Haemophilus ducreyiSapA Contributes to Cathelicidin Resistance and Virulence in Humans

Abstract

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.

Rapid Divergence of Two Classes of Haemophilus ducreyi

“…To expand this data set, we determined the sequences of the wecA, pal, murC, lspA2, and recA loci for 9 class I and class II strains and the ncaA locus for 6 class I strains of H. ducreyi (Table 1). This data set was further augmented by the sequences of the mtrC, sapA, and fgbA genes (3,29,35), kindly provided by Margaret Bauer. Thus, the total data set included sequences at 11 unlinked loci for at least 10 strains with broad global distributions ( Table 1).…”

Deletion of mtrC in Haemophilus ducreyi Increases Sensitivity to Human Antimicrobial Peptides and Activates the CpxRA Regulon

“…AP assays were performed as described previously (34,35). Briefly, H. ducreyi strains were grown to mid-logarithmic phase, harvested, washed, and diluted in 10 mM sodium phosphate buffer, pH 7.4, supplemented with 1% Columbia broth.…”

“…Bacterial strains are listed in Table 1. Except where noted, H. ducreyi strains were grown at 33°C with 5% CO 2 on chocolate agar plates supplemented with 1% IsoVitaleX and, for strains harboring plasmid vectors or antibiotic resistance cassettes, appropriate antibiotics, including spectinomycin (200 g/ml), kanamycin (20 g/ml), or streptomycin (100 g/ml) (35). For antimicrobial assays, strains were grown in Columbia broth supplemented with hemin (50 g/ml) (Aldrich Chemical Co., Milwaukee, WI), 5% heat-inactivated fetal bovine serum (HyClone, Logan, UT), 1% IsoVitaleX, and half the concentration of appropriate antibiotics.…”

“…Growth curves indicated no significant changes in growth rates compared with the parent strain (data not shown). Outer membrane protein preparations and Western blot analysis were performed as described previously (35). Densitometry of scanned immunoblots was performed using NIH ImageJ software (2).…”

“…The Sap uptake transporter in H. ducreyi confers resistance to LL-37 and contributes to virulence in humans (35). Interestingly, the H. ducreyi Sap transporter does not confer resistance to ␣-or ␤-defensins, suggesting that H. ducreyi possesses other mechanisms to survive attack by defensins (35).…”