA,KULeuven, Leuven, BELGIUM, 3Sidney Kimmel Cancer Center, SanDiego, CA

Chromatin immunoprecipitation (ChIP) combinedwith microarrays (chip) was applied to identifytranscriptional targets of the Salmonella enterica sv.Typhimurium HilA protein. HilA is the key regula-tor for the invasion of epithelial cells. Until recently,examining transcript profiles or screening thegenome for known regulatory motifs were bestsuited to expand this regulon on a high-throughputscale. But gene expression profiling does not distin-guish between direct and indirect effects of atranscription factor binding to target genes. On theother hand, in silico predictions often show a highnumber of false positives and potential false nega-tives. The ChIP-chip technique, however, allows a

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direct and in vivo identification of transcriptionfactor binding sites on a genome-wide scale. Theassay consists of crosslinking transcriptional regula-tors to their binding sites in vivo and isolating bothpartners out of the total cell content by immuno-precipitation. Bound DNA fragments are thenamplified and hybridized to a microarray ideallyrepresenting the whole Salmonella genome. The hilAgene was chromosomally tagged with a myc epitope.Cells were cultured under hilA inducing conditionsand three independent ChIP experiments wereperformed together with three independent mockexperiments. In the latter, a wildtype strain was usedto serve as a control for experimental errors. BoundDNA fragments were hybridized along with areference sample on six Salmonella cDNAmicroarrays. Although not optimally suited for thisapproach, an adequate result was expected due tothe overlap of the binding site-containing DNAfragments with the ORFs present on the array. Thereference sample is subjected to the same ChIPprotocol, but the immunoprecipitation step isomitted. The Wilcoxon rank-sum test was used tosearch for genes that show a high enrichment in theChIP experiment as compared to the mock experi-ment. Two known target genes, prgH and invF, wereamong the top scoring genes and provide a bench-mark for the applied technology. Other high scoringhits include genes involved in flagellar biosynthesis,invasion of epithelial cells, lipoprotein constitutionand genes with a putative or unknown function.These putative target genes are currently beingassayed on a low-throughput scale. Additionally,expression profiling and motif detection will beperformed to consolidate our genome-wide ap-proach. The integration of different high-through-put data should provide an effective means to dealwith noise in the individual data sources and com-bining the distinct aspects they unveil can lead to amore comprehensive view of the HilA regulon.

B89COORDINATE REGULATION OF THESALMONELLA PATHOGENICITY ISLAND 2SSAB-SSRA LOCUS.D. Walthers1, X. Feng2, L. J. Kenney1; 1University of Illinois at Chicago,Chicago, IL, 2Oregon Health & Science University, Portland, OR

Salmonella enterica serovar Typhimurium contains twopathogenicity islands that encode type III secretionsystems (TTSS) involved in distinct stages of viru-lence. Pathogenicity Island 1 (SPI-1) is required foruptake into non-phagocytic epithelial cells. Pathoge-nicity Island 2 (SPI-2) is required for survival andreplication in macrophages and systemic infectionin the murine model of typhoid fever. In additionto the TTSS apparatus, chaparones and secretedeffectors, SPI-2 encodes a two-component signaltransduction system (SsrA-SsrB) that regulatesgenes both within and outside of SPI-2. Regulationof this important regulatory locus is controlled byOmpR, SlyA and autoregulation by SsrB.The 400 bp intergenic region between ssrAB andthe ssaB operon contains binding regions for mul-tiple transcription factors. We have previouslyshown that ssrB was required for activation of ssrAand ssaB during growth in macrophages (Feng et al,Mol Microbiol. 48:1131-43, 2003). The location ofthe binding sites raise the question does SsrB alsofunction as a repressor? Although we haven’t yetobserved repression in Salmonella (presumablybecause we haven’t found the right conditions),when SsrB is over-expressed in E. coli, ssrA-lacZtranscription is repressed. Thus, it is appears thatSsrB may act as both an activator and a repressor ofthis regulatory locus. To clarify the contribution ofeach binding site to activation or repression, weconstructed precise deletions of the SsrB, SlyA andOmpR binding sites. Promoter fragments contain-ing the wild type fragment or a deletion mutationwere cloned, in either orientation, upstream of apromoter-less lacZ gene. Preliminary results indicatethat the ssrA-proximal binding site functions tonegatively regulate both ssrA and ssaB. In contrast,the ssaB-proximal binding site is required for posi-tive control of both ssrA and ssaB. We have alsodetermined that OmpR and SlyA contribute addi-


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tively to ssrA expression in E. coli (which lacks ssrB).This result suggests that the role of SlyA in activa-tion is not simply to displace SsrB from a putativerepressive site. The promoter fusions were movedinto an integrative vector and merodiploid back-grounds were constructed that leave the native SPI-2 regulatory locus intact. We are re-examining thepromoter activity of our fusions in wild type andmutant backgrounds during growth in macrophages.To understand coordinate control by SsrB and SlyA,we are testing the ability of each protein to com-pete for its overlapping binding site by performingDNase I protection assays in the presence of bothproteins. Our results should clarify the regulatoryhierarchy involved in activation of SPI-2 geneexpression under relevant in vivo conditions.

C90HILD, A SPI-1 TRANSCRIPTIONALREGULATOR, CONTROLS EXPRESSION OFTHE SPI-2 VIRULENCE REGULON INSALMONELLA ENTERICA SEROVARTYPHIMURIUMV. H. Bustamante1, L. A. Knodler2, F. J. Santana1, O. Steele-Mortimer2,J. L. Puente1; 1Departamento de Microbiología Molecular, Instituto deBiotecnología - UNAM., Cuernavaca, Morelos, MEXICO, 2Laboratory ofIntracellular Parasites, Rocky Mountain Laboratories, NIAID, NIH.,Hamilton, MT

Salmonella Typhimurium is a Gram-negative entericpathogen that causes human gastroenteritis and asystemic infection in mice similar to that producedby S. Typhi in humans. The two major SalmonellaPathogenicity Islands (SPI-1 and SPI-2) encode typeIII secretion systems that export bacterial effectorproteins into the host epithelial cells. SPI-1 isrequired for efficient invasion of the intestinalepithelium and for intestinal disease, while SPI-2 isessential for Salmonella replication and survivalwithin macrophages and for systemic infection.Several environmental signals, as well as global andspecific transcriptional regulators modulate, inde-pendently, the expression of SPI-1 and SPI-2 genesunder growth conditions resembling the extracellu-lar and intracellular environments, respectively. Apositive regulatory cascade formed by HilD, HilAand InvF, encoded in SPI-1, controls expression of

SPI-1 genes. The two-component regulatory systemSsrAB, encoded in SPI-2, controls expression ofSPI-2 genes. Recent studies have shown that SPI-2is necessary for complete intestinal virulence and,accordingly, that SPI-2 genes are expressed in theintestinal lumen. In this study, we are reporting anovel transcriptional cross-talk mechanism, whereHilD, a SPI-1-encoded regulator, has a critical rolein the activation of the SPI-2 regulon. HilD isessential for the expression of the SPI-2 transcrip-tional activator SsrB under specific growth condi-tions commonly used to mimic the extracellularenvironment, but not under those resembling theintracellular environment. However, OmpR, aglobal regulator known to be necessary for expres-sion of SPI-2 genes, is required for SsrB expressionin both growth conditions. Thus, our results indi-cate that SPI-2 genes are expressed by two regula-tory mechanisms in response to different environ-mental signals. We propose that HilD and OmpRcould coordinate the expression of SPI-2 genesbefore Salmonella penetrates the intestinal epithe-lium, which would be necessary for the putativeextracellular function of SPI-2 and for initiating theadaptation to the intracellular environment. OnceSalmonella is inside the epithelial cells, OmpR,together with other described transcriptional regula-tors, such as PhoP and SlyA, activate expression ofSPI-2 genes independently of HilD.

A91MOLECULAR ANALYSIS OF A GENECLUSTER ENCODING EUKARYOTIC-LIKEPROTEIN KINASES AND PHOSPHATASE INSALMONELLA ENTERICA SEROVAR TYPHIH. Le Moual1, S. Faucher2, S. Lai1, S. Leclerc1, F. Daigle2; 1Departmentof Miicrobiology and Immunology, McGill University, Montreal, PQ ,CANADA, 2Department of Miicrobiology and Immunology, University ofMontreal, Montreal, PQ , CANADA

A cluster of genes consisting of three open readingframes encoding proteins with homology to Ser/Thr protein kinases (prkY and prkX) and proteinphosphatases 2C (prpZ) was identified in the ge-nomes of Salmonella enterica serovar Typhi (Typhi)strains CT18 and Ty2. Recently, a microarray studyshowed that transcription of the prpZ gene de-

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creased by more than 3-fold upon cell exposure tohydrogen peroxide (H2O2). In this study, we deter-mined the transcriptional organization of this genecluster and showed that the transcription of thesegenes is affected by H2O2 and hypochlorous acid(HOCl) stresses. In the Typhi genome, the prpZ,prkY and prkX genes are adjacent and oriented inthe same direction, suggesting that they might becotranscribed as a polycistronic mRNA. To test thispossibility, reverse transcriptase PCR (RT-PCR) wasperformed on total RNA isolated from Typhi strainTy2. Unexpectedly, RT-PCR across the prpZ-prkYand prpY-prpX intergenic regions did not yield aPCR product, indicating that the three genes are nottranscriptionally linked. To determine whether theexpression of these genes is regulated by environ-mental cues, we created a prpZ-lacZ transcriptionalfusion in the chromosome of Typhi strain Ty2 andmeasured the beta-galactosidase activity underdifferent environmental conditions. LacZ activityfrom the prpZ promoter was unaffected by pH orthe addition of various antibiotics to the culturebroth. In contrast, we found that the LacZ activitywas drastically decreased by H2O2 and HOCl,although it was mostly unaffected by other formsof oxidative stress. Using real-time RT-PCR, wequantified the effects of H2O2 and HOCl on themRNA levels of prpZ, prkY and prkX. We foundthat HOCl decreased the expression of the threegenes by 5- to 10-fold. Although H2O2 also de-creased the expression of prpZ and prkY by 5-foldand 2-fold, respectively, it increased the expressionof prkX by 2-fold. We conclude that the geneproducts of prpZ, prkY and prkX might be involvedin a signaling pathway controlling resistance and/oradaptation of Typhi to H2O2 and HOCl stresses.

