Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC

INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.9.5626–5634.2001

Sept. 2001, p. 5626–5634 Vol. 69, No. 9

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC Mutantsin In Vitro and In Vivo Systems

DAVID A. LEWIS,1,2 MARLA K. STEVENS,1 JO L. LATIMER,1 CHRISTINE K. WARD,1 KAIPING DENG,1

ROBERT BLICK,1 SHERYL R. LUMBLEY,1 CATHERINE A. ISON,3 AND ERIC J. HANSEN1*

Department of Microbiology,1 University of Texas Southwestern Medical Center, Dallas, Texas 75235-9048,1 andDepartment of Genitourinary Medicine and Communicable Disease2 and Department of Microbiology,3

Imperial College School of Medicine, St. Mary’s Campus, London W2 1PG United Kingdom

Received 12 May 2000/Returned for modification 19 June 2000/Accepted 13 June 2001

Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that is encoded by the cdtABCgene cluster and can be detected in culture supernatant fluid by its ability to kill HeLa cells. The cdtA, cdtB,and cdtC genes of H. ducreyi were cloned independently into plasmid vectors, and their encoded proteins ex-pressed singly or in various combinations in an Escherichia coli background. All three gene products had to beexpressed in order for E. coli-derived culture supernatant fluids to demonstrate cytotoxicity for HeLa cells. Iso-genic H. ducreyi cdtA and cdtB mutants were constructed and used in combination with the wild-type parentstrain and a previously described H. ducreyi cdtC mutant (M. K. Stevens, J. L. Latimer, S. R. Lumbley, C. K.Ward, L. D. Cope, T. Lagergard, and E. J. Hansen, Infect. Immun. 67:3900–3908, 1999) to determine the rela-tive contributions of the CdtA, CdtB, and CdtC proteins to CDT activity. Expression of CdtA, CdtB, and CdtC ap-peared necessary for H. ducreyi-derived culture supernatant fluid to exhibit cytotoxicity for HeLa cells. Whole-cell sonicates and periplasmic extracts from the cdtB and cdtC mutants had no effect on HeLa cells, whereasthese same fractions from a cdtA mutant had a very modest cytotoxic effect on these same human cells. CdtAappeared to be primarily associated with the H. ducreyi cell envelope, whereas both CdtB and CdtC were pres-ent primarily in the soluble fraction from sonicated cells. Both the cdtA mutant and the cdtB mutant were foundto be fully virulent in the temperature-dependent rabbit model for experimental chancroid.

The genital ulcer disease chancroid, caused by the bacteriumHaemophilus ducreyi, is one of the most prevalent sexuallytransmitted diseases and is a major cause of morbidity in theresource-poor countries of Africa, Asia, and Latin America(50). In the pre-antibiotic era, chancroid ulcers were noted toheal at slow rates and often resolution was incomplete (38, 50).Bacterial factors, acting either directly or indirectly, may beresponsible for this observed chronicity of H. ducreyi genitalulceration. In particular, it is possible that one or both of thetwo toxins known to be elaborated by H. ducreyi could beinvolved in this effect. The hemolysin HhdA, which has beendescribed as being bacterial cell-associated, kills human fore-skin fibroblasts (HFF) in vitro (1, 28). The other H. ducreyicytotoxin, a member of the family of toxins known as cytolethaldistending toxins (CDTs) (33), has little effect on HFF cells butreadily kills other human cells, including HeLa cells in vitro(37, 44), and has been shown to cause Jurkat T cells to undergoapoptosis (14).

The vast majority of H. ducreyi strains (35) produce CDTthat can be readily detected in culture supernatant fluid. TheH. ducreyi CDT, first described phenotypically by Purven andLagergard (37), is chromosomally encoded by three adjacentgenes, cdtA, cdtB, and cdtC, which appear to be transcription-ally linked (8). Similar CDTs are expressed by a number ofdifferent enteric organisms, including Escherichia coli (18, 31,

40), Shigella species (17, 27), and Campylobacter species (19,32), as well as by Actinobacillus actinomycetemcomitans (25,46). CDT activity was originally characterized as causing rela-tively slow morphological changes in cultured epithelial cells,including progressive cellular distention and death within 96 to120 h (18). CDT exerts its effect through cell cycle arrest in theG2 phase of growth (7, 9, 30, 41, 46, 52). Most recently, it wasreported that CDT has intrinsic DNAse activity associated withthe CdtB polypeptide (11, 12, 22).

The exact composition of the active form of CDT is as yetunknown. All three gene products (i.e., CdtA, CdtB, and CdtC)appear to be necessary for the expression of E. coli CDT ac-tivity in culture supernatant fluid (31, 40). There is also evi-dence to support the involvement of the H. ducreyi cdtC geneproduct in the expression of cytotoxic activity because a mono-clonal antibody (MAb) to the H. ducreyi CdtC protein has beenshown to neutralize H. ducreyi CDT activity in vitro (8, 25),although it is unlikely that CdtC itself has toxic activity like thatattributed to CdtB (41).

In the present study, we report the construction and char-acterization of isogenic cdtA and cdtB mutants of H. ducreyi35000. The phenotypes of these two new mutants and that of aH. ducreyi cdtC mutant (44) were assessed through the use ofin vitro cytotoxicity assays with HeLa cells and in the temper-ature-dependent rabbit model for experimental chancroid. Cy-totoxic activity assays of whole-cell sonicates and periplasmicextracts prepared from these three isogenic mutants revealedthat these preparations from the cdtA mutant still possessedsome CDT activity, whereas the same preparations from thecdtB and cdtC mutants were devoid of CDT activity.

* Corresponding author. Mailing address: Department of Microbi-ology, University of Texas Southwestern Medical Center, 5323 HarryHines Blvd., Dallas, Texas 75390-9048. Phone: (214) 648-5974. Fax:(214) 648-5905. E-mail: [email protected].

