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.

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Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes sensitive to antimicrobial peptides (sap operon) in nontypeable Haemophilus influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild-type 35000HP and 35000HPsapA exposed to several human APs, including ␣-defensins, ␤-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HPsapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HPsapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P ‫؍‬ 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.

With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.Human inoculation experiments with many infectious agents have contributed to our understanding of transmission, pathogenesis, natural history, treatment, and vaccine development (12,25,31). After inoculation, some subjects may develop disease while others are asymptomatic or recover without treatment. Reinfection experiments usually have addressed whether experimental or natural infection with a pathogen affords protection against subsequent experimental challenge (5, 14,17,24,33). Reinfection trials generally have not addressed the issue of differences in host susceptibility to disease.To study Haemophilus ducreyi pathogenesis in humans, we developed an experimental model of infection in human volunteers (40). In the model, subjects are inoculated at multiple sites with strain 35000HP (HP, human passaged) via puncture wounds made in the skin of the upper arm by an allergy-testing device (6, 39). Within 24 h of inoculation, papules develop. These spontaneously resolve or progress to pustules in resemblance to the initial stages of natural chancroid. Lesion outcomes for an individual subject inoculated at multiple sites with identical suspensions of 35000HP sometimes differ in that a pustule may develop at one site while another site resolves (6, 39). Due to the fact that outcomes at different sites are not always the same, we initially used site as the unit of measurement for the calculation of papule and pustule formation rates. These analyses show a significant effect of the estimated delivered dose (EDD) on papule and pustule formation rates (4, 10). Although there is no effect of gender on papule formation, men are twice as likely to form pustules as women (consistent with the high male-to-female ratio in natural disease) (10).Throughout experimental infection, H. ducreyi colocalizes with collagen and fibrin and professional phagocytes (7,8). Fibrin and collagen deposition occur as part of the normal process of wound repair and provide a matrix for the infiltrating polymorphonuclear leukocytes (PMNs) and macrophages (7). The presence of fibrin suggests that serum transudates into the wounds. Interestingly, an isogenic mutant that lacks DsrA, an outer membrane protein which has several functions (including serum resistance) (13, 15), forms papules that do not progress to pustule formation (11). In pustules, the parent 35000HP strain is surrounded but not taken up by PMNs or macrophages. Thus, serum resistance and evasion of phagocytosis are virulence determinants in the model.In the model, some subjects form at least one pustule and other subjects reso...

A gene expression study of Haemophilus ducreyi identified the hypothetical lipoprotein HD0192, renamed here fibrinogen binder A (fgbA), as preferentially expressed in vivo. To test the role of fgbA in virulence, an isogenic fgbA mutant (35000HPfgbA) was constructed in H. ducreyi 35000HP, and six volunteers were experimentally infected with 35000HP and 35000HPfgbA. The overall pustule formation rate was 61.1% at parent sites and 22.2% at mutant sites (P = 0.019). Papules were significantly smaller at mutant sites than at parent sites (13.3 versus 37.9 mm2, P = 0.002) 24 h after inoculation. Thus, fgbA contributed significantly to virulence of H. ducreyi in humans. In vitro studies demonstrated that fgbA encodes a fibrinogen binding protein; no other fibrinogen binding proteins were identified in 35000HP. fgbA was conserved among clinical isolates of both class I and class II H. ducreyi strains, supporting the finding that fgbA is important for H. ducreyi infection.