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.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

Induction of melanin biosynthesis in Vibrio cholerae.

Vibrio cholerae synthesized the pigment melanin in response to specific physiological conditions that were stressful to the bacterium. Pigmentation was induced when V. cholerae was subjected to hyperosmotic stress in conjunction with elevated growth temperatures (above 30 degrees C). The salt concentration tolerated by V. cholerae was lowered by additional abiotic factors such as acidic starting pH of the growth medium and limitation of organic nutrients. Although the amount of toxin detected in the culture supernatant decreased significantly in response to stressful culture conditions, no correlation between the physiological conditions that induced melanogenesis and expression of OmpU or cholera toxin was detected. Since conditions that induce melanin production in V. cholerae occur in both the aquatic environment and the human host, it is possible that melanogenesis has a specific function with respect to the survival of the bacterium in these habitats.     

A glimpse into the expanded genome content of Vibrio cholerae through identification of genes present in environmental strains

Vibrio cholerae has multiple survival strategies which are reflected both in its broad distribution in many aquatic environments and its high genotypic diversity. To obtain additional information regarding the content of the V. cholerae genome, suppression subtractive hybridization (SSH) was used to prepare libraries of DNA sequences from two southern California coastal isolates which are divergent or absent in the clinical strain V. cholerae O1 El Tor N16961. More than 1,400 subtracted clones were sequenced. This revealed the presence of novel sequences encoding functions related to cell surface structures, transport, metabolism, signal transduction, luminescence, mobile elements, stress resistance, and virulence. Flanking sequence information was determined for loci of interest, and the distribution of these sequences was assessed for a collection of V. cholerae strains obtained from southern California and Mexican environments. This led to the surprising observation that sequences related to the toxin genes toxA, cnf1, and exoY are widespread and more common in these strains than those of the cholera toxin genes which are a hallmark of the pandemic strains of V. cholerae. Gene transfer among these strains could be facilitated by a 4.9-kbp plasmid discovered in one isolate, which possesses similarity to plasmids from other environmental vibrios. By investigating some of the nucleotide sequence basis for V. cholerae genotypic diversity, DNA fragments have been uncovered which could promote survival in coastal environments. Furthermore, a set of genes has been described which could be involved in as yet undiscovered interactions between V. cholerae and eukaryotic organisms.     

Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.     

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

Abstract Haemaglutinin/protease (HA/P) is one of the virulence factors of Vibrio cholerae O1 and pathogenic strains of V. cholerae non-O1. In this study, we examined protease activity of a new serogroup of Vibrio cholerae recently designated as O139 synonym Bengal. The protease activity was produced by all eight isolates of V. cholerae O139 from Bangladeshi patients. Purification and partial characterization of the protease from V. cholerae O139 demonstrated the purified protease (O139-P) was indistinguishable from that previously reported for HA/P of V. cholerae non-O1 (NAG-HA/P) and V. cholerae O1 (Vc-HA/P). These results prove that V. cholerae O139 produces a protease belonging to solHA/P, and suggest that the protease is another virulence factor found in newly emerged V. cholerae O139, as in V. cholerae O1.     

The mannose-sensitive hemagglutinin of Vibrio cholerae promotes adherence to zooplankton.

The bacterium Vibrio cholerae, the etiological agent of cholera, is often found attached to plankton, a property that is thought to contribute to its environmental persistence in aquatic habitats. The V. cholerae O1 El Tor biotype and V. cholerae O139 strains produce a surface pilus termed the mannose-sensitive hemagglutinin (MSHA), whereas V. cholerae O1 classical biotype strains do not. Although V. cholerae O1 classical does not elaborate MSHA, the gene is present and expressed at a level comparable to that of the other strains. Since V. cholerae O1 El Tor and V. cholerae O139 have displaced V. cholerae O1 classical as the major epidemic strains over the last fifteen years, we investigated the potential role of MSHA in mediating adherence to plankton. We found that mutation of mshA in V. cholerae O1 El Tor significantly diminished, but did not eliminate, adherence to exoskeletons of the planktonic crustacean Daphnia pulex. The effect of the mutation was more pronounced for V. cholerae O139, essentially eliminating adherence. Adherence of the V. cholerae O1 classical mshA mutant was unaffected. The results suggest that MSHA is a factor contributing to the ability of V. cholerae to adhere to plankton. The results also showed that both biotypes of V. cholerae O1 utilize factors in addition to MSHA for zooplankton adherence. The expression of MSHA and these additional, yet to be defined, adherence factors differ in a serogroup- and biotype-specific manner.     