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MATERIALS AND METHODS

Bacterial strains and culture conditions. The bacterial strains and plasmidsused in this study are listed in Table 1. All H. ducreyi strains were routinelycultivated on chocolate agar (CA) plates containing 1% (vol/vol) Iso VitaleX(BBL Microbiological Systems, Cockeysville, Md.) in a humidified atmosphere of95% air–5% CO2 at 33°C as previously described (23). Broth cultures of H. du-creyi were grown overnight (16 h) in modified Columbia broth (51) for thepreparation of whole-cell sonicates, periplasmic extracts, and culture supernatantfluids. Cells of E. coli strains were grown on Luria-Bertani medium (39) at 37°C.For antimicrobial supplementation, kanamycin was used at 30 mg/ml (for both E.coli and H. ducreyi), tetracycline was used at 15 mg/ml (for E. coli), and chlor-amphenicol was used at 0.5 mg/ml (for H. ducreyi) and 15 mg/ml (for E. coli).

MAbs. The H. ducreyi CdtA-reactive MAb 1G8, the CdtB-reactive MAb 20B2,and the CdtC-reactive MAb 8C9 have been described (44).

Recombinant DNA techniques. Standard techniques, including restriction en-zyme digests, ligation, transformation, and plasmid purification, have been de-scribed elsewhere (4, 39). An aphA-3 gene (kan1) lacking both its own promoterand transcriptional terminators was utilized for the construction of nonpolarinsertion mutations in the H. ducreyi cdtA and cdtB genes. This aphA-3 gene ispreceded by translational stop codons in all three reading frames and is imme-diately followed by a consensus ribosome binding site and a start codon. The useof appropriate restriction sites allows placement of the start codon (at the 39 endof this cassette) in the open reading frame of the stop codon of the mutated geneso that translation of the remaining 39 portion of the mutated gene will allow

translational coupling, if any, with a downstream gene(s). This aphA-3 gene wasexcised from pUC18K by digestion with SmaI (26). PCR was performed accord-ing to manufacturers’ instructions with either Taq DNA polymerase (Promega,Madison, Wis.) or Pfu DNA polymerase (Stratagene, La Jolla, Calif.). Boiledbacterial cell preparations (16) or purified H. ducreyi chromosomal DNA (5)were used as templates for PCR.

Nucleotide sequence analysis. DNA sequencing was performed using a model373A automated DNA sequencer (Applied Biosystems Inc., Foster City, Calif.).All PCR products used for complementation analyses were sequenced to confirmthe absence of PCR-derived mutations.

RT-PCR detection of H. ducreyi mRNA from rabbit lesions. A lesion from arabbit injected intradermally 48 h previously with 105 CFU of H. ducreyi 35000was excised and macerated in 1 ml of Ultraspec RNA isolation reagent (BiotecXLaboratories, Inc., Houston, Tex.) by using a glass homogenizer and then storedat 270°C until processed. After suspension of the purified RNA pellet in 50 mlof diethyl pyrocarbonate-H2O, the RNA sample was treated with 10 U of DNaseat 37°C for 30 min to degrade any residual DNA using the Message CleanR Kit(GenHunter Corporation, Nashville, Tenn.).

Reverse transcriptase (RT) PCR was performed using the Titan One TubeRT-PCR system (Boehringer-Mannheim Biochemicals, Indianapolis, Ind.) ac-cording to the manufacturer’s instructions. Test sample (lesion RNA), positivecontrol (H. ducreyi 35000 chromosomal DNA), no-RT control (reactions con-taining lesion RNA placed in the thermocycler at step 2), and negative control(no template) reactions were run simultaneously. The oligonucleotide primers

TABLE 1. Bacterial strains and plasmids used in this study

Strain or plasmid Genotype or description Referenceor source

E. coliDH5a Host strain for cloning experiments 39DH5a(pDL20-A pDL10-B pJL300-C) Contains the H. ducreyi cdtA, cdtB, and cdtC genes on three separate plasmids; ex-

presses CDT activity detectable in culture supernatant fluidThis study

DH5a(pDL19-2 pDL16-1 pLS88) Contains three different vectors; expresses no detectable CDT activity in culture super-natant fluid

This study

DH5a(pJL300) Contains H. ducreyi 35000 cdtABC gene cluster; expresses H. ducreyi CDT activitydetectable in culture supernatant fluid

44

DH5a(pJL301) Contains H. ducreyi 35000 cdtABC gene cluster with mutated cdtA gene; expresses nodetectable CDT activity in culture supernatant fluid

This study

DH5a(pJL302) Contains H. ducreyi 35000 cdtABC gene cluster with mutated cdtB gene; expresses nodetectable CDT activity in culture supernatant fluid

This study

DH5a(pJL303) Contains H. ducreyi 35000 cdtABC gene cluster with mutated cdtC gene; expresses nodetectable CDT activity in culture supernatant fluid

44

HB101 Host strain essential for propagating plasmids carrying mutated H. ducreyi DNA insertsused in H. ducreyi mutant construction

39

H. ducreyi35000 Wild-type strain isolated in Winnipeg, Manitoba, Canada; produces CDT 1535000.301 Isogenic cdtA mutant with kan1 cartridge inserted into cdtA gene; does not express

CDT activity detectable in culture supernatant fluidThis study

35000.302 Isogenic cdtB mutant with kan1 cartridge inserted into cdtB gene; does not expressCDT activity detectable in culture supernatant fluid

This study

35000.303 Isogenic cdtC mutant with cat cartridge inserted into cdtC gene; does not express CDTactivity detectable in culture supernatant fluid

