The CpxR response regulator mediates the virulence of <i>Klebsiella pneumoniae</i> by regulating the expression of virulence-associated genes

<i>Klebsiella pneumoniae</i> 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 of <i>K. pneumoniae</i> virulence via control of specific virulence-associated genes. The deletion of the <i>cpxR</i> gene resulted in reduced serum resistance and attenuated virulence in both <i>Galleria mellonella</i> larvae 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 <i>Galleria mellonella</i>. Among the genes identified with significantly diminished expression following <i>cpxR</i> deletion, <i>KPHS_28080</i> emerged as a novel candidate virulence-associated gene. The deletion of <i>KPHS_28080</i> impaired serum survival in both the carbapenem-resistant CRKp HS11286 and the hypervirulent hvKp ATCC43816 strains. Furthermore, the deletion of <i>KPHS_28080</i> in 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 of <i>KPHS_28080</i> harbors a conserved CpxR-binding motif, which enhances promoter activity and gene transcription upon CpxR binding. Sequence alignment revealed that <i>KPHS_28080</i> encodes a member of the short-chain dehydrogenase family, and this gene is highly conserved among <i>K. pneumoniae</i> strains. These results elucidate the pivotal role of CpxR in mediating virulence in <i>K. pneumoniae</i> and clarify its regulatory impact on virulence-associated gene expression.IMPORTANCEThe mechanisms underlying the pathogenicity of <i>Klebsiella pneumoniae</i>, 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 in <i>K. pneumoniae</i> remains elusive. In this study, we demonstrate that CpxR significantly enhances <i>K. pneumoniae</i> virulence in both <i>Galleria mellonella</i> and 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 <i>K. pneumoniae</i>, elucidating CpxR-dependent virulence regulation and its novel target provides a pivotal framework for developing therapeutic strategies against these challenging infections.