Vibrio cholerae O1 strains are facultative intracellular bacteria, able to survive and multiply symbiotically inside the aquatic free-living amoeba Acanthamoeba castellanii

Vibrio cholerae species are extracellular, waterborne, gram-negative bacteria that are overwhelmed by predators in aquatic environments. The unencapsulated serogroup V. cholerae O1 and encapsulated V. cholerae O139 cause epidemic and pandemic outbreaks of cholera. It has recently been shown that the aquatic and free-living amoeba Acanthamoeba castellanii is not a predator to V. cholerae O139; rather, V. cholerae O139 has shown an intracellular compatibility with this host. The aim of this study was to examine the ability of V. cholerae O1 classical and El Tor strains to grow and survive in A. castellanii. The interaction between A. castellanii and V. cholerae O1 strains was studied by means of amoeba cell counts and viable counts of the bacteria in the absence or presence of amoebae. The viable count of intracellularly growing bacteria was estimated by utilizing gentamicin assay. Confocal microscopy and electron microscopy were used to determine the intracellular localization of V. cholerae in A. castellanii. The results showed that V. cholerae O1 classical and El Tor strains grew and survived intracellularly in the cytoplasm of trophozoites, and that the bacteria were also found in the cysts of A. castellanii. The interaction showed a facultative intracellular behaviour of V. cholerae O1 classical and El Tor strains and a possible role of A. castellanii as an environmental host of V. cholerae species.     

Global impact of Vibrio cholerae interactions with chitin

The interaction of Vibrio cholerae with chitin exemplifies for microbial ecology a successful bacteria-substrate interaction with complex and significant influence on the lifestyle of the bacterium. Chitin is one of the most abundant polymers on earth and possibly the most abundant in the aquatic environment, where its association with V. cholerae has provided the microorganism with a number of advantages, including food availability, adaptation to environmental nutrient gradients, tolerance to stress and protection from predators. Emergent properties of V. cholerae-chitin interactions occur at multiple hierarchical levels in the environment and include cell metabolic and physiological responses e.g. chemotaxis, cell multiplication, induction of competence, biofilm formation, commensal and symbiotic relationship with higher organisms, cycling of nutrients, and pathogenicity for humans and aquatic animals. As factors mediating virulence of V. cholerae for humans and aquatic animals derive from mechanisms of adaptation to its environment, at different levels of hierarchical scale, V. cholerae interactions with chitin represent a useful model for examination of the role of primary habitat selection in the development of traits that have been identified as virulence factors in human disease.     

Biofilm acts as a microenvironment for plankton-associated Vibrio cholerae in the aquatic environment of Bangladesh

The role of biofilm as a microenvironment of plankton-associated Vibrio cholerae was investigated using plexiglass as a bait. A total of 72 biofilm samples were tested using culture, direct fluorescent antibody (DFA) and molecular techniques following standard procedures. Culturable V. cholerae (smooth and rugose variants) were isolated from 33% of the samples. V. cholerae O1 were detected by FA technique throughout the year except April and June. All V. cholerae O1 isolates were positive for tcpA, ctxA and ace genes while V. cholerae non-O1, non-O139 isolates lacked these genes. V. cholerae O1 (both Inaba and Ogawa) strains had identical ribotype pattern (R1), but V. cholerae non-O1, non-O139 had different ribotype patterns. All V. cholerae O1 strains were resistant to vibrio-static compound (O/129). All V. cholerae O1 except one were resistant to trimethoprime-sulphamethoxazole, streptomycin, nalidixic acid and furazolidone but sensitive to ciprofloxacin, and tetracycline. This study indicates that plexiglass can act as a bait to form biofilm, a microenvironment that provides shelter for plankton containing V. cholerae in the aquatic environment of Bangladesh.     

