Functional Study of Haemophilus ducreyi Cytolethal Distending Toxin Subunit B.

The Cytolethal Distending Toxin (CDT) is produced by many Gram-negative pathogenic bacteria responsible for major foodborne diseases worldwide. CDT induces DNA damage and cell cycle arrest in host-cells, eventually leading to senescence or apoptosis. According to structural and sequence comparison, the catalytic subunit CdtB is suggested to possess both nuclease and phosphatase activities, carried by a single catalytic site. However, the impact of each activity on cell-host toxicity is yet to be characterized. Here, we analyze the consequences of cell exposure to different CDT mutated on key CdtB residues, focusing on cell viability, cell cycle defects, and DNA damage induction. A first class of mutant, devoid of any activity, targets putative catalytic (H160A), metal binding (D273R), and DNA binding residues (R117A-R144A-N201A). The second class of mutants (A163R, F156-T158, and the newly identified G114T), which gathers mutations on residues potentially involved in lipid substrate binding, has only partially lost its toxic effects. However, their defects are alleviated when CdtB is artificially introduced inside cells, except for the F156-T158 double mutant that is defective in nuclear addressing. Therefore, our data reveal that CDT toxicity is mainly correlated to CdtB nuclease activity, whereas phosphatase activity may probably be involved in CdtB intracellular trafficking.

Haemophilus ducreyi Seeks Alternative Carbon Sources and Adapts to Nutrient Stress and Anaerobiosis during Experimental Infection of Human Volunteers.

Haemophilus ducreyi causes the sexually transmitted disease chancroid in adults and cutaneous ulcers in children. In humans, H. ducreyi resides in an abscess and must adapt to a variety of stresses. Previous studies (D. Gangaiah, M. Labandeira-Rey, X. Zhang, K. R. Fortney, S. Ellinger, B. Zwickl, B. Baker, Y. Liu, D. M. Janowicz, B. P. Katz, C. A. Brautigam, R. S. MunsonJr, E. J. Hansen, and S. M. Spinola, mBio 5:e01081-13, 2014, http://dx.doi.org/10.1128/mBio.01081-13) suggested that H. ducreyi encounters growth conditions in human lesions resembling those found in stationary phase. However, how H. ducreyi transcriptionally responds to stress during human infection is unknown. Here, we determined the H. ducreyi transcriptome in biopsy specimens of human lesions and compared it to the transcriptomes of bacteria grown to mid-log, transition, and stationary phases. Multidimensional scaling showed that the in vivo transcriptome is distinct from those of in vitro growth. Compared to the inoculum (mid-log-phase bacteria), H. ducreyi harvested from pustules differentially expressed ∼93 genes, of which 62 were upregulated. The upregulated genes encode homologs of proteins involved in nutrient transport, alternative carbon pathways (l-ascorbate utilization and metabolism), growth arrest response, heat shock response, DNA recombination, and anaerobiosis. H. ducreyi upregulated few genes (hgbA, flp-tad, and lspB-lspA2) encoding virulence determinants required for human infection. Most genes regulated by CpxRA, RpoE, Hfq, (p)ppGpp, and DksA, which control the expression of virulence determinants and adaptation to a variety of stresses, were not differentially expressed in vivo, suggesting that these systems are cycling on and off during infection. Taken together, these data suggest that the in vivo transcriptome is distinct from those of in vitro growth and that adaptation to nutrient stress and anaerobiosis is crucial for H. ducreyi survival in humans.

Distinct Roles for CdtA and CdtC during Intoxication by Cytolethal Distending Toxins.

