Pustule Formation Rates Research Articles

Formate production is dispensable for Haemophilus ducreyi virulence in human volunteers.

Haemophilus ducreyi is a causative agent of cutaneous ulcers in children who live in the tropics and of the genital ulcer disease chancroid in sexually active persons. In the anaerobic environment of abscesses and ulcers, anaerobic respiration and mixed acid fermentation (MAF) can be used to provide cellular energy. In Escherichia coli, MAF produces formate, acetate, lactate, succinate, and ethanol; however, MAF has not been studied in H. ducreyi. In human challenge experiments with H. ducreyi 35000HP, transcripts of the formate transporter FocA and pyruvate formate lyase (PflB) were upregulated in pustules compared to the inocula. We made single and double mutants of focA and pflB in 35000HP. Growth of 35000HPΔfocA was similar to 35000HP, but 35000HPΔpflB and 35000HPΔfocA-pflB had growth defects during both aerobic and anaerobic growth. Mutants lacking pflB did not secrete formate into the media. However, formate was secreted into the media by 35000HPΔfocA, indicating that H. ducreyi has alternative formate transporters. The pH of the media during anaerobic growth decreased for 35000HP and 35000HPΔfocA, but not for 35000HPΔpflB or 35000HPΔfocA-pflB, indicating that pflB is the main contributor to media acidification during anaerobic growth. We tested whether formate production and transport were required for virulence in seven human volunteers in a mutant versus parent trial between 35000HPΔfocA-pflB and 35000HP. The pustule formation rate was similar for 35000HP (42.9%)- and 35000HPΔfocA-pflB (62%)-inoculated sites. Although formate production occurs during in vitro growth and focA-pflB transcripts are upregulated during human infection, focA and pflB are not required for virulence in humans.

Dispensability of Ascorbic Acid Uptake and Utilization Encoded by ulaABCD for the Virulence of Haemophilus ducreyi in Humans.

Compared with wounded skin, ascorbic acid is enriched in pustules of humans experimentally infected with Haemophilus ducreyi. Compared with the broth-grown inocula, transcription of the H. ducreyi ulaABCD operon, which encodes genes for ascorbic acid uptake, is increased in pustules. We hypothesized that ascorbic acid uptake plays a role in H. ducreyi virulence. Five volunteers were infected with both H. ducreyi strain 35000HP and its isogenic ulaABCD deletion mutant at multiple sites; the papule and pustule formation rates of the mutant and parent strains were similar. Thus, ascorbic acid uptake is not essential for H. ducreyi virulence in humans.

Outer membrane protein P4 is not required for virulence in the human challenge model of Haemophilus ducreyi infection.

BackgroundBacterial lipoproteins often play important roles in pathogenesis and can stimulate protective immune responses. Such lipoproteins are viable vaccine candidates. Haemophilus ducreyi, which causes the sexually transmitted disease chancroid, expresses a number of lipoproteins during human infection. One such lipoprotein, OmpP4, is homologous to the outer membrane lipoprotein e (P4) of H. influenzae. In H. influenzae, e (P4) stimulates production of bactericidal and protective antibodies and contributes to pathogenesis by facilitating acquisition of the essential nutrients heme and nicotinamide adenine dinucleotide (NAD). Here, we tested the hypothesis that, like its homolog, H. ducreyi OmpP4 contributes to virulence and stimulates production of bactericidal antibodies.ResultsWe determined that OmpP4 is broadly conserved among clinical isolates of H. ducreyi. We next constructed and characterized an isogenic ompP4 mutant, designated 35000HPompP4, in H. ducreyi strain 35000HP. To test whether OmpP4 was necessary for virulence in humans, eight healthy adults were experimentally infected. Each subject was inoculated with a fixed dose of 35000HP on one arm and three doses of 35000HPompP4 on the other arm. The overall parent and mutant pustule formation rates were 52.4% and 47.6%, respectively (P = 0.74). These results indicate that expression of OmpP4 in not necessary for H. ducreyi to initiate disease or progress to pustule formation in humans. Hyperimmune mouse serum raised against purified, recombinant OmpP4 did not promote bactericidal killing of 35000HP or phagocytosis by J774A.1 mouse macrophages in serum bactericidal and phagocytosis assays, respectively.ConclusionsOur data suggest that, unlike e (P4), H. ducreyi OmpP4 is not a suitable vaccine candidate. OmpP4 may be dispensable for virulence because of redundant mechanisms in H. ducreyi for heme acquisition and NAD utilization.

