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. mimicusctx-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 (CTX^ET) carrying a novel ctxAB, tandem copies of environmental type pre-CTX prophage (pre-CTX^Env), and RS1 elements, which were organized as an RS1-CTX^ET-RS1-pre-CTX^Env-pre-CTX^Env 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 CTX^ET 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-CTXФ^Env and of a CTXФ^ET 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.

Keywords: CTXФ; El Tor biotype; Vibrio cholerae; Vibrio mimicus; cholera toxin; classical biotype; tcpA; toxT.

FIG 1

PFGE analysis of environmental ctx⁺ and reference (ATCC) strains of V. mimicus (VM) and of ctx⁺ non-O1/non-O139 (VCE 233), O1 El Tor (VC N16961), and O1 classical (VC O395) strains of V. cholerae. (A) Gel image of PFGE profiles of undigested gDNA showing similar sizes of the two chromosomes of ctx⁺ V. mimicus and ATCC V. mimicus strains. (B and C) Gel images showing PFGE patterns of NotI-digested (B) and SfiI-digested (C) gDNA of ctx⁺ V. mimicus and the reference strains. The ctx⁺ V. mimicus strains were clonal but differed in 1 to 2 bands, indicated by arrows. NotI- and SfiI-digested gDNA of ctx⁺ V. mimicus strains generated two (a and b) and three (I, II, and III) PFGE profiles, respectively. “MW” represents the molecular weight standard of the Hansenula wingei chromosomes (Bio-Rad) for undigested gDNA (A) and lambda ladder (Bio-Rad) for digested gDNA (B and C).

FIG 2

Genetic relatedness among ctxB and orfU genes of V. mimicus and V. cholerae strains. (A) Novel ctxB genotype 14 of V. mimicus showed the highest affiliation with genotype 12, reported to occur in a V. mimicus strain isolated from the United States. However, ctxB genotype 14 showed more closeness with both ctxB genotype 1 and ctxB genotype 7, representing classical and Haitian strains, respectively, in comparison to genotype 3, usually found in the typical El Tor strains of V. cholerae O1. (B) The orfU gene of V. mimicus of this study showed close affiliation to the strains grouped into the El Tor clade of V. cholerae.

FIG 3

Organization of CTXΦ^{El Tor}, RS1, and pre-CTXΦ^Env in V. mimicus strains. (A) Filled bars indicate the PCR arrays used to check probable locations of genes, and the sizes of PCR products are given at the top. Hashed bars indicate the genetic regions (names mentioned at the top) used as probes for Southern hybridization after restriction digestion with BglI or BglII enzymes; arrows indicate RS1, RS2, or core prophage data where dotted regions were analyzed by sequencing. Lines (filled and dotted) at the bottom show the distances between specific genetic locations determined by Southern hybridization analysis of the BglI- or BglII-digested genomic DNA performed using specific probes. (B) Signature sequences in the flanking region of pre-CTXΦ^Env and CTXΦ^{El Tor} in V. mimicus. The ctxAB promoter of CTXΦ^{El Tor} contains 5 heptamer (TTTTGAT) repeats, shown by filled black arrows, which is characteristic of classical type ctxAB. The phage integration site of the CTXΦ^{El Tor} in V. mimicus, the attR sequence, followed by XerC and XerD binding sites, starts at 106 bp downstream of ctxAB gene, which is similar to the reference El Tor strain, N16961, of V. cholerae. However, the phage integration site, characterized by these signature sequences, of pre-CTXΦ^Env in V. mimicus starts at 13 bp downstream of the zot gene.

FIG 4

Genetic relatedness of tcpA and toxT genes among strains of V. cholerae and V. mimicus of different serogroups. (A) The novel tcpA gene of V. mimicus of this study did not cluster in classical strains, El Tor strains, or other types of strains but formed a separate clade showing closeness to strains of serogroups O56 and O115 of V. cholerae. (B) The novel toxT gene of V. mimicus of this study did not group into the major cluster comprising the toxT genes of V. cholerae O1 classical, El Tor, O139, and non-O1/non-O139 strains or of other V. mimicus strains but grouped into a separate cluster with atypical toxT genes reported in a few non-O1/non-O139 strains of V. cholerae.

FIG 5

Variation in mRNA transcription levels of virulence genes and regulated genes between high-CT-producing and low-CT-producing strains of Vibrio mimicus. Transcriptional levels of various virulence-related genes were analyzed by qRT-PCR. The relative transcriptional level of each gene was normalized with the housekeeping recA gene. The mRNA transcription level of each gene in a low-CT-producing strain was compared with that of a high-CT-producing strain. The transcriptional level of each virulence-related gene of the high-CT-producing strain was arbitrarily assigned a value of 1 (relative arbitrary unit). Statistically significant differences were calculated using the two-sample t test. A P value of <0.05 was considered significant (***, P <0.005; **, P <0.01; *, P <0.05).