Cytolethal distending toxins (CDTs) are heterotrimeric protein exotoxins produced by a diverse array of Gram-negative pathogens. The enzymatic subunit, CdtB, possesses DNase and phosphatidylinositol 3-4-5 trisphosphate phosphatase activities that induce host cell cycle arrest, cellular distension and apoptosis. To exert cyclomodulatory and cytotoxic effects CDTs must be taken up from the host cell surface and transported intracellularly in a manner that ultimately results in localization of CdtB to the nucleus. However, the molecular details and mechanism by which CDTs bind to host cells and exploit existing uptake and transport pathways to gain access to the nucleus are poorly understood. Here, we report that CdtA and CdtC subunits of CDTs derived from Haemophilus ducreyi (Hd-CDT) and enteropathogenic E. coli (Ec-CDT) are independently sufficient to support intoxication by their respective CdtB subunits. CdtA supported CdtB-mediated killing of T-cells and epithelial cells that was nearly as efficient as that observed with holotoxin. In contrast, the efficiency by which CdtC supported intoxication was dependent on the source of the toxin as well as the target cell type. Further, CdtC was found to alter the subcellular trafficking of Ec-CDT as determined by sensitivity to EGA, an inhibitor of endosomal trafficking, colocalization with markers of early and late endosomes, and the kinetics of DNA damage response. Finally, host cellular cholesterol was found to influence sensitivity to intoxication mediated by Ec-CdtA, revealing a role for cholesterol or cholesterol-rich membrane domains in intoxication mediated by this subunit. In summary, data presented here support a model in which CdtA and CdtC each bind distinct receptors on host cell surfaces that direct alternate intracellular uptake and/or trafficking pathways.

Haemophilus ducreyi RpoE and CpxRA appear to play distinct yet complementary roles in regulation of envelope-related functions.

Haemophilus ducreyi causes the sexually transmitted disease chancroid and a chronic limb ulceration syndrome in children. In humans, H. ducreyi is found in an abscess and overcomes a hostile environment to establish infection. To sense and respond to membrane stress, bacteria utilize two-component systems (TCSs) and extracytoplasmic function (ECF) sigma factors. We previously showed that activation of CpxRA, the only intact TCS in H. ducreyi, does not regulate homologues of envelope protein folding factors but does downregulate genes encoding envelope-localized proteins, including many virulence determinants. H. ducreyi also harbors a homologue of RpoE, which is the only ECF sigma factor in the organism. To potentially understand how H. ducreyi responds to membrane stress, here we defined RpoE-dependent genes using transcriptome sequencing (RNA-Seq). We identified 180 RpoE-dependent genes, of which 98% were upregulated; a major set of these genes encodes homologues of envelope maintenance and repair factors. We also identified and validated a putative RpoE promoter consensus sequence, which was enriched in the majority of RpoE-dependent targets. Comparison of RpoE-dependent genes to those controlled by CpxR showed that each transcription factor regulated a distinct set of genes. Given that RpoE activated a large number of genes encoding envelope maintenance and repair factors and that CpxRA represses genes encoding envelope-localized proteins, these data suggest that RpoE and CpxRA appear to play distinct yet complementary roles in regulating envelope homeostasis in H. ducreyi.

Expression of the Flp proteins by Haemophilus ducreyi is necessary for virulence in human volunteers.

BACKGROUND: Haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp (fimbria like protein) operon that encodes proteins predicted to contribute to adherence and pathogenesis. H. ducreyi mutants that lack expression of Flp1 and Flp2 or TadA, which has homology to NTPases of type IV secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts (HFF). A tadA mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp2 mutant is virulent in rabbits. Whether a flp deletion mutant would cause disease in humans is not clear. RESULTS: We constructed 35000HPΔflp1-3, a deletion mutant that lacks expression of all three Flp proteins but has an intact tad secretion system. 35000HPΔflp1-3 was impaired in its ability to form microcolonies and to attach to HFF in vitro when compared to its parent (35000HP). Complementation of the mutant with flp1-3 in trans restored the parental phenotype. To test whether expression of Flp1-3 was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000HP (ranging from 54 to 67 CFU) on one arm and three doses of 35000HPΔflp1-3 (ranging from 63 to 961 CFU) on the other arm. The overall papule formation rate for the parent was 80% (95% confidence interval, CI, 55.2%-99.9%) and for the mutant was 70.0% (95% CI, 50.5%-89.5%) (P = 0.52). Mutant papules were significantly smaller (mean, 11.2 mm2) than were parent papules (21.8 mm2) 24 h after inoculation (P = 0.018). The overall pustule formation rates were 46.7% (95% CI 23.7-69.7%) at 30 parent sites and 6.7% (95% CI, 0.1-19.1%) at 30 mutant sites (P = 0.001). CONCLUSION: These data suggest that production and secretion of the Flp proteins contributes to microcolony formation and attachment to HFF cells in vitro. Expression of flp1-3 is also necessary for H. ducreyi to initiate disease and progress to pustule formation in humans. Future studies will focus on how Flp proteins contribute to microcolony formation and attachment in vivo.