B92STRUCTURAL ANALYSIS OF THESALMONELLA PATHOGENICITY ISLAND 2RESPONSE REGULATOR SSRBR. K. Carroll, X. Feng , X. Liao, L. J. Kenney; University of Illinois atChicago, Chicago, IL

The ability of Salmonella enterica serovarTyphimurium to survive and replicate inside host

cells is associated with a pathogenicity island lo-cated at 63 centisomes on the Salmonella chromo-some. This region, termed SPI-2 (Salmonella patho-genicity island 2) encodes components of a type IIIsecretion system as well as effector and chaperoneproteins. Also encoded within this locus are genesfor a two-component signal transduction systemthat controls expression of the genes within SPI-2.SsrA is a membrane bound tripartite histidinekinase, while SsrB is the cytoplasmic responseregulator. In response to some as yet unknownsignal, SsrA autophosphorylates, then transfers thephosphoryl group onto SsrB, thus activating it. Wehave examined the structure of SsrB and identifiedseveral residues that are important for its function.The phosphorylation site was identified as Asp 56and was shown to be required for transcriptionalactivation by SsrB. In addition, we have identifiedkey Cys residues that also are important for SsrBactivity. We have isolated the C-terminal DNAbinding domain of SsrB and shown that it has theability to bind DNA and activate transcription inthe absence of the N-terminus. We solved thestructure of this domain by NMR and have exam-ined chemical shift changes resulting from DNAbinding. We also constructed a series of N-terminaltruncates of SsrB and determined that whenoverexpressed in the presence of full length SsrB,they influence SsrB-dependent gene activation.

C93CD34/CD43 DEFICIENT MICE SHOWINCREASED RESISTANCE TO INFECTIONWITH SALMONELLA TYPHIMURIUMG. A. Grassl, E. Drew, Y. Valdez, K. M. McNagny, B. Finlay; Universityof British Columbia, Vancouver, BC, CANADA

Salmonella enterica serovar Typhimurium is a majorcause of bacterial enteritis worldwide. CD34 andCD43 are cell surface sialomucins expressed onhematopoetic progenitor cells and mast cells. Lossof these sialomucins prevents mast cellrepopulation and hematopoietic precursor reconsti-tution in vivo. We investigated the role of mast cellsdeficient for CD34 and CD43 in the response toSalmonella. Murine bone marrow was isolated andmucosal-type mast cells were derived. Salmonella


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actively invaded bone marrow derived mast cells(BMMCs) from wildype and CD34-/-CD43-/-doubleknockout (dko) mice while invasion-deficientSalmonella mutants were not efficiently phagocy-tosed. Salmonella survived inside BMMCs forseveral days but did not replicate. S. Typhimuriumtriggered IL-6 production but not degranulation ofwildtype and CD34-/-CD43-/- BMMCs. We foundthat CD34-/-CD43-/- dko mice are more resistantto oral infection with S. Typhimurium than C57/Bl6wildtype mice. CD34/43 dko mice have a lowerbacterial burden in spleen, liver and mesentericlymph nodes and the survival after infection with S.Typhimurium is significantly better than C57/Bl6wildtype control mice. Cecal mucosal mast cellnumbers increase in infected wildtype mice but notin CD34/43 dko mice. Furthermore serum levels ofIL-6 and IL-12p70 were higher in wildtype than inCD34/43 dko mice. The lower mucosal mast cellnumbers in the CD34/43 dko may result in a moremoderate disease development and thus be benefi-cial to the host.

A94HIGH PREVALENCE OF INCOMPATIBILITYGROUP INCA/C AMONG MULTI-DRUGRESISTANT SALMONELLA ENTERICASEROTYPE NEWPORT ISOLATED FROMDAIRY CATTLET. L. Poole, N. Ramlachan, T. S. Edrington, R. C. Anderson, D. J.Nisbet; USDA, Agricultural Research Service, College Station, TX

Multi-drug resistant (MDR) Salmonella enterica sero-type Newport exhibiting increased morbidity andmortality among man and animals has emerged inNorth America and Canada. Risk factors for infec-tion by MDR S. Newport have included exposure todairy farms, consumption of undercooked groundbeef or unpasteurized milk and cheese. One hun-dred fifteen S. Newport strains isolated from dairycattle in three states were phenotypically and geno-typically characterized to elucidate genetic relation-ships among the isolates. Susceptibility testing andbreakpoint determination was done according toCLSI (formerly NCCLS) protocols. Ninety-eight(85%) of these isolates were resistant to multipleantimicrobials (3 to 11). Eighty-four percent of the

MDR S. Newport strains (those resistant to 7 ormore antimicrobials) were resistant to expandedspectrum cephalosporins. Genotypic analysis in-cluded multiplex PCR amplification of 19 incom-patibility group broad host-range plasmids. Broadhost-range plasmids have the capacity to replicate inmany different bacterial genera; however, no twoplasmids of the same incompatibility group canreplicate in the same bacterial cell. This characteris-tic has been used to classify broad host-rangeplasmids into incompatibility groups (Inc). Acquisi-tion of resistance genes on these plasmids has beencumulative resulting, in some cases, to resistanceagainst nearly all antimicrobial classes. Althoughcharacterization of resistance traits carried by broadhost-range plasmids has been extensive, there hasbeen little characterization of plasmid incompatibil-ity groups among food borne pathogens. Ninety-two (94%) of the MDR S. Newport were positivefor IncA/C. Two isolates were positive for IncA/Cand IncN, and two were positive for IncA/C andIncI; other than these three, no other incompatibil-ity groups were detected. Six of the MDR S. New-port isolates did not amplify any of the incompat-ibility groups tested; however, IncA/C was detectedin 1 of the 17 pansusceptible isolates. This is thefirst known report describing a large reservoir ofIncA/C in any bacterial pathogen. The acquisitionof IncA/C may have contributed to the emergenceof MDR S. Newport in the last ten years. Addi-tional characterization of IncA/C from MDR S.Newport is underway to determine if virulence andresistance genes; particularly, to expanded spectrumcephalosporins are located on the plasmid.

B95DIFFERENTIALLY EXPRESSED GENES OFSALMONELLA ENTERICA SEROVAR TYPHIDETECTED FROM INFECTED HUMAN ANDMURINE MACROPHAGESM. Beland, F. Daigle; University of Montreal, Montreal, PQ ,CANADA

INTRODUCTION: Salmonella enterica serovar Typhi(Typhi) is the etiological agent of typhoid fever.This Gram-negative bacterium is a pathogen re-stricted to human and infects more than 16 million

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C96GENETIC CHARACTERIZATION ANDCORRELATION TO MULTIDRUGRESISTANCE IN ISOLATES OFSALMONELLA ENTERICA SEROVARNEWPORT DERIVED FROM BEEF ANDDAIRY CATTLEN. Ramlachan, T. L. Poole, T. S. Edrington, R. C. Anderson, D. J.Nisbet; USDA-ARS-FFSRU, College Station, TX

High-throughput genotyping of Salmonella isolatesobtained from food production animals is an essen-tial component of food safety to evaluate patho-genic and antimicrobial resistance profiles. A totalof 99 strains of Salmonella enterica serovar Newportwere studied by pulsed-field gel electrophoresis(PFGE) to elucidate genetic relationships amongstrains isolated from beef and dairy cattle slaughter-house hide and carcass samples. Antibiotic suscepti-bilities were determined and interpreted accordingto MIC interpretive standards for antibiotics includ-ing: amoxicillin/clavulanate, ampicillin, cefoxitin,ceftiofur, cephalothin, chloramphenicol, kanamycin,streptomycin, sulfamethoxazole, tetracycline andceftriaxone. Some isolates were found to be resis-tant to 10 or more antibiotics. PFGE of XbaI-digested chromosomal DNA and plasmid DNAfingerprinting profiles were used to assign Salmonellaisolates into clonal groups and to determine plasmidcontent on the basis of size. Cluster analysis basedon the unweighted pair group method using anarithmetic averages algorithm identified three mainclusters independent of plasmid content, indicatingthat the divergence of the PFGE subtypes wasprobably derived from genomic mutations. Themajority of pan-susceptible isolates grouped in thesame cluster had similar XbaI profiles. The dataconfirms that strains isolated from beef or dairycattle were of a unique clonal origin and that aparticular PFGE combination profile was predomi-nant and specific for pan-susceptible isolates frombeef and dairy cattle. Correlations between antimi-crobial resistance phenotype and genotype, as wellas predicted pathogenicity and presence of predis-posing conjugative elements and competitive fitnessfor each isolate tested revealed further differentia-tion within beef or dairy groups. Combined pheno-

persons causing over half million deaths per year.Understanding Typhi pathogenesis is limited by lackof virulence in animal models. Successful infectionof the host by Salmonella involves adaptation to itsmononuclear phagocytes. For instance, Typhiinvades and survives better in human macrophagesthan in murine macrophages. The capacity of Typhito survive, multiply and cause a systemic infectionin humans, may be due to the expression of specificgenes that are required for survival in human cells.OBJECTIVE: Our goal is to identify genes ex-pressed differentially in two different host cells.METHODS: The Differential Fluorescence Induc-tion (DFI) strategy was used to identify Typhi genesthat are more expressed in human macrophagesthan in murine macrophages by using the GreenFluorescent Protein (GFP) and by fluorescence-activated cell sorter (FACS) analysis. Small DNAfragments (0.4- 2 Kb) obtained by partial Sau3Adigestion of the Typhi chromosome were insertedinto a promoterless GFP vector and cloned intoTyphi. This library of Typhi clones was used toinfect macrophages. Human macrophages contain-ing fluorescent bacteria were sorted. These bacteriawere used to infect murine macrophages and mac-rophages that demonstrate low or no fluorescencewere sorted. A final passage of Typhi GFP clonesthrough human macrophages and sorting for fluo-rescent cells that were enriched for bacteria bearinga GFP fusion expressed in human macrophages wasperformed. To establish the ratio of fluorescenceinduction of GFP in human vs murine macroph-ages, the individual clones were used to infect bothtypes of macrophages. Then, clones that had ahuman cells/murine cells ratio superior to 1.5 weresequenced. RESULTS: We obtained 13 clonescorresponding to 11 different genes that show aratio above 1.5. Clone 2AB3 corresponding to theunknown gene STY1361 was further studied andcharacterized. CONCLUSION: The use of DFI hasallowed the identification of genes expressed byTyphi during infection of macrophages. This mayhelp to develop strategies that can be used toreduce the likelihood of Salmonella colonization andinfection.

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typic and genotypic profiles are necessary forepidemiological studies and public health investiga-tions on S. Newport. The use of such methodolo-gies for genetically determining multidrug resistancestatus, pathogenicity and ability to transfer and/oruptake such genes in a Salmonella spp. of publichealth concern, will allow us to elucidate themechanisms by which S. Newport increases inpathogenicity causing fatal disease in cattle as wellas serious cases of human food poisoning.

