Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence.

The function of the Vibrio 7(th) pandemic island-1 (VSP-1) in cholera pathogenesis has remained obscure. Utilizing chromatin immunoprecipitation sequencing and RNA sequencing to map the regulon of the master virulence regulator ToxT, we identify a TCP island-encoded small RNA that reduces the expression of a previously unrecognized VSP-1-encoded transcription factor termed VspR. VspR modulates the expression of several VSP-1 genes including one that encodes a novel class of di-nucleotide cyclase (DncV), which preferentially synthesizes a previously undescribed hybrid cyclic AMP-GMP molecule. We show that DncV is required for efficient intestinal colonization and downregulates V. cholerae chemotaxis, a phenotype previously associated with hyperinfectivity. This pathway couples the actions of previously disparate genomic islands, defines VSP-1 as a pathogenicity island in V. cholerae, and implicates its occurrence in 7(th) pandemic strains as a benefit for host adaptation through the production of a regulatory cyclic di-nucleotide.

Mapping the regulon of Vibrio cholerae ferric uptake regulator expands its known network of gene regulation.

ChIP coupled with next-generation sequencing (ChIP-seq) has revolutionized whole-genome mapping of DNA-binding protein sites. Although ChIP-seq rapidly gained support in eukaryotic systems, it remains underused in the mapping of bacterial transcriptional regulator-binding sites. Using the virulence-required iron-responsive ferric uptake regulator (Fur), we report a simple, broadly applicable ChIP-seq method in the pathogen Vibrio cholerae. Combining our ChIP-seq results with available microarray data, we clarify direct and indirect Fur regulation of known iron-responsive genes. We validate a subset of Fur-binding sites in vivo and show a common motif present in all Fur ChIP-seq peaks that has enhanced binding affinity for purified V. cholerae Fur. Further analysis shows that V. cholerae Fur directly regulates several additional genes associated with Fur-binding sites, expanding the role of this transcription factor into the regulation of ribosome formation, additional transport functions, and unique sRNAs.

DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine.

Ingested Vibrio cholerae pass through the stomach and colonize the small intestines of its host. Here, we show that V. cholerae requires at least two types of DNA repair systems to efficiently compete for colonization of the infant mouse intestine. These results show that V. cholerae experiences increased DNA damage in the murine gastrointestinal tract. Agreeing with this, we show that passage through the murine gut increases the mutation frequency of V. cholerae compared to liquid culture passage. Our genetic analysis identifies known and novel defense enzymes required for detoxifying reactive nitrogen species (but not reactive oxygen species) that are also required for V. cholerae to efficiently colonize the infant mouse intestine, pointing to reactive nitrogen species as the potential cause of DNA damage. We demonstrate that potential reactive nitrogen species deleterious for V. cholerae are not generated by host inducible nitric oxide synthase (iNOS) activity and instead may be derived from acidified nitrite in the stomach. Agreeing with this hypothesis, we show that strains deficient in DNA repair or reactive nitrogen species defense that are defective in intestinal colonization have decreased growth or increased mutation frequency in acidified nitrite containing media. Moreover, we demonstrate that neutralizing stomach acid rescues the colonization defect of the DNA repair and reactive nitrogen species defense defective mutants suggesting a common defense pathway for these mutants.

Role of iron in human serum resistance of the clinical and environmental Vibrio vulnificus genotypes.

We recently reported a simple PCR procedure that targets a sequence variation of the virulence-correlated gene locus vcg. It was found that 90% of all clinical isolates possessed the vcgC sequence variant, while 93% of all environmental isolates possessed the vcgE sequence variant. Here we report that the clinical genotype of Vibrio vulnificus is significantly better able to survive in human serum than is the environmental genotype. The presence of a siderophore-encoding gene, viuB, influenced serum survivability among all isolates of V. vulnificus tested. Those strains positive for viuB (all C-type strains but very few E-type strains) showed greater serum survivability than those lacking viuB (most E-type strains). The addition of iron (in the form of ferric ammonium citrate) to human serum restored the survival of E-type strains lacking viuB to levels not significantly different from those of C-type and E-type strains that possess viuB. These findings suggest that viuB may dictate serum survival in both C- and E-type strains of V. vulnificus and may explain why some strains (C- and E-type strains) are pathogenic and others (predominately E-type strains) are not. Additionally, C-type strains exhibited a cross-protective response against human serum, not exhibited by E-type strains, after incubation under nutrient and osmotic downshift conditions that mimicked estuarine waters. This suggests that the nutrient/osmotic environment may influence the survival of V. vulnificus following entry into the human body, leading to selection of the C genotype over the E genotype.

MetR-regulated Vibrio cholerae metabolism is required for virulence.

UNLABELLED: LysR-type transcriptional regulators (LTTRs) are the largest, most diverse family of prokaryotic transcription factors, with regulatory roles spanning metabolism, cell growth and division, and pathogenesis. Using a sequence-defined transposon mutant library, we screened a panel of V. cholerae El Tor mutants to identify LTTRs required for host intestinal colonization. Surprisingly, out of 38 LTTRs, only one severely affected intestinal colonization in the suckling mouse model of cholera: the methionine metabolism regulator, MetR. Genetic analysis of genes influenced by MetR revealed that glyA1 and metJ were also required for intestinal colonization. Chromatin immunoprecipitation of MetR and quantitative reverse transcription-PCR (qRT-PCR) confirmed interaction with and regulation of glyA1, indicating that misregulation of glyA1 is likely responsible for the colonization defect observed in the metR mutant. The glyA1 mutant was auxotrophic for glycine but exhibited wild-type trimethoprim sensitivity, making folate deficiency an unlikely cause of its colonization defect. MetJ regulatory mutants are not auxotrophic but are likely altered in the regulation of amino acid-biosynthetic pathways, including those for methionine, glycine, and serine, and this misregulation likely explains its colonization defect. However, mutants defective in methionine, serine, and cysteine biosynthesis exhibited wild-type virulence, suggesting that these amino acids can be scavenged in vivo. Taken together, our results suggest that glycine biosynthesis may be required to alleviate an in vivo nutritional restriction in the mouse intestine; however, additional roles for glycine may exist. Irrespective of the precise nature of this requirement, this study illustrates the importance of pathogen metabolism, and the regulation thereof, as a virulence factor. IMPORTANCE: Vibrio cholerae continues to be a severe cause of morbidity and mortality in developing countries. Identification of V. cholerae factors critical to disease progression offers the potential to develop or improve upon therapeutics and prevention strategies. To increase the efficiency of virulence factor discovery, we employed a regulator-centric approach to multiplex our in vivo screening capabilities and allow whole regulons in V. cholerae to be interrogated for pathogenic potential. We identified MetR as a new virulence regulator and serine hydroxymethyltransferase GlyA1 as a new MetR-regulated virulence factor, both required by V. cholerae to colonize the infant mouse intestine. Bacterial metabolism is a prerequisite to virulence, and current knowledge of in vivo metabolism of pathogens is limited. Here, we expand the known role of amino acid metabolism and regulation in virulence and offer new insights into the in vivo metabolic requirements of V. cholerae within the mouse intestine.