Temporal Transcriptional Analysis of the Early in vivo Initial Interactions of both Brucella & Bovine Host Carlos A. Rossetti, Brian Kamery, Sara Lawhon,

Temporal Transcriptional Analysis of the Early in vivo Initial Interactions of both

Brucella & Bovine Host

Carlos A. Rossetti, Brian Kamery, Sara Lawhon, Jairo Nunes, Tamara Gull, Cristi Galindo, Sangeeta Khare, Robin Everts, Mitch Magee, Harris Lewin, Stephen

Johnston, Harold Garner, Ken Drake & L. Garry Adams

Department of Veterinary Pathobiology

College of Veterinary MedicineTexas A&M University

Brucellosis Research Conference1 XII 07 Chicago

Pathogenesis of initial Pathogenesis of initial infectioninfection

Natural infections occur Natural infections occur primarily through adhesion primarily through adhesion and penetration of mucous and penetration of mucous membranes membranes - B. abortus & melitensis- B. abortus & melitensis: : alimentary tractalimentary tract-B. canis, B. ovis & B. suisB. canis, B. ovis & B. suis: : genital tractgenital tract

Macrophages, dendritic Macrophages, dendritic cells, & neutrophils cells, & neutrophils phagocytose free phagocytose free BrucellaBrucella in in the submucosal interstitiumthe submucosal interstitium

Metastasis to Metastasis to regional (primary) regional (primary) lymph nodeslymph nodes

Persistence of Persistence of infection in infection in reticuloendotelial reticuloendotelial systemsystem

The The goal goal of this study was to analyze of this study was to analyze the transcriptome of host the transcriptome of host andand BrucellaBrucella during their early during their early in vivoin vivo interactions in an effort to understand interactions in an effort to understand how this interaction modulates the how this interaction modulates the outcome of the infectious process.outcome of the infectious process.

ObjectiveObjective

METHODOLOGYMETHODOLOGY• Four - 12 hour ligated ileal Four - 12 hour ligated ileal

loop non-survival surgeries loop non-survival surgeries in 3-week old male in 3-week old male brucellosis-free beef calves brucellosis-free beef calves under BSL3 conditionunder BSL3 condition

• Abdominal wall incised under Abdominal wall incised under general anesthesiageneral anesthesia

• Twenty one – 6 cm ligated Twenty one – 6 cm ligated ileal loopsileal loops

- 7 inoculated with 3 ml of - 7 inoculated with 3 ml of LiveLive 1x10 1x1099 Wild Type (WT) Wild Type (WT) Bmel Bmel 16M 16M

- 7 inoculated with 3 ml of - 7 inoculated with 3 ml of Heat Inactivated Heat Inactivated (H-I)(H-I)1x101x1099 BmelBmel

- 7 injected with 3 ml of - 7 injected with 3 ml of media (control loops)media (control loops)

METHODOLOGYMETHODOLOGY

• Three loops (1 WT, 1 Three loops (1 WT, 1 H-I and 1 control) H-I and 1 control) excised at 0.25, 0.5, excised at 0.25, 0.5, 1, 2, 4, 8 & 12 h PI1, 2, 4, 8 & 12 h PI

• Sampled for tissue Sampled for tissue associated bacteria, associated bacteria, histopathology, histopathology, TEM, SEM & RNATEM, SEM & RNA

• Calves euthanatized Calves euthanatized at 12 h PIat 12 h PI

Peyer's patches colonization

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0.25 0.5 1 2 4 8 12

Time post-inoculation (h)

CFU/g of tissue (log

10)

BACTERIOLOGICAL RESULTSBACTERIOLOGICAL RESULTS

BACTERIOLOGICAL RESULTSBACTERIOLOGICAL RESULTS

• B. melitensisB. melitensis was recovered from was recovered from– Systemic blood within 30 min PISystemic blood within 30 min PI– Mesenteric LN & liver at 12 h PIMesenteric LN & liver at 12 h PI– Control & HI loops at 8 & 12 h PIControl & HI loops at 8 & 12 h PI

