PhoP-regulated VirK acts as an accessory factor to maintain virulence in polymyxin-resistant Klebsiella pneumoniae

The genetic connection between virulence and antibiotic resistance remains poorly understood. Our previous RNA-Seq analysis of a polymyxin-resistant Klebsiella pneumoniae ATCC BAA2146 mutant identified a highly expressed VirK/YbjX family gene (kpn2146_RS17285), encoding a conserved membrane protein, designated virK (virulence required for Klebsiella pneumoniae). While PhoP-dependent antibiotic resistance is mediated through established pathways such as arn/pmr, we identify VirK as a PhoP-regulated factor specifically contributing to virulence. VirK localizes to the outer membrane and, although not involved in lipopolysaccharide modification, its deletion modestly reduced bacterial virulence in a mouse systemic infection model. Transcriptional and electrophoretic mobility shift assays demonstrated that virK is directly activated by the PhoP protein. A strong positive correlation between virK and phoP expression (r = 0.98) was also observed in multidrug-resistant clinical isolates. Since the PhoP/PhoQ two-component system mediates polymyxin resistance, its direct regulation of VirK uncovers an adaptive mechanism coupling enhanced virulence with antibiotic resistance. These findings reveal a previously unrecognized PhoP/VirK regulatory pathway that contributes to pathogenicity in polymyxin-resistant Klebsiella pneumoniae, offering new insights into bacterial evolution and suggesting that targeting PhoP/PhoQ could provide an effective strategy to combat multidrug-resistant K. pneumoniae infections.