Bile Salts Differentially Enhance Resistance of Enterohemorrhagic Escherichia coli O157:H7 to Host Defense Peptides

Abstract

During passage through the human gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) is exposed to membrane-damaging bile in the small intestine. We previously reported that EHEC treatment with a physiological bile salt mixture upregulates basRS, encoding a two-component system, and arnBCADTEF, encoding the aminoarabinose lipid A modification pathway (J. V. Kus, A. Gebremedhin, V. Dang, S. L. Tran, A. Serbanescu, and D. Barnett Foster, J Bacteriol 193: 4509-4515, 2011, https://doi.org/10.1128/JB.00200-11). The present study examined the effect of bile salt mix (BSM) treatment on EHEC resistance to three human gastrointestinal defense peptides-HD-5, HNP-1, and LL-37-as well as the role of basRS and arnT in the respective responses. After BSM treatment, EHEC resistance to HD-5 and HNP-1 was significantly increased in a BSM-, defensin dose-dependent manner. The resistance phenotype was dependent on both basRS and arnT However, the BSM treatment did not alter EHEC resistance to LL-37, even when the ompT gene, encoding an LL-37 cleavage protease, was disrupted. Interestingly, enteropathogenic E. coli, a related pathogen that infects the small intestine, showed a similar BSM-induced resistance phenotype. Using a model of EHEC infection in Galleria mellonella, we found significantly lower survival rates in wax moth larvae infected with BSM-treated wild-type EHEC than in those infected with a BSM-treated basS mutant, suggesting that treatment with a physiological BSM enhances virulence through a basS-mediated pathway. The results of this investigation provide persuasive evidence that bile salts typically encountered during transit through the small intestine can serve as an environmental cue for EHEC, enhancing resistance to several key host defense peptides.

Keywords: antimicrobial resistance; bile salts; defensins; enterohemorrhagic E. coli; gastrointestinal infection; host defense peptides.

FIG 1

Bile salt treatment enhances resistance of EHEC and EPEC to HD-5 and HNP-1. Radial diffusion assays show resistance as a function of clear-zone size with various concentrations of host defense peptides. Clear zones show extent of killing of EHEC 86-24 with HD-5 (A), EHEC EDL933 with HD-5 (B), EHEC 86-24 with HNP-1 (C), EPEC E2348/69 with HD-5 (D), and EPEC E2348/64 with HNP-1 (E), where bacteria are cultured in the absence (black bars) or the presence (gray bars) of 1.5% BSM. Results are expressed as means and standard errors of the means (3 biological replicates, 3 technical replicates). Statistical analysis is 2× ANOVA with post hoc Tukey's multiple-comparison test. **, P < 0.01; *, P < 0.05.

FIG 2

BSM-induced resistance to HD-5 and HNP-1 depends on basS and basR. Clear-zone sizes show the extent of HD-5 (0.11 μg/μl) (A) and HNP-1 (0.11 μg/μl) (B) killing of EHEC 86-24, EHEC ΔbasS, EHEC ΔbasR, and complemented strains cultured in the absence (black bars) or the presence (gray bars) of 1.5% BSM. Results are expressed as means and standard errors of the means (3 biological replicates, 3 technical replicates). Statistical analysis was 2× ANOVA with post hoc Tukey's multiple-comparison test. **, P < 0.01.

FIG 3

BSM-induced resistance to HD-5 and HNP-1 is lost in the ΔarnT strain and restored in the complemented strain. Clear-zone sizes show the extent of HD-5 (0.11 μg/μl) (A) and HNP-1 (0.11 μg/μl) killing of EHEC 86-24, ΔarnT, and ΔarnT::arnT and HNP-1 dose-dependent killing of EHEC ΔarnT (C). All strains were cultured in the absence (black bars) or the presence (gray bars) of 1.5% BSM. Results are expressed as means and standard errors of the means (3 biological replicates, 3 technical replicates). Statistical analysis is 2× ANOVA with post hoc Tukey's multiple-comparison test. *, P < 0.05.

FIG 4

BSM treatment does not induce resistance to LL-37. Clear-zone sizes show extent of LL-37 (1.0 μg/ml) killing of EHEC 86-24 WT, ΔarnT, and ΔarnT::arnT (A) and EHEC EDL933, the ompT mutant (EDL 933), and EPEC E2348/69 (B), where bacteria were cultured in the absence (black bars) or the presence (gray bars) of 1.5% BSM. Results are expressed as means and standard errors of the means (at least 3 biological replicates, 3 technical replicates). Statistical analysis was 2× ANOVA with post hoc Tukey's multiple-comparison test, *, P < 0.05; ns, not significant.

FIG 5

Survival of Galleria mellonella larvae after injection with PBS or suspensions of 10⁸ (A), 10⁷ (B), and 10⁶ (C) cells of EHEC 86-24 or the ΔbasS mutant. Results are representative of 3 biological replicates. Statistical analysis of Kaplan-Meier curves used the log-rank test. *, P < 0.05 for WT versus mutant.

FIG 6

Survival of Galleria mellonella larvae after injection with PBS or suspensions of 10⁸ (A), 10⁷ (B), and 10⁶ (C) BSM-treated bacterial cells. EHEC 86-24 and EHEC ΔbasS were grown in M9 medium and subcultured in the same medium with 1.5% BSM. Results are representative of 3 biological replicates. Statistical analysis of Kaplan-Meier curves used the log-rank test. ****, P < 0.0001, and ***, P < 0.001, for WT versus mutant.