The ToxRS system is a member of a two-protein transmembrane transcriptional regulator family of proteins that act as critical environmental stress sensors and regulate virulence gene expression in some 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 Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxS has been shown to modulate the activity of its binding partner ToxR by binding bile salts, antimicrobial cholesterol metabolites secreted into the human gut. However, the molecular mechanism underlying this regulation is unclear. Here, we present the crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. Notably, the glycocholate-bound ToxSp structure forms a strand-swapped homodimer containing three bound glycocholate molecules. Modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp dimer provides a structure-based model for bile salt activation of the ToxRS system and suggests that ToxRS homologs may be regulated in a similar manner across diverse bacterial species. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, R21AI140740

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 β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the β-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.

Diarrheal disease caused by Gram-negative enteric pathogens, such as enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella spp., and Salmonella spp., is 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 effective 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 identified amino acid side chains predicted to be 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 block decanoic acid binding also produced a variant Rns that was fatty acid insensitive. We also observed that this variant is structurally more flexible than wildtype Rns bound to decanoic acid, suggesting that fatty acid binding contributes to structural rigidity. These studies demonstrate that Rns binding pocket residues are critical for binding fatty acids, which result in inhibition of DNA binding and support our hypothesis that fatty acids must bind in the binding pocket to inhibit other AraC regulators. Further work by us and others suggests that inhibition of AraC virulence regulators by fatty acids is a common paradigm among many bacterial pathogens. Therefore, understanding the molecular basis of inhibition lays the groundwork for the development of small molecule therapeutics targeting enteric disease. IMPORTANCE:As antimicrobial resistance increases, it is critical to develop new strategies to combat these infections. One area of concern is bacteria that cause intestinal disease such as Salmonella species, Vibrio cholerae, Shigella species, and enterotoxigenic Escherichia coli (ETEC). ETEC is a leading cause of travelers' diarrheal disease and a leading cause of mortality for children under 5 years old. To cause disease, ETEC requires the gene regulator Rns. Our previous work found that Rns was inhibited by a fatty acid. Here, we identify key features in the protein that are required for not only binding fatty acids but also for responding to them. This was done through a combination of microbiological as well as structural techniques of altered Rns proteins that can no longer bind fatty acid. Understanding how Rns is inhibited will lead to new ideas about how to target this class of proteins without causing antimicrobial resistance.

ABSTRACT Intestinal 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 regulation by CsrA, 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. IMPORTANCE Vibrio cholerae is uniquely adapted to marine environments as well as 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 the 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 in the human host, the nutrient-rich small intestinal mucosa.

One of the best studied aspects of pathogenic Vibrios are the virulence cascades that lead to the production of virulence factors and, ultimately, clinical outcomes. In this chapter, we will examine the regulation of Vibrio virulence gene networks from a structural and biochemical perspective. We will discuss the recent research into the numerous proteins that contribute to regulating virulence in Vibrio spp such as quorum sensing regulator HapR, the transcription factors AphA and AphB, or the virulence regulators ToxR and ToxT. We highlight how insights gained from these studies are already illuminating the basic molecular mechanisms by which the virulence cascade of pathogenic Vibrios unfold and contend that understanding how protein interactions contribute to the host-pathogen communications will enable the development of new antivirulence compounds that can effectively target these pathogens.

The chiral conformation that palmitoleic acid takes when it is bound to ToxT, the master regulator of virulence genes in the bacterial pathogen Vibrio cholerae, was used as inspiration to design a novel class of fatty acid mimetics. The best mimetic, based on a chiral hydrindane, was found to be a potent inhibitor of this target. The synthetic chemistry that enabled these studies was based on the sequential use of a stereoselective annulative cross-coupling reaction and dissolving metal reduction to establish the C13 and C9 stereocenters, respectively.

