sRNA-regulated S-glutathionylation controls Vibrio cholerae virulence

PROJECT SUMMARY Vibrio cholerae is a human pathogen that colonizes small intestines, resulting in the onset of a severe diarrheal disease known as cholera. Once reaching the small intestine, V. cholerae establishes colonization through virulence gene activation by the AraC-type master virulence regulator ToxT. Additionally, V. cholerae must cope with many host defense attacks during infection, including oxidative stress from reactive oxygen species (ROS). Small regulatory RNAs (sRNAs) are key posttranscriptional regulators that assist bacteria for rapid responses to stimuli. In our preliminary studies, we found that two sRNAs can repress expression of grxA, encoding a glutaredoxin that responds to ROS stress by catalysis of disulfide bond reduction and protein de- glutathionylation. S-glutathionylation, a reversible posttranslational protein modification of thiol groups of cysteine with glutathione, can alter protein function and/or stability. We also found that the master virulence regulator ToxT was glutathionylated (ToxT-SSG) and GrxA overproduction significantly reduced ToxT glutathionylation. Moreover, dysregulation of grxA reduced V. cholerae virulence gene expression in vitro and gut colonization in the infant mouse model, suggesting sRNA-mediated signaling cascades may contribute to V. cholerae virulence via protein S-glutathionylation. We therefore hypothesize that host signals, such as ROS, induce sRNA production, and sRNAs posttranscriptionally inhibit production of GrxA, a protein de- glutathionylation enzyme, protect the active pool of the S-glutathionylated virulence activator ToxT, thus contributing to the spatio-temporal regulation of V. cholerae pathogenesis. To test this hypothesis, we will first determine how sRNAs regulate grxA in Aim 1. We will identify and characterize sRNA regulators of grxA using a candidate approach and an unbiased systems approach rGRIL-Seq that relies on in vivo proximity ligation of sRNAs bound to their targets, selective enrichment and sequencing. We will also investigate host signals that activate sRNAs for grxA regulation. In Aim 2, we will examine the effects of GrxA-regulated S-glutathionylation on V. cholerae virulence. Our preliminary studies also found that exogenous glutathione (GSH) reduced virulence gene expression. GrxA uses GSH as reducing power to deglutathionate modified cysteines. We thus hypothesize that glutathionylation promotes ToxT function and V. cholerae use lumenal GSH as a spatio- temporal cue to guide its colonization. We will examine how glutathionylation affects ToxT function/stability. We will also examine GrxA impacts on glutathionylation of other proteins using targeted proteomics. Finally, we will use imaging mass cytometry to examine effects of the lumenal GSH on virulence. The proposed studies will reveal new posttranscriptional sRNA signaling in V. cholerae redox sensing and regulation and uncover the posttranslational protein S-glutathionylation in V. cholerae virulence control, expanding V. cholerae virulence control beyond the extensively studied gene transcription.