Midgett, C. | Search bioRxiv

bioRxiv2025

Characterization of the ligand binding pocket of the virulence regulator Rns, a Member of the AraC/XylS family of transcription factors

Diarrheal disease caused by Gram-negative enteric pathogens such as enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella spp., and, Salmonella spp. are a leading cause of morbidity and mortality of children; especially, in low resource nations. While progress has been made in reducing this burden, there remains a need to develop efficacious therapies. Recently, we determined the structure of Rns, a member of the AraC/XylS family that regulates the expression of pili and other virulence factors in ETEC. The structure revealed decanoic acid bound between the N- and C-terminal domains. To test the hypothesis that bound decanoic acid directly inhibits Rns, we used its structure to identify residues that are necessary for ligand binding. Removal of the positive side chains of R75 and H20 rendered Rns insensitive to fatty acid inhibition. Additionally, mutations designed to occlude decanoic acid binding also produced a variant that was fatty acid insensitive. We also observed that this variant is structurally more flexible than wildtype Rns with decanoic acid; suggesting that bound fatty acid contributes to structural rigidity. These studies precisely demonstrate Rns binding pocket residues critical for binding fatty acids and inhibition of DNA binding. This supports our hypothesis that fatty acids must bind in the binding pocket to inhibit AraC regulators. Further work by us and others suggests inhibition of AraC virulence regulators by fatty acids is a common paradigm among many bacterial pathogens. Therefore, understanding the molecular basis of this inhibition lays the groundwork for the development of small molecule therapeutics targeting enteric disease.

microbiology↗

bioRxiv2024

Vibrio cholerae CsrA controls ToxR levels by increasing the stability and translation of toxR mRNA

Regulation of colonization and virulence factor production in response to environmental cues is mediated through several regulatory factors in Vibrio cholerae, including the highly conserved RNA-binding global regulatory protein CsrA. We have shown previously that CsrA increases synthesis of the virulence-associated transcription factor ToxR in response to specific amino acids (NRES) and is required for the virulence of V. cholerae in the infant mouse model of cholera. In this study, we mapped the 5 untranslated region (5 UTR) of toxR and showed that CsrA can bind directly to an RNA sequence encompassing the 5 UTR, indicating that the regulation of ToxR levels by CsrA is direct. Consistent with this observation, the 5 UTR of toxR contains multiple putative CsrA binding sequences (GGA motifs), and mutating these motifs disrupted the CsrA-mediated increase in ToxR. Optimal binding of CsrA to a defined RNA oligonucleotide required the bridging of two GGA motifs within a single RNA strand. To determine the mechanism of CsrA regulation, we assayed toxR transcript levels, stability, and efficiency of translation. Both the amount of toxR mRNA in NRES and the stability of the toxR transcript were increased by CsrA. Using an in vitro translation assay, we further showed that synthesis of ToxR was greatly enhanced in the presence of purified CsrA, suggesting a direct role for CsrA in the translation of toxR mRNA. We propose a model in which CsrA binding to the 5 UTR of the toxR transcript promotes ribosomal access while precluding interactions with RNA-degrading enzymes. IMPORTANCEVibrio cholerae is uniquely adapted to life in marine environments as well as in the human intestinal tract. Global regulators such as CsrA, which help translate environmental cues into an appropriate cellular response, are critical for switching between these distinct environments. Understanding the pathways involved in relaying environmental signals is essential for understanding both the environmental persistence and the intestinal pathogenesis of this devastating human pathogen. In this study, we demonstrate that CsrA directly regulates synthesis of ToxR, a key virulence factor of V. cholerae. Under conditions favoring high levels of active CsrA in the cell, such as in the presence of particular amino acids, CsrA increases ToxR protein levels by binding to the toxR transcript and enhancing both its stability and translation. By responding to nutrient availability, CsrA is perfectly poised to activate the virulence gene regulatory cascade at the preferred site of colonization, the nutrient-rich small intestinal mucosa.

microbiology↗