Genetics of stress adaptation and virulence in toxigenic Vibrio cholerae

Vibrio cholerae, a Gram-negative bacterium belonging to the gamma-subdivision of the family Proteobacteriaceae is the etiologic agent of cholera, a devastating diarrheal disease which occurs frequently as epidemics. Any bacterial species encountering a broad spectrum of environments during the course of its life cycle is likely to develop complex regulatory systems and stress adaptation mechanisms to best survive in each environment encountered. Toxigenic V. cholerae, which has evolved from environmental nonpathogenic V. cholerae by acquisition of virulence genes, represents a paradigm for this process in that this organism naturally exists in an aquatic environment but infects human beings and cause cholera. The V. cholerae genome, which is comprised of two independent circular mega-replicons, carries the genetic determinants for the bacterium to survive both in an aquatic environment as well as in the human intestinal environment. Pathogenesis of V. cholerae involves coordinated expression of different sets of virulence associated genes, and the synergistic action of their gene products. Although the acquisition of major virulence genes and association between V. cholerae and its human host appears to be recent, and reflects a simple pathogenic strategy, the establishment of a productive infection involves the expression of many more genes that are crucial for survival and adaptation of the bacterium in the host, as well as for its onward transmission and epidemic spread. While a few of the virulence gene clusters involved directly with cholera pathogenesis have been characterized, the potential exists for identification of yet new genes which may influence the stress adaptation, pathogenesis, and epidemiological characteristics of V. cholerae. Coevolution of bacteria and mobile genetic elements (plasmids, transposons, pathogenicity islands, and phages) can determine environmental survival and pathogenic interactions between bacteria and their hosts. Besides horizontal gene transfer mediated by genetic elements and phages, the evolution of pathogenic V. cholerae involves a combination of selection mechanisms both in the host and in the environment. The occurrence of periodic epidemics of cholera in endemic areas appear to enhance this process.     

Virulence genes in environmental strains of Vibrio cholerae

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.     

Molecular ecology of toxigenic Vibrio cholerae

Toxigenic Vibrio cholerae is the etiological agent of cholera, an acute dehydrating diarrhea that occurs in epidemic form in many developing countries. Although V. cholerae is a human pathogen, aquatic ecosystems are major habitats of Vibrio species, which includes both pathogenic and nonpathogenic strains that vary in their virulence gene content. V. cholerae belonging to the 01 and 0139 serogroups is 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, which appear to constitute an environmental reservoir of virulence genes. Although the definitive roles of the virulence-associated factors in the environment, and the environmental selection pressures for V. cholerae-carrying virulence genes or their homologues is not clear, the potential for origination of new epidemic strains from environmental progenitors seems real. It is likely that the aquatic environment harbors different virulence-associated genes scattered among environmental vibrios, which possess a lower virulence potential than the epidemic strains. The ecosystem comprising the aquatic environment, V. cholerae, genetic elements mediating gene transfer, and the mammalian host appears to support the clustering of critical virulence genes in a proper combination leading to the origination of new V. cholerae strains with epidemic potential.     

Proteome comparison of Vibrio cholerae cultured in aerobic and anaerobic conditions

The pathogen Vibrio cholerae causes severe diarrheal disease in humans. This environmental inhabitant has two distinct life cycles, in the environment and in the human small intestine, in which it differs in its multiplication behavior and virulence expression. Anaerobiosis, limitation of some nutrient elements, and excess burden from host metabolism reactants are the major stresses for V. cholerae living in intestine, in comparison to conditions in the environment and laboratory medium. For an insight into the response of V. cholerae to different microenvironments, we cultured the bacteria in aerobic and anaerobic conditions, and compared the whole cell proteome by two-dimensional electrophoresis. Among the protein spots identified, some protein species involved in aerobic respiration and the nutrient carbohydrate transporters were found to be more abundant in aerobic conditions, and some enzymes for anaerobic respiration and some stress response proteins were found more abundant in anaerobic culture. One spot corresponding to flagellin B subunit was decreased in anaerobic conditions, which suggests correlation with the meticulous regulation of bacterial motility during infection in the host intestine. This proteome analysis is the starting point for in-depth understanding of V. cholerae behavior in different environments.     

Novel approaches to monitor bacterial gene expression in infected tissue and host

Elucidating the complex and dynamic host-microbe interactions during infection requires, among other things, detailed knowledge of microbial gene expression in vivo. Recently, advances in fluorescence and bioluminescence detection techniques, as well as recombinase-based in vivo expression technology, have rendered monitoring virulence gene expression in vivo a feasible task. These techniques have been adapted by several laboratories to study the spatial and temporal patterns of virulence gene expression by organisms such as Salmonella typhimurium, Listeria monocytogenes, Yersinia entercolitica and Vibrio cholerae during infection of tissue culture or animal models of infection.