44

PlasmidspBR322 Cloning vector, Ampr Tetr 39pUC18K pUC18 with kan1 gene ligated into multi-cloning site 26pACYC184 Cloning vector capable of replication in H. ducreyi; Chlorr, Tetr 6pLS88 Cloning vector capable of replication in H. ducreyi; Kanr, Smr, Sulr 10pJL300 pBR322 with 3-kb H. ducreyi 35000 DNA insert containing cdtABC gene cluster 44pJL301 pJL300 with kan1 cartridge inserted into RsrII site within cdtA gene This studypJL302 pJL300 with kan1 cartridge inserted into MscI site within cdtB gene This studypJL303 pJL300 with cat cartridge inserted into BseRI site within cdtC gene 44pDL19-2 Plasmid obtained by blunt-ending and self-ligating 3.6-kb PstI-EcoRI fragment from

pBR322; vector control for pDL20-AThis study

pDL20-A pDL19-2 with 1.1-kb PCR-derived PstI-EcoRI DNA fragment containing H. ducreyicdtA gene

This study

pDL16-1 Plasmid obtained by blunt-ending and self-ligating 4-kb SalI-BamHI fragment frompACYC184; vector control for pDL10-B

This study

pDL10-B pDL16-1 with 1.2-kb PCR-derived SalI-BamHI DNA fragment containing H. ducreyicdtB gene

This study

pJL300-C pLS88 with 813-bp PCR-derived insert containing H. ducreyi cdtC gene 44

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P1 (59-GCACCAGCATTTGTATTAACGGC-39) and P2 (59-CGGTTGAAACTTGGCTAACGC-39) were used in RT-PCR to obtain a predicted 355-bp prod-uct derived from the pal gene, which encodes a H. ducreyi lipoprotein (positivecontrol) (43). The following oligonucleotide primers were used in RT-PCR toobtain the corresponding products derived from the cdtABC genes: a 132-bpproduct from the cdtA gene using P3 (59-TTCGTAATTGGAAGATGG-39) andP4 (59-CGTGCTAATTTGTCCAGAC-39), a 460-bp product from the cdtB geneusing P5 (59-GGTTCCTTACCAAGTTCGGCAG-39) and P6 (59-TATTTTCACTCACTGCGG-39), and a 271-bp product from the cdtC gene using P7 (59-ATGTTTTGCTTTCCTGGG-39) and P8 (59-ACCCTGATTTCTTCGCAC-39).The RT-PCR cycle contained seven steps: step 1, 50°C (30 min); step 2, 94°C (2min); step 3, 94°C (1 min); step 4, 52°C (1 min); step 5, 68°C (1 min); step 6, 29cycles through steps 3 to 5; step 7, 68°C for 10 min.

Construction of isogenic H. ducreyi cdtA and cdtB mutants. The cdtA gene inthe cdtABC gene cluster in pJL300 (44) was inactivated by insertion of the kan1cartridge into the RsrII site (Fig. 1) (26) to produce pJL301. Inactivation of thecdtB gene was accomplished by inserting the same kan1 cartridge into the MscIsite within the cdtB gene in pJL300; the resultant construct was designatedpJL302. Selection for kanamycin resistance followed by nucleotide sequenceanalysis confirmed that the cartridge was inserted in the proper orientation ineach open reading frame. Plasmids pJL301 and pJL302 were purified from E. coliHB101 cells by using the Wizard Plus minipreps purification system (Promega),linearized by digestion with AatII, and used to electroporate H. ducreyi 35000.Transformants were selected on GC-heme plates supplemented with kanamycin(45). H. ducreyi transformants with potential mutations in the cdtA gene wereidentified by PCR-based amplification of chromosomal DNA from single colo-nies by using the oligonucleotide primers P9 (59-CCTTGTAGATTATTCACCGT-39) and P10 (59-AAAGTTTGATGGTTCAGACGG-39) and by the loss ofreactivity with the H. ducreyi CdtA-reactive MAb 1G8 using Western blot anal-ysis. Similarly, H. ducreyi transformants with potential mutations in the cdtB genewere identified by PCR-based amplification of chromosomal DNA from singlecolonies using oligonucleotide primers P11 (59-GCAAACCGAGTGAACTTAG-39) and P12 (59-TATTTTCACTCACTGCGG-39) and by loss of reactivity withthe H. ducreyi CdtB-reactive MAb 20B2. The isogenic H. ducreyi 35000 cdtCmutant used in this study has been described (44).

Construction of a plasmid containing the H. ducreyi cdtA gene for comple-mentation studies in E. coli. A 1.1-kb DNA fragment containing the cdtA genewas amplified from H. ducreyi 35000 chromosomal DNA by PCR using Pfu DNApolymerase and the oligonucleotide primers P13 (59-TACTGCAGTTCCCATA

CGCCAGGATAG-39) and P14 (59-TAGAATTCATAACATCACACAGAAAACCACAC-59); the underlined sequences indicate PstI and EcoRI sites, respec-tively. The PCR product was digested with PstI and EcoRI before ligation intothe 3.6-kb PstI-EcoRI fragment of the plasmid vector pBR322. The ligationmixture was used to transform E. coli DH5a; tetracycline-resistant transformantswere screened for expression of CdtA by Western blot analysis using MAb 1G8.Plasmid pDL20-A containing the H. ducreyi cdtA gene was obtained from one ofthese transformants. In addition, E. coli DH5a was transformed with the 3.6-kbpBR322-derived vector pDL19-2 (Table 1).