ConclusionsConclusions: : RapidRapid penetration of penetration of B. B. melitensismelitensis through Peyer’s patch & through Peyer’s patch &

metastasis via lymphatic vessels metastasis via lymphatic vessels followed by systemic bacteremia & followed by systemic bacteremia &

organ colonizationorgan colonization

Intracellular Intracellular B. melitensisB. melitensis gene gene expression profileexpression profile

• 4 biological replicas of 4 biological replicas of B. melitensisB. melitensis RNA were enriched & RNA were enriched & amplified from total RNA extracted from PP at 15 min to 4 amplified from total RNA extracted from PP at 15 min to 4 h PI & co-hybridized against h PI & co-hybridized against B. melitensisB. melitensis gDNA on 3.2 K gDNA on 3.2 K B. melitensisB. melitensis oligo-arrays oligo-arrays

• Original total RNA from WT Original total RNA from WT B. melitensisB. melitensis-infected PP -infected PP (non enriched, non amplified) was also co-hybridized (non enriched, non amplified) was also co-hybridized against against B. melitensisB. melitensis gDNA to gDNA to B. melitensisB. melitensis oligo-arrays - oligo-arrays - Oligospots with signal were considered Oligospots with signal were considered non-specificnon-specific & & eliminatedeliminated from all analyses to reduce false positive gene from all analyses to reduce false positive gene detectiondetection

• The intracellular The intracellular in vivo B. melitensisin vivo B. melitensis gene expression gene expression was compared to the gene expression in the inoculum was compared to the gene expression in the inoculum (cultures of (cultures of B. melitensisB. melitensis at late-log growth phase) at late-log growth phase)

• 4 biological replicas of 4 biological replicas of B. melitensisB. melitensis RNA were enriched & RNA were enriched & amplified from total RNA extracted from PP at 15 min to 4 amplified from total RNA extracted from PP at 15 min to 4 h PI & co-hybridized against h PI & co-hybridized against B. melitensisB. melitensis gDNA on 3.2 K gDNA on 3.2 K B. melitensisB. melitensis oligo-arrays oligo-arrays

• Original total RNA from WT Original total RNA from WT B. melitensisB. melitensis-infected PP -infected PP (non enriched, non amplified) was also co-hybridized (non enriched, non amplified) was also co-hybridized against against B. melitensisB. melitensis gDNA to gDNA to B. melitensisB. melitensis oligo-arrays - oligo-arrays - Oligospots with signal were considered Oligospots with signal were considered non-specificnon-specific & & eliminatedeliminated from all analyses to reduce false positive gene from all analyses to reduce false positive gene detectiondetection

• The intracellular The intracellular in vivo B. melitensisin vivo B. melitensis gene expression gene expression was compared to the gene expression in the inoculum was compared to the gene expression in the inoculum (cultures of (cultures of B. melitensisB. melitensis at late-log growth phase) at late-log growth phase)

In VivoIn Vivo Intracellular Intracellular B. melitensisB. melitensis expression profileexpression profile

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A B C D E F G H I J K L M N O P Q R

COGs functional categories

# of genes

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Down reg

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A B C D E F G H I J K L M N O P Q R

COGs functional categories

# of genes

Up reg

Down reg618618 genes differentially expressed genes differentially expressed

- 365 - 365 upup-regulated-regulated

- 253 - 253 downdown-regulated-regulated

Protein Protein biosynthesibiosynthesi

ss

Amino acid Amino acid transport transport

and and metabolismmetabolism

Energy Energy production and production and

conversionconversion

Cell wall/ Cell wall/ membrane membrane biogenesisbiogenesis

UnknowUnknown n function function

In Vivo In Vivo intracellular profile of intracellular profile of B. melitensisB. melitensis expression expression

• B. melitensis B. melitensis had a common had a common in vivoin vivo transcriptional profile in the first 4 h PItranscriptional profile in the first 4 h PI

