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

Abstract

Vibrio choleraeO1 and O139 isolates deploy cholera toxin (CT) and toxin-coregulated pilus (TCP) to cause the diarrhoeal disease cholera. ThectxABandtcpAgenes encoding CT and TCP are part of two acquired genetic elements, the CTX phage andVibriopathogenicity island-1 (VPI-1), respectively. ToxR and ToxT proteins are the key regulators of virulence genes ofV. choleraeO1 and O139.V. choleraeisolates 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 toxigenicV. choleraenon-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 ofV. choleraeO1 strains, including organization of the VPI-1 locus,ctxAB, tcpAandtoxTgenes, 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, ΔtoxRand ΔtoxTΔtoxRdeletion mutants of VCE232. Extensive genetic analyses of these mutants indicated that thetoxTandtoxRgenes of VCE232 are crucial for CT and TCP production. However, unlike O1 isolates, the presence of eithertoxTortoxRgene is sufficient for optimal CT production in VCE232. In addition, the VCE232 ΔtoxRmutant 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 toxigenicV. choleraenon-O1/non-O139 strain.