A97CLONING AND CHARACTERIZATION OFTHE STG FIMBRIAL OPERON FROMSALMONELLA ENTERICA SEROVAR TYPHIF. Daigle1, C. Forest1, S. Faucher1, K. Poirier1, S. Houle2, C. M. Dozois2;1University of Montreal, Montreal, PQ , CANADA, 2INRS-IAF, Laval,PQ , CANADA

Salmonella serovars express a wide variety of puta-tive fimbrial systems that may contribute to coloni-zation of specific niches. Salmonella enterica serovarTyphi is the etiologic agent of typhoid fever and is apathogen specific to humans. The complete genomesequence of serovar Typhi contains 13 putativefimbrial operons. In a previous study using Typhistrain ISP1820, we identified STY3920, a geneencoding for the usher StgC of the putative Stgfimbrial operon that is absent in serovarTyphimurium. Stg is a member of a distinct groupof related fimbriae that are located in the glmS-pstSintergenic region in certain E. coli and S. entericastrains. We cloned the stg fimbrial operon into anormally nonfimbriated E. coli K-12 strain anddemonstrated that Stg contributed to increasedadherence to human intestinal epithelial cells. Theproduction of Stg fimbriae was demonstrated bywestern blotting in the E. coli strain. Transcriptionalfusion assays in Typhi suggest that stg is preferen-tially expressed on minimal solid medium. Thedeletion of stg reduced adherence of Typhi toepithelial cells. Interestingly, the DELTAstg mutantwas highly phagocytosed by human macrophages,however the level of survival inside human mac-rophages was similar to the wild-type parent. Al-though the stgC gene contains a premature stop

codon that disrupts the expected ORF encoding theusher, and is therefore considered as pseudogene, itseems that Stg is still functional and able to conferadhesion to epithelial cells. The Stg fimbriae maytherefore mediate intestinal to colonization ofTyphi and potentially contribute to the initial stageof typhoid pathogenesis.

B98IDENTIFICATION OF A NOVEL PDZ-DOMAIN CONTAINING PROTEIN ON THESALMONELLA-CONTAINING VACUOLEL. Knodler, O. Steele-Mortimer ; Rocky Mountain Laboratories, NIAID,NIH, Hamilton, MT

Salmonella spp. translocate numerous type IIIeffectors into host cells via two type III secretionsystems (T3SS). Together, these effectors mediateinvasion and intracellular survival, both of whichare essential virulence determinants. One of theseeffectors, PipB, is translocated by T3SS2 and local-izes to the Salmonella-containing vacuole (SCV) andtubular extensions known as Salmonella-inducedfilaments (Sifs). PipB is required for virulence inbovine and avian models of infection, however thefunction of this effector remains unknown. Toidentify potential cellular targets of PipB, we iso-lated proteins that co-immunoprecipitated withEGFP-PipB that was ectopically expressed inmammalian cells. A protein with an apparent mo-lecular mass of ~150 kDa on SDS-PAGE wasidentified by mass spectrometry as PDZK8. Thishypothetical protein has an N-terminal signalpeptide, and PDZ and C1 domains, which arecommonly involved in protein and lipid interactionsrespectively. The PipB-PDZK8 interaction wasconfirmed by co-immunoprecipitation from trans-fected cells and immunoblotting using an antibodygenerated against a C-terminal peptide of PDZK8.Yeast two hybrid analysis demonstrated that thePipB-PDZK8 interaction is direct and requires theC-terminal 20 amino acid residues of PipB. Thespecificity of this interaction was further demon-strated in infected cells, as PDZK8 co-immunopre-cipitated with translocated PipB but not its effectorhomologue, PipB2. Immunofluorescence stainingof infected cells showed that PDZK8 is recruited to

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the SCV in a PipB-dependent manner. We arecurrently exploring the function of PDZK8 inuninfected and infected cells by siRNA and bio-chemical techniques. In summary, we have identi-fied PDZK8 as the first mammalian target of PipBand a novel protein that is recruited to the SCV.

C99ROLE OF MYOSIN II IN STABILITY ANDPOSITIONING OF THESALMONELLA-CONTAINING VACUOLEJ. A. Wasylnka, J. H. Brumell; Hospital for Sick Children, Toronto, ON,CANADA

Salmonella enterica serovar Typhimurium is a Gram-negative enteric pathogen that causes gastroenteritisin humans. Following entry into host cells, Salmo-nella reside in a unique compartment called theSalmonella-containing vacuole (SCV) segregatedfrom the normal endocytic pathway, which providesa replicative niche for the bacteria. Followingbiogenesis at the cell periphery, the SCV migrates tothe microtubule organizing center (MTOC) andmaintains this position within the host cell. Move-ment of organelles within eukaryotic cells is medi-ated by at least three types of cytoplasmic motors:dyneins and kinesins which use microtubules tomove their cargo, and myosins which use actinfilaments. Recent studies have shown that position-ing of the SCV is controlled by microtubule mo-tors, and that bacterial factors can influence theactivity of these motors. For example, kinesinactivity on the SCV is inhibited by SifA (Boucrot etal, 2005 Science). Filamentous actin structures havebeen observed on SCVs, suggesting that actin-basedmotors may also play a role in SCV membranedynamics (Meresse et al, Cell. Microbiol. 2001; Miaoet al, Cell. Microbiol. 2003). Here we examined non-muscle myosin II for its effects on SCV positioningand stability, since it is known to associate withmodel phagosomes in macrophages. We observedmyosin II recruitment to actin-rich ruffles at thesite of bacterial invasion. BDM (a general myosininhibitor) but not blebbistatin (a specific myosin IIinhibitor) prevented invasion of Salmonella intoHeLa cells, suggesting multiple myosins act redun-

dantly to promote bacterial uptake. Centripetalmovement of the SCV was inhibited byblebbistatin, indicating a specific role for myosin II.Similarly, myosin II was also required to maintainthe SCV at the peri-nuclear region: at 8 hours postinfection, blebbistatin treatment caused the cen-trifugal movement of SCVs to the cell periphery.Surprisingly, this centrifugal movement was notobserved in cells infected with a mutant lacking theSPI-2 type III secretion system. This suggests SPI-2effectors recruit motor proteins to direct centrifugalSCV movement, and that their activity is counter-acted by myosin II to maintain SCV positioning.Blebbistatin treatment also led to a decrease in SCVstability and a concomitant increase in the numberof intracellular bacteria (since the cytosol of HeLacells is permissive for Salmonella growth). Usingconfocal microscopy, we observed that myosin II islocalized to Salmonella-induced filaments (Sifs),tubular extensions of the SCV, in blebbistatintreated cells. We propose that myosin II cycles onand off Sifs in a manner that requires its ATPaseactivity. Together, these results indicate that myosinII plays a significant role in regulating SCV mem-brane dynamics.

A100THE ROLE OF SALMONELLA TYPE IIISECRETION SYSTEMS IN THECOLONIZATION OF POULTRYW. Köster1, E. Berberov1, T. Desin1, B. Koch1, S. Lam1, A. Wisner1, L.Krishnan2, B. Coombes3, M. Lowden3, B. Finlay3, S. Sad2, A. Potter1;1University of Saskatchewan, Saskatoon, SK, CANADA, 2NationalResearch Council of Canada, Ottawa, ON, CANADA, 3University ofBritish Columbia, Vancouver, BC, CANADA

The impact of food- and water-borne pathogenssuch as Salmonella enterica subsp. enterica serotypeEnteritidis (S. Enteritidis) and multidrug-resistantSalmonella enterica subsp. enterica serotypeTyphimurium (S. Typhimurium) on public healthand the economic livelihood of food producers issignificant and often underestimated. This is espe-cially true in the poultry industry even though theseorganisms do not cause significant disease in theiranimal hosts. Two distinct virulence-associated typeIII secretion systems (TTSS) are encoded within the


pathogenicity islands SPI-1 and SPI-2 of S. Enter-itidis and S. Typhimurium. For effective and persis-tent colonization of chickens, at least one TTSSseems to be essential. The objective of our researchis to determine the role(s) of SPI-1 and SPI-2 TTSSin colonization of chickens by Salmonella speciesand also to determine the vaccine potential ofstructural components as well as effector molecules.S. Enteritidis knockout mutants were constructed inseveral genes of interest by allelic replacement inorder to compare the wild type strain and theresulting mutant strains for their ability to colonizethe chicken gut as well as liver and spleen. S.Typhimurium SPI-1 and SPI-2 secretions systemsalso play a role in colonization of poultry. In orderto test the vaccine potential of TTSS and theireffectors from S. Typhimurium, pools of SPI-1 andSPI-2 proteins were used to immunize chickens.Neither vaccine formulation had any effect on cecalcolonization following oral infection with S.Typhimurium. However, a significant reduction incolonization of the liver and spleen was observed inbirds immunized with SPI-2 antigens deliveredalone or in combination with SPI-1 proteins. Similarexperiments are currently underway using S. Enter-itidis TTSS vaccine formulations.

B101AKT RECRUITMENT TOSALMONELLA-INDUCED MEMBRANERUFFLES IS PI 3-KINASE INDEPENDENTR. Ireland, O. Steele-Mortimer ; Rocky Mountain Laboratory, Hamilton,MT

Salmonella sp have two type III secretion systemsthat are used to translocate bacterial effector pro-teins into the host cell where they can directlyinterfere with host cell processes such as membranetrafficking or cell survival. Effectors translocated bythe invasion-associated type III secretion system(T3SS1) act cooperatively to induce bacterial uptakeby nonphagocytic cells. One of these effectors,SopB/SigD, is an inositol phosphatase that alsoinduces activation of the prosurvival kinase Akt ininfected epithelial cells. Canonical Akt-activation isdependent on phosphatidylinositol (PI) 3-kinase

and elevated levels of its products PI(3,4,5)P3 andPI(3,4)P2, since Akt preferentially associates withthese phospholipids in the plasma membrane. Onceat the membrane Akt associates with anotherphospholipid-binding protein kinase, PDK1, result-ing in Akt phosphorylation on Thr308. Full activa-tion of Akt requires phosphorylation at Ser473,which appears to be mediated by a complex con-taining mTOR, GBetaL and rictor. Here we haveinvestigated the role of PI 3-kinases in SopB-dependent Akt activation in HeLa cells using thewell-characterized chemical inhibitors Wortmannin(WTM) and LY294002 (LY29). Immunoblot analy-sis using phospho-specific antibodies showed thatLY29, but not WTM, blocked SopB-dependent Aktphosphorylation. In contrast, immunofluorescencemicroscopy revealed that ruffle formation and Aktrecruitment was insensitive to either LY29 or WTM.However, we also found that the Akt kinases, PDK1and rictor, are required for SopB-dependent Aktactivation and are translocated to ruffles. Altogetherthese results suggest that SopB-dependent Aktactivation does not involve the canonical PI 3-kinase pathway.