• 618 genes (19.3 % of 618 genes (19.3 % of B. melitensis B. melitensis genome) were identified as differentially genome) were identified as differentially expressed in at least 4 of 5 time points expressed in at least 4 of 5 time points evaluated evaluated

• Most of the functional categories were Most of the functional categories were over expressed, except transcription, over expressed, except transcription, defense, motility, intracellular trafficking defense, motility, intracellular trafficking & secretion& secretion

• 37.5% of the genes differentially 37.5% of the genes differentially expressed lacked functional annotationexpressed lacked functional annotation

• B. melitensis B. melitensis had a common had a common in vivoin vivo transcriptional profile in the first 4 h PItranscriptional profile in the first 4 h PI

• 618 genes (19.3 % of 618 genes (19.3 % of B. melitensis B. melitensis genome) were identified as differentially genome) were identified as differentially expressed in at least 4 of 5 time points expressed in at least 4 of 5 time points evaluated evaluated

• Most of the functional categories were Most of the functional categories were over expressed, except transcription, over expressed, except transcription, defense, motility, intracellular trafficking defense, motility, intracellular trafficking & secretion& secretion

• 37.5% of the genes differentially 37.5% of the genes differentially expressed lacked functional annotationexpressed lacked functional annotation

In vivoIn vivo Bovine Peyer’s patch Bovine Peyer’s patch gene expression profilegene expression profile

• Total RNA was extracted from 4 Total RNA was extracted from 4 calves at 15 min to 4 h post-calves at 15 min to 4 h post-infection from WT, H-I and control infection from WT, H-I and control loops (n=60)loops (n=60)

• RNA was co-hybridized against RNA was co-hybridized against bovine reference RNA & 13K bovine reference RNA & 13K custom bovine arrays (UIUC)custom bovine arrays (UIUC)

• Total RNA was extracted from 4 Total RNA was extracted from 4 calves at 15 min to 4 h post-calves at 15 min to 4 h post-infection from WT, H-I and control infection from WT, H-I and control loops (n=60)loops (n=60)

• RNA was co-hybridized against RNA was co-hybridized against bovine reference RNA & 13K bovine reference RNA & 13K custom bovine arrays (UIUC)custom bovine arrays (UIUC)

B. melitensis-B. melitensis-infected Bovine infected Bovine Peyer’s patch transcriptional Peyer’s patch transcriptional

profileprofile

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GO biological processes

# of genes

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# of genes

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A B C D E F G H I J K L M N O P Q R S T U V W


# of genes

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# of genes

Up-reg

Down-reg

15 m - 1 h 15 m - 1 h PIPI

1 h - 4 h 1 h - 4 h PIPI

224224 genes differentially expressed genes differentially expressed

- 196 196 UpUp-regulated-regulated

- 28 28 DowDown-regulatedn-regulated

11631163 genes differentially expressed genes differentially expressed


- 704 704 DownDown-regulated-regulated

B. melitensisB. melitensis infected bovine infected bovine Peyer’s patch transcriptional Peyer’s patch transcriptional

profileprofile

• Two different expression profiles were observed Two different expression profiles were observed in the bovine Peyer’s patch during the first 4 h in the bovine Peyer’s patch during the first 4 h PIPI– Up-regulation of the transcriptome in the first hour PI Up-regulation of the transcriptome in the first hour PI

(86%)(86%)– Down-regulation of the transcriptome between the 1 Down-regulation of the transcriptome between the 1

and the 4 h PI (63%)and the 4 h PI (63%)

• Interesting findings Interesting findings – Anti-chemo attractant PMN and monocyte responseAnti-chemo attractant PMN and monocyte response– Pro-apoptotic responsePro-apoptotic response– Pro-abortive transcriptional response Pro-abortive transcriptional response – Arresting of the cell cycle and inhibition of cell Arresting of the cell cycle and inhibition of cell

proliferation and differentiationproliferation and differentiation

• Two different expression profiles were observed Two different expression profiles were observed in the bovine Peyer’s patch during the first 4 h in the bovine Peyer’s patch during the first 4 h PIPI– Up-regulation of the transcriptome in the first hour PI Up-regulation of the transcriptome in the first hour PI

