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

Abstract

Atypical El Tor strains of Vibrio cholerae O1 harboring variant ctxB genes of cholera toxin (CT) have gradually become a major cause of recent cholera epidemics. Vibrio mimicus occasionally produces CT, encoded by ctxAB 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 V. mimicus ctx-positive (ctx+) strains (i.e., V. mimicus strains containing ctx) 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 as an RS1-CTXET-RS1-pre-CTXEnv-pre-CTXEnv array. Additionally, novel variants of tcpA and toxT, respectively, showing phylogenetic lineage to a clade of V. cholerae non-O1 and to a clade of V. cholerae non-O139, were identified. The V. mimicus strains lacked the RTX (repeat in toxin) and TLC (toxin-linked cryptic) elements and lacked Vibrio seventh-pandemic islands of the El Tor strains but contained five heptamer (TTTTGAT) repeats in ctxAB promoter region similar to those seen with some classical strains of V. cholerae O1. Pulsed-field gel electrophoresis (PFGE) analysis showed that all the ctx+ V. mimicus strains were clonally related. However, their in vitro CT production and in vivo 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 V. mimicus strains act as a potential reservoir of atypical virulence factors, including variant CT and ToxT regulons, and may contribute to the evolution of V. cholerae hybrid strains. IMPORTANCE Natural diversification of CTXФ 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 report, showing the occurrence of CTXET comprising a novel variant of ctxAB in V. mimicus, points out a previously unnoticed evolutionary event that is independent of the evolutionary event associated with the El Tor strains of V. cholerae. Identification and cluster analysis of the newly discovered alleles of tcpA and toxT suggest their horizontal transfer from an uncommon clone of V. cholerae. The genomic contents of ToxT regulon and of tandemly arranged multiple pre-CTXФEnv and of a CTXФET in V. mimicus probably act as salient raw materials that induce natural recombination among the hallmark virulence genes of hybrid V. cholerae strains. This report provides valuable information to enrich our knowledge on the evolution of new variant CT and ToxT regulons.

Core and Accessory Genome Analysis of Vibrio mimicus

“…The main differences found in this study were on virulence genes, hypothetical proteins, and transcriptional regulators. These differences in virulence content could explain the distinctiveness of the pathogenic potential of each species [ 48 ]. Several virulence genes were identified and classified in this study for V. mimicus strains.…”

<p>Non-O1, Non-O139 <em>Vibrio cholerae</em> (NOVC) Bacteremia: Case Report and Literature Review, 2015–2019</p>

“…NOVC strains typically did not carry the main virulence factors (cholera toxin and toxin-coregulated pilus), but clinical isolates expressed various synergistic factors, including hemolysin (hlyA), repeats in toxin (rtx), hemagglutinin protease (hapA), toxin regulatory gene (toxR), outer membrane proteins (omp), and the type III (T3SS) and type VI (T6SS) secretion systems. 29,30 Furthermore, several new virulence gene variants were reported in some environmental NOVC strains. 23 2018 A study suggested that NOVC can release outer membrane vesicles that induce miR-146a to induce colonization.…”

Population Analysis of Vibrio cholerae in Aquatic Reservoirs Reveals a Novel Sister Species ( Vibrio paracholerae sp. nov.) with a History of Association with Humans

“…One of them was the occasional human pathogen Vibrio mimicus , which was proposed as a new species in 1981 based on phenotypic characteristics ( 3 ). Later, genome-based studies established the molecular basis of its importance as a pathogen and close association and exchange of important virulence genes with V. cholerae ( 4 , 5 ). Two other closely related novel species, Vibrio sp.…”