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Abstract
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 ΔenvCEc or ΔzapBEc 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 ΔenvCSe 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 ΔenvCEc mutant, but not the ΔenvCSe 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 ΔenvCSe 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.
Highlights
Antimicrobial peptides are an essential part of innate immune systems in vertebrates [1]
We attempted to uncover genes required for intrinsic resistance to human neutrophil peptide 1 (HNP-1), and we identified two genes previously unknown to be involved in resistance to cationic antimicrobial peptides (CAMPs): envC and zapB
To identify the gene(s) involved in bacterial intrinsic resistance to human neutrophil peptide 1 (HNP-1), we screened the E. coli Keio collection of 3,985 nonessential deletion mutants by using a previously described microarray-based genomic method called monitoring of gene knockouts (MGK) [21], which simultaneously monitors the abundances of individual mutants within a library of mutants grown together as a population
Summary
Antimicrobial peptides are an essential part of innate immune systems in vertebrates [1]. Structurally distinct, ␣-helical LL-37 and -sheet HNP-1 (a representative ␣-defensin) share common properties, such as amphipathicity and cationicity, and both belong to a large family of cationic antimicrobial peptides (CAMPs). Due to their overall positive charges and amphipathic properties, CAMPs are thought to interact electrostatically with negatively charged molecules on the bacterial cell surface, e.g., lipopolysaccharides (LPS) in Gram-negative bacteria, and subsequently to target negatively charged cytoplasmic membranes to cause bacterial cell death [2]. PhoP-activated genes confer resistance to a broader spectrum of CAMPs, including polymyxin B, certain ␣-helical CAMPs (such as LL-37 and C18G) [14], and the -sheet rabbit defensin NP-1 [15]
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