(86%)(86%)– Down-regulation of the transcriptome between the 1 Down-regulation of the transcriptome between the 1

and the 4 h PI (63%)and the 4 h PI (63%)

• Interesting findings Interesting findings – Anti-chemo attractant PMN and monocyte responseAnti-chemo attractant PMN and monocyte response– Pro-apoptotic responsePro-apoptotic response– Pro-abortive transcriptional response Pro-abortive transcriptional response – Arresting of the cell cycle and inhibition of cell Arresting of the cell cycle and inhibition of cell

proliferation and differentiationproliferation and differentiation

H-I H-I B. melitensisB. melitensis-inoculated bovine -inoculated bovine Peyer’s patch expression profilePeyer’s patch expression profile

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# of genes

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140140 genes differentially genes differentially expressedexpressed


- 62 62 DownDown-regulated-regulated

Inflammatory and Inflammatory and immune responseimmune response

UnknowUnknown n functiofunctionn

Mathematical modeling predictive analysis framework for mechanistic

discovery• Data fusionData fusion

– Prior biological knowledge (qualitative data)Prior biological knowledge (qualitative data)– MetadataMetadata– Data extracted from the actual experiment Data extracted from the actual experiment

(quantitative data)(quantitative data)

Multi-conditional Multi-conditional analysisanalysis

Biosystem Biosystem modeling modeling

and and discoverydiscovery

Mathematical modeling predictive analysis framework for mechanistic

discovery• Data fusionData fusion

– Prior biological knowledge (qualitative data)Prior biological knowledge (qualitative data)– MetadataMetadata– Data extracted from the actual experiment (quantitative data)Data extracted from the actual experiment (quantitative data)

• Pathways & GO comparative analysis: Identify significantly Pathways & GO comparative analysis: Identify significantly expressed genes associated with known metabolic and regulatory expressed genes associated with known metabolic and regulatory pathways & map significant changed genes to GO categoriespathways & map significant changed genes to GO categories

• Data fusionData fusion– Prior biological knowledge (qualitative data)Prior biological knowledge (qualitative data)– MetadataMetadata– Data extracted from the actual experiment (quantitative data)Data extracted from the actual experiment (quantitative data)

• Pathways & GO comparative analysis: Identify significantly Pathways & GO comparative analysis: Identify significantly expressed genes associated with known metabolic and regulatory expressed genes associated with known metabolic and regulatory pathways & map significant changed genes to GO categoriespathways & map significant changed genes to GO categories

Multi-conditional Multi-conditional analysisanalysis

Biosystem Biosystem modeling modeling

and and discoverydiscovery

B. melitensisB. melitensis bio-signature mechanistic bio-signature mechanistic candidate genes candidate genes in vivoin vivo

Dynamic Bayesian modeling: Dynamic Bayesian modeling: - 11 highly activated GO - 11 highly activated GO biological groups containing biological groups containing 78 mechanistic candidate 78 mechanistic candidate genes in the first 4 h PIgenes in the first 4 h PI- 17 top pathways analyzed - 17 top pathways analyzed containing 119 mechanistic containing 119 mechanistic candidate genescandidate genes


B. melitensisB. melitensis bio-signature mechanistic bio-signature mechanistic candidate genes candidate genes in vivoin vivo



