Interactions of the Skin Pathogen Haemophilus ducreyi With the Human Host.

The obligate human pathogen Haemophilus ducreyi causes both cutaneous ulcers in children and sexually transmitted genital ulcers (chancroid) in adults. Pathogenesis is dependent on avoiding phagocytosis and exploiting the suppurative granuloma-like niche, which contains a myriad of innate immune cells and memory T cells. Despite this immune infiltrate, long-lived immune protection does not develop against repeated H. ducreyi infections-even with the same strain. Most of what we know about infectious skin diseases comes from naturally occurring infections and/or animal models; however, for H. ducreyi, this information comes from an experimental model of infection in human volunteers that was developed nearly three decades ago. The model mirrors the progression of natural disease and serves as a valuable tool to determine the composition of the immune cell infiltrate early in disease and to identify host and bacterial factors that are required for the establishment of infection and disease progression. Most recently, holistic investigation of the experimentally infected skin microenvironment using multiple "omics" techniques has revealed that non-canonical bacterial virulence factors, such as genes involved in central metabolism, may be relevant to disease progression. Thus, the immune system not only defends the host against H. ducreyi, but also dictates the nutrient availability for the invading bacteria, which must adapt their gene expression to exploit the inflammatory metabolic niche. These findings have broadened our view of the host-pathogen interaction network from considering only classical, effector-based virulence paradigms to include adaptations to the metabolic environment. How both host and bacterial factors interact to determine infection outcome is a current focus in the field. Here, we review what we have learned from experimental H. ducreyi infection about host-pathogen interactions, make comparisons to what is known for other skin pathogens, and discuss how novel technologies will deepen our understanding of this infection.

A Class I Haemophilus ducreyi Strain Containing a Class II hgbA Allele Is Partially Attenuated in Humans: Implications for HgbA Vaccine Efficacy Trials.

Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.

Putative vaccine candidates and drug targets identified by reverse vaccinology and subtractive genomics approaches to control Haemophilus ducreyi, the causative agent of chancroid.

Chancroid is a sexually transmitted infection (STI) caused by the Gram-negative bacterium Haemophilus ducreyi The control of chancroid is difficult and the only current available treatment is antibiotic therapy; however, antibiotic resistance has been reported in endemic areas. Owing to recent outbreaks of STIs worldwide, it is important to keep searching for new treatment strategies and preventive measures. Here, we applied reverse vaccinology and subtractive genomic approaches for the in silico prediction of potential vaccine and drug targets against 28 strains of H. ducreyi We identified 847 non-host homologous proteins, being 332 exposed/secreted/membrane and 515 cytoplasmic proteins. We also checked their essentiality, functionality and virulence. Altogether, we predicted 13 candidate vaccine targets and three drug targets, where two vaccines (A01_1275, ABC transporter substrate-binding protein; and A01_0690, Probable transmembrane protein) and three drug targets (A01_0698, Purine nucleoside phosphorylase; A01_0702, Transcription termination factor; and A01_0677, Fructose-bisphosphate aldolase class II) are harboured by pathogenicity islands. Finally, we applied a molecular docking approach to analyse each drug target and selected ZINC77257029, ZINC43552589 and ZINC67912117 as promising molecules with favourable interactions with the target active site residues. Altogether, the targets identified here may be used in future strategies to control chancroid worldwide.

Host Polymorphisms in TLR9 and IL10 Are Associated With the Outcomes of Experimental Haemophilus ducreyi Infection in Human Volunteers.

BACKGROUND: In humans inoculated with Haemophilus ducreyi, there are host effects on the possible clinical outcomes-pustule formation versus spontaneous resolution of infection. However, the immunogenetic factors that influence these outcomes are unknown. Here we examined the role of 14 single-nucleotide polymorphisms (SNPs) in 7 selected pathogen-recognition pathways and cytokine genes on the gradated outcomes of experimental infection. METHODS: DNAs from 105 volunteers infected with H. ducreyi at 3 sites were genotyped for SNPs, using real-time polymerase chain reaction. The participants were classified into 2 cohorts, by race, and into 4 groups, based on whether they formed 0, 1, 2, or 3 pustules. χ(2) tests for trend and logistic regression analyses were performed on the data. RESULTS: In European Americans, the most significant findings were a protective association of the TLR9 +2848 GG genotype and a risk-enhancing association of the TLR9 TA haplotype with pustule formation; logistic regression showed a trend toward protection for the TLR9 +2848 GG genotype. In African Americans, logistic regression showed a protective effect for the IL10 -2849 AA genotype and a risk-enhancing effect for the IL10 AAC haplotype. CONCLUSIONS: Variations in TLR9 and IL10 are associated with the outcome of H. ducreyi infection.