C102IDENTIFICATION OF NEW SALMONELLASSRB AND PHOP REGULATED GENESUSING A FLOW CYTOMETRY SCREENO. Gal-Mor, B. B. Finlay; University of British Columbia, Vancouver,BC, CANADA

As a facultative intracellular pathogen, Salmonellaenterica utilizes two pivotal two-component regula-tory systems necessary for its intracellular life-style,known as PhoPQ and SsrAB pathways. Althoughthe environmental signals that activate these path-ways might be complex, it seems that both systemsrespond to Mg2+ starvation. It is believed that bymonitoring the availability of extracellular Mg2+,these two-component systems allow Salmonella tosense the transition from an extracellular environ-ment to a subcellular location and to activate a setof virulence factors essential for intracellularsurvival. In order to identify PhoP and SsrB regu-lated genes, we preformed a flow cytometry screen

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of a S. typhimurium genomic DNA library fused to apromoterless gfp gene under lowMg2+concentrations and acidic pH conditions. Thisscreen resulted in the identification of 48 genes thathad high expression levels under these conditions.The identified genes are known to be involved indifferent aspects of Salmonella biology includingvarious metabolic pathways, gene regulation, viru-lence, stress response and other functions. Amongthese identified genes, at least seven genes (ppsA,yfbE4, STM3595, prmD, pagO, pSLT046, and ybjX)were previously described or predicted (based onpromoter analyses) to belong to the PhoPQregulon. Eight identified genes were further charac-terized by constructing translational lacZ fusionsand analyzing them in different genetic back-grounds. This analysis confirmed that pagO,pSLT046 and ybjX are activated by PhoP and re-vealed a previously unknown PhoP activated gene,ybaJ. In addition, we identified at least two othernovel genes that appeared multiple times in ourscreen to be activated by both PhoP and SsrB, thephage gene sb26 and STM2287. The expression ofthese genes was also found to be lower in an ompRmutant strain and during growth in LB medium (incomparison to MgM minimal medium). Theseresults suggest that ybaJ, sb26, and STM2287 mayplay a role in the intracellular phase of Salmonella,and reinforce the linkage between the PhoPQ andSsrAB pathways as regulators that respond tosimilar environmental signals and may affect theexpression of common target genes.

A103REGULATION OF THE MISLAUTOTRANSPORTER PROTEIN INSALMONELLA ENTERICA SEROTYPETYPHIMURIUMC. Tukel1, M. Akcelik2, A. Baumler1; 1UC Davis, Davis, CA, 2AnkaraUniversity, Ankara, TURKEY

MisL is an autotransporter protein located onSalmonella pathogeniciy island-3 (SPI-3). MisL hasbeen shown to bind to the extracellular matrixproteins, fibronectin and collagen IV. AlthoughMisL is expressed in vivo as suggested byseroconversion to this antigen during infection of

mice, this adhesin is not expressed in vitro. Toinvestigate the regulatory mechanisms that lead toinduction of misL expression in vivo, a suicidevector, pFUSE, was used to generate a chromo-somal misL::lacZYA transcriptional fusion in S.Typhimurium strain LT2. To identify genes regulat-ing misL expression, we performed transposonmutagenesis using T-POP. Among approximately40.000 transductants, two mutants were identifiedthat expressed the misL::lacZYA transcriptionalfusion, as indicated by a blue colony phenotype onagar plates supplemented with 5-bromo-4-chloro-3-indolyl-Beta-D-galactopyranoside (X-gal). Expres-sion of misL detected by a Beta-galactosidase assayor by Western blot with anti-MisL serum wastetracycline-dependent, suggesting that, in thesemutants, the tetracycline promoter of T-POP maydrive the expression of a positive regulatory ele-ment. Cloning of transposon-flanking DNA byinverse PCR revealed that in both independentmutants T-pop was inserted in the second codon ofthe marT gene, which is also located in SPI-3.When the marT gene was cloned behind an arabi-nose-inducible promoter from E. coli, MisL wasexpressed in a dose dependent manner with increas-ing concentrations of arabinose as determined byBeta-galactosidase and Western blot assays. Theseresults show that the marT gene encodes an activa-tor of the misL gene and suggest a possible role ofMarT in inducing in vivo expression of anautotransporter protein involved in binding to theextracellular matrix of host tissue.

B104ROLE OF THE VI CAPSULAR ANTIGEN OFSALMONELLA ENTERICA SEROTYPE TYPHIIN ELICITING RESPONSES IN ANTIGENPRESENTING CELLSR. P. Wilson, D. Chessa, M. Raffatellu, C. Tukel, A. Baumler; UCDavis, Davis, CA

A massive neutrophil influx in the intestine is thehistopathological hallmark of S. enterica serotypeTyphimurium infection in humans; however, neu-trophils are scarce in intestinal infiltrates of patientsinfected with S. enterica serotype Typhi. The S.

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Typhi Vi capsular antigen, which is absent in S.Typhimurium, is thought to be a key virulencefactor that prevents the production of neutrophilchemoattractants, such as interleukin (IL)-8. Ourgroup has previously shown that the Vi capsularantigen inhibits IL-8 production by blocking Toll-like Receptor (TLR) signaling, thereby preventingneutrophil recruitment. However, TLR signaling isalso important for responses leading to the develop-ment of adaptive immunity, such as cytokines andcostimulatory molecules, which polarize T cellresponses. The purpose of this study was to investi-gate whether expression of the Vi-antigen wouldalter adaptive immune responses during typhoidfever. We investigated the effects of Vi capsularantigen on the production of CD80, CD86, IL-12,IL-23, IL-10, IL-1beta, TNF-alpha and IL-6, usingJ774 cells, a macrophage like mouse cell line. To dothis, we cloned the Vi capsule biosynthesis genecluster (viaB region) on a low copy number vectorand the resulting plasmid (pDC5) was introducedinto a S. Typhi viaB mutant and into S.Typhimurium. Expression of the Vi capsular anti-gen in S. Typhi Ty2 (wild type), S. Typhi viaB(pDC5) and S. Typhimurium (pDC5) reduced TNF-alpha and IL-6 protein secretion and surface expres-sion of CD80. We are currently investigatingcytokine expression in Jaws II cells, an immaturemouse dendritic cell line, and in a mouse model.

C105MUCOSAL PRIMING WITH SALMONELLATYPHI EXPRESSING Y. PESTIS AND B.ANTHRACIS PROTECTIVE ANTIGENSFOLLOWED BY PARENTERAL SUBUNITVACCINE BOOST CAN SERVE AS ANEFFECTIVE BIODEFENSE VACCINESTRATEGY FOR EARLY LIFEIMMUNIZATIONK. Ramirez, A. Capozzo, S. Lloyd, J. Galen, M. Sztein, J. Nataro, M.Pasetti; University of Maryland, Baltimore, MD

Neonates have a limited capacity to generate pro-tective immunity in response to vaccination. Theirresponses are usually feeble and Th2-biased, whichhas been attributed, in part, to the immaturity ofneonatal dendritic cells (DC) that leads to inad-

equate antigen presentation and T cell stimulation.Neonatal Th1-type responses, however, can beinduced using the appropriate antigen/stimulation.We have shown that Salmonella-based live vectorvaccines are excellent candidates to induce immuneresponses against a foreign antigen at very early lifestages. Attenuated S. Typhi vaccine strainsACAMBCVD 948 and CVD 908-htrA were engi-neered to express Yersinia pestis F1 capsule andBacillus anthracis protective antigen (PA) domain 4(D4). Recognizing that biodefense vaccines will beneeded not only for healthy adults but also for high-risk groups such as young infants and children, weinvestigated the immunogenicity of these vaccinecandidates in a neonatal mouse model that parallelsimmune responses in humans. Newborn mice wereimmunized intranasally with S. Typhi expressing F1or PA-D4 on day 7 after birth, and they wereboosted 14 days later (day 21 after birth) with F1-or PA-alum. Control groups received 2 doses of S.Typhi(F1) or S. Typhi(PA-D4), 2 doses of F1- orPA-alum, or one dose of S. Typhi (alone) followedby F1- or PA-alum. Newborns primed with S.Typhi(F1) or S. Typhi(PA-D4) and boosted with F1-or PA-alum induced high levels of F1 and PA-specific IgG antibodies. Live vector priming elicitedstrong cell-mediated immunity (CMI) including Tcell proliferation and IFN-ã secretion, and mucosalantibody secreting cells, during the true neonatalperiod. Newborns immunized with F1- or PA-alum,on the contrary, failed to induce strong CMI andmucosal immunity. Mice primed with S. Typhi(F1)or S. Typhi(PA-D4) exhibited F1 and PA-specificplasma cells and memory B cells in the bone mar-row (BM). We hypothesized that one reason for theenhanced responses observed could be the capacityof Salmonella to activate and enhance maturation ofneonatal DC and T cell stimulation. We tested thishypothesis incubating DC derived from the BM ofneonatal (7-day-old) mice with S. Typhi CVD 908-htrA. S. Typhi-exposed CD11c+ DC showed in-creased expression of cell surface markers CD80,CD86, CD40 and MHC class II molecules, andsecretion of Th1-type/proinflamatory cytokines IL-12, TNF-á, IFN-ã, IL-6 and MCP-1. DC infectedwith S. Typhi(F1) induced strong proliferation of


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F1-specific T cells, in contrast to DC incubatedwith F1 or DC alone. S. Typhi-induced maturationof neonatal DC was confirmed using human cord-blood cells. Responses primed by S. Typhi express-ing F1 or PA increased significantly after F1- andPA-alum boost. We conclude that S. Typhi express-ing plague and anthrax protective antigens followedby parenteral subunit vaccine boost could serve asan effective biodefense vaccine strategy for younghosts.

A106REGULATION OF SALMONELLA TYPE IIISECRETED EFFECTORSY. Dieye1, B. M. Ahmer2; 1Arizona State University, Tempe, AZ, 2TheOhio State University, Columbus, OH

The regulation of the type three secretion systems(TTSS) in Salmonella is known to be complex withseveral positive and negative regulatory proteinsoperating to allow the expression in desired com-partments during infection of the host. We per-formed a systematic analysis of the regulation oftype III secreted effectors during growth in vitro.We constructed a multi-mutant strain that lacksseven TTSS regulatory genes (hilC, hilD, hilA, invF,sprB, ssrB, sirA). In this strain we made lacZ chromo-somal transcription fusions to genes encodingsecreted effectors (sopA, sopB, sopD, sopE2, sspH2,sifA, sifB, sseI, sseG, slrP, sspH1). We then introducedplasmids encoding one of the missing regulators.The fusions to sopA, sopB, and sopE2 were activatedin the presence of the plasmid encoding invF andsicA, and the fusions to sspH2, sifB, sseI and sseGwere activated by ssrB during growth in LB medium.The fusion to sifA was activated by ssrB whengrown in a SPI2-inducing medium but not in LB.The slrP gene was regulated by plasmid-encoded butnot chromosomal sprB and invF. No regulator wasfound to activate the sopD and sspH1 fusions. Weexamined the regulation of sifA in the mouse usingthe RIVET approach and found that it is expressedin the intestinal lumen and in the spleen, with ssrBbeing the main regulator in both compartments.

