Novel cholera toxin variant and ToxT regulon in environmentalVibrio mimicusstrains: potential resources for the evolution ofVibrio choleraehybrid strains

Atypical El Tor strains ofVibrio choleraeO1 harboring variantctxBgenes of cholera toxin (CT) are gradually becoming a major cause of recent cholera epidemics.Vibrio mimicusoccasionally contains virulence factors associated with cholera, e.g., CT, encoded byctxABon CTXФ genome; and TCP, the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon inctx+vestrains ofV. mimicusisolated from the Bengal coast. Southern hybridization, PCR, and DNA sequencing of virulence related-genes revealed the presence of an El Tor type CTX prophage (CTXET) carrying a novelctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTXEnv), and RS1 elements, which were organized in an array of RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv. Additionally, a novel variant oftcpAandtoxTrespectively, showing clonal lineage to a phylogenetic clade ofV. choleraenon-O1/O139, was identified. TheV. mimicusstrains lacked the RTX and TLC elements, andVibrioseventh pandemic islands of the El Tor strains, but contained five heptamer (TTTTGAT) repeats inctxABpromoter region like some classical strains ofV. choleraeO1. PFGE analysis showed all thectx+veV. mimicusstrains were clonally related. However, theirin vitroCT production andin vivotoxigenecity were variable, which could be explained by differential transcription of virulence genes along with ToxR regulon. Taken together, our findings strongly suggest that environmentalV. mimicusstrains act as potential reservoir of atypical virulence factors, including variant CT and ToxT regulon, and may contribute to the evolution ofV. choleraehybrid strains.IMPORTANCENatural diversification of CTXФ andctxABgenes certainly influences disease severity and shifting patterns in major etiological agents of cholera, e.g., the overwhelming emergence of hybrid El Tor variants, replacing the prototype El Tor strains ofV. cholerae. This study showing the occurrence of CTXETcomprising a novel variant ofctxABinV. mimicuspoints out a previously unnoticed evolutionary event, independent to that of the El Tor strains ofV. cholerae. Identification and cluster analysis of the newly-discovered alleles oftcpAandtoxTindicates their horizontal transfer from an uncommon clone ofV. cholerae. The genomic content of ToxT regulon, and tandemly arranged multiple pre-CTXФEnvand a CTXФETinV. mimicusprobably act as salient raw materials inducing natural recombination among the hallmark virulence genes of hybridV. choleraestrains. This study will facilitate deeper understanding of the evolution of new variant CT and ToxT regulon, influencing cholera epidemiology.