The capacity to produce cholera exotoxin in natural strains of Vibrio cholerae

The study of 27 V. cholerae strains, isolated from cholera patients and found to be hemolytically inactive, with a view to establish their capacity for the production of cholera toxin has revealed that 4 strains (V. cholerae cholerae Dacca 35, V. cholerae cholerae Dacca 3, V. cholerae cholerae B1307, V. cholerae cholerae J89) produce this protein. The quantitative determination of enterotoxin has been made with the use of GM1 ELISA technique. Strain Dacca 35 has been found to be highly toxigenic and, as regards the amount of exotoxin it produces, no different from V. cholerae cholerae strain 569B, a well-known producer of cholera toxin. In strain Dacca 35 correlation between the capacity of the cells for toxin production and the morphology of colonies has been established. The study has revealed that the chromosome of strain Dacca 35 contains two copies of gene vctAB responsible for the synthesis of cholera toxin.     

Analysis of residues determining specificity of Vibrio cholerae TonB1 for its receptors

In gram-negative organisms, high-affinity transport of iron substrates requires energy transduction to specific outer membrane receptors by the TonB-ExbB-ExbD complex. Vibrio cholerae encodes two TonB proteins, one of which, TonB1, recognizes only a subset of V. cholerae TonB-dependent receptors and does not facilitate transport through Escherichia coli receptors. To investigate the receptor specificity exhibited by V. cholerae TonB1, chimeras were created between V. cholerae TonB1 and E. coli TonB. The activities of the chimeric TonB proteins in iron utilization assays demonstrated that the C-terminal one-third of either TonB confers the receptor specificities associated with the full-length TonB. Single-amino-acid substitutions near the C terminus of V. cholerae TonB1 were identified that allowed TonB1 to recognize E. coli receptors and at least one V. cholerae TonB2-dependent receptor. This indicates that the very C-terminal end of V. cholerae TonB1 determines receptor specificity. The regions of the TonB-dependent receptors involved in specificity for a particular TonB protein were investigated in experiments involving domain switching between V. cholerae and E. coli receptors exhibiting different TonB specificities. Switching the conserved TonB box heptapeptides at the N termini of these receptors did not alter their TonB specificities. However, replacing the amino acid immediately preceding the TonB box in E. coli receptors with an aromatic residue allowed these receptors to use V. cholerae TonB1. Further, site-directed mutagenesis of the TonB box -1 residue in a V. cholerae TonB2-dependent receptor demonstrated that a large hydrophobic amino acid in this position promotes recognition of V. cholerae TonB1. These data suggest that the TonB box -1 position controls productive interactions with V. cholerae TonB1.     

多重PCR方法检测霍乱弧菌的研究

霍乱弧菌是霍乱的病原体,可以分为O1群、O139群和非O1/非O139群。O1群和O139群霍乱弧菌产生的霍乱肠毒素(也称霍乱毒素)是产生霍乱的主要原因,也只有O1群和O139群霍乱弧菌可引起霍乱。其他群的霍乱弧菌毒性不高,但在食品中也不允许被检出。实验以霍乱胶原酶基因和霍乱毒素基因为目的基因,试图建立一种PCR方法对霍乱弧菌进行检测研究,结果表明此方法可以用于食品中的霍乱弧菌检测。     

How Vibrio cholerae survive during starvation

Vibrio cholerae, a Gram-negative, motile, aquatic bacterium, is the causal agent of the diarrheal disease cholera. Cholera is a serious epidemic disease that has killed millions of people and continues to be a major health problem world-wide. The hypothesis that V. cholerae occupies an ecological niche in the estuarine environment requires that this organism is able to survive the dynamics of physiochemical stresses, including nutrient starvation. As a result of these stresses, bacteria in nature often exist in non-growth or very slow growth states with a low metabolic activity. Because microorganisms have little ability to control their environment, environmental changes have led to changes in cell function and structure. Such cellular responses can originate in one of two ways: by changes in genetic constitution or by phenotypic adaptation. In this review, we will focus on the phenotypic responses of V. cholerae of a given genotype to starvation stress.     