Construction of a plasmid containing the H. ducreyi cdtB gene for comple-mentation studies in E. coli. A 1.2-kb DNA fragment containing the cdtB genewas amplified from H. ducreyi 35000 chromosomal DNA as described aboveusing the oligonucleotide primers P15 (59-TAGGATCCTGGTGCGGTTGTCATTAAAAG-39) and P16 (59-ATTAGTCGACGAGGAGGTGATAACTCTACATCAGG-39); the underlined sequences indicate BamHI and SalI sites, respec-tively. The PCR product was digested with BamHI and SalI before ligation intothe 4-kb BamHI-SalI fragment of the plasmid vector pACYC184 (6). The liga-tion mixture was used to transform E. coli DH5a; chloramphenicol-resistanttransformants were screened for expression of CdtB by Western blot analysisusing MAb 20B2. Plasmid pDL10-B was obtained from one of these transfor-mants (Table 1). Plasmid pACYC184 was digested with both BamHI and SalIand, after blunt-ending, was self-ligated to form the vector pDL16-1.

Plasmid pDL20-A containing the cdtA gene (Table 1), pDL10-B containingthe cdtB gene (Table 1), and pJL300-C containing the cdtC gene (reference 44and Table 1), in addition to their respective vector controls (pDL19-2 forpDL20-A, pDL16-1 for pDL10-B, and pLS88 for pJL300-C), were used incomplementation studies with E. coli cells (Table 1). E. coli DH5a cells wereelectroporated with pDL20-A, pDL10-B, or pJL300-C alone or in various com-binations in order to examine the contributions of CdtA, CdtB, and CdtC toCDT activity as assayed in the HeLa cell cytotoxicity assay.

Preparation of bacterial extracts for cytotoxicity testing. E. coli culture super-natant fluid preparations were prepared as previously described (44). To obtainH. ducreyi culture supernatant fluid preparations, 16-h overnight broth cultureswere subjected to centrifugation at 7,600 3 g for 20 min to collect the bacterialcells which were then used to prepare whole-cell sonicates and periplasmicextracts as described below. The resultant supernatant was sterilized by filtrationthrough a cellulose acetate filter (0.2-mm pore size; Nalgene, Rochester, N.Y.)and then subjected to centrifugation at 219,000 3 g for 1.5 h. The final super-natant fluid from this last centrifugation step was filter sterilized again and used

FIG. 1. Partial restriction map of the H. ducreyi 35000 chromosomal DNA insert in pJL300 and related plasmids. Restriction sites inparentheses indicate vector cloning sites. The arrows indicate the direction of transcription. Plasmid pJL300 was derived from the pBR322 vector;both it and pJL303 have been described (44). The kan1 cartridge was blunt-ended and ligated into the similarly treated RsrII site in cdtA toconstruct pJL301 and into the similarly treated MscI site of cdtB to produce pJL302. Plasmid pDL20-A is a modified pBR322 vector containingthe 1.1-kb PstI- and EcoRI-digested PCR product with the cdtA gene. Plasmid pDL10-B is a modified pACYC184 vector containing the 1.2-kb SalI-and BamHI-digested PCR product with the cdtB gene. Plasmid pJL300-C is pLS88 containing the cdtC gene (44).

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as the culture supernatant fluid for cytotoxicity testing. To prepare whole-cellsonicates, the bacterial cells collected by centrifugation from the broth culturewere washed once with pH 7.3 phosphate-buffered saline and collected again bycentrifugation. The cell pellet was suspended in 20 mM Tris-HCl (pH 8.0)containing 20% (wt/vol) sucrose, using 5 ml of this solution per gram (wetweight) of cells. This suspension was sonicated five times (1-min sonicationfollowed by a 1-min cooling period each time) using a Branson model 450sonifier (Branson Sonic Power Co., Danbury, Conn.) and a tapered microtip onpower setting no. 10 and at 60% duty. The whole-cell sonicate was subjected tocentrifugation at 27,000 3 g for 20 min to remove unbroken cells and debris. Theresultant whole-cell sonicate was sterilized by filtration through a cellulose ace-tate filter (0.2-mm pore size) and stored at 4°C overnight prior to performing thecytotoxicity assay. Periplasmic extracts were prepared as previously described(23), filter sterilized, and stored at 4°C until used to perform cytotoxicity assays.To confirm the presence of periplasmic proteins in the periplasmic extract, MAb3F1, which is specific for the H. ducreyi periplasmic ZnuA protein (23), was usedin Western blot analysis. In addition, periplasmic extracts and culture superna-tant fluids were tested for the presence of cell envelopes by using MAb 3F12,specific for the H. ducreyi major outer membrane protein (MOMP) (21). Thesepreparations were also tested for the presence of cytosolic contamination byassaying the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (24).

Cytotoxicity assays. Cytotoxicity assays for CDT activity used HeLa cells(ATCC CCL-2) grown in supplemented Dulbecco’s modified Eagle’s medium(GIBCO-BRL, Gaithersburg, Md.) in 24-well plates as previously described (44).After incubation of freshly seeded HeLa cells (3 3 104 cells/well) in 0.5 ml oftissue culture medium overnight, either a sample of bacterial culture supernatantfluid (0.5 ml) or a sample of H. ducreyi periplasmic extract or whole-cell sonicate(50 mg of protein in 0.5 ml of tissue culture medium) was added to the HeLa cellsfor 3 h before removal of the fluid and replacement with fresh tissue culturemedium. Serial twofold dilutions of these three preparations were made in tissueculture medium to determine CDT titers. The plates were incubated for 96 h at37°C in an atmosphere of 95% air–5% CO2. At the 96 h time point, the wellswere either stained with Giemsa and photographed or the CellTiter 96Aqueous

One Solution Cell Proliferation Assay (Promega) was used to determine theextent of killing (i.e., which wells still had viable cells). The CDT titer was definedas the greatest dilution which did not produce detectable killing of the HeLacells.

