Biology subjects

Kull, F. J.

Publications and source records attributed to Kull, F. J..

5 recordsLinked to original sources

bioRxiv2026

Crystal structure of the V. parahaemolyticus ToxS dimer suggests a model for bile-induced ToxRS activation

The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxR is a DNA-binding regulator associated in the periplasm with ToxS, a protein of poorly understood function. ToxS modulates the activity of its binding partner ToxR in the presence of bile salts, antimicrobial cholesterol metabolites secreted into the gut. To date, the molecular mechanism underlying this regulation remains unclear. We present crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken {beta}-barrel with a central -helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the {beta}-barrel fold, while the binding pocket tolerates substantial variation, consistent with binding hydrophobic ligands. Strikingly, we discovered that Vp-ToxSp binds three molecules of glycocholate and the presence of this bile salt leads to the formation a strand-swapped ToxS homodimer. Finally, modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp homodimer provides a structure-based model for bile salt-mediated heterotetramerization of the ToxRS system. Overall, our study addresses a major longstanding question in the field of Vibrio virulence regulation providing a scenario that could apply to other pathogens that utilize these membrane-bound family transcriptional regulators. AUTHOR SUMMARYThe ability to sense the environment is essential for bacterial pathogens to successfully colonize the human host. Many pathogenic bacteria utilize members of the family of co-component transmembrane transcription regulators (coTTRs) to regulate pathogenesis in response to host signals. The ToxRS system is a coTTR regulatory pair that is essential for host colonization and virulence regulation of the enteric pathogens Vibrio cholerae and Vibrio parahaemolyticus. This system encompasses a transcription factor, ToxR, and a binding partner of enigmatic function, ToxS. Here, we solved the structure of the ToxS periplasmic domain V. parahaemolyticus and found that it belongs to a larger family of chaperone-like proteins present in many organisms. Strikingly, we discovered that the presence of the bile salt glycocholate leads to the dimerization of the periplasmic domain of ToxS. We propose that bile salt-induced ToxS dimerization brings two ToxR cognates together forming a heterotetramer activating the regulatory system. Our results and model address a major longstanding question in the fields of Vibrio virulence regulation and could apply to other pathogens that utilize these membrane bound family transcriptional regulators.

biochemistry↗

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↗

bioRxiv2020

The structure of Rns from enterotoxigenic Escherichia coli reveals a small molecule inhibitor

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrheal disease worldwide and a frequent cause of travelers diarrhea. In addition to the production of enterotoxins, studies with human volunteers established ETEC virulence is dependent upon the production of proteinaceous adhesive pili for attaching to the intestinal wall. Although pilins are highly immunogenic, vaccines incorporating them have yet to be proven efficacious. An additional challenge for vaccines is the heterogeneity of ETEC pili, as 20 different pilus types have been identified. However, the expression of a significant number of pilus types is dependent upon Rns, an AraC family transcription factor. Furthermore, Rns also regulates the expression of the virulence factor CexE, an outer membrane coat protein. To determine how Rns functions and is regulated we solved its structure by X-ray crystallography to 3 [A] resolution. Rns forms a dimer via its N-terminal domain and its structure is consistent with the dimer binding looped DNA. Our analyses also revealed a fatty acid, decanoic acid, bound within the Rns structure. Although Rns was not known to specifically bind small molecule ligands, biochemical analysis showed decanoic acid specifically stabilized Rns in a dose dependent manner. Lac reporter assays further showed that decanoic acid inhibits Rns function at both activated and repressed promoters. In situ, exogenous decanoic acid inhibited the expression of Rns-dependent CFA/I pili and CexE in different ETEC strains. Thus, our study reveals for the first time a naturally occurring small molecule ligand specifically inhibits Rns activity and potently suppresses the expression of ETEC virulence factors. Our findings provide an alternative approach to vaccines for inhibiting ETEC pathogenesis by using the Rns structure as a framework for rational drug design.

biochemistry↗

bioRxiv2020

A disulfide constrains the ToxR periplasmic domain structure, altering its interactions with ToxS and bile-salts

ToxR is a transmembrane transcription factor that, together with its integral membrane periplasmic binding partner ToxS, is conserved across the Vibrio family. In some pathogenic Vibrios, including V. parahaemolyticus and V. cholerae, ToxR is required for bile resistance and virulence, and ToxR is fully activated and protected from degradation by ToxS. ToxS achieves this in part by ensuring formation of an intra-chain disulfide bond in the C-terminal periplasmic domain of ToxR (dbToxRp). In this study, biochemical analysis showed dbToxRp to have a higher affinity for the ToxS periplasmic domain than the non-disulfide bonded conformation. Analysis of our dbToxRp crystal structure showed this is due to disulfide bond stabilization. Furthermore, dbToxRp is structurally homologous to the V. parahaemolyticus VtrA periplasmic domain. These results highlight the critical structural role of disulfide bond in ToxR and along with VtrA define a domain fold involved in environmental sensing conserved across the Vibrio family.

microbiology↗