The CpxR response regulator mediates the virulence ofby regulating the expression of virulence-associated genes.

Abstract

is a prominent human pathogen that has developed resistance to multiple antibiotics. While the roles of capsules and siderophores are well established, the identification of additional virulence determinants remains limited. In this study, we hypothesize that the two-component system response regulator CpxR is integral to the regulation ofvirulence via control of specific virulence-associated genes. The deletion of thegene resulted in reduced serum resistance and attenuated virulence in bothlarvae and murine infection models compared to the wild-type strain. To elucidate the repertoire of virulence-associated genes regulated by CpxR, a multidisciplinary workflow was employed, integrating RNA sequencing, real-time quantitative PCR, gene knockout strategies, serum resistance assays, and infection experiments utilizing. Among the genes identified with significantly diminished expression followingdeletion,emerged as a novel candidate virulence-associated gene. The deletion ofimpaired serum survival in both the carbapenem-resistant CRKp HS11286 and the hypervirulent hvKp ATCC43816 strains. Furthermore, the deletion ofin hvKp ATCC43816 led to significantly decreased colonization and impaired dissemination to multiple organs in murine models, corresponding with an overall reduction in virulence. The promoter region ofharbors a conserved CpxR-binding motif, which enhances promoter activity and gene transcription upon CpxR binding. Sequence alignment revealed thatencodes a member of the short-chain dehydrogenase family, and this gene is highly conserved amongstrains. These results elucidate the pivotal role of CpxR in mediating virulence inand clarify its regulatory impact on virulence-associated gene expression.IMPORTANCEThe mechanisms underlying the pathogenicity of, particularly the discovery of novel virulence factor genes, remain poorly understood. CpxR, a response regulator of the two-component system, is critical for mediating envelope stress responses. While CpxR has been implicated in the virulence of diverse bacterial pathogens, its role inremains elusive. In this study, we demonstrate that CpxR significantly enhancesvirulence in bothand murine infection models. Furthermore, we identify a previously uncharacterized virulence-associated gene, encoding a short-chain dehydrogenase/reductase oxidoreductase family member, whose expression is upregulated by CpxR. Given the clinical prevalence and antimicrobial resistance of, elucidating CpxR-dependent virulence regulation and its novel target provides a pivotal framework for developing therapeutic strategies against these challenging infections.