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.

We examined the susceptibility of Haemophilus ducreyi to antimicrobial peptides likely to be encountered in vivo during human infection. H. ducreyi was significantly more resistant than Escherichia coli to the bactericidal effects of all peptides tested. Class I and II H. ducreyi strains exhibited similar levels of resistance to antimicrobial peptides.Haemophilus ducreyi, an extracellular pathogen of human skin, encounters several cell types during human infection, including neutrophils, macrophages, and keratinocytes (3-5) that secrete cationic antimicrobial peptides (APs) (recently reviewed in reference 10). APs secreted by neutrophils include the ␣-defensins human neutrophil peptides 1 to 4 (HNP-1 to -4) and cathelicidin LL-37 (1, 7). Macrophages secrete human ␤-defensin 1 (HBD-1) and HBD-2 and LL-37, and keratinocytes secrete HBD-1 to -4 and LL-37 (1, 9, 21). Vaginal epithelial cells also secrete the ␣-defensin human defensin 5 (HD-5) (14,15). We previously demonstrated that HNP-1 to -3 are present within natural chancroidal ulcers (5). Because H. ducreyi multiplies in an environment with APs, we hypothesized that H. ducreyi resists the bactericidal effects of human APs encountered in vivo.Bactericidal assay. H. ducreyi 35000HP and Escherichia coli ML35 and their growth conditions have been described previously (2,8,12). Protegrin 1 (PG-1) was provided by Robert I. Lehrer. Other APs were purchased from PeproTech (HNP-1, HBD-2, and HBD-3) (Rocky Hill, NJ), Sigma Aldrich (HNP-2) (St. Louis, MO), Peptides International (HNP-3 and HD-5) (Louisville, KY), AnaSpec (HBD-4) (San Jose, CA), and Phoenix Pharmaceuticals (LL-37) (Belmont, CA).Mid-logarithmic-phase bacteria were suspended in 10 mM sodium phosphate buffer (pH 7.4) with 1% brain-heart infusion broth. Bacteria were mixed with the indicated peptide concentrations in a 96-well polypropylene plate (Costar 3790) and incubated for 1 h at 33°C (H. ducreyi) or 37°C (E. coli), and the remaining bacteria were quantified by plate count. Survival in the presence of APs was calculated as a percentage of the rate of survival in control wells without APs. Results were subjected to a mixed-model statistical analysis, and the Sidak adjustment was used to control for multiple comparisons (17). P values of Ͻ0.05 were considered statistically significant.Differential susceptibilities of H. ducreyi and E. coli to APs. H. ducreyi is susceptible to killing by PG-1, a porcine AP with no human homolog (8). In our assay, both H. ducreyi and E. coli exhibited Ͻ1% survival at a PG-1 concentration of 0.2 g/ml (data not shown). Thus, our assay detected AP-mediated bactericidal activity.In assays with ␣-defensins, E. coli was sensitive to HNP-1 to -3, demonstrating 10 to 30% survival at 20 g/ml (Fig. 1A to C). HD-5 was more potent against E. coli, with Ͻ1% survival at 20 g/ml and 16% survival at 2 g/ml (Fig. 1D). In contrast, H. ducreyi exhibited Ͼ88% survival at all concentrations of HNP-1 to -3 and HD-5 ( Fig. 1) and was significantly more resistant than E. coli to ␣-defensin-mediate...