Chancroid transmission dynamics: a mathematical modeling approach.

Mathematical models have long been used to better understand disease transmission dynamics and how to effectively control them. Here, a chancroid infection model is presented and analyzed. The disease-free equilibrium is shown to be globally asymptotically stable when the reproduction number is less than unity. High levels of treatment are shown to reduce the reproduction number suggesting that treatment has the potential to control chancroid infections in any given community. This result is also supported by numerical simulations which show a decline in chancroid cases whenever the reproduction number is less than unity.

Deletion of mtrC in Haemophilus ducreyi increases sensitivity to human antimicrobial peptides and activates the CpxRA regulon.

Haemophilus ducreyi resists killing by antimicrobial peptides encountered during human infection, including cathelicidin LL-37, α-defensins, and ß-defensins. In this study, we examined the role of the proton motive force-dependent multiple transferable resistance (MTR) transporter in antimicrobial peptide resistance in H. ducreyi. We found a proton motive force-dependent effect on H. ducreyi's resistance to LL-37 and ß-defensin HBD-3, but not α-defensin HNP-2. Deletion of the membrane fusion protein MtrC rendered H. ducreyi more sensitive to LL-37 and human ß-defensins but had relatively little effect on α-defensin resistance. The mtrC mutant 35000HPmtrC exhibited phenotypic changes in outer membrane protein profiles, colony morphology, and serum sensitivity, which were restored to wild type by trans-complementation with mtrC. Similar phenotypes were reported in a cpxA mutant; activation of the two-component CpxRA regulator was confirmed by showing transcriptional effects on CpxRA-regulated genes in 35000HPmtrC. A cpxR mutant had wild-type levels of antimicrobial peptide resistance; a cpxA mutation had little effect on defensin resistance but led to increased sensitivity to LL-37. 35000HPmtrC was more sensitive than the cpxA mutant to LL-37, indicating that MTR contributed to LL-37 resistance independent of the CpxRA regulon. The CpxRA regulon did not affect proton motive force-dependent antimicrobial peptide resistance; however, 35000HPmtrC had lost proton motive force-dependent peptide resistance, suggesting that the MTR transporter promotes proton motive force-dependent resistance to LL-37 and human ß-defensins. This is the first report of a ß-defensin resistance mechanism in H. ducreyi and shows that LL-37 resistance in H. ducreyi is multifactorial.

On the evolution of the sexually transmitted bacteria Haemophilus ducreyi and Klebsiella granulomatis.

Haemophilus ducreyi and Klebsiella (Calymmatobacterium) granulomatis are sexually transmitted bacteria that cause characteristic, persisting ulceration on external genitals called chancroid and granuloma inguinale, respectively. Those ulcers are endemic in developing countries or exist, as does granuloma inguinale, only in some geographic "hot spots."H. ducreyi is placed in the genus Haemophilus (family Pasteurellacae); however, this phylogenetic position is not obvious. The multiple ways in which the bacterium may be adapted to its econiche through specialized nutrient acquisitions; defenses against the immune system; and virulence factors that increase attachment, fitness, and persistence within genital tissue are discussed below. The analysis of K. granulomatis phylogeny demonstrated a high degree of identity with other Klebsiella species, and the name K. granulomatis comb. nov. was proposed. Because of the difficulty in growing this bacterium on artificial media, its characteristics have not been sufficiently defined. More studies are needed to understand bacterial genetics related to the pathogenesis and evolution of K. granulomatis.

Regulation of expression of the Haemophilus ducreyi LspB and LspA2 proteins by CpxR.

