Identification of EnvC and Its Cognate Amidases as Novel Determinants of Intrinsic Resistance to Cationic Antimicrobial Peptides

Cationic antimicrobial peptides (CAMPs) are an essential part of the innate immune system. Some Gram-negative enteric pathogens, such as Salmonella enterica, show intrinsic resistance to CAMPs. However, the molecular basis of intrinsic resistance is poorly understood, largely due to a lack of information about the genes involved. In this study, using a microarray-based genomic technique, we screened the Keio collection of 3,985 Escherichia coli mutants for altered susceptibility to human neutrophil peptide 1 (HNP-1) and identified envC and zapB as novel genetic determinants of intrinsic CAMP resistance. In CAMP killing assays, an E. coli ΔenvC(Ec) or ΔzapB(Ec) mutant displayed a distinct profile of increased susceptibility to both LL-37 and HNP-1. Both mutants, however, displayed wild-type resistance to polymyxin B and human β-defensin 3 (HBD3), suggesting that the intrinsic resistance mediated by EnvC or ZapB is specific to certain CAMPs. A corresponding Salmonella ΔenvC(Se) mutant showed similarly increased CAMP susceptibility. The envC mutants of both E. coli and S. enterica displayed increased surface negativity and hydrophobicity, which partly explained the increased CAMP susceptibility. However, the ΔenvC(Ec) mutant, but not the ΔenvC(Se) mutant, was defective in outer membrane permeability, excluding this defect as a common factor contributing to the increased CAMP susceptibility. Animal experiments showed that the Salmonella ΔenvC(Se) mutant had attenuated virulence. Taken together, our results indicate that the role of envC in intrinsic CAMP resistance is likely conserved among Gram-negative enteric bacteria, demonstrate the importance of intrinsic CAMP resistance for full virulence of S. enterica, and provide insight into distinct mechanisms of action of CAMPs.