Enteric infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae , Shigella flexneri , and Salmonella enterica are among the most common and affect billions of people each year. These bacteria control expression of virulence factors using a network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae . In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 Å crystal structure of Rns and show it closely resembles ToxT. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Furthermore, the structure of Rns revealed the presence of a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Together, these results support our hypothesis that fatty acids regulate virulence controlling AraC family members in a common manner across a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting the ETEC Rns regulon, providing a basis for development of therapeutics against this deadly human pathogen.

Infections caused by the gram-negative bacteria enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, Shigella flexneri, and Salmonella enterica are among the most common enteric pathogens and infect billions of people each year. These bacteria control expression of virulence factors using a genetic network of transcriptional regulators, some of which are modulated by small molecules as has been shown for ToxT, an AraC family member from V. cholerae. In ETEC the expression of many types of adhesive pili is dependent upon the AraC family member Rns. We present here the 3 A crystal structure of Rns and show it closely resembles ToxT. Furthermore, Rns contains a ligand, decanoic acid, that inhibits its activity in a manner similar to the fatty acid mediated inhibition observed for ToxT and the S. enterica homologue HilD. Rns crystallized as a dimer via an interface similar to that observed in other dimeric AraC’s. Together, these results support our hypothesis that virulence controlling AraC family members are regulated by fatty acids in a common manner in a number of enteric pathogens. Furthermore, for the first time this work identifies a small molecule capable of inhibiting ETEC virulence, providing a basis for development of therapeutics against this deadly human pathogen.

ToxR is a transmembrane transcription factor that, together with its integral membrane periplasmic binding partner ToxS, is conserved across the Vibrionaceae 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 Vibrionaceae family.

Vibrio cholerae is an infectious bacterium that causes activation of transmembrane ion channels responsible for mediating the tonicity of cells in the small intestine. Activation of these ion channels results in osmotic movement into the small intestine, thus dehydrating cells and the infected individual. The transcription factor, ToxT, upregulates production of cholera toxin (CT) and the toxin coregulated pilus (TCP), key virulence factors in the progression of cholera. ToxT, a member of the AraC family of transcriptional regulators, has been crystallized in the presence of an unsaturated fatty acid (UFA) revealing a repressed form of the transcription factor in a conformation that prevents DNA binding. Two □‐helices in the DNA binding domain (DBD), □6 and □9, must be parallel to bind consecutive major grooves in the DNA. In the UFA‐bound structure, these helices are non‐parallel, supporting the model that ligand binding to ToxT prevents DNA binding. ToxT has been shown biochemically to be homodimeric when bound to DNA. However, crystallization of the DNA bound form of ToxT has been challenging due to instability of the complex. Recently, a natural variant of ToxT with increased solubility has been crystallized in the presence and absence of UFA. Crystallization of the apo form of ToxT required a lysine to alanine substitution at position 231 in the ligand‐binding pocket of the protein to prevent UFA binding. Although the structure of apo‐ToxT is highly similar to the UFA‐bound structure, the apo version was shown to be more flexible than ligand‐bound ToxT, supporting a model in which □6 and □9 in each subunit of dimeric ToxT are parallel and capable of binding to DNA. Using the structures described in this study, The Pingry School SMART (Students Modeling A Research Topic) Team used a 3D‐printer from the Milwaukee School of Engineering (MSOE) to model the apo structure and the dimeric DNA‐bound model of ToxT. These models support an in‐depth analysis of ToxT structural conformations in support of the proposed mechanism of regulation. A detailed structural understanding of ToxT may support future development of vaccines and therapeutics for cholera and other pathogens whose toxicity is regulated by AraC family members.

The AraC/XylS-family transcriptional regulator ToxT is the master virulence activator of Vibrio cholerae, the gram-negative bacterial pathogen that causes the diarrheal disease cholera. Unsaturated fatty acids (UFAs) found in bile inhibit the activity of ToxT. Crystal structures of inhibited ToxT bound to UFA or synthetic inhibitors have been reported, but no structure of ToxT in an active conformation had been determined. Here we present the 2.5 Å structure of ToxT without an inhibitor. The structure suggests release of UFA or inhibitor leads to an increase in flexibility, allowing ToxT to adopt an active conformation that is able to dimerize and bind DNA. Small-angle X-ray scattering was used to validate a structural model of an open ToxT dimer bound to the cholera toxin promoter. The results presented here provide a detailed structural mechanism for virulence gene regulation in V. cholerae by the UFA components of bile and other synthetic ToxT inhibitors.