Preparation of soluble cell proteins and Sarkosyl-insoluble cell fractions. CAplate-grown H. ducreyi cells were subjected to sonication as described above. Thesonicate was centrifuged at 3,000 3 g for 10 min to remove whole cells and grossdebris; the supernatant fluid from this centrifugation step was then subjected tocentrifugation at 40,000 3 g for 1 h to collect cell envelopes. The supernatantfluid from this second centrifugation step was subjected to ultracentrifugation at156,000 3 g for 2 h to remove any membrane fragments; the final supernatantfluid was designated as the soluble cell fraction. The cell envelopes were washedtwice with phosphate-buffered saline and then treated with Sarkosyl as previouslydescribed (13) to obtain the Sarkosyl-insoluble fraction.

Sodium dodecylsulfate-polyacrylamide gel electrophoresis and Western blot(immunoblot) methods. Proteins present in various cell fractions and concen-trated culture supernatant fluids were resolved by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis and transferred to nitrocellulose paper for West-ern blot analysis with MAbs as previously described (44).

Virulence testing. The relative virulence of the three H. ducreyi cdt mutantsused in this study was determined in a blinded manner using the temperature-dependent rabbit model for experimental chancroid (34). Lesion characteristicson days 2, 4, and 7 postinfection were scored with the following numeric values:0 5 no change, 1 5 erythema, 2 5 induration, 3 5 nodule, 4 5 pustule ornecrosis. On day 7 postinfection, material excised from lesions caused by injec-tion of 105 CFU was cultured on CA plates. Statistical analyses were performedas previously described (2, 45).

RESULTS

Complementation studies with E. coli. In a preliminary effortto evaluate the contribution of the CdtA, CdtB, and CdtC pro-teins to the cytotoxic activity of H. ducreyi CDT, E. coli wasused as the host for various combinations of the H. ducreyicdtA, cdtB, and cdtC genes carried individually on plasmids.Two new plasmids with H. ducreyi cdtA (i.e., pDL20-A) andcdtB (i.e., pDL10-B) gene inserts were constructed for this pur-pose (Table 1 and Fig. 1). Plasmid pJL300-C containing a

functional H. ducreyi cdtC gene (44) was also used (Table 1).Western blot analysis of whole-cell lysates of the various re-combinant E. coli strains with CdtA-reactive, CdtB-reactive,and CdtC-reactive MAbs confirmed that each construct ex-pressed the expected cdt gene product (data not shown).Assays for CDT activity were performed using culture super-natant fluids obtained from overnight cultures of strains pos-sessing each gene individually as well as all possible double ortriple combinations of the three cdt genes and their relevantvector controls. In addition, E. coli strains DH5a(pBR322) andDH5a(pJL300) (Table 1) were used as negative and positivecontrols, respectively, in the cytotoxicity assays.

HeLa cell cytotoxicity was observed only with culture super-natant fluids from E. coli DH5a(pJL300), which containedthe cdtABC gene cluster, and from E. coli DH5a(pDL20-ApDL10-B pJL300-C), which contained the individual cdtA,cdtB, and cdtC genes on separate plasmids (data not shown).Culture supernatant fluids derived from the correspondingtransformants DH5a(pBR322) and DH5a(pDL19-2 pDL16-1pLS88) (Table 1), which contained only vector, were not cyto-toxic to HeLa cells. Culture supernatant fluids from E. colistrains containing plasmids expressing either one or two of theCdtA, CdtB, and CdtC proteins failed to exhibit cytotoxicityfor HeLa cells (data not shown).

Construction of isogenic H. ducreyi cdtA and cdtB mutants.To construct an isogenic H. ducreyi cdtA mutant, pJL301 (Fig.1) was linearized and used to electroporate H. ducreyi 35000. Akanamycin-resistant transformant which failed to react withthe H. ducreyi CdtA-reactive MAb 1G8 was selected for furtherstudy and designated strain 35000.301. Western blot analysisrevealed that this cdtA mutant expressed no detectable CdtAprotein (Fig. 2A, lane 2) and readily detectable levels of bothCdtB (Fig. 2B, lane 2) and CdtC (Fig. 2C, lane 2).

An isogenic cdtB mutant was constructed in H. ducreyi 35000by the method described above except that linearized pJL302(Fig. 1) was used to electroporate H. ducreyi 35000 cells. Akanamycin-resistant H. ducreyi transformant that failed to re-act with the H. ducreyi CdtB-reactive MAb 20B2 was desig-nated 35000.302. Western blot analysis showed that this cdtBmutant expressed no detectable CdtB protein (Fig. 2B, lane 3)but did express both CdtA (Fig. 2A, lane 3) and CdtC (Fig.2C, lane 3). The level of CdtC expressed by this cdtB mutantwas lower than that expressed by the wild-type parent strain.PCR and Southern blot analyses confirmed that the desiredallelic exchanges had occurred in both strains 35000.301 and35000.302 (data not shown).

Cytotoxic activity of the wild-type and mutant H. ducreyistrains. Initially, culture supernatant fluids from the wild-typestrain 35000, the cdtA mutant 35000.301, the cdtB mutant35000.302, and the cdtC mutant 35000.303 (44) were tested forHeLa cell cytotoxicity. These culture supernatant fluids werefree of cytosolic contamination as determined by the absenceof detectable glucose-6-phosphate dehydrogenase activity (datanot shown) and did not contain either periplasmic proteins orouter membranes, as evidenced by their lack of reactivity withthe ZnuA-specific MAb 3F1 and the MOMP-specific MAb3F12, respectively (data not shown). Cytotoxicity was observedwith culture supernatant fluids from the wild-type strain only(Fig. 3A); culture supernatant fluids from the three isogenicmutants, 35000.301, 35000.302, and 35000.303, all failed to



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exhibit cytotoxic activity. When CdtA-, CdtB-, and CdtC-di-rected MAbs were used in Western blot analysis to probeculture supernatant fluid obtained from the wild-type parentstrain, none of the Cdt proteins could be detected (data notshown). Fortyfold concentrations of these supernatant fluidsstill did not allow detection of Cdt proteins by Western blotanalysis (data not shown).