Defining Potential Vaccine Targets of Haemophilus ducreyi Trimeric Autotransporter Adhesin DsrA.

Haemophilus ducreyi is the causative agent of the sexually transmitted genital ulcer disease chancroid. Strains of H. ducreyi are grouped in two classes (I and II) based on genotypic and phenotypic differences, including those found in DsrA, an outer membrane protein belonging to the family of multifunctional trimeric autotransporter adhesins. DsrA is a key serum resistance factor of H. ducreyi that prevents binding of natural IgM at the bacterial surface and functions as an adhesin to fibronectin, fibrinogen, vitronectin, and human keratinocytes. Monoclonal antibodies (MAbs) were developed to recombinant DsrA (DsrA(I)) from prototypical class I strain 35000HP to define targets for vaccine and/or therapeutics. Two anti-DsrAI MAbs bound monomers and multimers of DsrA from genital and non-genital/cutaneous H. ducreyi strains in a Western blot and reacted to the surface of the genital strains; however, these MAbs did not recognize denatured or native DsrA from class II strains. In a modified extracellular matrix protein binding assay using viable H. ducreyi, one of the MAbs partially inhibited binding of fibronectin, fibrinogen, and vitronectin to class I H. ducreyi strain 35000HP, suggesting a role for anti-DsrA antibodies in preventing binding of H. ducreyi to extracellular matrix proteins. Standard ELISA and surface plasmon resonance using a peptide library representing full-length, mature DsrAI revealed the smallest nominal epitope bound by one of the MAbs to be MEQNTHNINKLS. Taken together, our findings suggest that this epitope is a potential target for an H. ducreyi vaccine.

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

UNLABELLED: Haemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways. IMPORTANCE: Phagocytosis is a critical component of the immune system that enables pathogens to be contained and cleared. A number of bacterial pathogens have developed specific strategies to either physically evade phagocytosis or block the intracellular signaling required for phagocytic activity. Haemophilus ducreyi, a sexually transmitted pathogen, secretes a 4,153-amino-acid (aa) protein (LspA1) that effectively inhibits FcγR-mediated phagocytic activity. In this study, we show that a 294-aa domain within this bacterial protein binds to C-terminal Src kinase (Csk) and stimulates its catalytic activity, resulting in a significant attenuation of Src kinase activity and consequent inhibition of phagocytosis. The ability to inhibit phagocytosis via Csk is not unique to H. ducreyi, because we found that the Helicobacter pylori CagA protein also inhibits phagocytosis in a Csk-dependent manner. Harnessing Csk to subvert the FcγR-mediated phagocytic pathway represents a new bacterial effector mechanism for circumventing the innate immune response.

Trimeric autotransporter DsrA is a major mediator of fibrinogen binding in Haemophilus ducreyi.

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. In both natural and experimental chancroid, H. ducreyi colocalizes with fibrin at the base of the ulcer. Fibrin is obtained by cleavage of the serum glycoprotein fibrinogen (Fg) by thrombin to initiate formation of the blood clot. Fg binding proteins are critical virulence factors in medically important Gram-positive bacteria. H. ducreyi has previously been shown to bind Fg in an agglutination assay, and the H. ducreyi Fg binding protein FgbA was identified in ligand blotting with denatured proteins. To better characterize the interaction of H. ducreyi with Fg, we examined Fg binding to intact, viable H. ducreyi bacteria and identified a novel Fg binding protein. H. ducreyi bound unlabeled Fg in a dose-dependent manner, as measured by two different methods. In ligand blotting with total denatured cellular proteins, digoxigenin (DIG)-Fg bound only two H. ducreyi proteins, the trimeric autotransporter DsrA and the lectin DltA; however, only the isogenic dsrA mutant had significantly less cell-associated Fg than parental strains in Fg binding assays with intact bacteria. Furthermore, expression of DsrA, but not DltA or an empty vector, rendered the non-Fg-binding H. influenzae strain Rd capable of binding Fg. A 13-amino-acid sequence in the C-terminal section of the passenger domain of DsrA appears to be involved in Fg binding by H. ducreyi. Taken together, these data suggest that the trimeric autotransporter DsrA is a major determinant of Fg binding at the surface of H. ducreyi.