The LspA1, LspA2, and LspB proteins of Haemophilus ducreyi comprise a two-partner secretion system that has been shown to be necessary for H. ducreyi to inhibit phagocytosis by immune cells in vitro. Inactivation of lspA1 resulted in increased levels of LspA2, suggesting that these two proteins are differentially controlled (C. J. Ward et al., Infect. Immun. 71:2478-2486, 2003). Expression of LspA2 but not LspA1 was shown to be both growth phase dependent and affected by the presence of fetal calf serum (FCS) in the growth medium. In addition, neither LspA1 nor LspA2 could be detected in culture supernatant fluid in the absence of FCS. DNA microarray analysis revealed that 324 H. ducreyi genes were differentially regulated after growth in the presence of FCS. Among these, the CpxRA two-component sensory transduction system was downregulated by the presence of FCS. Inactivation of cpxR resulted in increased expression of both LspB and LspA2. Electrophoretic mobility shift assays showed that a recombinant H. ducreyi CpxR protein bound the promoter region of the lspB-lspA2 operon. The cpxR and cpxA genes were shown to be part of an operon containing two additional genes in H. ducreyi 35000HP. This is the first description of a two-component sensory transduction system regulating a proven virulence factor of H. ducreyi.

Experimental infection with Haemophilus ducreyi in persons who are infected with HIV does not cause local or augment systemic viral replication.

We infected 11 HIV-seropositive volunteers whose CD4(+) cell counts were >350 cells/ microL (7 of whom were receiving antiretrovirals) with Haemophilus ducreyi. The papule and pustule formation rates were similar to those observed in HIV-seronegative historical control subjects. No subject experienced a sustained change in CD4(+) cell count or HIV RNA level. The cellular infiltrate in biopsy samples obtained from the HIV-seropositive and HIV-seronegative subjects did not differ with respect to the percentage of leukocytes, neutrophils, macrophages, or T cells. The CD4(+):CD8(+) cell ratio in biopsy samples from the HIV-seropositive subjects was 1:3, the inverse of the ratio seen in the HIV-seronegative subjects (P<.0001). Although CD4(+) cells proliferated in lesions, in situ hybridization and reverse-transcription polymerase chain reaction for HIV RNA was negative. We conclude that experimental infection in HIV-seropositive persons is clinically similar to infection in HIV-seronegative persons and does not cause local or augment systemic viral replication. Thus, prompt treatment of chancroid may abrogate increases in viral replication associated with natural disease.

FrankSum: new feature selection method for protein function prediction.

In the study of in silico functional genomics, improving the performance of protein function prediction is the ultimate goal for identifying proteins associated with defined cellular functions. The classical prediction approach is to employ pairwise sequence alignments. However this method often faces difficulties when no statistically significant homologous sequences are identified. An alternative way is to predict protein function from sequence-derived features using machine learning. In this case the choice of possible features which can be derived from the sequence is of vital importance to ensure adequate discrimination to predict function. In this paper we have successfully selected biologically significant features for protein function prediction. This was performed using a new feature selection method (FrankSum) that avoids data distribution assumptions, uses a data independent measurement (p-value) within the feature, identifies redundancy between features and uses an appropriate ranking criterion for feature selection. We have shown that classifiers generated from features selected by FrankSum outperforms classifiers generated from full feature sets, randomly selected features and features selected from the Wrapper method. We have also shown the features are concordant across all species and top ranking features are biologically informative. We conclude that feature selection is vital for successful protein function prediction and FrankSum is one of the feature selection methods that can be applied successfully to such a domain.

Inhibition of phagocytosis by Haemophilus ducreyi requires expression of the LspA1 and LspA2 proteins.

Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

The lbgAB gene cluster of Haemophilus ducreyi encodes a beta-1,4-galactosyltransferase and an alpha-1,6-DD-heptosyltransferase involved in lipooligosaccharide biosynthesis.

