# sRNA-regulated S-glutathionylation controls Vibrio cholerae virulence

> **NIH NIH R21** · UNIVERSITY OF PENNSYLVANIA · 2024 · $200,925

## Abstract

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 virule...

## Key facts

- **NIH application ID:** 10763882
- **Project number:** 5R21AI175874-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Jun Zhu
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $200,925
- **Award type:** 5
- **Project period:** 2023-01-15 → 2025-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10763882

## Citation

> US National Institutes of Health, RePORTER application 10763882, sRNA-regulated S-glutathionylation controls Vibrio cholerae virulence (5R21AI175874-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10763882. Licensed CC0.

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