HapR is a TetR-family transcriptional regulator that controls quorum sensing in Vibrio cholerae, the causative agent of cholera. HapR regulates the expression of hemagglutinin protease, virulence and biofilm genes. The crystal structure of wild-type HapR from V. cholerae strain O1 El Tor C6706 has previously been solved. In this study, the structure of a DNA-binding-deficient variant of HapR (HapRV2) derived from the protease-deficient V. cholerae serotype O37 strain V2 is reported. The structure reveals no structural differences compared with wild-type HapR. However, structural alignment of HapRV2 with the TetR-family member QacR in complex with its operator DNA suggests that the aspartate residue located between the regulatory and DNA-binding domains may clash with and electrostatically repel the phosphate backbone of DNA to prevent binding.

We have previously designed and synthesized small-molecule inhibitors that reduce Vibrio cholerae virulence in vitro by targeting the transcription factor ToxT. Here we report the synthesis and biological activity of derivatives of our previous bicyclic, fatty acid-like inhibitors. All of the synthesized derivatives show antivirulence activity in vitro. For the most potent compounds, a concentration of 5 μM completely inhibited ToxT-mediated tcpA expression as measured in the β-galactosidase assay. One indole compound, 3-(1-butyl-1 H-indol-7-yl)propanoic acid (8), was also effective at inhibiting intestinal colonization in the infant mouse. These modified compounds may serve as good candidates for further anti-cholera drug development.

Vibrio cholerae is responsible for the diarrheal disease cholera that infects millions of people worldwide. While vaccines protecting against cholera exist, and oral rehydration therapy is an effective treatment method, the disease will remain a global health threat until long-term solutions such as improved sanitation and access to clean water become widely available. Because of this, there is a pressing need for potent therapeutics that can either mitigate cholera symptoms, or act prophylactically to prevent the virulent effects of a cholera infection. Here we report the design, synthesis, and characterization of a set of compounds that bind and inhibit ToxT, the transcription factor that directly regulates the two primary V. cholerae virulence factors. Using the folded structure of the monounsaturated fatty acid observed in the X-ray structure of ToxT as a template, we designed ten novel compounds that inhibit the virulence cascade to a greater degree than any known inhibitor. Our findings provide a structural and functional basis for the development of viable antivirulence therapeutics that combat cholera and, potentially, other forms of bacterial pathogenic disease.

AphB is a LysR-type transcriptional regulator (LTTR) that cooperates with a second transcriptional activator, AphA, at the tcpPH promoter to initiate expression of the virulence cascade in Vibrio cholerae. Because it is not yet known whether AphB responds to a natural ligand in V. cholerae that influences its ability to activate transcription, we used a computational approach to identify small molecules that influence its activity. In silico docking was used to identify potential ligands for AphB, and saturation transfer difference nuclear magnetic resonance was subsequently employed to access the validity of promising targets. We identified a small molecule, BP-15, that specifically binds the C-terminal regulatory domain of AphB and increases its activity. Interestingly, molecular docking predicts that BP-15 does not bind in the putative primary effector-binding pocket located at the interface of RD-I and RD-II as in other LTTRs, but rather at the dimerization interface. The information gained in this study helps us to further understand the mechanism by which transcriptional activation by AphB is regulated by suggesting that AphB has a secondary ligand binding site, as observed in other LTTRs. This study also lays the groundwork for the future design of inhibitory molecules to block the V. cholerae virulence cascade, thereby preventing the devastating symptoms of cholera infection.