Cytotoxicity of subcellular fractions from wild-type and mu-tant strains. Both periplasmic extracts and whole-cell sonicatesfrom the wild-type strain and the cdtA, cdtB, and cdtC mutantswere tested for their ability to kill HeLa cells. The periplasmicextracts were free of cytosolic contamination (data not shown)and outer membrane fragments (Fig. 2E, lanes 5 to 8) as de-termined above and did contain the periplasmic protein ZnuA(Fig. 2D, lanes 5 to 8). The relevant Cdt proteins could be de-tected readily in the whole-cell sonicates (Fig. 2A to C, lanes 1to 4). However, in most cases, Cdt proteins could not be de-tected by Western blot analysis in the periplasmic extracts (Fig.2A to C, lanes 5 to 8), even with the wild-type strain. Bothwhole-cell sonicates (Fig. 3C) and periplasmic extracts (Fig.

3B) from wild-type strain 35000 caused marked killing of theHeLa cells, whereas the same preparations from the cdtA mu-tant 35000.301, the cdtB mutant 35000.302, and the cdtC mu-tant 35000.303 appeared to lack killing activity as determinedby a tetrazolium dye reduction test (described in Materials andMethods). However, microscopy of the HeLa monolayers ex-posed to the most concentrated samples (i.e., 50 mg of pro-tein in 0.5 ml) of whole-cell sonicate from the cdtA mutant35000.301 (Fig. 4B) revealed the presence of many distendedor rounded HeLa cells. These distended cells were not presentin the monolayers treated with whole-cell sonicates from thecdtB mutant (Fig. 4C) and the cdtC mutant (Fig. 4D). No vi-

FIG. 2. Western blot-based detection of H. ducreyi CdtA, CdtB,and CdtC proteins expressed by wild-type and mutant H. ducreyistrains. Whole-cell sonicates (lanes 1 to 4) and periplasmic extracts(lanes 5 to 8) of these four strains were probed with the H. ducreyiCdtA-reactive MAb 1G8 (A), the H. ducreyi CdtB-reactive MAb 20B2(B), the H. ducreyi CdtC-reactive MAb 8C9 (C), the ZnuA-reactiveMAb 3F1 (D), and the MOMP-reactive MAb 3F12 (E). Equivalentamounts of whole-cell sonicate and periplasmic extract were loadedinto each lane. Lanes: 1 and 5, wild-type H. ducreyi 35000; 2 and 6,H. ducreyi cdtA mutant 35000.301; 3 and 7, H. ducreyi cdtB mutant35000.302; 4 and 8, H. ducreyi cdtC mutant 35000.303. Molecular sizemarkers (in kilodaltons) are indicated on the right.

FIG. 3. Killing of HeLa cells by CDT in subcellular fractions andculture supernatant fluids from wild-type and mutant strains of H. du-creyi. Cytotoxic activity was assessed in culture supernatant fluids (A),periplasmic extracts (B), and whole-cell sonicates (C) obtained fromthe wild-type H. ducreyi strain 35000, the cdtA mutant 35000.301, thecdtB mutant 35000.302, and the cdtC mutant 35000.303. HeLa cellswere exposed to filter-sterilized culture supernatant fluids, periplasmicextracts, or whole-cell sonicates as described in Materials and Meth-ods. After 96 h, wells in which viable cells were present were detectedby the use of the tetrazolium reduction-based assay described in Ma-terials and Methods. An optical density at 492 nm (OD492) greaterthan 1.8 indicates wells in which all of the HeLa cells were viable.



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able HeLa cells were present in monolayers treated with thewhole-cell sonicate from the wild-type parent strain (Fig. 4A).The periplasmic extract from the cdtA mutant also causedHeLa cell rounding and distention whereas the periplasmicextracts from the cdtB and cdtC mutants did not affect thesecells (data not shown).

In a preliminary effort to localize the Cdt proteins in H. du-creyi, we prepared a whole-cell sonicate (Fig. 5A to D, lanes 1)from the wild-type strain 35000 and separated the soluble cellcontents (Fig. 5, lanes 2) from the cell envelopes (Fig. 5, lanes3) and then treated the cell envelopes with Sarkosyl to obtainthe Sarkosyl-insoluble fraction of the cell envelopes (Fig. 5,lanes 4). The amounts of soluble cell contents, cell envelopes,and Sarkosyl-insoluble material were equivalent to the numberof cells used to prepare the sample contained in Fig. 5, lane 1.The bulk of the CdtB (Fig. 5B, lane 2) and CdtC (Fig. 5C, lane2) proteins appeared to be soluble, whereas most of the CdtAprotein (Fig. 5A, lane 3) was associated with the cell envelopes.These cell envelopes did contain the MOMP (Fig. 5D, lane 3).When these envelopes were extracted with Sarkosyl, the CdtAprotein was not detectable in the Sarkosyl-insoluble fraction(Fig. 5A, lane 4), which did contain the MOMP (Fig. 5D, lane 4).

Virulence testing. After confirming that the three isogenicmutants 35000.301, 35000.302, and 35000.303 each grew at arate indistinguishable from that of the wild-type strain 35000in broth (data not shown), all four strains were tested in thetemperature-dependent rabbit model. The cdtA and cdtB mu-tants both proved to be as virulent as the wild-type parentstrain with regard to lesion production (Table 2). As seen pre-viously (44), the cdtC mutant 35000.303 also was no less viru-lent than the wild-type strain (Table 2). In addition, viableH. ducreyi organisms were isolated from the lesions resultingfrom an injection of 105 CFU for all four strains (data notshown). The numbers of CFUs of each of the four strainsrecovered from these lesions were similar (data not shown).

