Gain-of-function mutation in the sensor kinase CpxS modulates the antimicrobial resistance and virulence of Pseudomonas aeruginosa.

The CpxRA two-component system is a key regulator of envelope stress responses in Gram-negative bacteria. In this study, a gain-of-function mutant in Pseudomonas aeruginosa was generated by truncating the periplasmic sensing domain (PSD) of the sensor kinase CpxS in PAO1 (PAO1::cpxSΔ32-147), leading to constitutive activation of its cognate response regulator CpxR. The mutant exhibited a 2- to 4-fold increase in antimicrobial resistance while showing significantly attenuated virulence traits: swimming and swarming motility decreased by 44% and 34%, respectively, and production of pyoverdine, rhamnolipids, pyocyanin, and biofilm was reduced by 1.8-, 6.7-, 1.9-, and 3.5-fold compared to the wild-type PAO1. Cellular virulence was also impaired. The mutant exhibited significantly reduced cytotoxicity toward A549 lung epithelial cells, with the relative inhibition rate decreasing from 79.6% to 25.4% at multiplicities of infection (MOI) 100. Additionally, its intracellular replication in MH-S macrophages was reduced by 85%. In a murine acute pneumonia model, infection with PAO1::cpxSΔ32-147 resulted in only 10% lethality at 72 h, contrasting with 100% for PAO1. This attenuation correlated with lower lung bacterial burdens, reduced levels of inflammatory cytokines (TNF-α, IL-6) in bronchoalveolar lavage fluid, decreased oxidative damage, and higher antioxidant enzyme activity. All observed phenotypes were strictly CpxR-dependent. Transcriptomic analysis further revealed coordinated downregulation of genes associated with secretion systems, iron acquisition, and quorum sensing, indicating multi-pathway repression of virulence. Collectively, this study identifies the CpxRS system as a central regulator of pathogenicity in P. aeruginosa and supports its therapeutic potential as an anti-virulence target in multidrug-resistant infections.