Bile Salts Promote ToxR Regulon Activation during Growth under Virulence-Inducing Conditions.

Cholera is an epidemic disease caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae is found in aquatic ecosystems and infects people through the consumption of V. cholerae-contaminated food or water. Following ingestion, V. cholerae responds to host cues to activate the expression of critical virulence genes that are under the control of a hierarchical regulatory system called the ToxR regulon. The ToxR regulon is tightly regulated and is expressed in vitro only under special growth conditions referred to as AKI conditions. AKI conditions have been instrumental in elucidating V. cholerae virulence regulation, but the chemical cues within AKI medium that activate virulence gene expression are unknown. In this study, we fractionated AKI medium on a reverse-phase chromatography column (RPCC) and showed that the virulence-activating molecules were retained on the RPCC column and recovered in the eluate. Liquid chromatography-high-resolution mass spectrometry (LC-HRMS) analysis of the eluate revealed the presence of a known ToxR regulon activator, taurocholate, and other bile salts. The RPCC eluate activated the ToxR regulon when added to noninducing medium and promoted TcpP dimerization in a two-hybrid system, consistent with taurocholate being responsible for the virulence-inducing activity of AKI medium. Additional experiments using purified bile salts showed that the ToxR regulon was preferentially activated in response to primary bile acids. The results of this study shed light on the chemical cues involved in V. cholerae virulence activation and suggested that V. cholerae virulence genes are modulated in response to regionally specific bile acid species in the intestine.

Vibrio cholerae TolC Is Required for Expression of the ToxR Regulon.

Vibrio cholerae is a Gram-negative bacterium that causes the enteric disease cholera. V. cholerae colonization of the human intestine is dependent on the expression of both virulence genes and environmental adaptation genes involved in antimicrobial resistance. The expression of virulence genes, including the genes encoding the main virulence factors cholera toxin (CT) and the toxin-coregulated pilus (TCP), are coordinately regulated by the ToxR regulon. Tripartite transport systems belonging to the ATP binding cassette, major facilitator, and resistance-nodulation-division families are critical for V. cholerae pathogenesis. Transport systems belonging to these families contribute to myriad phenotypes, including protein secretion, antimicrobial resistance, and virulence. TolC plays a central role in bacterial physiology by functioning as the outer membrane pore protein for tripartite transport systems. Consistent with this, V. cholerae tolC was previously found to be required for MARTX toxin secretion and antimicrobial resistance. Here, we investigated the contribution of TolC to V. cholerae virulence. We documented that tolC was required for CT and TCP production in O1 El Tor V. cholerae. This phenotype was linked to repression of the critical ToxR regulon transcription factor aphA. Decreased aphA transcription correlated with increased expression of the LysR-family transcription factor leuO. Deletion of leuO restored aphA expression, and CT and TCP production, in a tolC mutant. The collective results document that tolC is required for ToxR regulon expression and further suggest that tolC participates in an efflux-dependent feedback circuit to regulate virulence gene expression.

ToxR Mediates the Antivirulence Activity of Phenyl-Arginine-ß-Naphthylamide To Attenuate Vibrio cholerae Virulence.

Multidrug efflux systems belonging to the resistance-nodulation-cell division (RND) family are ubiquitous in Gram-negative bacteria and are critical for antimicrobial resistance. This realization has led to efforts to develop efflux pump inhibitors (EPI) for use as adjuvants for antibiotic treatment of resistant organisms. However, the functions of RND transporters extend beyond antimicrobial resistance to include physiological functions that are critical for pathogenesis, suggesting that EPIs could also be used as antivirulence therapeutics. This was documented in the enteric pathogen Vibrio cholerae, in which EPIs were shown to attenuate the production of the critical virulence factors cholera toxin (CT) and the toxin-coregulated pilus (TCP). In this study, we investigated the antivirulence mechanism of action of the EPI phenyl-arginine-ß-naphthylamide (PAßN) on V. cholerae. Using bioassays, we documented that PAßN inhibited virulence factor production in three epidemic V. cholerae isolates. Transcriptional reporter studies and mutant analysis indicated that PAßN initiated a ToxR-dependent regulatory circuit to activate leuO expression and that LeuO repressed the expression of the critical virulence activator aphA to attenuate CT and TCP production. The antivirulence activity of PAßN was found to be dependent on the ToxR periplasmic sensing domain (PPD), suggesting that a feedback mechanism was involved in its activity. Collectively, the data indicated that PAßN inhibited V. cholerae virulence factor production by activating a ToxR-dependent metabolic feedback mechanism to repress the expression of the ToxR virulence regulon. This suggests that efflux pump inhibitors could be used as antivirulence therapeutics for the treatment of cholera and perhaps that of other Gram-negative pathogens.

NexusLIMS: A Laboratory Information Management System for Shared-Use Electron Microscopy Facilities.

This work introduces NexusLIMS, an electron microscopy laboratory information management system designed and implemented by the Office of Data and Informatics and the Materials Science and Engineering Division at NIST for a multi-user electron microscopy co-op facility. NexusLIMS comprises network infrastructure, shared information technology resources, a custom software package to harvest and extract experimental information and construct experimental metadata records, and an intuitive web-based user-facing interface for searching, browsing, and examining research data. These metadata records conform to the Nexus Experiment schema, which is introduced in this work. The NexusLIMS suite of tools requires minimal input and adjustments to user behavior, instead relying on existing organizational procedures and the collection of information from a multitude of sources to construct a complete picture and record of a research experiment. The underlying infrastructure and design considerations for a multi-user data management system are discussed. The core codebase for NexusLIMS is made publicly available alongside this work, and its modular design encourages the adaptation of the presented methods at other research organizations.

Construction of a tetracycline inducible expression vector and characterization of its use in Vibrio cholerae.

We report the construction of a tetracycline inducible expression vector that allows regulated gene expression in the enteric pathogen Vibrio cholerae. The expression vector, named pXB300, contains the tetracycline regulatory elements from Tn10, a multiple cloning site downstream of the tetA promoter and operator sequences, a ColE1 origin of replication, a ß-lactamase resistance gene for positive selection, and the hok/sok addiction system for selection in the absence of antibiotic. The function of the tetracycline expression system was demonstrated by cloning lacZ under control of the tetA promoter and quantifying ß-galactosidase expression in Escherichia coli and V. cholerae. The utility for pXB300 was documented by complementation of V. cholerae virulence mutants during growth under virulence inducing conditions. The results showed that pXB300 allowed high-level expression of recombinant genes with linear induction in response to the exogenous concentration of the inducer anhydrotetracycline. We further show that pXB300 was reliably maintained in V. cholerae during growth in the absence of antibiotic selection.