Top Scoring Top Scoring GO Biological GO Biological

GroupsGroups

Top Scoring Top Scoring PathwaysPathways

1. Amino acid 1. Amino acid biosynthesisbiosynthesis

2.2. Adaptation to Adaptation to atypical conditionsatypical conditions

3. 3. Cell envelopeCell envelope 4. Regulation of 4. Regulation of

transcriptiontranscription 5. 5. TranscriptionTranscription6. Transport: Cations 6. Transport: Cations

and iron carrying and iron carrying compoundscompounds

1. Oxidative 1. Oxidative phosphorylationphosphorylation

2. Protein export2. Protein export3. ABC 3. ABC

transporterstransporters 4. Pyruvate 4. Pyruvate

metabolismmetabolism5. TCA cycle5. TCA cycle 6. Two component 6. Two component

systemsystem

Bovine bio-signature mechanistic Bovine bio-signature mechanistic candidate genes candidate genes in vivoin vivo

Dynamic Bayesian modeling: Dynamic Bayesian modeling: - 47 highly activated GO - 47 highly activated GO biological groups containing 52 biological groups containing 52 mechanistic candidate genes in mechanistic candidate genes in the first 4 h PIthe first 4 h PI - 16 top pathways analyzed - 16 top pathways analyzed containing 37 mechanistic containing 37 mechanistic candidate genescandidate genes


Bovine bio-signature mechanistic Bovine bio-signature mechanistic candidate genes candidate genes in vivoin vivo



Top Scoring GO Top Scoring GO Biological Biological

GroupsGroups

Top Scoring Top Scoring PathwaysPathways

1. Positive regulation 1. Positive regulation of TNFa of TNFa biosynthesisbiosynthesis

2.2. Proteolysis and Proteolysis and peptidolysispeptidolysis

3. 3. MHC II MHC II presentation presentation4. Inflammatory 4. Inflammatory

responseresponse 5. 5. Coupled receptor Coupled receptor

protein signaling protein signaling pathwaypathway

6. Regulation of cell 6. Regulation of cell cyclecycle

1. NK cells 1. NK cells mediated mediated cytotoxicitycytotoxicity

2. Cytokine-2. Cytokine-cytokine receptor cytokine receptor interactioninteraction

3. MAPK signaling3. MAPK signaling 4. Cell adhesion4. Cell adhesion5. Ca signaling 5. Ca signaling

pathwaypathway6. Leukocyte 6. Leukocyte

transendotelial transendotelial migrationmigration

ConclusionsConclusions

• BrucellaBrucella invade the host via intestinal Peyer’s patches invade the host via intestinal Peyer’s patches followed by metastasis & systemic distribution and organ followed by metastasis & systemic distribution and organ colonization via blood and lymphatic vessels colonization via blood and lymphatic vessels

• NO histopathological changes in early infected tissuesNO histopathological changes in early infected tissues

• A common transcriptional profile was identified in A common transcriptional profile was identified in B. B. melitensis melitensis in the first 4 h PI in the first 4 h PI in vivoin vivo

• Two different transcriptional profiles were observed in the Two different transcriptional profiles were observed in the bovine host early in the infectionbovine host early in the infection

• This study provides specific genes & pathways to further This study provides specific genes & pathways to further elucidate how both host and elucidate how both host and BrucellaBrucella interact interact in vivoin vivo during during the early infectious process to the eventual benefit of the the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve hostpathogen and to the detriment of the naïve host

• BrucellaBrucella invade the host via intestinal Peyer’s patches invade the host via intestinal Peyer’s patches followed by metastasis & systemic distribution and organ followed by metastasis & systemic distribution and organ colonization via blood and lymphatic vessels colonization via blood and lymphatic vessels

• NO histopathological changes in early infected tissuesNO histopathological changes in early infected tissues

• A common transcriptional profile was identified in A common transcriptional profile was identified in B. B. melitensis melitensis in the first 4 h PI in the first 4 h PI in vivoin vivo

• Two different transcriptional profiles were observed in the Two different transcriptional profiles were observed in the bovine host early in the infectionbovine host early in the infection

• This study provides specific genes & pathways to further This study provides specific genes & pathways to further elucidate how both host and elucidate how both host and BrucellaBrucella interact interact in vivoin vivo during during the early infectious process to the eventual benefit of the the early infectious process to the eventual benefit of the pathogen and to the detriment of the naïve hostpathogen and to the detriment of the naïve host

