Characterization of CarR‐CarS Two‐Component System Involved in Polymyxin B Resistance of Vibrio vulnificus

Enteropathogenic bacteria inevitably encounter cationic antimicrobial peptides (CAMPs) produced by the host immune defense system. The CAMPs bind to negatively charged lipid A molecules of lipopolysaccharide on the bacterial cell surface through electrostatic interaction, leading to increased bacterial membrane permeability and cell lysis. It is known that a fulminating human pathogen Vibrio vulnificus is resistant to the CAMP polymyxin B (Pm). However, the mechanisms by which V. vulnificusdevelops Pm resistance are still elusive. To identify transcription factors conferring Pm resistance on V. vulnificus, a library of knock-out mutants lacking putative transcription-factor genes was constructed and screened for the Pm-sensitive mutants. From the screening, the VVMO6_RS15995 gene (carRVv ) encoding a response regulator homologous to Vibrio cholerae CarR (CarRVc ) was discovered to be critical for the Pm resistance of V. vulnificus. CarRVv composes a two-component system with a histidine kinase CarSVv whose gene VVMO6_RS15990 is located next to carRVv . Of note, homologues of CarRVc -regulated almEFG that enhance V. cholerae Pm resistance through glycine modification of lipid A were not found in V. vulnificus genome. Instead, qRT-PCR revealed that CarRVv acts as a positive regulator for the expression of VVMO6_RS15980 encoding a putative phosphoethanolamine transferase EptA as well as VVMO6_RS15985 and VVMO6_RS15975 genes that exist as an operon adjacent to carSVv . Biochemical analyses demonstrated that the products of this eptA operon are required for Pm resistance of V. vulnificus presumably via lipid A modification. Electrophoretic mobility shift assay and DNase I protection assay indicated that CarRVv regulates the expression of the eptA operon by directly binding to its upstream region. In addition, mutational analyses showed that each amino acid residue involved in the phosphorelay signal transduction of the CarR-CarS two-component system plays an important role in the activation of the eptA operon and the development of Pm resistance. The combined results suggest that the CarR-CarS two-component system of V. vulnificus could contribute to the bacterial survival against host-derived CAMPs by mechanisms different from those of V. cholerae.