Haemophilus ducreyi Is Resistant to Human Antimicrobial Peptides

Abstract

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...

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

“…We have previously shown that LL-37 and ␤-defensin resistance in the class II strain ATCC CIP542 is similar to that of 35000HP (34), suggesting that the class II mtrC allele is functional. The finding of a truncated mtrC allele in two class I clinical isolates raised the question of whether these isolates would exhibit increased sensitivity to APs and constitutive activation of CpxRA, as was observed in 35000HPmtrC.…”

“…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.…”

“…APs play a protective role by controlling bacterial infection; however, successful pathogens have developed a variety of mechanisms to resist AP-mediated killing (13,25,27). H. ducreyi is resistant to human APs found during infection, including ␣-defensins, ␤-defensins, and cathelicidin LL-37 (34). The Sap uptake transporter in H. ducreyi confers resistance to LL-37 and contributes to virulence in humans (35).…”

Haemophilus ducreyiSapA Contributes to Cathelicidin Resistance and Virulence in Humans

“…The 96-well AP bactericidal assay was performed as previously described (38). Briefly, bacteria were grown to mid-logarithmic phase and suspended in 10 mM sodium phosphate (pH 7.4) supplemented with 0.1% brain heart infusion broth (BD).…”

“…Dual staining for H. ducreyi and HNP-1, HNP-2, and HNP-3 demonstrate that H. ducreyi is exposed to APs at the papular, pustular, and ulcerative stages of disease (10; C. A. Townsend and M. E. Bauer, unpublished data). H. ducreyi resists the bactericidal effects of several human APs, including those predicted to be at the site of infection; this resistance has been observed in representative members of two phenotypic H. ducreyi classes (38,51). The mechanism(s) by which H. ducreyi evades AP-mediated killing is unknown.…”