RfaH is Essential for Virulence and Adaptive Responses in Yersinia pseudotuberculosis Infection

We previously suggested that increased expression of the gene encoding transcriptional antiterminator RfaH during Yersinia pseudotuberculosis transcriptional reprogramming is necessary for adapting to persistent infection. In this study, we examined the role of RfaH in virulence and bacterial physiology under infection-relevant stress conditions, and identified genes differentially regulated in the absence of RfaH in Y. pseudotuberculosis. We employed a mouse infection model and phenotypic assays to test RfaH's role in virulence and physiology, as well as RNA sequencing, including O-antigen biosynthesis-deficient strains. Our findings demonstrate that loss of RfaH significantly attenuates virulence, reducing the capacity of Y. pseudotuberculosis to establish persistent infection. RfaH expression is increased during the stationary growth phase and under various stress conditions, such as high osmolarity and bile salts, which are known to induce envelope stress. Functional assays revealed that the ?rfaH strain displayed defects in motility and increased clumping, indicating altered surface properties affecting motility. Moreover, transcriptomic profiling of the ?rfaH strain revealed a specific RfaH-dependent gene set after filtering out genes affected by O-antigen-related mutations, thereby minimizing confounding effects from surface structure alterations. These results suggest that RfaH influences a broader set of virulence and adaptation pathways beyond O-antigen regulation. Collectively, our findings suggest that RfaH is essential for the virulence and adaptive capacity of Y. pseudotuberculosis to colonize the host. This study provides insights into regulatory mechanisms that facilitate bacterial survival in hostile environments and highlights the importance of RfaH and its regulatory targets in the pathogenesis of Y. pseudotuberculosis. Overall design: We performed phenotypic assays to dissect RfaH-dependent changes during bacterial growth and establishment of persistent infection in the host. We performed massive parallel sequencing of various mutants (?rfaH, m-ops, and ?O-aB) and the wild-type (wt) strain at control (26°C) and temperature treatment (37°C) on the Illumina MiSeq sequencing platform to offer an in-depth transcriptome scenario of Y. pseudotuberculosis in response to environmental stress. The transcriptome data obtained was then used to pinpoint common as well as specific mutation related genes and pathways that may shed more light on the potential molecular mechanisms of RfaH in Y. pseudotuberculosis persistent infection