All Haemophilus ducreyi strains examined contain a lipooligosaccharide (LOS) consisting of a single but variable branch oligosaccharide that emanates off the first heptose (Hep-I) of a conserved Hep(3)-phosphorylated 3-deoxy-D-manno-octulosonic acid-lipid A core. In a previous report, identification of tandem genes, lbgA and lbgB, that are involved in LOS biosynthesis was described (Stevens et al., Infect. Immun. 65:651-660, 1997). In a separate study, the same gene cluster was identified and the lbgB (losB) gene was found to be required for transfer of the second sugar, D-glycero-D-manno-heptose (DD-Hep), of the major branch structure (Gibson et al., J. Bacteriol. 179:5062-5071, 1997). In this study, we identified the function of the neighboring upstream gene, lbgA, and found that it is necessary for addition of the third sugar in the dominant oligosaccharide branch, a galactose-linked beta1-->4, to the DD-Hep. LOS from an lbgA mutant and an lbgAB double mutant were isolated and were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, carbohydrate analysis, mass spectrometry, and nuclear magnetic resonance spectroscopy. The results showed that the mutant strains synthesize truncated LOS glycoforms that terminate after addition of the first glucose (lbgAB) or the disaccharide DD-Hepalpha1-->6Glcbeta1 (lbgA) that is attached to the heptose core. Both mutants show a significant reduction in the ability to adhere to human keratinocytes. Although minor differences were observed after two-dimensional gel electrophoresis of total proteins from the wild-type and mutant strains, the expression levels of the vast majority of proteins were unchanged, suggesting that the differences in adherence and invasion are due to differences in LOS. These studies add to the mounting evidence for a role of full-length LOS structures in the pathophysiology of H. ducreyi infection.

Haemophilus ducreyi requires the flp gene cluster for microcolony formation in vitro.

Haemophilus ducreyi, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in H. ducreyi that encoded predicted protein products with significant homology to those encoded by the tad (for tight adhesion) locus in Actinobacillus actinomycetemcomitans that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this H. ducreyi gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the tad locus; this 15-gene cluster in H. ducreyi was designated flp. RT-PCR analysis indicated that the H. ducreyi flp gene cluster was likely to be a polycistronic operon. Mutations within the flp gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An H. ducreyi mutant with an inactivated tadA gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another H. ducreyi mutant with inactivated flp-1 and flp-2 genes was as virulent as the wild-type parent strain. These results indicate that the flp gene cluster is essential for microcolony formation by H. ducreyi, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.

Haemophilus ducreyi: clinical disease and pathogenesis.

Haemophilus ducreyi causes the sexually transmitted disease chancroid, which facilitates the transmission of HIV infection. This review focuses on recent advances in the epidemiology, diagnosis, treatment and pathogenesis of this disease.

Haemophilus ducreyi inhibits phagocytosis by U-937 cells, a human macrophage-like cell line.

Haemophilus ducreyi is a gram-negative obligate human pathogen that causes the genital ulcer disease chancroid. Chancroid lesions are deep necrotic ulcers with an immune cell infiltrate that includes macrophages. Despite the presence of these phagocytic cells, chancroid ulcers can persist for months and live H. ducreyi can be isolated from these lesions. To analyze the interaction of H. ducreyi with macrophages, we investigated the ability of H. ducreyi strain 35000 to adhere to, invade, and survive within U-937 cells, a human macrophage-like cell line. We found that although H. ducreyi strain 35000 adhered efficiently to U-937 cells, few bacteria were internalized, suggesting that H. ducreyi avoids phagocytosis by human macrophages. The few bacteria that were phagocytosed in these experiments were rapidly killed. We also found that H. ducreyi inhibits the phagocytosis of a secondary target (opsonized sheep red blood cells). Antiphagocytic activity was found in logarithmic, stationary-phase, and plate-grown cultures and was associated with whole, live bacteria but not with heat-killed cultures, sonicates, or culture supernatants. Phagocytosis was significantly inhibited after a 15-min exposure to H. ducreyi, and a multiplicity of infection of approximately 1 CFU per macrophage was sufficient to cause a significant reduction in phagocytosis by U-937 cells. Finally, all of nine H. ducreyi strains tested were antiphagocytic, suggesting that this is a common virulence mechanism for this organism. This finding suggests a mechanism by which H. ducreyi avoids killing and clearance by macrophages in chancroid lesions and inguinal lymph nodes.

The effect of temperature on the interaction of Haemophilus ducreyi with human epithelial cells.

