CpxAR two-component system contributes to virulence properties of Cronobacter sakazakii

Cronobacter sakazakii (C. sakazakii) is a foodborne pathogen which threaten susceptible hosts including infants. CpxA/CpxR, a regulatory two-component system (TSC), contributes to stress response and virulence in various Gram-negative pathogens, but its role in C. sakazakii has not been thoroughly studied. In this study, we con-structed CpxA, CpxR, CpxAR deletion and complementation strains. The mutants showed weakened bacterial adhesion to and invasion of HBMEC and Caco-2, reduced intracellular survival and replication of C. sakazakii within RAW264.7 macrophages, and decreased translocation of HBMEC and Caco-2 monolayers. Mutants demonstrated lower levels of tight junction proteins disruption and reduced apoptosis and cytotoxicity in Caco-2 monolayer compared to wild type strain. CpxAR TCS deletion mutants demonstrate attenuated virulence in newborn mice, which was evidenced by fewer bacterial cells loads in tissues and organs, lower levels of intestinal epithelial barrier dysfuction and milder damages in intestinal tissues. All these phenotypes were recovered in complemented strains. In addition, qRT-PCR results showed that CpxAR TCS of C. sakazakii played roles in regulating the expression of several genes associated with bacterial virulence and cellular invasion. These findings indicate that CpxAR TCS is an important regulatory mechanism for virulence of C. sakazakii, which enrich our understanding of genetic determinants of pathogenicity of the pathogen.