ToxR is a transmembrane transcription factor that is essential for virulence gene expression and human colonization by Vibrio cholerae. ToxR requires its operon partner ToxS, a periplasmic integral membrane protein, for full activity. These two proteins are thought to interact through their respective periplasmic domains, ToxRp and ToxSp. In addition, ToxR is thought to be responsive to various environmental cues, such as bile salts and alkaline pH, but how these factors influence ToxR is not yet understood. Using NMR and reciprocal pull down assays, we present the first direct evidence that ToxR and ToxS physically interact. Furthermore, using NMR and DSF, it was shown that the bile salts cholate and chenodeoxycholate interact with purified ToxRp and destabilize it. Surprisingly, bile salt destabilization of ToxRp enhanced the interaction between ToxRp and ToxSp. In contrast, alkaline pH, which is one of the factors that leads to ToxR proteolysis, decreased the interaction between ToxRp and ToxSp. Taken together, these data suggest a model whereby bile salts or other detergents destabilize ToxR, increasing its interaction with ToxS to promote full ToxR activity. Subsequently, as V. cholerae alkalinizes its environment in late stationary phase, the interaction between the two proteins decreases, allowing ToxR proteolysis to proceed.

Myosins are essential for many cellular processes including cell motility and muscle contraction. These motors have active site motifs (P-loop, switch-1, switch-2) that bind ATP and a divalent metal cofactor to coordinate ATP hydrolysis. ATP hydrolysis is activated through allosteric communication between the ATP and polymer binding sites to couple the free energy of ATP hydrolysis to force generation. Our lab seeks to understand how changes in enzymatic properties leads to different mechanical properties in molecular motors. We hypothesize that myosins depend on active site closure via switch-1 to regulate the motor-actin interaction during the ATPase cycle for productive force generation. We developed a strategy to control enzymatic activity and motility of myosin by substituting a switch-1 serine with cysteine, diminishing its interaction with the Mg+2 metal thus inhibiting ATPase activity. Substituting Mg+2 with Mn+2, which strongly interacts with cysteine, restores metal interaction and ATPase activity. Thus swapping of the divalent metals provides a direct and experimentally reversible link between switch-1 and the actin binding cleft. We have shown that the S237C mutant of non-muscle myosin II has rapid basal steady-state ATP turnover, but upon binding actin, its MgATPase is inhibited. Mn+2 relieves this inhibition and restores ATP turnover near WT values. The acto•S237C complex has slow, rate limiting MgATP binding which is rescued to near WT activity in the presence of Mn+2. Mant-ADP release experiments show the need for switch-1 interaction with the metal for tight ADP binding. Finally, to test whether this allosteric communication is necessary for force generation, we will test S237C motility in the presence of Mg2+ and Mn2+. These results support a strong reciprocal coupling of nucleotide and F-actin binding in myosin.

Formins are a class of actin binding proteins that regulate the elongation and nucleation of actin filaments and they are key to maintaining cellular morphology. The mammalian formin‐like 3 (FMNL3) protein is essential for the formation of filopodia, projections of the plasma membrane that enable cell motility. Formins contain a highly conserved formin homology 2 (FH2) domain that dimerizes through interactions of the lasso and post subdomains. These dimers bind to actin filament barbed ends to mediate elongation and prevent inhibition of filament growth by capping proteins, thereby ensuring the formation of long, linear actin filaments. The FH2 dimer is able to remain associated with the growing end of the filament during elongation through the flexibility of linker regions that connect the FH2 subunits. A previous structural study of the yeast formin Bni1p FH2 domain was found to form a complex with three adjacent actin subunits, supporting a simple stepping model of formin‐mediated actin polymerization. The X‐ray crystal structure of the FMNL3 FH2 domain was solved in complex with monomeric actin. In this structure, the FH2 dimer forms a symmetric and stable complex with only two actin monomers through primary interactions between the knob and coiled‐coil FH2 subdomains and actin. These recent findings support the existence of an additional step in the formin‐mediated processive elongation mechanism. The Pingry School 2014‐2015 SMART team has created a model to further examine the structure and function of formin FH2 domains in complex with actin.