B107A S.I.M.P.L.E. HUNT FOR REGULATORS OFFIMBRIAL EXPRESSIONS. Nuccio, D. Chessa, A. J. Baumler ; University of California at Davis,Davis, CA

Surface structures known as fimbriae often play acritical role in the pathogenesis of a bacterium,from the initial colonization of the host to its long-term persistence. While the roles in the host-pathogen interaction of a couple of the thirteenfimbrial operons of Salmonella Typhimurium havebeen well described, the rest lack thorough func-tional analysis — a deficiency attributable to thecurrent inability to provide in vitro growth condi-tions suitable to induce their expression. In order tounderstand which media components and environ-ments might be employed, we undertook a study ofthe regulatory mechanisms controlling fimbrialexpression. The pefBACD encoded fimbria, Pef, waschosen as a starting point for study as it has aknown weakly inducing environmental signal, acidicpH, but no known correlate regulator of expres-sion. A T-POP transposon mutant library wasgenerated and screened at neutral pH for the sur-face expression of PefA, the major fimbrial subunit,utilizing a novel method coined S.I.M.P.L.E.: Screenwith Immunomagnetic Particles for Ligand Expres-sion. Superparamagnetic beads were coated withanti-PefA serum and used to purify mutants ex-pressing PefA on their surface from the mutantpools grown at neutral pH. An SR-11 delta-fimstrain was chosen as the screen background to avoidposited complications with steric hindrance fromFim, the dominantly expressed fimbria in liquidculture, impeding efficient antibody binding tomutants expressing Pef. Serendipitously, this strainexhibited a marked improvement in Pef expressionover Wild Type at acidic pH, though it retained thepH-regulated phenotype. Four insertion mutantsexpressing PefA at neutral pH were subsequentlyisolated with this S.I.M.P.L.E. method, all within orjust upstream of hns. We are currently analyzing theexpression of other fimbriae in these mutant back-grounds and employing the screen to identifymutants expressing fimbriae under different growthconditions.



C108ADMINISTRATION OF A HIGHLYCONSERVED ENTERIC BACTERIAL OUTERSURFACE PROTEIN (OSPA) INDUCESHETEROPHIL-MEDIATED INNATE IMMUNEFUNCTION AND PROTECTION AGAINSTORGAN INVASION BY SALMONELLAENTERICA SEROVAR ENTERITIDIS INYOUNG CHICKENSM. Kogut1, K. J. Genovese1, H. He1, T. H. Richardson2, R. Brown2, G.Djordjevic2, L. Hickle2; 1USDA-ARS, College Station, TX, 2DiversaCorporation, San Diego, CA

A Rickettsial outer surface membrane protein hasbeen shown to be an efficacious vaccine against thesalmon pathogen, Piscirickettsia salmonis. Entericbacteria possess a homolog of this antigen whichhas been isolated and purified for evaluation as apromiscuous vaccine candidate against entericbacteria in chickens. The ideal vaccine would inducea heterophil-mediated innate response to controlearly infection and regulate the proper acquiredmemory response. The objective of the presentstudies was to evaluate the enteric homolog of theOspA membrane protein as a modulator of theinnate response and inducer of protection againstextraintestinal infection by Salmonella enterica serovarEnteritidis (SE). Day-of-hatch chickens were sepa-rated into one of 5 treatment groupss: (1) nonvaccinated, non-challenged control, (2) non-vacci-nated, challenged, (3) mock-vaccinated, challengedcontrol, (4) carrier protein vaccinated, challengedcontrol, and (5) OspA vaccinated, challenged. Allvaccinated birds were injected sub-Q with 200 μgprotein/chicken. Both carrier protein and OspAwere produced in E.coli and were free of LPS. Fourhours after vaccination, the birds were orally chal-lenged with 5 x 107 cfu/ml SE. Twenty-four hourslater, from each chicken, the liver and spleen wereaseptically removed and combined in 50 mltetrathionate broth for enrichment of SE. A highlysignificant reduction in SE organ invasion wasfound only in the OspA injected group whencompared to all of the controls. In addition, asignificant increase in circulating peripheral bloodheterophils in the OspA-injected group was found.The functions of heterophils isolated from chickens

injected with OspA was significantly up-regulatedwhen compared to heterophils isolated from chick-ens given either PBS or PBS + carrier protein asdetermined with phagocytosis, oxidative burst, anddegranulation assays. This is the first report of apotential vaccine from the outer surface of entericbacteria inducing the up-regulation of the avianinnate immune response that provides protectionagainst extraintestinal Salmonella infections. Thesignificance of these data is that OspA stimulate theinnate response at a time of immunologic ineffi-ciency and increased susceptibility to bacterialinfections (first week posthatch). Because of thenon-specific nature of the innate response, theOpsA antigen given during the first week posthatchcould potentially provide increased protectionagainst a variety of enteric food safety bacteria.

A109INNATE DEFENSE REGULATORS PROTECTHOST CELLS FROM BACTERIALPATHOGENS BY INDUCTION OFPROTECTIVE ELEMENTS OF THE INNATEIMMUNE RESPONSEE. M. Dullaghan1,2, H. Wang1, O. Donini1, M. Guarna1, B. B. Finlay2,R. E. Hancock2, M. G. Scott1; 1Inimex pharmaceuticals, Vancouver, BC,CANADA, 2University of British Columbia, Vancouver, BC, CANADA

The innate immune response is the first line ofdefence against pathogens. It involves an interactivenetwork of cellular and molecular systems thatrespond to pathogen-associated molecular patterns.We are investigating novel innate defense regulators(IDR) that selectively up-regulate the infection-clearing aspects of innate immunity without harm-ful inflammatory responses. The objectives of thesestudies were twofold; firstly, to understand the hostresponse to challenge by Salmonella enterica serovarTyphimurium and secondly to further elucidate themechanism of peptide action. We utilizedmicroarrays to study the global human gene expres-sion patterns in PMA treated THP-1 cells in re-sponse to Salmonella Typhimurium infection in thepresence and absence of an IDR. The resultsprovide further characterization of the host re-sponse to bacterial infections and the mechanism bywhich IDR’s can alter the immune balance in favor


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of the host during an infection.

B110

A YEAST TWO-HYBRID GENOMIC LIBRARYOF SALMONELLA ENTERITIDIS TOANALYSE PROTEIN-PROTEININTERACTIONSS. Holbert, J. Trotereau, Y. Fardini, P. Velge, I. Virlogeux-Payant;INRA, Nouzilly, FRANCE

The yeast two-hybrid system (Y2H) is a powerfulsystem to study the protein networks. This system isused to (1) study the interaction between twoproteins which are expected to interact and/or (2)find prey proteins in a library by large screen with abait of interest. Previously, this method was suc-cessfully used to analyse networks of protein inter-actions that mediate assembly of molecular ma-chines and dynamic cellular pathways. We haveinitiated a project to identify protein-protein inter-actions involved in the pathogenicity of Salmonella.For this purpose, we constructed a genomic Y2Hlibrary of Salmonella Enteritidis. Five restrictionenzymes, AciI, Hinp1I, MaeII, MspI and TaqI, wereused for the partial restriction of genomic DNA togenerate a highly complex library. DNA fragmentswere cloned into the pGAD-C1, pGAD-C2 orpGAD-C3 vectors which differ in the translationalreading frame of the polylinker sites, generating 15sub-libraries (one out of 5 enzymes with one out of3 vectors). Recombinants plasmids were thendirectly introduced in the yeast Saccharomycescerevisiae Y187 without passaging through E. coli asthis step was previously shown to increase theretention of functional fusions to toxic genes. Foreach sub-library, the number of independent clones,the distribution of the insert size and the percent-age of vector without insert were determined. Inthe final pooled library, inserts in the size ranges of1 to 400 bp and 400 to 3000 bp were present inabout equal proportions (49% versus 51%). Finally,we screened the whole genomic library with theempty pGBKT7 bait vector in order to identifyclones that on their own lead to activation of theHIS3 and lacZ reporter genes. The DNA inserts ofthe autoactivator clones derived from only 4 S.

Enteritidis genes : amyH, talC, STM3142 and adi.This two-hybrid library is a new tool allowing theidentification of protein-protein interactions in thebiology of Salmonella but also in the interaction ofthis bacterium with its hosts.

C111PHYLOGENY OF THE SALMONELLA SUBSPECIES USING FOUR HOUSEKEEPINGGENES AND EVIDENCE OF A LATERALGENE TRANSFER EVENTJ. R. McQuiston1, S. Herrera-Leon2, E. Chaitkin3, J. Doyle3, P. I.Fields1, R. V. Tauxe1, J. M. Logsdon4; 1CDC, Atlanta, GA, 2LaboratorioNacional de Referencia de Salmonella y Shigella, Centro Nacional deMicrobiologia, Madrid, SPAIN, 3Emory University, Atlanta, GA, 4Centerfor Comparative Genomics, University of Iowa, Atlanta, IA

The Salmonellae are a diverse group within the familyEnterobacteriaceae that includes two species, Salmonellaenterica and Salmonella bongori. Salmonella enterica isfurther divided into seven subspecies (subsp.)designated by Roman numerals in addition to theirtaxonomic names. Subsp. I, II, IIIb and VI containserotypes that are generally diphasic, expressing twocoordinately regulated loci, fliC and fljB, encodingthe flagellin. The Subsp. IIIa, IV, VII and Salmonellabongori are monophasic or contain only the fliClocus, thus expressing only one flagellin. Previousstudies have determined phylogenies of Salmonellabased on different methods producing differentconclusions; however these studies used very fewisolates or were limited to the most common sub-species. To ascertain whether the phylogeny ofSalmonella was universal across Salmonella and as aninitial step to determine the history of the acquisi-tion of the fljB locus, we analyzed four housekeep-ing genes from 71 taxa of Salmonella representing awide diversity of subspecies and serotypes. Thegenes gapA, phoP, mdh and recA, were sequenced andanalyzed to determine the phylogenetic relation-ships among these Salmonellae. Sixty-one isolatesrepresenting the most common serotypes of theseven subspecies of S. enterica and six isolates of S.bongori as well as genes from four published genomesequences were included in the study. Phylogeneticanalysis showed that each subspecies formed a



distinct clade, and that the diphasic and monophasicsubspecies clustered separately. This leads us toconclude that the acquisition of the fljB locus likelyoccurred as a single event.We also determined that the gene recA has beenhorizontally transferred from Subsp. IIIb to Subsp.IIIa and rapidly evolved in the latter since the event.This study defines the phylogeny of the Salmonellaspecies and subspecies based on the four genesgapA, phoP, mdh and recA, across all subspecies withmany serotypes, providing a solid scaffold forfurther analyses of the events in the history ofSalmonella.

