Novel cholera toxin variant and ToxT regulon in environmental Vibrio mimicus strains: potential resources for the evolution of Vibrio cholerae hybrid strains

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) are gradually becoming a major cause of recent cholera epidemics. Vibrio mimicus occasionally contains virulence factors associated with cholera, e.g., CT, encoded by ctxAB on CTX[FE] genome; and TCP, the CTX[FE]-specific receptor. This study carried out extensive molecular characterization of CTX[FE] and ToxT regulon in ctx+ve strains of V. mimicus isolated 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 novel ctxAB, 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 of tcpA and toxT respectively, showing clonal lineage to a phylogenetic clade of V. cholerae non-O1/O139, was identified. The V. mimicus strains lacked the RTX and TLC elements, and Vibrio seventh pandemic islands of the El Tor strains, but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region like some classical strains of V. cholerae O1. PFGE analysis showed all the ctx+ve V. mimicus strains were clonally related. However, their in vitro CT production and in vivo toxigenecity were variable, which could be explained by differential transcription of virulence genes along with ToxR regulon. Taken together, our findings strongly suggest that environmental V. mimicus strains act as potential reservoir of atypical virulence factors, including variant CT and ToxT regulon, and may contribute to the evolution of V. cholerae hybrid strains.\n\nIMPORTANCENatural diversification of CTX[FE] and ctxAB genes 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 of V. cholerae. This study showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus points out a previously unnoticed evolutionary event, independent to that of the El Tor strains of V. cholerae. Identification and cluster analysis of the newly-discovered alleles of tcpA and toxT indicates their horizontal transfer from an uncommon clone of V. cholerae. The genomic content of ToxT regulon, and tandemly arranged multiple pre-CTX[FE]Env and a CTX[FE]ET in V. mimicus probably act as salient raw materials inducing natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This study will facilitate deeper understanding of the evolution of new variant CT and ToxT regulon, influencing cholera epidemiology.