Expression of the Flp proteins by Haemophilus ducreyiis necessary for virulence in human volunteers

BackgroundHaemophilus 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.ResultsWe 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).ConclusionThese 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.

A Haemophilus ducreyi CpxR Deletion Mutant Is Virulent in Human Volunteers

Haemophilus ducreyi 35000HP contains a homolog of the CpxRA 2-component signal transduction system, which controls the cell envelope stress response system in other gram-negative bacteria and regulates some important H. ducreyi virulence factors. A H. ducreyi cpxR mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule formation rate in 5 volunteers was 33% (95% confidence interval [CI], 1.3%-65.3%) at 15 parent sites and 40% (95% CI, 18.1%-61.9%) at 15 mutant sites (P = .35). Thus, the cpxR mutant was not attenuated for virulence. Inactivation of the H. ducreyi cpxR gene did not reduce the ability of this mutant to express certain proven virulence factors, including the DsrA serum resistance protein and the LspA2 protein, which inhibits phagocytosis. These results expand our understanding of the involvement of the CpxRA system in regulating virulence expression in H. ducreyi.

Haemophilus ducreyiSapA Contributes to Cathelicidin Resistance and Virulence in Humans

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 alpha-defensins, beta-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.

Inactivation of theHaemophilus ducreyi luxSGene Affects the Virulence of This Pathogen in Human Subjects

Haemophilus ducreyi 35000HP contains a homologue of the luxS gene, which encodes an enzyme that synthesizes autoinducer 2 (AI-2) in other gram-negative bacteria. H. ducreyi 35000HP produced AI-2 that functioned in a Vibrio harveyi-based reporter system. A H. ducreyi luxS mutant was constructed by insertional inactivation of the luxS gene and lost the ability to produce AI-2. Provision of the H. ducreyi luxS gene in trans partially restored AI-2 production by the mutant. The luxS mutant was compared with its parent for virulence in the human challenge model of experimental chancroid. The pustule-formation rate in 5 volunteers was 93.3% (95% confidence interval, 81.7%-99.9%) at 15 parent sites and 60.0% (95% confidence interval, 48.3%-71.7%) at 15 mutant sites (1-tailed P < .001). Thus, the luxS mutant was partially attenuated for virulence. This is the first report of AI-2 production contributing to the pathogenesis of a genital ulcer disease.

A Fibrinogen-Binding Lipoprotein Contributes to the Virulence ofHaemophilus ducreyiin Humans

A gene expression study of Haemophilus ducreyi identified the hypothetical lipoprotein HD0192, renamed here "fibrinogen binder A" (FgbA), as being preferentially expressed in vivo. To test the role played by fgbA in virulence, an isogenic fgbA mutant (35000HPfgbA) was constructed using H. ducreyi 35000HP, and 6 volunteers were experimentally infected with 35000HP or 35000HPfgbA. The overall pustule-formation rate was 61.1% at parent sites and 22.2% at mutant sites (P = .019). Papules were significantly smaller at mutant sites than at parent sites (13.3 vs. 37.9 mm(2); P = .002) 24 h after inoculation. Thus, fgbA contributed significantly to the virulence of H. ducreyi in humans. In vitro experiments 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 II H. ducreyi strains, supporting the finding that fgbA is important for H. ducreyi infection.

Evaluation of the Repertoire of the TonB‐Dependent Receptors ofHaemophilus ducreyifor Their Role in Virulence in Humans

Haemophilus ducreyi contains 3 TonB-dependent receptors: the hemoglobin receptor HgbA, which is required for virulence in humans; the heme receptor TdhA; and an uncharacterized conserved hypothetical protein TdX (HD0646). A double tdX/tdhA mutant (FX527) was constructed on the background of a human-passaged variant of strain 35000 (35000HP). Six volunteers were infected with 35000HP at 3 sites on one arm and with FX527 at 3 sites on the other. The pustule formation rate was 55.6% (95% confidence interval [CI], 35.7%-75.4%) at 18 parent-strain sites and 44.4% (95% CI, 15.0%-73.9%) at 18 mutant-strain sites (P = .51). Similar amounts of 35000HP and FX527 were recovered from pustules in semiquantitative culture. Thus, TdX and TdhA are not necessary for virulence, whereas HgbA is both necessary and sufficient for virulence in humans. The data suggest that hemoglobin is the sole source of heme/iron used by H. ducreyi in vivo and has implications for the potential of HgbA as a vaccine.

Experimental Infection withHaemophilus ducreyiin 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.