A112NEAR COMPLETE CONSERVATION OFENDONUCLEASE CLEAVAGE SITES TODEFINE THE BASIC PHYLOGENETIC UNITOF SALMONELLALiu, G.-R.,1,2 W.-Q. Liu,3 G.X. Zhao,1 R. N. Johnston,4 L. Wang ,1 K. E.Sanderson,3 S.-L. Liu.1,2* 1Department of Microbiology, Peking UniversityHealth Science Center, Beijing , China; Departments of 2Microbiology andInfectious Diseases, 3Biological Sciences, and 4Biochemistry and MolecularBiology, University of Calgary, AB, Canada

Definition of a bacterial species has been arbitrary,largely because of lack of a theory-based scientificspecies concept. The polyphasic taxonomy strategydefines a bacterial species as a category that circum-scribes a (preferably) genomically coherent groupof individual isolates/strains sharing a high degreeof similarity in many independent features, com-paratively tested under highly standardized condi-tions. In this definition, no definite criteria are givenon a strict phylogenetic basis. Theoretically, mem-bers of the same bacterial species should own someunique genome features, clearly absent in membersof any other species. Based on this theoreticalprediction, we attempted to find such genomefeatures. Having compared the genomes of thesequenced S. typhi strains CT18 and Ty2 and find-ing that cleavage sites for a number of endonu-cleases were nearly completely conserved, wecompared Salmonella gallinarum and Salmonellapullorum strains by mapping. We purposely chose S.gallinarum strains that had drastically differentoverall genome structures and one S. pullorum

strain that had a similar genome structure to one ofthe S. gallinarum strains in the comparisons. Wefound that cleavage sites of the selected endonu-cleases were nearly completely conserved betweenthe S. gallinarum strains but not between S.gallinarum and S. pullorum strains. We concludethat a Salmonella serovar is a basic phylogeneticunit equivalent to a phylogenetic species, membersof which share the same gene pool and occupy thesame niche. Once a member obtains better fitness,it will increase in population size and graduallyreplace all other populations of the species. As aresult, all members occupying the same niche andsharing the same gene pool will relatively retain thenucleotide identity throughout the genome asreflected by the nearly complete conservation ofthe endonuclease cleavage sites. When anothermember acquires better fitness, it will replace allpopulations that may have accumulated somemutations involving endonuclease cleavage sites.

B113USING ANTIBIOTIC SUSCEPTIBILITYPATTERNS AND PULSE FIELD GELELECTROPHORESIS TO DESCRIBE THEDISSEMINATION OF SALMONELLAENTERICA SUBSPECIES ENTERICASEROVAR TYPHIMURIUM IN A DAIRY SHEDA. C. B. Berge, W. M. Sischo, University of California, Davis, Veteri-nary Medicine Teaching and Research Center (VMTRC), Tulare, CA

Multi-drug resistant (MDR) Salmonella enterica sub-species enterica serovar Typhimurium (S.Typhimurium) is a public and animal health hazard.Antibiotic resistance (ABR) and pulse field gelelectrophoresis (PFGE) patterns of 194 S.Typhimurium isolates collected between January2000 and April 2003 from the San Joaquin valley inCalifornia were determined. The isolates came fromdairy cattle clinical samples, bulk tank milk samples,fecal and environmental samples from farms,human clinical samples, municipal water treatmentplants, and waterways. Antibiotic resistance to 18antibiotics was determined using a disk diffusionassay. PFGE patterns were determined using asingle enzyme (XbaI). The isolate set contained 14PFGE clusters (excluding singleton clusters) and 18

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antibiotic resistance (ABR) clusters. The dominantABR-PFGE cluster pattern contained more thanhalf of the isolates and demonstrated the pheno-typic traits of S. Typhimurium DT104 with chro-mosomally mediated resistance to at least 5 antibiot-ics that has shown a world-wide clonal dissemina-tion. No other ABR-PFGE patterns were sharedbetween animal and human sources. This studytherefore confirms the clonal spread of the ABR-PFGE cluster associated with S. TyphimuriumDT104, but does not indicate that other strains ofS. Typhimurium show a clonal spread.

C114YDEI IS AN RCS-REGULATED,PERIPLASMIC PROTEIN IMPORTANT FORPERSISTENT INFECTIONK.D. Erickson and C. S. Detweiler, University of Colorado at BoulderMolecular, Cellular and Developmental Biology Boulder, Colorado

Bacteria utilize phosphorelay systems to respond toenvironmental or intracellular stimuli. Salmonellaenterica encodes a four-step phosphorelay systemthat involves two sensor kinase proteins, RcsC andRcsD, and a response regulator, RcsB. The physi-ological stimulus for Rcs phosphorelay activation isunknown however Rcs-regulated genes can beinduced in vitro by osmotic shock, low temperatureand antimicrobial peptide exposure. We investi-gated the role of the Rcs pathway using phyloge-netic analysis and experimental techniques. Phylo-genetic analysis determined that full-length RcsCand RcsD-like proteins are found only in the Entero-bacteriaceae family, specifically in species that have apathogenic or commensal relationship with thehost. Experimental data show that RcsD and RcsB,in addition to RcsC, are important for systemicinfection in mice and polymyxin B resistance in vitro.To identify Rcs-regulated genes that confer thesephenotypes, we took advantage of our observationthat RcsA, a transcription factor and binding part-ner of RcsB, is not required for polymyxin B resis-tance or survival in mice. S. Typhimurium oligo-nucleotide microarrays were used to identify 18 locithat are activated by RcsC, RcsD, and RcsB but notRcsA. Five of the 18 loci encode genes that con-

tribute to polymyxin B resistance. One of thesegenes, ydeI, was shown by quantitative real-timePCR to be regulated by the Rcs pathway indepen-dently of RcsA. In vivo infections indicate that ydeImutants are out-competed by wild-type 10- to 100-fold after oral inoculation, but are only modestlyattenuated after intraperitoneal inoculation. Thesedata indicate that ydeI is an Rcs-activated gene thatplays an important role in persistent infection ofmice, possibly by increasing bacterial resistance toantimicrobial peptides.

A115HORIZONTAL TRANSMISSION OF SALMONELLA ENTERICA SEROVARTYPHIMURIUM USING THE MURINEPERSISTENCE MODEL: IDENTIFICATIONOF SPI1 AS A TRANSMISSION FACTORT. D. Lawley and D. M. Monack,Stanford University Medical Center

We recently described a mouse model of Salmonellapersistence in which Salmonella enterica serovarTyphimurium (serovar Typhimurium) colonizes129X1/SvJ (Nramp+/+) mice for up to a year and isshed in feces for extended periods. Here we de-scribe the use of this model to study horizontaltransmission via the natural fecal-oral route.Serovar Typhimurium was rapidly transmitted fromorogastrically infected mice to cohoused naïve mice.During transmission, the naïve mice are colonizedby serovar Typhimurium at both the mucosal andsystemic sites at levels comparable to theorogastrically infected mice. In addition, miceinfected by the fecal-oral route developed a serovarTyphimurium mucosal IgA response with similarkinetics to that of orogastrically inoculated mice.As few as one orogastrically infected mouse couldtransmit serovar Typhimurium to naïve mice if fecalshedding reached levels of at least 107 cfu/g. Fur-thermore, the higher the level of bacteria shed inthe feces, the more rapidly transmission occurred toa naïve host. To test the possible role of SalmonellaPathogenicity Island 1 (SPI-1) in transmission,which is required for Peyer’s patch colonization, weinfected mice with the effector/translocator mutantsipB::Cm. The levels of the sipB::Cm mutant thatwere shed in the feces were similar to wild-type



bacteria. However, the sipB::Cm mutant bacteriawere not transmitted to naïve mice, even whencohoused for 45 days with orogastrically infectedmice that were shedding sipB::Cm mutant bacteria atlevels that allowed 100% transmission of wild-typebacteria. Thus, we have described a mouse modelthat mimics the natural fecal-oral route of transmis-sion in wild-type (Nramp1+/+, pIgR+/+) mice andprovides a basis to genetically dissect the horizontaltransmission of Salmonella. Finally, we have shownthat SPI1 is required for transmission via the fecal-oral route.

B116CONSTRUCTION OF A LIVE-ATTENUATEDSALMONELLA ENTERICA SEROVARPARATYPHI A VACCINEC. J. Vindurampulle, E. M. Barry and M. M. Levine,Center for Vaccine Development, University of Maryland School of Medicine,Baltimore, MD , *Current Address: Department of Microbiology andImmunology, University of Melbourne

Infectious Salmonellosis remains a significant threaton human morbidity and mortality, particularly indeveloping countries. Incidences of enteric fevercaused by Salmonella enterica serovar Paratyphi A areon the rise due to the emergence and spread ofantibiotic resistant variants. We have utilizedlambda Red-mediated mutagenesis to delete variouschromosomal loci from S. Paratyphi A 9150 in aneffort to create a live-attenuated vaccine strain.Resulting mutants were tested in the murine mucinmodel of Salmonella virulence. We first introduced adeletion genes coding for proteins involved inguanine biosynthesis (guaBA) based on CVD 1208,the live-attenuated Shigella flexneri 2a vaccine strainwhich has successfully been trialled in humanvolunteers. This lesion was complemented in transboth in vitro and in vivo with a low copy numberplasmid encoding guaBA. Secondary deletions ineither clpP (protease) or clpX (ATPase), which codefor a protease complex, were demonstrated tofurther attenuate Salmonella virulence. Conversely,deletions in clpB (chaperone) and degQ (periplasmicprotease) did not. Mutants containing lesions inguaBA and either clpP or clpX were complemented intrans using bicistronic plasmid constructs. Addi-

tional preclinical characterization studies are ongo-ing.

