Interlaboratory performance and quantitative PCR data acceptance metrics for NIST SRM® 2917.

Surface water quality quantitative polymerase chain reaction (qPCR) technologies are expanding from a subject of research to routine environmental and public health laboratory testing. Readily available, reliable reference material is needed to interpret qPCR measurements, particularly across laboratories. Standard Reference Material® 2917 (NIST SRM® 2917) is a DNA plasmid construct that functions with multiple water quality qPCR assays allowing for estimation of total fecal pollution and identification of key fecal sources. This study investigates SRM 2917 interlaboratory performance based on repeated measures of 12 qPCR assays by 14 laboratories (n = 1008 instrument runs). Using a Bayesian approach, single-instrument run data are combined to generate assay-specific global calibration models allowing for characterization of within- and between-lab variability. Comparable data sets generated by two additional laboratories are used to assess new SRM 2917 data acceptance metrics. SRM 2917 allows for reproducible single-instrument run calibration models across laboratories, regardless of qPCR assay. In addition, global models offer multiple data acceptance metric options that future users can employ to minimize variability, improve comparability of data across laboratories, and increase confidence in qPCR measurements.

Indole Inhibits ToxR Regulon Expression in Vibrio cholerae.

Indole is a degradation product of tryptophan that functions as a signaling molecule in many bacteria. This includes Vibrio cholerae, where indole was shown to regulate biofilm and type VI secretion in nontoxigenic environmental isolates. Indole is also produced by toxigenic V. cholerae strains in the human intestine, but its significance in the host is unknown. We investigated the effects of indole on toxigenic V. cholerae O1 El Tor during growth under virulence inducing conditions. The indole transcriptome was defined by RNA sequencing and showed widespread changes in the expression of genes involved in metabolism, biofilm production, and virulence factor production. In contrast, genes involved in type VI secretion were not affected by indole. We subsequently found that indole repressed genes involved in V. cholerae pathogenesis, including the ToxR virulence regulon. Consistent with this, indole inhibited cholera toxin and toxin-coregulated pilus production in a dose-dependent manner. The effects of indole on virulence factor production and biofilm were linked to ToxR and the ToxR-dependent regulator LeuO. The expression of leuO was increased by exogenous indole and linked to repression of the ToxR virulence regulon. This process was dependent on the ToxR periplasmic domain, suggesting that indole was a ToxR agonist. This conclusion was further supported by results showing that the ToxR periplasmic domain contributed to indole-mediated increased biofilm production. Collectively, our results suggest that indole may be a niche-specific cue that can function as a ToxR agonist to modulate virulence gene expression and biofilm production in V. cholerae.

The Vibrio cholerae RND efflux systems impact virulence factor production and adaptive responses via periplasmic sensor proteins.

Resistance-nodulation-division (RND) efflux systems are ubiquitous transporters in Gram-negative bacteria that are essential for antibiotic resistance. The RND efflux systems also contribute to diverse phenotypes independent of antimicrobial resistance, but the mechanism by which they affect most of these phenotypes is unclear. This is the case in Vibrio cholerae where the RND systems function in antimicrobial resistance and virulence factor production. Herein, we investigated the linkage between RND efflux and V. cholerae virulence. RNA sequencing revealed that the loss of RND efflux affected the activation state of periplasmic sensing systems including the virulence regulator ToxR. Activation of ToxR in an RND null mutant resulted in ToxR-dependent transcription of the LysR-family regulator leuO. Increased leuO transcription resulted in the repression of the ToxR virulence regulon and attenuated virulence factor production. Consistent with this, leuO deletion restored virulence factor production in an RND-null mutant, but not its ability to colonize infant mice; suggesting that RND efflux was epistatic to virulence factor production for colonization. The periplasmic sensing domain of ToxR was required for the induction of leuO transcription in the RND null mutant, suggesting that ToxR responded to metabolites that accumulated in the periplasm. Our results suggest that ToxR represses virulence factor production in response to metabolites that are normally effluxed from the cell by the RND transporters. We propose that impaired RND efflux results in periplasmic metabolite accumulation, which then activates periplasmic sensors including ToxR and two-component regulatory systems to initiate the expression of adaptive responses.

Vibrio cholerae LeuO Links the ToxR Regulon to Expression of Lipid A Remodeling Genes.

Vibrio cholerae is an intestinal pathogen that causes the diarrheal disease cholera. Colonization of the intestine depends upon the expression of genes that allow V. cholerae to overcome host barriers, including low pH, bile acids, and the innate immune system. ToxR is a major contributor to this process. ToxR is a membrane-spanning transcription factor that coordinates gene expression in response to environmental cues. In previous work we showed that ToxR upregulated leuO expression in response to bile salts. LeuO is a LysR family transcription factor that contributes to acid tolerance, bile resistance, and biofilm formation in V. cholerae Here, we investigated the function of ToxR and LeuO in cationic antimicrobial peptide (CAMP) resistance. We report that ToxR and LeuO contribute to CAMP resistance by regulating carRS transcription. CarRS is a two-component regulatory system that positively regulates almEFG expression. AlmEFG confers CAMP resistance by glycinylation of lipid A. We found that the expression of carRS and almEFG and the polymyxin B MIC increased in mutants lacking toxRS or leuO Conversely, leuO overexpression decreased the polymyxin B MIC. Furthermore, we found that LeuO directly bound to the carRS promoter and that ToxR-dependent activation of leuO transcription regulated carRS transcription in response to bile salts. Our results suggest that LeuO functions downstream of ToxR to modulate carRS expression in response to environmental cues. This study extends the functional role of ToxR and LeuO in environmental adaptation to include cell surface remodeling and CAMP resistance.

Vibrio cholerae leuO Transcription Is Positively Regulated by ToxR and Contributes to Bile Resistance.

UNLABELLED: Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCE: The success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance.

Substrate-dependent activation of the Vibrio cholerae vexAB RND efflux system requires vexR.

Vibrio cholerae encodes six resistance-nodulation-division (RND) efflux systems which function in antimicrobial resistance, virulence factor production, and intestinal colonization. Among the six RND efflux systems, VexAB exhibited broad substrate specificity and played a predominant role in intrinsic antimicrobial resistance. The VexAB system was encoded in an apparent three gene operon that included vexR; which encodes an uncharacterized TetR family regulator. In this work we examined the role of vexR in vexRAB expression. We found that VexR bound to the vexRAB promoter and vexR deletion resulted in decreased vexRAB expression and increased susceptibility to VexAB antimicrobial substrates. Substrate-dependent induction of vexRAB was dependent on vexR and episomal vexR expression provided a growth advantage in the presence of the VexAB substrate deoxycholate. The expression of vexRAB increased, in a vexR-dependent manner, in response to the loss of RND efflux activity. This suggested that VexAB may function to export intracellular metabolites. Support for this hypothesis was provided by data showing that vexRAB was upregulated in several metabolic mutants including tryptophan biosynthetic mutants that were predicted to accumulate indole. In addition, vexRAB was found to be upregulated in response to exogenous indole and to contribute to indole resistance. The collective results indicate that vexR is required for vexRAB expression in response to VexAB substrates and that the VexAB RND efflux system modulates the intracellular levels of metabolites that could otherwise accumulate to toxic levels.