Haemophilus ducreyi infection induces activation of the NLRP3 inflammasome in nonpolarized but not in polarized human macrophages.

Recognition of microbial infection by certain intracellular pattern recognition receptors leads to the formation of a multiprotein complex termed the inflammasome. Inflammasome assembly activates caspase-1 and leads to cleavage and secretion of the proinflammatory cytokines interleukin-1 beta (IL-1ß) and IL-18, which help control many bacterial pathogens. However, excessive inflammation mediated by inflammasome activation can also contribute to immunopathology. Here, we investigated whether Haemophilus ducreyi, a Gram-negative bacterium that causes the genital ulcer disease chancroid, activates inflammasomes in experimentally infected human skin and in monocyte-derived macrophages (MDM). Although H. ducreyi is predominantly extracellular during human infection, several inflammasome-related components were transcriptionally upregulated in H. ducreyi-infected skin. Infection of MDM with live, but not heat-killed, H. ducreyi induced caspase-1- and caspase-5-dependent processing and secretion of IL-1ß. Blockage of H. ducreyi uptake by cytochalasin D significantly reduced the amount of secreted IL-1ß. Knocking down the expression of the inflammasome components NLRP3 and ASC abolished IL-1ß production. Consistent with NLRP3-dependent inflammasome activation, blocking ATP signaling, K(+) efflux, cathepsin B activity, and lysosomal acidification all inhibited IL-1ß secretion. However, inhibition of the production and function of reactive oxygen species did not decrease IL-1ß production. Polarization of macrophages to classically activated M1 or alternatively activated M2 cells abrogated IL-1ß secretion elicited by H. ducreyi. Our study data indicate that H. ducreyi induces NLRP3 inflammasome activation via multiple mechanisms and suggest that the heterogeneity of macrophages within human lesions may modulate inflammasome activation during human infection.

Haemophilus ducreyi-induced interleukin-10 promotes a mixed M1 and M2 activation program in human macrophages.

During microbial infection, macrophages are polarized to classically activated (M1) or alternatively activated (M2) cells in response to microbial components and host immune mediators. Proper polarization of macrophages is critical for bacterial clearance. To study the role of macrophage polarization during Haemophilus ducreyi infection, we analyzed a panel of macrophage surface markers in skin biopsy specimens of pustules obtained from experimentally infected volunteers. Lesional macrophages expressed markers characteristic of both M1 and M2 polarization. Monocyte-derived macrophages (MDM) also expressed a mixed M1 and M2 profile of surface markers and cytokines/chemokines upon infection with H. ducreyi in vitro. Endogenous interleukin 10 (IL-10) produced by infected MDM downregulated and enhanced expression of several M1 and M2 markers, respectively. Bacterial uptake, mediated mainly by class A scavenger receptors, and activation of mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways were required for H. ducreyi-induced IL-10 production in MDM. Compared to M1 cells, IL-10-polarized M2 cells displayed enhanced phagocytic activity against H. ducreyi and similar bacterial killing. Thus, IL-10-modulated macrophage polarization may contribute to H. ducreyi clearance during human infection.

Oral immunization using HgbA in a recombinant chancroid vaccine delivered by attenuated Salmonella typhimurium SL3261 in the temperature-dependent rabbit model.

Chancroid, a sexually transmitted genital ulcer disease caused by the Gram-negative bacterium Haemophilus ducreyi, facilitates the acquisition and transmission of HIV. An effective vaccine against chancroid has not been developed. In this preliminary study, the gene encoding the H. ducreyi outer membrane hemoglobin receptor HgbA was cloned into the plasmid pTETnir15. The recombinant construct was introduced into the attenuated Salmonella typhimurium SL3261 strain and stable expression was induced in vitro under anaerobic conditions. The vaccine strain was delivered into the temperature-dependent rabbit model of chancroid by intragastric immunization as a single dose, or as three doses administered at two-weekly intervals. No specific antibody to HgbA was elicited after either dose schedule. Although the plasmid vector survived in vivo passage for up to 15 days following single oral challenge, HgbA expression was restricted to plasmid isolates recovered one day after immunization. Rabbits inoculated with the 3-dose booster regimen achieved no protective immunity from homologous challenge. These results emphasize that refinements in plasmid design to enhance a durable heterologous protein expression are necessary for the development of a live oral vaccine against chancroid.

Passive immunization with a polyclonal antiserum to the hemoglobin receptor of Haemophilus ducreyi confers protection against a homologous challenge in the experimental swine model of chancroid.

