Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P ‫؍‬ 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

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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...

In women the disease will resolve and not progress to the pustular stage of disease as often as in men. The high male-to-female ratio in naturally occurring chancroid may in part reflect biological differences in gender susceptibility to disease progression, although the mechanisms responsible for this difference are unclear.

Haemophilus ducreyi produces a periplasmic copper-zinc superoxide dismutase (Cu-Zn SOD), which is thought to protect the organism from exogenous reactive oxygen species generated by neutrophils during an inflammatory response. We had previously identified the gene, sodC, responsible for the production and secretion of Cu-Zn SOD and constructed an isogenic H. ducreyi strain with a mutation in the sodC gene