To investigate if temperature affects the interaction of Haemophilus ducreyi with human epithelial cells, nine strains were used to evaluate the adhesion kinetics of the organism at 33 degrees C and 37 degrees C. The effect of the free toxin on the epithelial cells at those temperatures was also assessed. The cyto-adherence kinetics of H. ducreyi to the epithelial cells was significantly greater at 33 degrees C (10 times more) than at 37 degrees C in all seven clinical isolates tested. There was a significant difference in cell-associated H. ducreyi at 33 degrees C as compared with 37 degrees C. Control strains showed similar adhesion properties at both temperatures. However, the virulent strain CIP542 adhered in larger amounts than the avirulent strain A77. Electron microscopy revealed that there was more tissue necrosis at the lower than the higher temperature. The effect of the free toxin was the same at each temperature. However, strain A77 had significantly lower toxicity than strain CIP542 and the clinical isolates. These results suggest that H. ducreyi displays a temperature-dependent interaction with human epithelial cells, and this feature may play a role in the virulence of the organism in vivo. While the overall toxic effect of viable bacteria depends on the metabolic activity of the bacteria and is, therefore, higher at 33 degrees C than at 37 degrees C withthe same initial inoculum, the effect of the extracted toxin at molecular level with fixed concentrations is a temperature-independent event.

Haemophilus ducreyi associates with phagocytes, collagen, and fibrin and remains extracellular throughout infection of human volunteers.

In a previous study, Haemophilus ducreyi was found in the pustule and dermis of samples obtained at the clinical end point in the human model of infection. To understand the kinetics of localization, we examined infected sites at 0, 24, and 48 h after inoculation and at the clinical end point. Immediately after inoculation, bacteria were found predominantly in the dermis but also in the epidermis. Few bacteria were detectable at 24 h; however, by 48 h, bacteria were readily seen in the pustule and dermis. H. ducreyi was associated with polymorphonuclear leukocytes and macrophages in the pustule and at its base, but was not associated with T cells, Langerhans' cells, or fibroblasts. H. ducreyi colocalized with collagen and fibrin but not laminin or fibronectin. Association with phagocytes, collagen, and fibrin was seen as early as 48 h and persisted at the pustular stage of disease. Optical sectioning by confocal microscopy and transmission electron microscopy both failed to demonstrate intracellular H. ducreyi. These data identify collagen and fibrin as potentially important targets of adherence in vivo and strongly suggest that H. ducreyi remains extracellular throughout infection and survives by resisting phagocytic killing in vivo.

Binding of Haemophilus ducreyi to extracellular matrix proteins.

We developed an enzyme-linked immunosorbent assay-based assay to assess Haemophilus ducreyi binding to extracellular matrix (ECM) proteins. H. ducreyi 35000HP bound to fibronectin, laminin, and type I and III collagen but not to type IV, V, or VI collagen or elastin. Isogenic strains with mutations in ftpA or losB bound as well as the parent, suggesting that neither pili nor full-length lipooligosaccharide is required for H. ducreyi to bind to ECM proteins.

GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells.

The Haemophilus ducreyi homolog of GroEL, a 58.5-kDa heat shock protein (Hsp), is a dominant protein produced not only in response to heat stress but also under in vitro growth conditions. Extracellular localization of the 58.5-kDa Hsp was investigated by whole-cell enzyme-linked immunosorbent assay (ELISA) and immunoelectron microscopy and in supernatants of washed bacteria by immunoblotting with a Haemophilus ducreyi GroEL-specific mouse monoclonal antibody (BB11). To investigate binding of the Hsp to eukaryotic cells, the 58.5-kDa Hsp was purified by ion-exchange and size exclusion chromatography; incubated with HEp-2 cells, HeLa cells, and human fibroblasts; and then analyzed by immunoblotting. Direct involvement of the 58.5-kDa Hsp in the adherence of H. ducreyi to HEp-2 cells was investigated by using an inhibition assay. An epitope of the 58.5-kDa Hsp was detected by whole-cell ELISA on all of the strains tested, suggesting that it is associated with the cell surface. This was also supported by immunoelectron microscopy results. In supernatants of washed bacteria, the 58.5-kDa Hsp was detected by immunoblotting after 10 h of cultivation. The 58.5-kDa Hsp bound to the eukaryotic cells tested but exerted only limited (about 20%) inhibition of H. ducreyi adherence to HEp-2 cells. These results demonstrate that the 58.5-kDa Hsp of H. ducreyi is associated with the bacterial surface, binds to eukaryotic cells, and partially influences H. ducreyi adherence to HEp-2 cells, indicating possible involvement of the 58.5-kDa Hsp in the attachment of bacteria to host cells and to each other.