Haemophilus ducreyi, the etiologic agent of chancroid, has an obligate requirement for heme. Heme is acquired by H. ducreyi from its human host via TonB-dependent transporters expressed at its bacterial surface. Of 3 TonB-dependent transporters encoded in the genome of H. ducreyi, only the hemoglobin receptor, HgbA, is required to establish infection during the early stages of the experimental human model of chancroid. Active immunization with a native preparation of HgbA (nHgbA) confers complete protection in the experimental swine model of chancroid, using either Freund's or monophosphoryl lipid A as adjuvants. To determine if transfer of anti-nHgbA serum is sufficient to confer protection, a passive immunization experiment using pooled nHgbA antiserum was conducted in the experimental swine model of chancroid. Pigs receiving this pooled nHgbA antiserum were protected from a homologous, but not a heterologous, challenge. Passively transferred polyclonal antibodies elicited to nHgbA bound the surface of H. ducreyi and partially blocked hemoglobin binding by nHgbA, but were not bactericidal. Taken together, these data suggest that the humoral immune response to the HgbA vaccine is protective against an H. ducreyi infection, possibly by preventing acquisition of the essential nutrient heme.

Haemophilus ducreyi lipooligosaccharides induce expression of the immunosuppressive enzyme indoleamine 2,3-dioxygenase via type I interferons and tumor necrosis factor alpha in human dendritic cells.

Haemophilus ducreyi causes chancroid, a genital ulcer disease. In human inoculation experiments, most volunteers fail to clear the bacteria despite the infiltration of innate and adaptive immune cells to the infected sites. The immunosuppressive protein indoleamine 2,3-dioxygenase (IDO) is a rate-limiting enzyme in the L-tryptophan-kynurenine metabolic pathway. Tryptophan depletion and tryptophan metabolites contribute to pathogen persistence by inhibiting T cell proliferation, inducing T cell apoptosis, and promoting the expansion of FOXP3(+) regulatory T (Treg) cells. We previously found that FOXP3(+) Treg cells are enriched in experimental lesions and that H. ducreyi induced IDO transcription in dendritic cells (DC) derived from blood of infected volunteers who developed pustules. Here, we showed that enzymatically active IDO was induced in DC by H. ducreyi. Neutralizing antibodies against interferon alpha/beta receptor 2 chain (IFNAR2) and tumor necrosis factor alpha (TNF-α) inhibited IDO induction. Inhibitors of the mitogen-activated protein kinase (MAPK) p38 and nuclear factor-κB (NF-κB) also inhibited IDO expression. Neither bacterial contact with nor uptake by DC was required for IDO activation. H. ducreyi culture supernatant and H. ducreyi lipooligosaccharides (LOS) induced IDO expression, which required type I interferons, TNF-α, and the three MAPK (p38, c-Jun N-terminal kinase, and extracellular signal regulated kinase) and NF-κB pathways. In addition, LOS-induced IFN-ß activated the JAK-STAT pathway. Blocking the LOS/Toll-like receptor 4 (TLR4) signaling pathway greatly reduced H. ducreyi-induced IDO production. These findings indicate that H. ducreyi-induced IDO expression in DC is largely mediated by LOS via type I interferon- and TNF-α-dependent mechanisms and the MAPK, NF-κB, and JAK-STAT pathways.

Rapid divergence of two classes of Haemophilus ducreyi.

Haemophilus ducreyi, the etiologic agent of chancroid, expresses variants of several key virulence factors. While previous reports suggested that H. ducreyi strains formed two clonal populations, the differences between, and diversity within, these populations were unclear. To assess their variability, we examined sequence diversity at 11 H. ducreyi loci, including virulence and housekeeping genes, augmenting published data sets with PCR-amplified genes to acquire data for at least 10 strains at each locus. While sequences from all 11 loci place strains into two distinct groups, there was very little variation within each group. The difference between alleles of the two groups was variable and large at 3 loci encoding surface-exposed proteins (0.4 < K(S) < 1.3, where K(S) is divergence at synonymous sites) but consistently small at genes encoding cytoplasmic or periplasmic proteins (K(S) < 0.09). The data suggest that the two classes have recently diverged, that recombination has introduced variant alleles into at least 3 distinct loci, and that these alleles have been confined to one of the two classes. In addition, recombination is evident among alleles within, but not between, classes. Rather than clones of the same species, these properties indicate that the two classes may form distinct species.

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.

