Molecular basis of bile-salt- and iron-induced enterohaemorrhagic E. coli resistance to cationic antimicrobial peptides

Colonization of the gastrointestinal tract by enterohaemorrhagic Escherichia coli (EHEC) is critically dependent on its ability to sense and respond to various microenvironments within the host. EHEC exposure to physiologically relevant levels of bile salts upregulates the two component system, pmrAB(-), and the arnBCADTEF operon, resulting in lipopolysaccharide modification and increased resistance to the cationic antimicrobial peptide, polymyxin B (PMB). A similar pmrAB and arn-dependent PMB resistance has been observed in Salmonella enterica in the presence of ferric iron. Limiting magnesium levels and mild acid can also induce Salmonella resistance to PMB through another twocomponent system, PhoPQ and the connector protein, PmrD. This study aims to evaluate the relative contributions of a bile salt mix (BSM), iron, limiting magnesium as well as the roles of pmrAB, phoPQ and pmrD to EHEC's resistance to PMB. Killing assays show that EHEC treatment with the BSM or iron under excess magnesium and neutral pH conditions induces a pmrAB-dependent, phoPindependent PMB resistance. By contrast, exposure to limiting magnesium triggers a pmrB(-), phoPand pmrDdependent PMB resistance. The ironinduced PMB resistance is independent of phoP and pmrD under limiting magnesium conditions while the bile saltinduced PMB resistance is independent of pmrD only under non- PhoPinducing conditions. GFP-pmrD transcriptional reporter studies reveal that the limiting magnesium enhances pmrD expression, which is repressed upon additional exposure to either BSM or iron. Our results also show that exposure to mild acid enhances PMB resistance in a pmrDindependent manner and GFP reporter results confirm minimal expression of pmrD at this pH regardless of the magnesium level. This study provides novel insights into how EHEC differentially employs PmrAB, PhoPQ and PmrD to monitor and respond to bile salts, iron, acidic pH and magnesium typically encountered within the gastrointestinal tract in order to modulate its survival against cationic antimicrobial peptides.