C117TRANSFER OF ANTIBIOTIC RESISTANCEFROM S. TYPHIMURIUM DT104 TO E. COLIK12 AND S. AGONA IN FOODC. Walsh, G. Duffy, R O’Mahony, E.S. O’Regan, D. A. McDowell and S.Fanning; Centre for Food Safety, University College Dublin, Ireland

Multi-antibiotic resistant Salmonella TyphimuriumDT104 has emerged as a global public health andfood safety problem over the last two decades. Itstypical antibiotic resistance profile (ACSSuT) hasbeen reported in other bacteria, suggesting themovement of all or some of this set of resistantgenes among other serovars and species. This studyinvestigated the transfer of ampicillin resistancefrom a donor S. Typhimurium DT104 strain to arecipient strain (E. coli K12 or S. Agona). Antibi-otic resistance transfer experiments were conductedin broth, milk and mince-meat (ground beef) atincubation temperatures of 4, 15, 25 and 37oC for18 and 36 h. Similar frequencies of antibioticresistance transfer were observed into both recipi-ent strains in all transfer media (broth, milk andmince-meat) at 25 and 37oC (10-4 - 10-7 log10 CFU ml-

1or g-1, transconjugants per recipient) over an 18 hperiod. At 15oC, antibiotic resistance transfer wasobserved in mince-meat for both recipient strains(10-5 -10-6, log10 CFU g-1, transconjugants/ perrecipients cells), but not in broth or milk. At 4oC,there was no antibiotic resistance transfer intoeither of the recipient strains, in any of the matricesexamined. Further analysis by PCR (polymerasechain reaction) showed the presence of the b-lactamase genes (conferring ampicillin resistance)carb and tem in the E. coli K12 transconjugant, butnot in the S. Agona transconjugant. Ampicillinresistance was shown to be stable in the E. coli K12transconjugant but was unstable in the S. Agonatransconjugant and this strain rapidly reverted tobeing susceptible to ampicillin. This instability mayhave been due to the presence of the IncP plasmidin the recipient S. Agona isolate, resulting in plas-mid “incompatibility”. This study demonstrates the

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transfer of the gene encoding for ampicilin resis-tance between bacteria serovars and species in foodmatrices at mid-range temperatures.

A118LONG-TERM SURVIVAL AND PERSISTENCEOF SALMONELLAA. P. White, D. L. Gibson, S. L. Stocki, W. W. Kay and M. G. Surette;University of Calgary, Calgary, Alberta, Canada

Salmonella spp. are common infectious agents inmost areas of the world and are proposed to havecyclic lifestyles, consisting of passage through ahost, into the environment and back into a suscep-tible host. The persistence of Salmonella in non-host environments is not as well studied as patho-genesis but may be of similar importance in learn-ing how to combat and prevent infection. It is wellknown that cells of most Salmonella spp. have thepropensity to aggregate, which can be observed asformation of rough and dry (rdar) colonies withelaborate surface patterns when grown for severaldays on standard laboratory agar media. Cells inrdar colonies are encased in an extracellular matrixcomprised of several polymeric substances: thinaggregative fimbriae (Tafi; curli), cellulose and atleast two novel extracellular polysaccharides (EPS).We analyzed the benefits of extracellular matrixproduction in our wt strain (S. enterica serovarTyphimurium ATCC14028) for resistance to desic-cation and treatment with four common sanitizingagents compared to mutants that are deficient inTafi, cellulose and/or EPS production. Extracellu-lar matrix polymers positively contributed to Salmo-nella survival in all tests, providing at least four logsof protection against each sanitizing agent. Regu-lation of extracellular matrix production primarilyfeeds through AgfD (CsgD), the transcriptionalactivator for Tafi (curli) biosynthesis. Using lu-ciferase-transcriptional fusions, we have shown thatagfD expression is conserved throughout all Salmo-nella subgroups, except subgroup IIIa (arizonae).Our results suggest that extracellular matrix produc-tion could play a key role in the survival and persis-tence of Salmonella spp. in non-host environments.

B119

SIGMA E-CONTROLLED SRNAS MAINTAINENVELOPE HOMEOSTASIS ANDACCELERATE SELECTIVE OMP MRNADECAY IN RESPONSE TO MEMBRANESTRESS

V. Pfeiffer, K. Papenfort, S. Lucchini*, J. C. Hinton*, J. Vogel, MaxPlanck Institute for Infection Biology, RNA Biology, Berlin, Germany;*Molecular Microbiology Group, Institute of Food Research, NorwichResearch Park, Norwich, UK

How bacteria selectively degrade no longer wantedmRNAs in the course of a rapid stress response islittle understood. Few ribonucleases exhibit ex-tended sequence-specificity, hence these enzymeswill require additional specificity factors if only adefined subset of mRNAs is to be degraded. Smallnoncoding RNAs (sRNAs) have recently emergedas a new class of axillary factors that help thegeneral RNA decay machinery to recognize specificmRNAs for accelerated turn-over. We have hereidentified two sRNAs, RybB and MicA, as novelmembers of the óE-mediated envelope stress re-sponse of Salmonella typhimurium. The highly con-served 90 nt RybB RNA of hitherto unknownfunction, is shown to be essential for rapid removalof nearly all mRNAs that encode for major outermembrane proteins (OMPs) when cells face enve-lope stress. The rapid removal of bulk omp mRNAsis a hallmark of the óE-response, and we show thatin the absence of RybB, this specific decay is de-layed and incomplete. Collectively, our data suggeststhat the two sRNAs serve dual functions. In nor-mally growing cells, they form an autoregulatoryloop with the óE regulon that limits OMP biogen-esis to prevent the accumulation of misfoldedintermediates. Upon sudden environmental chal-lenges that require adjustments to the bacterialenvelope, the two sRNAs ensure fast remodellingof the outer membrane.



Abd E L Ghany, M. S6:5Abedi, D. A13Adams, G. S1:7Adams, L. G. A76Adkins, J. N. S3:7Ahmad, N. A67Ahmer, B. M. S1:2, A106, S1:7Akcelik, M. A103Alegado, R. A. S2:7Alston, M. S1:6Altier, C. C72, S1:7Alvarez, S. C33Ammendola, S. B17, B74Anderson, B. A25Anderson, R. C. A31, A82

A94, B32, C96Andrews-Polymenis, H. S1:2Arrach, N. S1:2Asghari, G. A13Axel, C. C42Baazov, T. C39Bailey, M. C63Barrow, P. S2:6Barry, E.M. B116Barton, M. D. C15Battista, P. B44Battistoni, A. B17, B74Baumler, A. J. A103, B104, A76,

B107, B35, B77S2:1

Beijersbergen, R. A1Beland, M. B95Ben-Barak Zelas, Z. C60Ben-Shalom, L. B53Benoit, D. C42Berberov, E. A100Berge, A.C. B. B113Betancor, L. B59, B83Betts, R. P. A61Birtles, R. C12Blattner, F. R. A25Blondel, C. C33Boerlin, P. A70Bohez, L. S6:6Bottreau, E. C84Bowen, A. J. A79Boyle, E. C. S4:6Brady, J. C57Brandtzaeg, P. A55Bravo, D. C33Brown, K. A. C24Brown, N. S3:4Brown, N. F. S4:6Brown, R. C108Brumell, J. H. C21, C99Bruni, G. B44Bryant, C. A10Bumgarner, T. E. A37Burkhardt, D. T. C69Burland, V. A25Bustamante, V. H. C90Cabot, E. A25Callaway, T. R. A31, B32Camarda, A. B44

Cannu, N. A. A4Capozzo, A. C105Cardona Castro, N. M. A4Carroll, R. K. B92Carter, J. C33Casey, T. A. A82Cavet, J. S. C66,S5:7Cerquetti, M. C. B5, C3Chabalgoity, J. A. B59, B83Chaitkin, E. C111Chanana, V. B41Charleston, B. S2:5Chen, H. C30Chen, M. B26Chen, S. C48Chen, T. A16Chen, T. C. B26Chen, Y. C48Cheng-Hsun, C. C42Chessa, D. A76, B104, B107

B35, B77Chi-Shih, C. C42Chinh, N. Tran. B38Chiou, C. C48Chiu, C. C30, C48Chiu, T. H. B26Christianson, S. B2Chu, C. C48Circella, E. B44Clifton, S. A34, S1:2Cloeckaert, A. B2, B62Cobb, B. A. B35Coburn, B. S3:4Coleman, J. R. S3:7Contreras, I. C33Cookson, B. T. S5:4Coombes, B. A100, S3:4Copass, M. K. S6:4Crawford, R. W. S5:5Curtiss, R. K.A., S1:7D’Orazio, M. B74Daigle, F. A91, A97

B95, S3:6Danchin, A. C81Danino, V. E. C63, B80De Coster, D. C87De Keersmaecker, S. C.

A85, A88, C87De Moor, B. A85Delehaunty, K. A34Demartin, M. B62Deng, W. S3:4Desin, T. A100Detweiler, C.S. C114Dieye, Y. A106Djordjevic, G. C108Dobrindt, U. B80Dohnkova, A. S3:7Dolecek, C. S7:4Donini, O. A109Dordari, S. C6Dorman, C. J. S1:5Doublet, B. B2, B62

Dougan, G. B83, C24, S6:5 S7:4

Doyle, J. C111Doyle, M. S1:5Dozois, C. M. A97Drecktrah, D. C75Drew, E. C93Ducatelle, R. A28, S6:6Duffy, G. C117Dullaghan, E. M. A109Dung, N. S7:4Dunstan, S. J. B38, S7:4Dyszel, J. L. S1:7Edrington, T. S. A31, A94

B32, C96Eeckhaut, V. A28, S6:6Egan, D. A1Egbejule, E. B8Ellermeier, C. D. S1:7Ellermeier, J. R. C18Emery, D. A. C69Emody, L. B80Engelen, K. A88, C87Erickson, K.D. C114Fadda, A. A85Falegan, A. A7Falkow, S. S7:4Fanning, S. C117Fang, F. C. A37, A43, S5:3Fardini, Y. B110, C84Farrar, J. J. B38, S7:4Faucher, S. P. A91, A97, S3:6Fazly Bazzaz, B. B29Feng, X. B89, B92Field, T. R. S4:4Fields, P. I. A34, C111Figueiredo, J. F. A76Fink, S. L.. S5:4Finlay, B. A100, C93,S3:4

A109, C102, S4:6Florea, L. S1:2Fookes, M. B83, S1:5Forbes, J. S3:7Forest, C. A97Fortune, D. R. C72Foster, G. L. A10, S2:3Gal-Mor, O. C102Galan, J. E. S4:3Galen, J. C105Galic, M. S7:3Galyov, E. E. S4:4Gantois, I. A28García Cattaneo, A. C3Garcia-del Portillo, F. S3:3Geddes, K. S4:5Geluk, A. A1Genovese, K. J. A31, A58, B14