Detoxified Haemophilus ducreyi cytolethal distending toxin and induction of toxin specific antibodies in the genital tract.

Haemophilus ducreyi causes genital ulceration (chancroid), a sexually transmitted infection and still an important factor which contributes to the spread of HIV in developing countries. The bacterium produces a cytolethal distending toxin (HdCDT) causing cell cycle arrest and apoptosis/necrosis of human cells and contributes to the aggravation of ulcers. The aim of the study was to induce toxin-neutralizing antibodies in the genital tract of mice. Repeated subcutaneous (sc) immunisations with 5-10microg active HdCDT induced low levels of serum anti-HdCDT IgG without neutralizing capacity. High levels of specific IgG1 antibodies in serum and genital tract were generated after sc immunisations with 10microg formaldehyde detoxified HdCDT toxoid alone and the addition of aluminium salts or RIBI (based on the lipid A moiety) as adjuvant further increased the level of serum antibodies. A high correlation was found between elevated levels of anti-HdCDT IgG in sera, the level of neutralizing activity and the antibody level in genital tract (r=0.8). Thus, induction of high antibody levels specific to HdCDT in the genital tissue can be achieved by parenteral immunisation with the toxoid. The HdCDT toxoid can be considered as a candidate component in vaccine against chancroid.

Immunization with the Haemophilus ducreyi hemoglobin receptor HgbA with adjuvant monophosphoryl lipid A protects swine from a homologous but not a heterologous challenge.

Haemophilus ducreyi, the etiological agent of chancroid, has a strict requirement for heme, which it acquires from its only natural host, humans. Previously, we showed that a vaccine preparation containing the native hemoglobin receptor HgbA purified from H. ducreyi class I strain 35000HP (nHgbAI) and administered with Freund's adjuvant provided complete protection against a homologous challenge. In the current study, we investigated whether nHgbAI dispensed with monophosphoryl lipid A (MPL), an adjuvant approved for use in humans, offered protection against a challenge with H. ducreyi strain 35000HP expressing either class I or class II HgbA (35000HPhgbAI and 35000HPhgbAII, respectively). Pigs immunized with the nHgbAI/MPL vaccine were protected against a challenge from homologous H. ducreyi strain 35000HPhgbAI but not heterologous strain 35000HPhgbAII, as evidenced by the isolation of only strain 35000HPhgbAII from nHgbAI-immunized pigs. Furthermore, histological analysis of the lesions showed striking differences between mock-immunized and nHgbAI-immunized animals challenged with strains 35000HPhgbAI but not those challenged with strain 35000HPhgbAII. Mock-immunized pigs were not protected from a challenge by either strain. The enzyme-linked immunosorbent assay (ELISA) activity of the nHgbAI/MPL antiserum was lower than the activity of antiserum from animals immunized with the nHgbAI/Freund's vaccine; however, anti-nHgbAI from both studies bound whole cells of 35000HPhgbAI better than 35000HPhgbAII and partially blocked hemoglobin binding to nHgbAI. In conclusion, despite eliciting lower antibody ELISA activity than the nHgbAI/Freund's, the nHgbAI/MPL vaccine provided protection against a challenge with homologous but not heterologous H. ducreyi, suggesting that a bivalent HgbA vaccine may be needed.

Role played by CD4+FOXP3+ regulatory T Cells in suppression of host responses to Haemophilus ducreyi during experimental infection of human volunteers.

Haemophilus ducreyi causes chancroid, a genital ulcer disease. Among human volunteers, the majority of experimentally infected individuals fail to clear the infection and form pustules. Here, we investigated the role played by CD4(+)FOXP3(+) regulatory T (T(reg)) cells in the formation of pustules. In pustules, there was a significant enrichment of CD4(+)FOXP3(+) T cells, compared with that in peripheral blood. The majority of lesional FOXP3(+) T cells were CD4(+), CD25(+), CD127(lo/-), and CTLA-4(+). FOXP3(+) T cells were found throughout pustules but were most abundant at their base. Significantly fewer lesional CD4(+)FOXP3(+) T cells expressed interferon gamma, compared with lesional CD4(+)FOXP3(-) effector T cells. Depletion of CD4(+)CD25(+) T cells from the peripheral blood of infected and uninfected volunteers significantly enhanced proliferation of H. ducreyi-reactive CD4(+) T cells. Our results indicate that the population of CD4(+)CD25(+)CD127(lo/-)FOXP3(+) T(reg) cells are expanded at H. ducreyi-infected sites and that these cells may play a role in suppressing the host immune response to the bacterium.

