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. IMPORTANCEVibrio 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. Vibrio cholerae, a Gram-negative curved bacillus possessing a single polar flagellum, is the causative agent of cholera. Cholera is a diarrheal disease contracted by consuming contaminated food or water; it is characterized by severe diarrhea that leads to dehydration and can ultimately cause death if left untreated. Each year, there are an estimated 1.4 million to 4.3 million cholera cases and 20,000 to 142,000 deaths from the disease (1, 2). To cause disease, V. cholerae must colonize the upper small intestine, where it expresses virulence genes, including those that encode the t...

SUMMARY Ail, a multifunctional outer membrane protein of Yersinia pestis, confers cell binding, Yop delivery, and serum resistance activities. Resistance to complement proteins in serum is critical for survival of Y. pestis during the septicemic stage of plague infections. Bacteria employ a variety of tactics to evade the complement system, including recruitment of complement regulatory factors, such as factor H, C4b-binding protein (C4BP), and vitronectin (Vn). Y. pestis Ail interacts with the regulatory factors Vn and C4BP, and Ail homologs from Y. enterocolitica and Y. pseudotuberculosis recruit factor H. Using co-sedimentation assays, we demonstrate that two surface-exposed amino acids, F80 and F130, are required for interaction of Y. pestis Ail with Vn, factor H, and C4BP. However, although Ail-F80A/F130A fails to interact with these complement regulatory proteins, it still confers 10,000-fold more serum resistance than a Δail strain and prevents C9 polymerization, potentially by directly interfering with MAC assembly. Using site-directed mutagenesis we further defined this additional mechanism of complement evasion conferred by Ail. Finally, we find that at Y. pestis concentrations reflective of early-stage septicemic plague, Ail weakly recruits Vn and fails to recruit factor H, suggesting that this alternative mechanism of serum resistance may be essential during plague infection.

bThe 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.

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. Vibrio cholerae is the etiological agent of the severe diarrheal disease cholera. Cholera disease is characterized by extreme water loss and dehydration due to diarrhea and if left untreated can result in death. The bacteria are usually ingested through contaminated food or water and colonize the upper small intestine (1). When the V. cholerae bacterium is in the optimal environment within the intestine, it begins producing the major virulence factors responsible for causing disease, cholera toxin (CT) and toxincoregulated pilus (TCP) (2-4). CT is an ADP-ribosylating toxin composed of five binding B subunits and one enzymatic A subunit (5). After binding the GM 1 ganglioside via the B subunits, the A subunit is translocated into the intestinal epithelial cell, where it modifies G s ␣ 1 , leading to aberrant secretion of chloride, water, and other electrolytes (6). TCP is a type IV bundle-forming pilus that is responsible for bacterium-bacterium interactions that result in microcolony formation during intestinal colonization (4,7,8).TCP and CT are produced via a virulence regulatory cascade known as the ToxR regulon. The expression of CT and TCP is directly activated by the major virulence transcription regulator, ToxT (9, 10). ToxT binds "toxbox" motifs in the promoters of ctxAB and tcpA, as well as in the promoters of other accessory virulence factors, such as acfA, acfD, tagA, aldA, and tcpI, and small regulatory RNAs tarA and tarB, resulting in the expression of these genes under appropriate conditions (9,(11)(12)(13)(14)(15)(16)(17). ToxT is a 276-amino-acid protein that is part of the AraC/XylS family of transcription regulators (18). ToxT consists of two domains, the...

Objectives To evaluate the ability of different esthetic archwires to retain oral biofilms in vitro. Materials and Methods Seven different brands of coated orthodontic archwires were tested: two epoxy coated, two polytetrafluoroethylene coated, two rhodium coated, and one silver plus polymer coated. Conventional uncoated metallic archwires were used as controls. Streptococus mutans adherence to archwires was quantified by colony count following 24 hours of biolfilm growth, and total wire-associated biofilm was measured using a crystal violet staining assay. For both tests, two conditions were used: 0% sucrose and 3% sucrose. For statistical analysis, P < .05 was considered as statistically significant. Results For S. mutans colony forming units per biofilm, there were no statistically significant differences among the various archwires (P = .795 for 0% sucrose; P = .905 for 3% sucrose). Regarding total biofilm formed on archwires in the 3% sucrose condition, there were statistically significant differences in crystal violet staining only for the comparison between Niti Micro Dental White and Copper Ni-Ti wires (P < .05). Conclusions The clinical use of esthetic-coated orthodontic wires may be considered to have similar risks as uncoated archwires for biofilm retention.