Detection of cdtA, cdtB, and cdtC mRNA produced in vivo.The fact that the mutants lacking CDT activity were as virulentas the wild-type strain in the rabbit model prompted us toconfirm that the cdtABC genes were actually transcribed in thecells of the wild-type parent strain growing in vivo. RNA wasprepared from pustular lesion material obtained 48 h afterinfection of rabbits with the wild-type strain, 35000. RT-PCRconfirmed the presence of mRNA transcribed from the cdtA,cdtB, and cdtC genes. Specific primers amplified a 132-bp cdtA-encoded product, a 460-bp cdtB-encoded product, and a 271-bp cdtC-encoded product from the RNA (Fig. 6, lane 3). A355-bp pal-encoded product was amplified from the RNA as apositive control (Fig. 6, lane 3); the H. ducreyi pal gene encodesan unprocessed 18-kDa lipoprotein (43). H. ducreyi 35000chromosomal DNA was used as a template positive control(Fig. 6, lane 1). No PCR product was seen in a reaction devoid

FIG. 4. Cytopathic effect of CDT from wild-type and mutantstrains of H. ducreyi. HeLa cells were exposed to whole-cell sonicatesfrom the wild-type strain 35000 (A), the H. ducreyi cdtA mutant35000.301 (B), the H. ducreyi cdtB mutant 35000.302 (C), and theH. ducreyi cdtC mutant 35000.303 (D), and 96 h later the wells werephotographed at an original magnification of 340. (E) HeLa cellsexposed to the buffer used for the preparation of the whole-cell soni-cates were used as the negative control.


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of template (Fig. 6, lane 2) or in a reaction in which the RTstep was omitted (Fig. 6, lane 4).

DISCUSSION

Johnson and Lior (18) originally described CDT activity as atoxic factor produced by certain E. coli strains which woulddistend and kill CHO cells in vitro. CDT production in severalunrelated pathogenic bacteria was subsequently demonstrated(33). CDT can affect a number of different cell types in vitro,including keratinocytes (9, 44), HeLa cells, HEp-2 cells, Verocells (20), Caco-2 cells (52), hamster lung fibroblasts (9), and

both CD41 and CD81 human T cells (42). While new datahave appeared recently concerning the mechanism of action ofCDT (7, 9, 30, 46, 52), there is little information about thecomposition of the CDT holotoxin.

To date, there have been only two reports describing thepurification of a CDT. Working with H. ducreyi, Lagergard andcolleagues (36) used a MAb specific for CdtC in an immuno-affinity-based purification method and concluded that H. du-creyi CDT consisted of only CdtC. In contrast, Shenker et al.(42) purified an immunosuppressive factor from A. actino-

TABLE 2. Lesion formation by wild-type and mutant H. ducreyistrains in the temperature-dependent rabbit modela

StrainInoculum

size(CFU)

Mean (6SD) lesion scorebP

valuecDay 2 Day 4 Day 7

35000 (wild type) 105 4.00 (0) 4.00 (0) 4.00 (0)35000.301 (cdtA mutant) 105 4.00 (0) 4.00 (0) 4.00 (0)35000.302 (cdtB mutant) 105 3.88 (0.35) 4.00 (0) 4.00 (0)35000.303 (cdtC mutant) 105 4.00 (0) 4.00 (0) 4.00 (0)

35000 (wild type) 104 3.50 (0.53) 4.00 (0) 4.00 (0)35000.301 (cdtA mutant) 104 3.38 (0.52) 3.75 (0.46) 4.00 (0) 0.079635000.302 (cdtB mutant) 104 3.25 (0.46) 3.38 (0.52) 3.75 (0.46) 0.025635000.303 (cdtC mutant) 104 3.63 (0.52) 3.75 (0.46) 4.00 (0) 0.6845

a Eight rabbits were used in this experiment.b Lesion characteristics were scored as follows: 0, no change; 1, erythema; 2,

induration; 3, nodule; 4, pustule or necrosis.c P values were calculated for the difference between wild-type and mutant

strain lesion scores. P values were calculated using the lesion scores from the104-CFU inoculum size on 3 days. Using the Bonferroni correction necessitatedby the number of comparisons between strains in this analysis, the P valueneeded for significance is 0.0083. Therefore, there was no significant differencein lesion scores produced by the three isogenic mutants when they were com-pared in a pairwise manner.

FIG. 5. Detection of Cdt proteins in soluble and insoluble fractionsfrom H. ducreyi cells. Whole-cell sonicates were prepared from thewild-type strain 35000 as described in Materials and Methods by usingcentrifugation at 3,000 3 g to remove whole cells and gross debris.Equivalent amounts of whole-cell sonicate (lane 1), soluble cell con-tents (lane 2), cell envelopes (lane 3), and Sarkosyl-insoluble material(lane 4) were probed by Western blot analysis using the CdtA-reactiveMAb 1G8 (A), the CdtB-reactive MAb 20B2 (B), the CdtC-reactiveMAb 8C9 (C), and the MOMP-reactive MAb 3F12 (D). Molecular sizeposition markers (in kilodaltons) are on the right.

FIG. 6. Agarose gel electrophoresis of H. ducreyi multiplex RT-PCR products. The sizes of the predicted RT-PCR products were asfollows: pal, 355 bp; cdtA, 132 bp; cdtB, 460 bp; cdtC, 271 bp. Tem-plates included the following: lane 1, 100 ng of H. ducreyi 35000 ge-nomic DNA (positive control); lane 2, no template (negative control);lanes 3 and 4, 2 mg of RNA isolated from H. ducreyi-infected rabbitlesions. The reaction mixture loaded in lane 4 was not subjected to thereverse transcription step of the RT-PCR protocol and served as acontrol to detect DNA contamination of the RNA template. Lane M,DNA size markers (in base pairs).



