Haemophilus ducreyi SapA 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 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.

FIG. 1.

Analysis and mutagenesis of the sap genes in H. ducreyi 35000HP. (A) Genetic map of sap-containing loci. An operon containing tyrR and sapABCD was located between ORFs HD1228 and HD1236, and the unlinked sapF-containing locus was located between ORFs HD0861 and HD0866. Arrowheads show the direction of transcription, stalked arrows represent predicted promoters, and ball-and-stick structures indicate predicted stem-loop transcriptional terminators. Primer binding sites are indicated by small arrows. (B) Operon mapping of the sapABCD locus. RT-PCR was used to amplify mRNA across junctions of the indicated adjacent genes. Lanes 1, chromosomal DNA; lanes 2, no-template control; lanes 3, RNA samples with RT; lanes 4, RNA samples with no RT added. (C) Insertion/deletion mutagenesis of sapA. A 676-bp fragment of sapA (hatched box) was replaced with a 840-bp nonpolar Kan^r cassette (gray box). This construct was inserted into the chromosome by allelic replacement as described in Materials and Methods. (D) Expression of sapA downstream of its native promoter for trans-complementation. sapA was cloned downstream of 237 bp of the untranslated sequence 5′ of tyrR for expression from the native promoter. The cloned 5′ sequence contained a predicted −35 and −10 region and ribosome binding site. Panels A, C, and D are drawn at the same scale.

FIG. 2.

sapA is expressed in vivo and conserved among H. ducreyi strains. (A) Nested RT-PCR of sapA transcripts was performed on RNA from broth culture (lanes 3 and 4) or a pustule biopsy specimen from the human model (lanes 5 and 6). Controls were included in which no RT was added to reaction mixtures (lanes 4 and 6) or no template was used (lane 2). Genomic DNA (lane 1) served as a positive control. (B) PCR amplification of sapA-containing sequence from class I and class II clinical isolates of H. ducreyi (see Table 1). A 2.032-kbp fragment was amplified from genomic DNA of each strain using primers flanking the sapA ORF in H. ducreyi 35000HP. Lanes 1 to 6 contain class I strains as follows: lane 1, 35000HP; 2, HD183; 3, HD188; 4, 82-029362; 5, 6644; 6, HD85-023233. Lanes 7 to 10 contain class II strains as follows: lane 7, CIP542-ATCC; 8, HMC112; 9, 33921; 10, DMC64. nt, no-template control.

FIG. 3.

H. ducreyi SapA confers resistance to the human cathelicidin LL-37, but not to human α- or β-defensins. Bactericidal assays comparing the percent survival of H. ducreyi 35000HP, 35000HPsapA, and 35000HPsapA/psapA exposed to the indicated concentrations of LL-37 (A and B), α-defensin HD-5 (C), or β-defensin HBD-4 (D). Data represent the means plus standard errors (error bars) for three independent assays. Percent survival values that are significantly different (P ≤ 0.01) from those of the parent strain are indicated by an asterisk.