Jeffrey H. Withey

Bicarbonate Induces Vibrio cholerae Virulence Gene Expression by Enhancing ToxT Activity

ABSTRACT Vibrio cholerae is a gram-negative bacterium that is the causative agent of cholera, a severe diarrheal illness. The two biotypes of V. cholerae O1 capable of causing cholera, classical and El Tor, require different in vitro growth conditions for induction of virulence gene expression. Growth under the inducing conditions or infection of a host initiates a complex regulatory cascade that results in production of ToxT, a regulatory protein that directly activates transcription of the genes encoding cholera toxin (CT), toxin-coregulated pilus (TCP), and other virulence genes. Previous studies have shown that sodium bicarbonate induces CT expression in the V. cholerae El Tor biotype. However, the mechanism for bicarbonate-mediated CT induction has not been defined. In this study, we demonstrate that bicarbonate stimulates virulence gene expression by enhancing ToxT activity. Both the classical and El Tor biotypes produce inactive ToxT protein when they are cultured statically in the absence of bicarbonate. Addition of bicarbonate to the culture medium does not affect ToxT production but causes a significant increase in CT and TCP expression in both biotypes. Ethoxyzolamide, a potent carbonic anhydrase inhibitor, inhibits bicarbonate-mediated virulence induction, suggesting that conversion of CO2 into bicarbonate by carbonic anhydrase plays a role in virulence induction. Thus, bicarbonate is the first positive effector for ToxT activity to be identified. Given that bicarbonate is present at high concentration in the upper small intestine where V. cholerae colonizes, bicarbonate is likely an important chemical stimulus that V. cholerae senses and that induces virulence during the natural course of infection.

Zebrafish as a Natural Host Model for Vibrio cholerae Colonization and Transmission

ABSTRACT The human diarrheal disease cholera is caused by the aquatic bacterium Vibrio cholerae. V. cholerae in the environment is associated with several varieties of aquatic life, including insect egg masses, shellfish, and vertebrate fish. Here we describe a novel animal model for V. cholerae, the zebrafish. Pandemic V. cholerae strains specifically colonize the zebrafish intestinal tract after exposure in water with no manipulation of the animal required. Colonization occurs in close contact with the intestinal epithelium and mimics colonization observed in mammals. Zebrafish that are colonized by V. cholerae transmit the bacteria to naive fish, which then become colonized. Striking differences in colonization between V. cholerae classical and El Tor biotypes were apparent. The zebrafish natural habitat in Asia heavily overlaps areas where cholera is endemic, suggesting that zebrafish and V. cholerae evolved in close contact with each other. Thus, the zebrafish provides a natural host model for the study of V. cholerae colonization, transmission, and environmental survival.

Mechanism for Inhibition of Vibrio cholerae ToxT Activity by the Unsaturated Fatty Acid Components of Bile

UNLABELLED The Gram-negative curved bacillus Vibrio cholerae causes the severe diarrheal illness cholera. During host infection, a complex regulatory cascade results in production of ToxT, a DNA-binding protein that activates the transcription of major virulence genes that encode cholera toxin (CT) and toxin-coregulated pilus (TCP). Previous studies have shown that bile and its unsaturated fatty acid (UFA) components reduce virulence gene expression and therefore are likely important signals upon entering the host. However, the mechanism for the bile-mediated reduction of TCP and CT expression has not been clearly defined. There are two likely hypotheses to explain this reduction: (i) UFAs decrease DNA binding by ToxT, or (ii) UFAs decrease dimerization of ToxT. The work presented here elucidates that bile or UFAs directly affect DNA binding by ToxT. UFAs, specifically linoleic acid, can enter V. cholerae when added exogenously and are present in the cytoplasm, where they can then interact with ToxT. Electrophoretic mobility shift assays (EMSAs) with ToxT and various virulence promoters in the presence or absence of UFAs showed a direct reduction in ToxT binding to DNA, even in promoters with only one ToxT binding site. Virstatin, a synthetic ToxT inhibitor, was previously shown to reduce ToxT dimerization. Here we show that virstatin affects DNA binding only at ToxT promoters with two binding sites, unlike linoleic acid, which affects ToxT binding promoters having either one or two ToxT binding sites. This suggests a mechanism in which UFAs, unlike virstatin, do not affect dimerization but affect monomeric ToxT binding to DNA. IMPORTANCE Vibrio cholerae must produce the major virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause cholera. CT and TCP production depends on ToxT, the major virulence transcription activator. ToxT activity is negatively regulated by unsaturated fatty acids (UFAs) present in the lumen of the upper small intestine. This study investigated the mechanism for inhibition of ToxT activity by UFAs and found that UFAs directly reduce specific ToxT binding to DNA at virulence promoters and subsequently reduce virulence gene expression. UFAs inhibit ToxT monomers from binding DNA. This differs from the inhibitory mechanism of a synthetic ToxT inhibitor, virstatin, which inhibits ToxT dimerization. Understanding the mechanisms for inhibition of virulence could lead to better cholera therapeutics.