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mycetemcomitans cells that killed human T cells and HeLacells and indicated that this purified toxin was comprised ofCdtB. (The protein products of the A. actinomycetemcomitanscdtABC genes are 92 to 97% identical to those of the H. ducreyicdtABC genes [25].) In neither case was the purity of the pro-tein preparation demonstrated conclusively; in particular, theabsence of the other two cdt gene products was not proven.

Data derived from the use of individually cloned H. ducreyicdtA, cdtB, and cdtC genes in an E. coli background demon-strated that CDT activity in culture supernatant fluid was de-tectable only when all three cdt gene products were expressed.This result reinforces results obtained in earlier studies of thecdtABC gene clusters from two different E. coli strains (31, 40).Similarly, culture supernatant fluids from isogenic H. ducreyicdtA, cdtB, and cdtC mutants all failed to kill HeLa cells,whereas culture supernatant fluid from the wild-type parentstrain caused extensive cell killing (Fig. 3). In contrast to theresults obtained with culture supernatant fluids, both whole-cell sonicates and periplasmic extracts prepared from theH. ducreyi cdtA mutant produced visible cytopathic effectsin HeLa cells, with cell distention being most prominent. Itshould be noted that cell distention caused by CDT precedeskilling (18, 20), such that the cell distention effect of the whole-cell sonicate (Fig. 4B) and periplasmic extract of the cdtAmutant likely reflects the presence of either a small quantity ofactive CDT or a partially inactive CDT. More importantly, thewhole-cell sonicates and periplasmic extracts prepared fromthe H. ducreyi cdtB and cdtC mutants showed no evidence ofcytotoxic activity in the HeLa cell assay.

The composition of active H. ducreyi CDT remains to bedetermined. The results described above indicate that muta-tions in cdtB and cdtC eliminate CDT activity completely,whereas some very limited CDT activity is expressed by thecdtA mutant. These results, taken together with the facts thatMAbs to CdtC can neutralize CDT activity (8, 25, 36) and thatthe CdtB protein has cytotoxic DNAse activity (11, 12, 22),suggest that both CdtB and CdtC are likely present in the CDTholotoxin. Our finding that a cdtA mutant still expressed de-tectable CDT activity does not preclude the presence of CdtAin the CDT holotoxin. The very weak cytotoxic effect detect-able in the cdtA mutant would be consistent with CdtA beinga structural component essential for fully active toxin. Alter-natively, CdtA could function to posttranslationally modifyeither CdtB or CdtC or both proteins to enable them to func-tion optimally.

This study also confirmed our previous observation that aH. ducreyi cdtC mutant was as virulent as its wild-type parentstrain in the temperature-dependent rabbit model (44); thismutant was also recently shown to form pustules in humanvolunteers at a rate similar to that obtained with its wild-typeparent strain (53). In addition, we were unable to demonstrateany decrease in virulence associated with either a cdtA or cdtBmutation (Table 2). RT-PCR analysis of RNA prepared fromrabbit lesion material confirmed that all three genes of theH. ducreyi cdtABC cluster were transcribed in rabbit skin dur-ing infection. In vivo transcription of at least cdtB was alsoshown to occur in human volunteers infected with wild-typeH. ducreyi (48). These findings suggest that the failure to ob-serve diminished virulence with the H. ducreyi cdtA, cdtB, andcdtC mutants in rabbits and with the cdtC mutant in humans is

unlikely to be due to a lack of expression of the relevant geneproducts in vivo.

The apparent lack of effect of mutations in the cdtABC genecluster on virulence expression by H. ducreyi in both humanand animal models of infection indicates that CDT does notplay a role in the development of the pustule that forms in theearly stages of chancroid. The determination of whether CDTmight be involved in either ulcer development or the retarda-tion of healing of the chancroidal ulcer cannot be accom-plished in these model systems. In the former, safety concernspreclude studies involving ulcer development while in the lat-ter model, the lack of ulcer production by the wild-type strainin rabbits will not allow either issue to be addressed. However,data from recent in vitro experiments suggest that CDT couldaffect the immune response to this pathogen. It has beenshown that the spectrum of H. ducreyi CDT activity also in-cludes human T cells (i.e., the Jurkat T-cell line) (14), and thisfinding is reinforced by a recent study in which H. ducreyi CDTwas shown to inhibit proliferation of human T cells and B cellsin vitro (47). These results are complemented by studies whichindicated that the immunosuppressive factor produced byA. actinomycetemcomitans is a CDT that causes G2 arrest inhuman T cells in vitro (42). Therefore, it is conceivable thatH. ducreyi CDT might be responsible at some level for the de-layed healing of chancroidal ulcers observed in the pre-antibi-otic era (38, 50) and for the apparent lack of protective immu-nity after chancroid (3). Whether H. ducreyi CDT can exertimmunosuppressive activity in the human host remains a mat-ter of speculation at this time.

ACKNOWLEDGMENTS

This study was supported by U.S. Public Health Service grantAI32011 to E.J.H. and by a Wellcome Training Fellowship in ClinicalTropical Medicine (reference number 049246/Z/96) to D.A.L. underthe joint sponsorship of J. N. Weber (Department of GenitourinaryMedicine and Communicable Disease) and D. B. Young (Departmentof Microbiology) at Imperial College School of Medicine, St. Mary’sCampus, London, United Kingdom. C.K.W. was supported by Na-tional Research Service Award F32-AI09845.

The kan1 cartridge used in this study was kindly provided by JamesB. Kaper.

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Characterization of Haemophilus ducreyi cdtA, cdtB, and cdtC