Host-pathogen interplay of Haemophilus ducreyi.

PURPOSE OF REVIEW: Haemophilus ducreyi, the causative agent of the sexually transmitted infection chancroid, is primarily a pathogen of human skin. During infection, H. ducreyi thrives extracellularly in a milieu of professional phagocytes and other antibacterial components of the innate and adaptive immune responses. This review summarizes our understanding of the interplay between this pathogen and its host that leads to development and persistence of disease. RECENT FINDINGS: H. ducreyi expresses key virulence mechanisms to resist host defenses. The secreted LspA proteins are tyrosine-phosphorylated by host kinases, which may contribute to their antiphagocytic effector function. The serum resistance and adherence functions of DsrA map to separate domains of this multifunctional virulence factor. An influx transporter protects H. ducreyi from killing by the antimicrobial peptide LL37. Regulatory genes have been identified that may coordinate virulence factor expression during disease. Dendritic cells and natural killer cells respond to H. ducreyi and may be involved in determining the differential outcomes of infection observed in humans. SUMMARY: A human model of H. ducreyi infection has provided insights into virulence mechanisms that allow this human-specific pathogen to survive immune pressures. Components of the human innate immune system may also determine the ultimate fate of H. ducreyi infection by driving either clearance of the organism or an ineffective response that allows disease progression.

Immunogenic and adjuvant properties of Haemophilus ducreyi lipooligosaccharides.

Haemophilus ducreyi, the chancroid-causing bacterium, produces lipooligosaccharides (HdLOS) that comprise 5-11 partially sialylated monosaccharides. Subcutaneous immunisation of mice with 5 microg of HdLOS purified from H. ducreyi strains 4438 and 7470 induced high levels of anti-HdLOS IgG. The antibody responses displayed T-cell-independent features, and were dependent upon Toll-like receptor 4/MyD88 signalling pathways as demonstrated using knockout mice. The immunogenicity of HdLOS was found to require the intact lipid A moiety. The specificity studies of the anti-HdLOS antibodies, as revealed by absorption studies, antibody detection in ELISA, and immune thin-layer chromatography, indicated that the majority of the anti-LOS antibodies were specific for the inner core of the HdLOS. Antibodies to HdLOS failed to inhibit LOS induction of TNF-alpha release from human mononuclear cells. The adjuvanticity of HdLOS7470 was assessed in BALB/c mice that were immunised with bovine serum albumin (BSA) with or without the addition of HdLOS. The addition of 5 microg HdLOS resulted in a 10-fold increase in the total anti-BSA IgG antibody level as estimated by ELISA. The highest increase was noted for IgG2b, which contrasted with the predominantly IgG1 subclass response to immunisation with BSA alone, indicating an immunomodulatory activity of the HdLOS.

Haemophilus ducreyi LspA proteins are tyrosine phosphorylated by macrophage-encoded protein tyrosine kinases.

The LspA proteins (LspA1 and LspA2) of Haemophilus ducreyi are necessary for this pathogen to inhibit the phagocytic activity of macrophage cell lines, an event that can be correlated with a reduction in the level of active Src family protein tyrosine kinases (PTKs) in these eukaryotic cells. During studies investigating this inhibitory mechanism, it was discovered that the LspA proteins themselves were tyrosine phosphorylated after wild-type H. ducreyi cells were incubated with macrophages. LspA proteins in cell-free concentrated H. ducreyi culture supernatant fluid could also be tyrosine phosphorylated by macrophages. This ability to tyrosine phosphorylate the LspA proteins was not limited to immune cell lineages but could be accomplished by both HeLa and COS-7 cells. Kinase inhibitor studies with macrophages demonstrated that the Src family PTKs were required for this tyrosine phosphorylation activity. In silico methods and site-directed mutagenesis were used to identify EPIYG and EPVYA motifs in LspA1 that contained tyrosines that were targets for phosphorylation. A total of four tyrosines could be phosphorylated in LspA1, with LspA2 containing eight predicted tyrosine phosphorylation motifs. Purified LspA1 fusion proteins containing either the EPIYG or EPVYA motifs were shown to be phosphorylated by purified Src PTK in vitro. Macrophage lysates could also tyrosine phosphorylate the LspA proteins and an LspA1 fusion protein via a mechanism that was dependent on the presence of both divalent cations and ATP. Several motifs known to interact with or otherwise affect eukaryotic kinases were identified in the LspA proteins.