Characterization of VPI Pathogenicity Island and CTXφ Prophage in Environmental Strains of Vibrio cholerae

Abstract

Environmental isolates of Vibrio cholerae of eight randomly amplified polymorphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to gain insights into V. cholerae evolution. We found that each isolate contained a variant form of the VPI pathogenicity island. Distinguishing features included (i) four new alleles of tcpF (which encodes secreted virulence protein; its exact function is unknown), 20 to 70% divergent (at the protein level) from each other and canonical tcpF; (ii) a new allele of toxT (virulence regulatory gene), 36% divergent (at the protein level) in its 5 half and nearly identical in its 3 half to canonical toxT; (iii) a new tcpA (pilin) gene; and (iv) four variant forms of a regulatory sequence upstream of toxT. Also found were transpositions of an IS903-related element and function-unknown genes to sites in VPI. Cholera toxin (ctx) genes were found in isolates of two RAPD types, in each case embedded in CTX-like prophages. Fragments that are inferred to contain only putative repressor, replication, and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters found in some nonepidemic V. cholerae strains, suggest that some of these genes contribute to fitness in nature, and emphasize the potential importance of interstrain gene exchange in the evolution of this species.Vibrio cholerae is a genetically diverse species that lives in warm-water environments, often associated with plankton and other aquatic organisms (9, 15, 31). Strains of just two of the approximately 200 currently known O-antigen serogroups (50; T. Shimada, personal communication), O1 and O139, cause epidemic cholera-the acute, devastating diarrheal disease that afflicts many thousands of people annually, especially in developing countries. Although certain other serogroups cause sporadic diarrheal disease (30, 37), most V. cholerae organisms probably do not infect humans. Epidemic strains are also distinguished from most others by their production of cholera toxin and a toxin-coregulated pilus (TCP) (15,21).Epidemic strains of V. cholerae have a complex natural history in areas of cholera endemicity, involving rapid transmission to humans via contaminated food and water during each year's cholera season and persistence or proliferation in aquatic organisms or abiotic niches at other times. Several recent major changes in patterns of epidemic cholera may stem in large part from a combination of human factors and environmental fluctuation that could affect the distribution of aquatic host species. The bacterial changes include (i) the replacement of classical biotype O1 strains beginning in 1961 by strains of the new El Tor O1 biotype (15), (ii) the reemergence of classical biotype strains in parts of Bangladesh i...

Distribution of virulence markers in clinical and environmental Vibrio cholerae non-O1/non-O139 strains isolated in Brazil from 1991 to 2000

“…However, our results contradict the assumption that most cholera toxin-producing strains are also positive for TCP, since TCP is known to be the receptor for CTXφ infection of V. cholerae. In fact, our results appear to be consistent with data from recent studies that report the presence of the two virulence genes found in epidemic strains, ctxAB and tcpA, in environmental and clinical strain of serogroups other than O1 and O139 5,10,27,28,31,32 .…”

Genetic diversity and virulence potential of environmental Vibrio cholerae population in a cholera-endemic area

“…Several studies have suggested a role of virulence factors or their homologues in the symbiotic association of V. cholerae with a variety of aquatic organisms. Besides gene clusters associated with pandemic strains, recent studies have recognized the existence of different alleles of virulence genes in environmental V. cholerae strains, including different alleles of tcpA, tcpF, and toxT genes and different alleles of the CTX⌽ prophage repressor rstR in vibrios of various nonepidemic serogroups (9,10). It has been suggested that the different environmental alleles of virulence genes may have evolved in response to selective pressures that vary between the environment and the host (36).…”

“…Included were 15 strains that colonized mice and caused fluid accumulation in rabbits despite the absence of TCP and CT genes. Recent studies have identified genetic variants of tcpA gene encoding the major pilus subunit in environmental strains (9). These 15 strains were also negative for the known environmental variants of tcpA gene.…”

“…Included are PCR assays specific for the tcpA, tcpI, and acfB genes of the toxin-coregulated pilus (TCP) pathogenicity island (22)(23)(24), environmental tcpA variant (9), genes of the RS1-element (rstR and rstC), hemolysin (hlyA), RTX toxin gene cluster (rtxA and rtxC), and mshA for mannose-sensitive hemagglutinin (25)(26)(27)(28)(29). PCR reagents and kits were obtained either from Perkin-Elmer or Invitrogen, and PCR was performed essentially as described previously.…”

“…V. cholerae O1 and O139 are commonly known to carry a set of virulence genes necessary for pathogenesis in humans. Recent studies have indicated that virulence genes or their homologues are also dispersed among environmental strains of V. cholerae belonging to diverse serogroups that appear to constitute an environmental reservoir of virulence genes (9,10). Although the roles of virulence-associated factors in the environment and the selection pressures for environmental V. cholerae carrying virulence genes is not clear, it is possible that these strains may be precursors of pathogenic strains or may participate in genetransfer events leading to the origination of pathogenic strains.…”