Zebrafish as a model for zoonotic aquatic pathogens.

Aquatic habitats harbor a multitude of bacterial species. Many of these bacteria can act as pathogens to aquatic species and/or non-aquatic organisms, including humans, that come into contact with contaminated water sources or colonized aquatic organisms. In many instances, the bacteria are not pathogenic to the aquatic species they colonize and are only considered pathogens when they come into contact with humans. There is a general lack of knowledge about how the environmental lifestyle of these pathogens allows them to persist, replicate and produce the necessary pathogenic mechanisms to successfully transmit to the human host and cause disease. Recently, the zebrafish infectious disease model has emerged as an ideal system for examining aquatic pathogens, both in the aquatic environment and during infection of the human host. This review will focus on how the zebrafish has been used successfully to analyze the pathogenesis of aquatic bacterial pathogens.

Flexibility of Vibrio cholerae ToxT in transcription activation of genes having altered promoter spacing.

Cholera, a severe diarrheal disease, is caused by ingestion of the gram-negative bacterium Vibrio cholerae. Expression of V. cholerae virulence factors is highly regulated at the transcriptional and posttranscriptional levels by a complex network of proteins and small noncoding RNAs. The direct activator of transcription of most V. cholerae virulence genes is the ToxT protein. ToxT binds to a 13-bp sequence, the toxbox, located upstream of genes in its regulon. However, the organization of toxboxes relative to each other and to the core promoter elements at different genes varies dramatically. At different ToxT-activated genes a single toxbox may be necessary and sufficient for full activation, or pairs of toxboxes organized as either inverted or direct repeats may be required for full activation. Although all toxboxes are located at positions consistent with a class I promoter architecture, the locations of toxboxes relative to the transcription start site also vary from gene to gene. To further assess the ability of ToxT to activate transcription from different configurations relative to the core promoter elements, we constructed promoter-lacZ fusions having altered spacing both between toxbox pairs and between the promoter-proximal toxbox and the -35 box at five different ToxT-activated promoters. Our results suggest that that ToxT has remarkable flexibility in its positioning as a transcription activator and that different interactions between ToxT and RNA polymerase occur during transcription activation of promoters having different toxbox configurations.

A Metalloprotease Secreted by the Type II Secretion System Links Vibrio cholerae with Collagen

Vibrio cholerae is autochthonous to various aquatic niches and is the etiological agent of the life-threatening diarrheal disease cholera. The persistence of V. cholerae in natural habitats is a crucial factor in the epidemiology of cholera. In contrast to the well-studied V. cholerae-chitin connection, scarce information is available about the factors employed by the bacteria for the interaction with collagens. Collagens might serve as biologically relevant substrates, because they are the most abundant protein constituents of metazoan tissues and V. cholerae has been identified in association with invertebrate and vertebrate marine animals, as well as in a benthic zone of the ocean where organic matter, including collagens, accumulates. Here, we describe the characterization of the V. cholerae putative collagenase, VchC, encoded by open reading frame VC1650 and belonging to the subfamily M9A peptidases. Our studies demonstrate that VchC is an extracellular collagenase degrading native type I collagen of fish and mammalian origin. Alteration of the predicted catalytic residues coordinating zinc ions completely abolished the protein enzymatic activity but did not affect the translocation of the protease by the type II secretion pathway into the extracellular milieu. We also show that the protease undergoes a maturation process with the aid of a secreted factor(s). Finally, we propose that V. cholerae is a collagenovorous bacterium, as it is able to utilize collagen as a sole nutrient source. This study initiates new lines of investigations aiming to uncover the structural and functional components of the V. cholerae collagen utilization program.

