Virulence genes in environmental strains of Vibrio cholerae

Abstract

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.

FIG. 1

PCR analysis of tcpA and ctxA of genomic DNA from representative environmental strains of V. cholerae isolated from enriched APW (pH 8.5) samples. Lane 1, environmental strain of V. cholerae (SCE5) possessing tcpA-C; lane 2, environmental strain of V. cholerae (SCE340) possessing tcpA-E; lane 3, environmental strain of V. cholerae (SCE188) possessing ctxA; lane 4, V. cholerae O1 Ogawa, El Tor (positive control for ctxA and tcpA-E); lane 5, V. cholerae O1, Ogawa, classical (positive control for ctxA and tcpA-C).

FIG. 2

Southern blot hybridization of PstI-digested genomic DNA from environmental strains of V. cholerae using tcpA (A) and ctxA (B) probes.

FIG. 3

BglI restriction patterns of rRNA genes in environmental strains of V. cholerae isolated in Calcutta. Ribotype patterns A through J produced by different strains are shown (see Table 1 for details). Numbers indicating the molecular sizes of bands correspond to a 1-kb DNA ladder (Bethesda Research Laboratories).

FIG. 4

Multiple alignment of pilin amino acid sequence of TcpA-C, TcpA of SCE5, and TcpA-E. The alignment was created by the DNAsis (Hitachi) program. The shaded areas indicate identical residues, while unshaded areas indicate dissimilar residues. The GenBank accession numbers for tcpA-C and tcpA-E are M33514 and U89807, respectively.

FIG. 5

Electron micrographs of pili of environmental V. cholerae strain SCE5. Bacteria were cultured at 25°C for 24 h on CFA agar and negatively stained. (A) Single pilus. Magnification, ×40,000. (B) After 36 h, TCP bundles. Magnification, 147,000. P, pili; F, flagellum.