UC Santa Cruz

Cationic antimicrobial peptides (CAMPs) are key components of innate immunity that provide defense against bacterial pathogens. Vibrio cholerae, the causative agent of cholera, colonizes the small intestine, where it encounters CAMPs produced by the host epithelium. Although several CAMP-responsive pathways have been characterized, the full complement of genetic determinants that enable V. cholerae to sense, adapt to, and survive CAMP stress remains incompletely understood. Bacterial responses to environmental stress often involve two-component signaling (TCS) systems, including the CarRS system, which is critical for CAMP resistance and is one of the few regulators known to negatively regulate biofilm formation in V. cholerae. However, the mechanisms underlying CarRS-mediated antimicrobial resistance and biofilm regulation remain poorly understood. To comprehensively define the genetic and regulatory mechanisms underlying the V. cholerae CAMP response, we combined whole-genome expression profiling with genome-wide random-barcode transposon-site sequencing (RB-Tn-seq). We challenged transposon libraries with subinhibitory concentrations of polymyxin B to identify genes required for fitness during CAMP stress. This approach identified known CAMP resistance determinants, including carRS, almEFG, and lpxN, as well as previously unreported genes involved in lipopolysaccharide core and O-antigen biosynthesis, outer membrane biogenesis, and envelope homeostasis. To further investigate CarRS, we examined the phosphorylation states of CarS and CarR to determine their influence on antimicrobial resistance and biofilm formation. These findings show that CarRS phosphorylation partially influences both phenotypic outcomes, linking this TCS activity to CAMP resistance and biofilm maintenance. Together, these findings expand the genetic framework of the V. cholerae CAMP stress response and provide new insight into how envelope homeostasis and CarRS signaling coordinate antimicrobial resistance and biofilm regulation.