Glycogen contributes to the environmental persistence and transmission of Vibrio cholerae

Pathogenic Vibrio cholerae cycle between the nutrient-rich human intestinal tract and nutrient-poor aquatic environments and currently few bacterial factors are known that aid in the transition between these disparate environments. We hypothesized that the ability to store carbon as glycogen would facilitate both bacterial fitness in the aquatic environment and transmission of V. cholerae to new hosts. To investigate the role of glycogen in V. cholerae transmission, we constructed mutants that cannot store or degrade glycogen. Here, we provide the first report of glycogen metabolism in V. cholerae and demonstrate that glycogen prolongs survival in nutrient-poor environments that are known ecological niches of V. cholerae , including pond water and rice-water stool. Additionally, glycogen contributes to the pathogenesis of V. cholerae in a transmission model of cholera. A role for glycogen in the transmission of V. cholerae is further supported by the presence of glycogen granules in rice-water stool vibrios from cholera patients, indicating that glycogen is stored during human infection. Collectively, our findings indicate that glycogen metabolism is critical for V. cholerae to transition between host and aquatic environments.     

The absence of a flagellum leads to altered colony morphology, biofilm development and virulence in Vibrio cholerae O139

Throughout most of history, epidemic and pandemic cholera was caused by Vibrio cholerae of the serogroup O1. In 1992, however, a V. cholerae strain of the serogroup O139 emerged as a new agent of epidemic cholera. Interestingly, V. cholerae O139 forms biofilms on abiotic surfaces more rapidly than V. cholerae O1 biotype El Tor, perhaps because regulation of exopolysaccharide synthesis in V. cholerae O139 differs from that in O1 El Tor. Here, we show that all flagellar mutants of V. cholerae O139 have a rugose colony morphology that is dependent on the vps genes. This suggests that the absence of the flagellar structure constitutes a signal to increase exopolysaccharide synthesis. Furthermore, although exopolysaccharide production is required for the development of a three-dimensional biofilm, inappropriate exopolysaccharide production leads to inefficient colonization of the infant mouse intestinal epithelium by flagellar mutants. Thus, precise regulation of exopolysaccharide synthesis is an important factor in the survival of V. cholerae O139 in both aquatic environments and the mammalian intestine.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

In situ survival of Vibrio cholerae and Escherichia coli in tropical coral reefs.

Vibrio cholerae and Escherichia coli were inoculated into membrane diffusion chambers and placed around two small coral reef islands in Puerto Rico and monitored for 5 days. Several chambers were also buried in the sands of one of the reefs. Both E. coli and V. cholerae densities declined by 2 orders of magnitude, as measured by direct particle counts with a Coulter Counter (Coulter Electronics, Inc., Hialeah, Fla.). However, the density of neither bacteria changed dramatically when the same samples were analyzed by epifluorescent direct counts. Differences in the two direct count methods were accounted for by changes in cell morphology that occurred in both bacteria after exposure to seawater. Morphological changes occurred more rapidly in E. coli compared with those in V. cholerae. Bacteria in chambers exposed to sediment did not show significant changes in morphology and had only a slight decline in density. Physiological activity declined by more than 40% for both bacteria within 24 h. The decline in activity was less severe in the sediments. Tropical coral reef sands and turtle grass beds were shown to be less stressful environments for V. cholerae and E. coli than would have been predicted from temperature and microcosm studies. V. cholerae can survive the in situ conditions of a tropical coral reef and could become a source of bacterial contamination for fish and shellfish in this environment. The simultaneous monitoring of E. coli levels established that this bacteria can not be used as an indicator of V. cholerae or other fecal-borne pathogens in coral reef environments because of the greater stress these environments put on E. coli. Both bacteria could be of greater public health importance in tropical marine areas than previously imagined.     

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.