Molecular mechanism underlying YibT-mediated RpoS regulation of virulence variation in Salmonella typhimurium.

Abstract

Salmonella typhimurium, a Gram-negative pathogen widely distributed in the environment, poses a serious threat to public health by contaminating food and causing foodborne diseases. Our preliminary study found that under thymol stress, the expression of YibT, a poorly characterized factor in salmonella, was significantly reduced, and deletion of the yibT gene markedly impaired biofilm formation. However, the biological role of yibT in salmonella pathogenesis remains unclear. In this study, the λ-Red homologous recombination system and pET28a vector were used to construct the single deletion mutant STΔrpoS, the double deletion mutant STΔrpoSΔyibT, complementation strains, and an overexpression strain. Promoter reporter vectors were constructed using pKP302. Growth characteristics, biofilm formation, adhesion, invasion, virulence, and pathogenicity were evaluated. The regulatory relationship between rpoS and yibT was examined by β-galactosidase assays. Results showed that deletion of rpoS and yibT led to slow growth, reduced biofilm formation, and decreased flagella and surface appendages under transmission electron microscopy. qRT-PCRshowed that there is a negative feedback loop between yibT and rpoS, where RpoS positively regulates yibT transcription and YibT negatively regulates RpoS expression. Preliminary detection of β-galactosidase confirms that yibT may be located downstream of rpoS. Cellular infection models demonstrated that deletion of yibT and rpoS significantly reduced salmonella adhesion, invasion, and intracellular survival. In mouse infection experiments, bacterial loads in the liver, spleen, and ileum were markedly decreased in mutant-infected mice, with further reduction under quercetin intervention. Flow cytometry analysis of T-cell subsets suggested attenuated immune modulation by the mutant strains, indicating reduced pathogenicity. Collectively, yibT contributes to regulate adhesion and invasion of S. typhimurium, and its deletion attenuates bacterial virulence and host pathogenicity. YibT may participate in the virulence regulatory network by interacting with the transcriptional regulator RpoS. This study provides a theoretical basis for elucidating the molecular mechanism by which YibT modulates salmonella pathogenicity.