Bicarbonate Increases Binding Affinity of Vibrio cholerae ToxT to Virulence Gene Promoters

The major Vibrio cholerae virulence gene transcription activator, ToxT, is responsible for the production of the diarrhea-inducing cholera toxin (CT) and the major colonization factor, toxin coregulated pilus (TCP). In addition to the two primary virulence factors mentioned, ToxT is responsible for the activation of accessory virulence genes, such as aldA, tagA, acfA, acfD, tcpI, and tarAB. ToxT activity is negatively modulated by bile and unsaturated fatty acids found in the upper small intestine. Conversely, previous work identified another intestinal signal, bicarbonate, which enhances the ability of ToxT to activate production of CT and TCP. The work presented here further elucidates the mechanism for the enhancement of ToxT activity by bicarbonate. Bicarbonate was found to increase the activation of ToxT-dependent accessory virulence promoters in addition to those that produce CT and TCP. Bicarbonate is taken up into the V. cholerae cell, where it positively affects ToxT activity by increasing DNA binding affinity for the virulence gene promoters that ToxT activates regardless of toxbox configuration. The increase in ToxT binding affinity in the presence of bicarbonate explains the elevated level of virulence gene transcription.

Termination of Vibrio cholerae virulence gene expression is mediated by proteolysis of the major virulence activator, ToxT

Vibrio cholerae is the causative agent of cholera, a severe diarrhoeal illness. V. cholerae produces two major virulence factors: the cholera toxin, which directly causes diarrhoea, and the toxin‐coregulated pilus, which is required for intestinal colonization. Production of these virulence factors is dependent on the major virulence regulator, ToxT. Under virulence‐inducing growth conditions, transcription factors ToxR and TcpP initially activate transcription of toxT. However, once ToxT has been expressed, it produces more of itself independent of ToxR and TcpP by activating transcription of the long tcpA operon, within which toxT is located. It is known that V. cholerae terminates virulence gene expression prior to escape from the host, but it is unknown how this ToxT‐positive feedback loop is broken, an essential step in terminating virulence gene expression. To better understand how ToxT protein activity is regulated, we monitored ToxT accumulation and activity under virulence‐inducing and ‐repressing growth conditions. Our results suggest that ToxT protein undergoes proteolytic degradation to terminate virulence gene expression. This directed degradation of ToxT supports a model for terminating V. cholerae virulence gene expression late in infection, with both ToxT and TcpP undergoing proteolysis prior to escape from the host.

Conjugated linoleic acid reduces cholera toxin production in vitro and in vivo by inhibiting Vibrio cholerae ToxT activity

ABSTRACT The severe diarrheal disease cholera is endemic in over 50 countries. Current therapies for cholera patients involve oral and/or intravenous rehydration, often combined with the use of antibiotics to shorten the duration and intensity of the disease. However, as antibiotic resistance increases, treatment options will become limited. Linoleic acid has been shown to be a potent negative effector of V. cholerae virulence that acts on the major virulence transcription regulator protein, ToxT, to inhibit virulence gene expression. ToxT activates transcription of the two major virulence factors required for disease, cholera toxin (CT) and toxin-coregulated pilus (TCP). A conjugated form of linoleic acid (CLA) is currently sold over the counter as a dietary supplement and is generally recognized as safe by the U.S. Food and Drug Administration. This study examined whether CLA could be used as a new therapy to reduce CT production, which, in turn, would decrease disease duration and intensity in cholera patients. CLA could be used in place of traditional antibiotics and would be very unlikely to generate resistance, as it affects only virulence factor production and not bacterial growth or survival.

A Small Unstructured Region in Vibrio cholerae ToxT Mediates the Response to Positive and Negative Effectors and ToxT Proteolysis

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. The production of the virulence factors that are required for human disease is controlled by a complex network of transcriptional and posttranscriptional regulators. ToxT is the transcription regulator that directly controls the production of the two major virulence factors, toxin-coregulated pilus (TCP) and cholera toxin (CT). The solved crystal structure of ToxT revealed an unstructured region in the N-terminal domain between residues 100 and 110. This region and the surrounding amino acids have been previously implicated in ToxT proteolysis, resistance to inhibition by negative effectors, and ToxT dimerization. To better characterize this region, site-directed mutagenesis was performed to assess the effects on ToxT proteolysis and bile sensitivity. This analysis identified specific mutations within this unstructured region that prevent ToxT proteolysis and other mutations that reduce inhibition by bile and unsaturated fatty acids. In addition, we found that mutations that affect the sensitivity of ToxT to bile also affect the sensitivity of ToxT to its positive effector, bicarbonate. These results suggest that a small unstructured region in the ToxT N-terminal domain is involved in multiple aspects of virulence gene regulation and response to human host signals.