Novel Cholera Toxin Variant and ToxT Regulon in Environmental Isolates: Potential Resources for the Evolution of Hybrid Strains

Atypical El Tor strains of O1 harboring variant genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. occasionally produces CT, encoded by on CTXФ genome; toxin-coregulated pilus (TCP), a major intestinal colonization factor; and also the CTXФ-specific receptor. This study carried out extensive molecular characterization of CTXФ and ToxT regulon in -positive () strains (i.e., strains containing ) 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 (CTX) carrying a novel , tandem copies of environmental type pre-CTX prophage (pre-CTX), and RS1 elements, which were organized as an RS1-CTX-RS1-pre-CTX-pre-CTX array. Additionally, novel variants of and , respectively, showing phylogenetic lineage to a clade of non-O1 and to a clade of non-O139, were identified. The strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in promoter region similar to those seen with some classical strains of O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the strains were clonally related. However, their CT production and toxigenicity characteristics were variable, which could be explainable by differential transcription of virulence genes along with the ToxR regulon. Taken together, our findings strongly suggest that environmental strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of hybrid strains. Natural diversification of CTXФ and 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 This report, showing the occurrence of CTX comprising a novel variant of in , points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of Identification and cluster analysis of the newly discovered alleles of and suggest their horizontal transfer from an uncommon clone of The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФ and of a CTXФ in probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.