A DltA Mutant of Haemophilus ducreyi Is Partially Attenuated in Its Ability To Cause Pustules in Human Volunteers

Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

Expression of OmpP2A and OmpP2B is not required for pustule formation by Haemophilus ducreyi in human volunteers

Haemophilus ducreyi express two porin proteins, termed OmpP2A and OmpP2B. To test whether expression of OmpP2A and OmpP2B was necessary for virulence in humans, eight volunteers were experimentally infected with the parent (35000HP) in one arm and a double OmpP2A OmpP2B mutant (35000HP::P2AB) in the other arm. The pustule formation rates were 58.3% (95% CI, 33.2–83.5%) for the parent and 41.7% (95% CI, 19.3–64.0%) for the mutant ( P=0.25). Biopsy of 35000HP and 35000HP::P2AB-infected sites yielded similar amounts of bacteria in quantitative culture. These results indicate that expression of OmpP2A and OmpP2B is not necessary to initiate disease or to progress to pustule formation in humans.

Expression of the LspA1 and LspA2 proteins by Haemophilus ducreyi is required for virulence in human volunteers.

Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HP Omega 12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm(2)) than at parent sites (mean size, 39.1 mm(2)) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

A superoxide dismutase C mutant of Haemophilus ducreyi is virulent in human volunteers.

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 (35000HP-sodC-cat). Compared to the parent, the mutant does not survive in the presence of exogenous superoxide (L. R. San Mateo, M. Hobbs, and T. H. Kawula, Mol. Microbiol. 27:391-404, 1998) and is impaired in the swine model of H. ducreyi infection (L. R. San Mateo, K. L. Toffer, P. E. Orndorff, and T. H. Kawula, Infect. Immun. 67:5345-5351, 1999). To test whether Cu-Zn SOD is important for bacterial survival in vivo, six human volunteers were experimentally infected with 35000HP and 35000HP-sodC-cat and observed for papule and pustule formation. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule formation rates were 75% (95% confidence intervals [CI], 43 to 95%) at 12 parent-inoculated sites and 67% (95% CI, 41 to 88%) at 18 mutant-inoculated sites (P = 0.47). There was no significant difference in levels of H. ducreyi recovery from mutant- and parent-inoculated biopsy sites. These results suggest that expression of Cu-Zn SOD does not play a major role in the survival of this pathogen in the initial stages of experimental infection of humans.

Men are more susceptible than women to pustule formation in the experimental model of Haemophilus ducreyi infection.

Naturally occurring chancroid is usually more prevalent in men than in women. To examine whether there were gender differences in susceptibility to Haemophilus ducreyi infection by analyzing the papule and pustule formation rates for men and women who were experimentally inoculated with Haemophilus ducreyi. Ninety volunteers were included in the analysis. A total of 189 sites were available for estimation of the papule formation rate, and 166 sites for estimation of the pustule formation rates using logistic regression modeling. Although there were no gender differences in papule formation rates, the women had significantly lower rates of pustule formation than the men after adjustment for the estimated delivered dose. 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 lipooligosaccharide mutant defective in expression of beta-1,4-glucosyltransferase is virulent in humans.

The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.

Expression of cytolethal distending toxin and hemolysin is not required for pustule formation by Haemophilus ducreyi in human volunteers.

Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

DsrA-deficient mutant of Haemophilus ducreyi is impaired in its ability to infect human volunteers.

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.

Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection.

Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n = 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n = 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

An isogenic hemoglobin receptor-deficient mutant of Haemophilus ducreyi is attenuated in the human model of experimental infection.

Haemophilus ducreyi expresses a conserved hemoglobin-binding outer-membrane protein (HgbA). To test the role of HgbA in pathogenesis, we infected 9 adults with isolate 35000 and its isogenic hgbA-inactivated mutant (FX504) on their upper arms in a double-blinded, escalating dose-response study. Papules developed at similar rates at sites inoculated with the mutant or parent. The pustule-formation rate was 55% (95% confidence interval [CI], 30. 8%-78.5%) at parent sites and 0 (95% CI, 0-10.5%) at mutant sites (P<.0001). The recovery rate of H. ducreyi from surface cultures was 16% (n=142) from parent sites and 0 (n=213) from mutant sites (P<. 0001). H. ducreyi was recovered at biopsy from 6 of 7 parent sites and from 0 of 3 mutant sites. The results indicate that hemoglobin may be a critical source of heme or iron for the establishment of H. ducreyi infection in humans.