Haemophilus ducreyi Is Resistant to Human Antimicrobial Peptides

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 <0.05 were considered statistically significant.

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 <1% survival at a PG-1 concentration of 0.2 μg/ml (data not shown). Thus, our assay detected AP-mediated bactericidal activity.

In assays with α-defensins, E. coli was sensitive to HNP-1 to -3, demonstrating 10 to 30% survival at 20 μg/ml (Fig. 1A to C). HD-5 was more potent against E. coli, with <1% survival at 20 μg/ml and 16% survival at 2 μg/ml (Fig. 1D). In contrast, H. ducreyi exhibited >88% survival at all concentrations of HNP-1 to -3 and HD-5 (Fig. 1) and was significantly more resistant than E. coli to α-defensin-mediated killing.

In β-defensin assays, <7% of E. coli survived a peptide concentration of 20 μg/ml that 26 to 66% of H. ducreyi survived (Fig. 2). At a concentration of 2 μg/ml, only 25 to 30% of E. coli but >84% of H. ducreyi survived exposure to HBD-3 and HBD-4 (Fig. 2B and C). At a peptide concentration of 20 μg/ml, which represents 5 μM of α-defensin or 2 to 4 μM of β-defensin, the β-defensins exhibited greater bactericidal activity than the α-defensins against H. ducreyi. Nevertheless, H. ducreyi was significantly more resistant than E. coli to killing by HBD-2 to -4 (Fig. 2).

LL-37 exhibited qualitatively greater activity than the defensins against E. coli and H. ducreyi. Nonetheless, the rate of survival of H. ducreyi (16%) was significantly greater than the rate of survival of E. coli (5%) at a dose of 2 μg/ml peptide (Fig. 3A). When LL-37 activity was assessed with serial twofold dilutions, the rate of survival of H. ducreyi (43%) was significantly higher than the rate of survival of E. coli (3%) at a dose of 0.5 μg/ml (Fig. 3B). These data shown that, relative to the level of resistance of E. coli, H. ducreyi resists LL-37 activity.

H. ducreyi strains comprise two phenotypic classes (20). We compared the susceptibilities of the class I strain 35000HP and the class II strain CIP542 ATCC (8) to PG-1, HNP-1, HBD-3, and LL-37. As with 35000HP, CIP542 ATCC was susceptible to PG-1, with <2% survival at a dose of 0.2 μg/ml (data not shown). 35000HP was significantly more resistant than CIP542 ATCC to HBD-3 but less resistant than CIP542 ATCC to LL-37 (Fig. 4A and B). Importantly, both the class I and class II strain exhibited resistance to all human APs tested (Fig. 4), indicating that AP resistance may represent a conserved mechanism of H. ducreyi survival.

Bactericidal assays showed low levels of killing of H. ducreyi with the β-defensins and LL-37 (Fig. 2 and 3). We thus assessed the activities of these APs in a radial diffusion assay (RDA), which enabled the calculation of a minimum effective concentration (MEC) of each peptide against each strain. The RDA was performed as previously described (8) except that salts were omitted from the agarose underlay to prevent the inactivation of salt-sensitive APs. The units of AP activity were derived from measuring the zones of inhibition surrounding AP-impregnated wells. The MEC of each peptide was defined as the x intercept of plots of AP activity over the AP concentration range (Table 1) (8, 18). If less than two concentrations exhibited activity against a strain, no x intercept could be defined, and the MEC was estimated as >158 μg/ml, the upper limit of measurable MEC in this assay.

All peptides exhibited activity against E. coli, with MECs between 2 and 18 μg/ml (Table 1). The MECs of PG-1 against the H. ducreyi strains were not significantly different than the PG-1 activity against E. coli (Student's t test; P = 0.1). With LL-37 and the β-defensins, the dose curves from the RDAs showed significantly less activity against either H. ducreyi strain than against E. coli (P < 0.001; data not shown). The MECs of LL-37 and the β-defensins against H. ducreyi exceeded the upper limits of the assay, while the MECs of the human APs against E. coli were <20 μg/ml (Table 1). These data confirm that both known classes of H. ducreyi are significantly more resistant than E. coli to LL-37 and HBD-2 to -4.

We have demonstrated that H. ducreyi is resistant to human APs likely encountered during infection. Control assays confirmed previous studies demonstrating that the organism is susceptible to PG-1, which H. ducreyi does not naturally encounter (8). The differential susceptibility of H. ducreyi to human and animal APs may contribute to both its limited host range (humans) and differences in the organism's survival in human and animal models of infection (16, 19), although a much broader panel of APs tested against more H. ducreyi strains is needed to address this hypothesis.

Pathogens have evolved various mechanisms to overcome bactericidal APs, including modulating AP production, inactivating APs, pumping APs out of the cell or into the cytoplasm, and repelling APs electrostatically (6, 11, 13). The expression of multiple AP-resistance strategies in pathogens such as Neisseria gonorrhoeae, Salmonella enterica, and Staphylococcus aureus demonstrates the importance to pathogenesis of combating APs. Future work will focus on elucidating the mechanism(s) of AP resistance in H. ducreyi.

We thank Robert I. Lehrer for providing PG-1 and helpful discussions; Stanley M. Spinola for the H. ducreyi strains; Anna Pulliam, Linden Horton, and Ashley Alexander for technical assistance; and Susan Ofner for statistical support. We thank Stanley M. Spinola and X. Frank Yang for helpful discussions and critical review of the manuscript.

This work was supported by the Developmental Awards Program of the National Institutes of Health NIAID Sexually Transmitted Infections and Topical Microbicide Cooperative Research Centers (STI-TM CRC) grants to the University of Washington (AI 31448) and Indiana University (AI 31494) and by the Indiana Genomics Initiative of Indiana University, which is supported in part by Lilly Endowment, Inc.

All authors report no financial conflicts of interest.