Future stepsFuture steps

• Develop of modern Develop of modern softwaresoftware and and modeling modeling approaches that help connect approaches that help connect BrucellaBrucella effectors with host targetseffectors with host targets

• Laser Capture Micro-dissectionLaser Capture Micro-dissection (LCM) analysis (LCM) analysis to study the temporal expression profile of to study the temporal expression profile of both, both, BrucellaBrucella and the host more precisely, and the host more precisely, providing an approach of how providing an approach of how BrucellaBrucella modify modify their transcriptome inside different cell types their transcriptome inside different cell types & how these cells respond to & how these cells respond to BrucellaBrucella infection infection

• Discovery of novel genes & important Discovery of novel genes & important pathways critical to the host response in the pathways critical to the host response in the pathogen virulence to determine pathogen virulence to determine potential potential targetstargets for subsequent therapeutic and for subsequent therapeutic and vaccine researchvaccine research

• Develop of modern Develop of modern softwaresoftware and and modeling modeling approaches that help connect approaches that help connect BrucellaBrucella effectors with host targetseffectors with host targets

• Laser Capture Micro-dissectionLaser Capture Micro-dissection (LCM) analysis (LCM) analysis to study the temporal expression profile of to study the temporal expression profile of both, both, BrucellaBrucella and the host more precisely, and the host more precisely, providing an approach of how providing an approach of how BrucellaBrucella modify modify their transcriptome inside different cell types their transcriptome inside different cell types & how these cells respond to & how these cells respond to BrucellaBrucella infection infection

• Discovery of novel genes & important Discovery of novel genes & important pathways critical to the host response in the pathways critical to the host response in the pathogen virulence to determine pathogen virulence to determine potential potential targetstargets for subsequent therapeutic and for subsequent therapeutic and vaccine researchvaccine research

• Dr. Adams’ labDr. Adams’ lab- Tiffany Fausett- Tiffany Fausett- Josely Figueiredo- Josely Figueiredo- Tamara Gull- Tamara Gull- Doris Hunter- Doris Hunter- Sangeeta Khare- Sangeeta Khare- Sara Lawhon- Sara Lawhon- Jairo Nunes- Jairo Nunes- Alan Patranella- Alan Patranella- Roberta Pugh- Roberta Pugh- Quynhtien Tran- Quynhtien Tran

• B. melitensisB. melitensis microarray microarray printingprinting- Dr. Mitchell McGee and Dr. - Dr. Mitchell McGee and Dr. Stephen A. Johnston from the Stephen A. Johnston from the Center for Innovations in Center for Innovations in Medicine, A.S.UMedicine, A.S.U

• Bovine microarray printingBovine microarray printing- Dr. Robin Everts and Dr. - Dr. Robin Everts and Dr. Harris Lewin from UIUCHarris Lewin from UIUC

AcknowledgementsAcknowledgements

• Microarray analysisMicroarray analysis- Dr. Cristi L. Galindo and Dr. - Dr. Cristi L. Galindo and Dr. Harold Garner (UTSWMS – Harold Garner (UTSWMS – Dallas)Dallas)- Dr. Bryan Kamery and Dr. - Dr. Bryan Kamery and Dr. Ken Drake (Seralogix, Inc.)Ken Drake (Seralogix, Inc.)

• Financial support: I.N.T.A.-Financial support: I.N.T.A.-Fulbright Argentina Fulbright Argentina scholarship, NIH/NIAID scholarship, NIH/NIAID Western Regional Center of Western Regional Center of Excellence, & DHS – FAZD Excellence, & DHS – FAZD grantsgrants

Documents

Temporal Transcriptional Analysis of the Early in vivo Initial Interactions of both Brucella & Bovine Host Carlos A. Rossetti, Brian Kamery, Sara Lawhon,