Genetic and mutational analysis of virulence traits and their modulation in an environmental toxigenic Vibrio cholerae non-O1/non-O139 strain, VCE232.

Vibrio cholerae O1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. The ctxAB and tcpA genes encoding CT and TCP are part of two acquired genetic elements, the CTX phage and Vibrio pathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes of V. cholerae O1 and O139. V. cholerae isolates belonging to serogroups other than O1/O139, called non-O1/non-O139, are usually devoid of virulence-related elements and are non-pathogenic. Here, we have analysed the available whole genome sequence of an environmental toxigenic V. cholerae non-O1/non-O139 strain, VCE232, carrying the CTX phage and VPI-1. Extensive bioinformatics and phylogenetic analyses indicated high similarity of the VCE232 genome sequence with the genome of V. cholerae O1 strains, including organization of the VPI-1 locus, ctxAB, tcpA and toxT genes, and promoters. We established that the VCE232 strain produces an optimal amount of CT at 30 °C under AKI conditions. To investigate the role of ToxT and ToxR in the regulation of virulence factors, we constructed ΔtoxT, ΔtoxR and ΔtoxTΔtoxR deletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that the toxT and toxR genes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of either toxT or toxR gene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxR mutant showed differential regulation of the major outer membrane proteins, OmpT and OmpU. This is the first attempt to explore the regulation of expression of major virulence genes and regulators in an environmental toxigenic V. cholerae non-O1/non-O139 strain.

Functional Insights Into the Role of gppA in (p)ppGpp Metabolism of Vibrio cholerae.

The stringent response, an adaptive response to nutrient limitation and exposure to xenobiotics in bacteria, is mediated by two intracellular signaling molecules, pppGpp and ppGpp, together called (p)ppGpp. The cellular level of (p)ppGpp in bacterial cells is controlled by the Rel/Spo family of proteins. In the cholera pathogen, Vibrio cholerae, (p)ppGpp metabolism is regulated by the products of at least three genes relA, spoT, and relV. In this study, we identify and characterize the function of the guanosine-5'-triphosphate 3'-diphosphate pyrophosphatase A (GppA) encoding gene gppA of V. cholerae. Genomic analysis indicates that the gppA locus is conserved in vibrios and organized as a bicistronic operon along with the rhlB gene. We engineered the genome of V. cholerae to develop different mutants devoid of GppA and/or other phosphate metabolic enzymes. Our findings indicate that in V. cholerae, GppA plays an important role in the conversion of pppGpp to ppGpp during amino acid deprivation but not during glucose starvation. Quantitative analyses of the gppA transcript level reveal its differential expression pattern at different growth phases and starvation conditions. It has been observed that the GppA deficiency during amino acid starvation condition could be complemented by overexpressing the exopolyphosphatase coding gene ppx of V. cholerae. By deletion analysis, we further demonstrate that the amino and carboxy terminal sequences flanking the Ppx-GppA motif of the GppA protein of V. cholerae are also important for its enzymatic function.