B23, C108Ghokasian, K. A13Giacomodonato, M. N. B5, C3Gibson, D. L. S5:5, A118Gilberthorpe, N. J. A22Glasner, J. D. A25Godfrey, J. A34

Golding, G. R. B2Golubeva, Y. A. C45Gordon, M. A. S7:5Gordon, S. B. S7:5Graham, M. R. B2Grant, A. A10, S1:4, S2:3Grassl, G. A. C93Greene, J. M. A25Groisman, E. S4:2Gros, P. S3:7Guarna, M. A109Gunn, J. S. S5:5Hacker, J. B80Haesebrouck, F. A28, S6:6Hampton, T. A25Hancock, R. E. A109Hardt, W. D. S2:2, A64, B65

B86Hart, C. A. S6:2Hartog, E. B50, B53Hautefort, I. A28, A67He, H. A58, B14

B23, C108Heath, W. C57Heffron, F. S3:7, S4:5Hensel, M. S3:2Herrera-Leon, S. C111Heuzenroeder, M. W. C15Hickle, L. C108Hien, T. S7:4, B38Hinton, J. C.D. C63, S1:6, A28

A52, A61, A67B56, B80, C51

S1:3, S3:5, B119Hoare, A. C33Holbert, S. B110Holden, D. W. A40Homauni, M. B29Horrocks, S. M. A82Houle, S. A97House, D. S7:4Hradecka, H. B20Hsiu-Ling, C. C42Hu, Q. S3:4Hu, Y. Chi. B26Huang, J. C81Hue, N. T. B38Hume, M. E. A31Humphrey, T. C63Ireland, R. B101Ivens, A. B83, S1:5Jalali, M. A13Janssen, L. A1Jepson, M. A. S4:7Jiang, Y. B14Johansen, F. A55Johnston, R.N. A112Jones, M. S2:6Kaeppeli, R. A64Kaparakis, M. C57Karine, P. C42Kariuki, S. S6:2Kay, W. W. S5:5, A118Kenney, L. J. B89, B92

INDEX

INDEX

102 ASM Conferences

Kiiru, J. S6:2Kim, B. S5:6Kingsley, R. A. C24, S6:5Knodler, L.A. B98, C75, C90Koch, B. A100Kochetkova, I. S6:7Kocsis, B. B80Kogut, M. H. C108, A58

B14, B23Konjufca, V. S1:7Köster, W. A100Kremer, M. A64Krishnakumar, R. S5:6Krishnan, L. A100Kuijl, C. A1Kuznetsova, N. A. A73Lai, S. A91Lam, S. A100Lanh, M. N. B38Lawhon, S. D. A76, S1:7Lawley, T.D. A115Layton, A. N. S4:4Le Moual, H. A91, B71Le Sage, V. B71Leclerc, S. A91Leong, M. C81Levine, M.M. B116Lew, A. C57Lewis, C. J. B35Li, Y. S3:4Liao, X. B92Libby, S. J. A37, A43Lin, D. A49Lin, J. Sheng. C9Lin-Xian, K. C42Liss, P. A25Liu, D. C81Liu, G.-R. A112Liu, S. C30Liu, S.-L. A112Liu, W.Q. A112Lloyd, S. C105Logsdon, J. M. C111Long, F. S1:2Lott, P. C12Lowden, M. A100Lucchini, S. A52, A67, B56

C51, S1:6, S3:5B119

Maguire, M. E. B68Main-Hester, K. L. A37, A43Majumdar, S. B41Malcova, M. B20Manes, N. P. S3:7Mantena, R. R. C27Marchal, K. A85, A88, C87Marsman, M. A1Martell, R. A25Martin, L. A70Martinez, A. B59, B83Martinez, C. H. A31Maskell, D. A10, B83

S1:4, S2:3Mastroeni, P. A10, S1:4, S2:3

Matiasovicova, J. B20Matthews, K. R. B50, B53McClelland, M.F. A88, S1:2, S3:6

A34McDermott, P. B56McDowell, D.A. C117McNagny, K. M. C93McQuiston, J. R. C111Meinel, K. M. S7:6Menager, N. S2:3Menashe, O. C39Mika, F. S3:5Miller, S. S3:1Molyneux, M. E. S7:5Monack, D. S2:4, S7:4, C78Monsieurs, P. A85, C87Morgan, E. A79, S4:4

Mota, L. J. A40Mottaz, H. M. S3:7Muckle, A. A70Müller, A. J. A64, B86Mulvey, M. R. B2Musaya, L. S7:5Mwituria, J. S6:2Nagy, G. B80Nataro, J. C105Navarre, W. W. A37Neefjes, J. J. A1Neeno-Eckwall, E. A25Nisbet, D. J. A31, A82, A94

B32, C96Norbeck, A. D. S3:7Noto Llana, M. B5Nuccio, S. B107, B77Ochoa-Repáraz, J. S6:7Ogunlowo, O. B8Olawuyi, O. I. A7Olekhnovich, I. N. B11Oliver, C. E. A82Olson, A. B. B2O’Mahony, R. C117O’Regan, E.S. C117Osman, D. C66, S5:7Ottenhoff, T. H.M. A1Overkleeft, H. A1Pacello, F. B74Pang, J. C. C9Panthel, K. S7:6Papenfort, K. B86, S3:5, B119Papp-Wallace, K. M. B68Park, Y. A46Parker, M. L. A67Pascual, D. W. S6:7Pasetti, M. C105Pasmans, F. A28, S6:6Pasquali, P. B17Paulin, S. M. S2:5Peck, M. W. A61Pennelli, D. B44Perkins, T. S7:4Perna, N. T. A25Perrett, C. A. S4:7Pfeiffer, V. B86, S3:5, B119

Phuong, L. T. B38Pickard, D. B83Pin, C. A61Plunkett, G. A25Poirier, K. A97Ponder, M. A. A34Poole, R. K. A22Poole, T. L. A94, C96Poppe, C. A70Porwollik, S. A88, S1:2, S3:6Pot, D. A25Potter, A. A100, S3:4Praud, K. B62Price, J. C57, S7:3Puente, J. L. C90Pullinger, G. D. S2:5Purvine, S. O. S3:7Qiu, Y. A25Raffatellu, M. A76, B104, B35Rakov, A. V. A73, B47Ramirez, K. C105Ramlachan, N. A94, C96Ramsden, A. E. A40Read, R. C. A22, S7:5Restif, O. S1:4Revathi, G. S6:2Rhen, M. A67Richardson, T. H. C108Rishi, P. B41Robbins, J. B. B35Rodland, K. D. S3:7Rolfe, M. A52Rolfe, M. D. A61Ross, T. T. A31Rotilio, G. B74Rowley, G. C51Rubino, S. A4, C3Rusch, M. A25Rüssmann, H. B35, S7:6Rychlik, I. B20Sad, S. A100Sait, L. C63Sajid, S. U. C36Sanchez Jimenez, M. M. A4Sanderson, K.E. A112Sandstrom, J. D. C69Santana, F. J. C90Santiviago, C. S1:2Santos, R. L. A76Sarnacki, S. H. B5, C3Savage, N. D.L. A1Schaull, L. A25Schelotto, F. B83Schepmoes, A. A. S3:7Scher, K. B50Schlumberger, M. B65, A64Schwerk, P. A52Scott, M. G. A109Sfeir, R. A25Shachar, D. B53Shaker, M. A25Shepherd, C. C12Sheppard, M. S1:4Shetty, P. A25

Shi, L. S3:7Shubin, F. N. A73, B47Simpfendorfer, K. A55, S7:3Sischo, W.M. B113Sittka, A. B86Slauch, J. M. A49, C18, C45

S1:7, S5:6Slinden, L. M. C69Smallwood, H. S. S3:7Smith, D. J. A82Smith, J. N. S1:7Smith, R. D. S3:7Solnik-Isaac, H. C54Sonck, K. C87Squier, T. C. S3:7Starnbach, M. N. S6:4Steele-Mortimer, O. B101, B98

C75, C90, S5:2Stevens, M. P. A79, S2:5Stocki, S.L. A118Straub, D. E. C69Stringer, M. F. A61Strugnell, R. A. A55, C57

S7:3, C27Surette, M.G. C78, A118Suyemoto, M. C72Swaggerty, C. L. A58Szeto, J. C21Sztein, M. S7:1,C105Szu, S. C. B35Tabak, M. B50Taghipor, M. A13Tan, M. S2:7Tan, S. C15Tang, W. C30Tauxe, R. V. C111Taylor, C. M. C66, S5:7Taylor, J. D. C24Tchikindas, M. L. B50Tedin, K. A52Thijs, G. A85Thijs, I. M. A88, C87Thomas, G. A. A43Thompson, A. A28, A52, A61

A67, B56, S1:6Thompson, L. J. S7:4Trotereau, J. B110, C84Trunkle, T. S6:7Tsai, C. C. C9Tsen, H. C9Tsen, H. Y. B26Tuin, E. A1Tukel, C. A103, B104Turnbull, A.L. C78Uppington, H. S2:3Uren, T. A55Ureta, A. R. B59Uzzau, S. A4, C3, S3:7Valdez, Y. C93Valvano, M. A. B5, C33van Boxel, N. C87van den Eeden, S. J. A1van der Velden, A. W. S6:4Van Immerseel, F. A28, S6:6

INDEX INDEX


van Marcel, G. A1Vanderleyden, J. A85Vanderleyden, J. A88, C87Velge, P. B110, C84Vignoli, R. B59Villarreal-Ramos, B. S2:5Vindurampulle, C.J. B116Vinh, H. B38Virlogeux Payant, I. C84, B110Vogel, J. B86, S3:5, B119Wain, J. S1:5Wallis, T. S. A79, S2:5Walsh, C. C117Walthers, D. B89Wang, H. A109, S3:7Wang, J. C81Wang, L. A112Wang, N. C48Wang, Y. A16Wasylnka, J. A. C99Watson, P. R. S2:5Watt, R. M. C81Weill, F. B62Weinberger, M. C54Weir, E. A70Wetter, M. A64White, A.P. A118Wigley, P. C12, S2:6Wijburg, O. A55, C57, S7:3Wijburg, O. L.C. C27Wilson, R. A34Wilson, R. Paul. A76, B104Winnen, B. B65Winter, M. B77Winter, S. E. A76, B35Wisner, A. A100Wonderling, L. D. C69Wong, M. A25Yaron, S. B50, B53, C39

C54Yeh, C. C48Yeh, K. A16Ygberg-Eriksson, S. A67Yim, L. B59, B83Yoon, W. A46Young, G. M. S1:7Zaldivar, M. C33Zhan, Y. C57Zhao, G.X. A112Zhao, H. C87

INDEX INDEX

104 ASM Conferences

NOTES NOTES


NOTES

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