Genetic variation in the Vibrio vulnificus group 1 capsular polysaccharide operon

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.     

Deletion of valR,a homolog of Vibrio harveyis´ luxR generates an intermediate colony phenotype between opaque/rugose and translucent/smooth in Vibrio alginolyticus

A previous study has shown that Vibrio alginolyticus ZJ-51 undergoes colony phase variation between opaque/rugose (Op) and translucent/smooth (Tr). The AI-2 quorum-sensing master regulator ValR, a homolog to V. harveyi LuxR, was suggested to be involved in the transition. To investigate the role of ValR in the variation and in biofilm formation, an in-frame deletion of valR in both Op and Tr backgrounds was carried out. The mutants in both backgrounds showed an intermediate colony morphotype, where the colonies were less opaque/rugose but not fully translucent/smooth either. They also showed an intermediate level of motility. However, biofilm formation was severely decreased in both mutants and polar flagella were depleted also. Quantitative PCR showed that most of the genes related to flagellar and polysaccharide biosynthesis were upregulated in the mutant of Op background (ΔvalR/Op) but downregulated in the mutant of Tr background (ΔvalR/Tr) compared with their parental wild-type strains. This suggests that ValR may control biofilm formation by regulating flagellar biosynthesis and affect the expression of the genes involved in colony phase variation in V. alginolyticus.     

Capsular polysaccharide phase variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

Phenotypic and genetic differences between opaque and translucent colonies of Vibrio alginolyticus

Many pathogens undergo phase variation between rugose and smooth colony morphology or between opaque and translucent colony morphology, which is mainly due to the variation in the surface polysaccharides. In this study, Vibrio alginolyticus ZJ-51 displayed phase variation between opaque, rugose colonies (Op) and translucent, smooth colonies (Tr). Unlike the vibrios reported previously, Tr cells of ZJ-51 enhanced biofilm formation and motility, but they did not differ from Op cells in the quantity of surface polysaccharides produced. Real time PCR was used to analyze the expression of the genes involved in polysaccharide biosynthesis, flagellar synthesis, and the AI-2 quorum-sensing system. The results revealed that the K-antigen capsule gene cluster (which consists of homologs to the cpsA-K in Vibrio parahaemolyticus) and O-antigen polysaccharide gene cluster (which contains homologs to the wza-wzb-wzc) were significantly more transcribed in Tr cells. The AI-2 quorum-sensing genes showed enhanced expression in the Tr variant which also exhibited greater expression of genes associated with polar flagellar biosynthesis. These results suggest that colony phase variation might affect the virulence and survival ability in the stressful environment inhabited by V. alginolyticus.     

Capsular Polysaccharide Phase Variation in Vibrio vulnificus

Commonly found in raw oysters, Vibrio vulnificus poses a serious health threat to immunocompromised individuals and those with serum iron overload, with a fatality rate of approximately 50%. An essential virulence factor is its capsular polysaccharide (CPS), which is responsible for a significant increase in virulence compared to nonencapsulated strains. However, this bacterium is known to vary the amount of CPS expressed on the cell surface, converting from an opaque (Op) colony phenotype to a translucent (Tr) colony phenotype. In this study, the consistency of CPS conversion was determined for four strains of V. vulnificus. Environmental conditions including variations in aeration, temperature, incubation time, oxidative stress, and media (heart infusion or modified maintenance medium agar) were investigated to determine their influence on CPS conversion. All conditions, with the exception of variations in media and oxidative stress, significantly affected the conversion of the population, with high ranges of CPS expression found even within cells from a single colony. The global quorum-sensing regulators RpoS and AI-2 were also examined. While RpoS was found to significantly mediate phenotypic conversion, quorum sensing was not. Finally, 12 strains that comprise the recently found clinical (C) and environmental (E) genotypes of V. vulnificus were examined to determine their rates of population conversion. C-genotype strains, which are most often associated with infection, had a significantly lower rate of population conversion from Op to Tr phenotypes than did E-genotype strains (ca. 38% versus ca. 14%, respectively). Biofilm capabilities of these strains, however, were not correlated with increased population conversion.     

Phase variation, capsular polysaccharide, pilus and flagella contribute to uptake of Vibrio vulnificus by the Eastern oyster (Crassostrea virginica)

Vibrio vulnificus infections are associated with raw oyster consumption, and disease reservoirs are determined by the ability of this bacterium to infect and persist in oysters. Surface structures, such as capsular polysaccharide (CPS), pili and flagella, function as virulence factors in mouse infection models. Furthermore, virulence is related to phase variation in colony morphology, which reflects CPS expression and includes opaque (encapsulated, virulent), translucent (reduced encapsulation, avirulent) and rugose (wrinkled, biofilm-enhanced) colony types. The role of these factors in environmental survival is unknown; therefore, mutational analysis and phase variation of V. vulnificus were examined in an oyster infection model. Oysters ( Crassostrea virginica ) were pre-treated with tetracycline to reduce background bacteria and subsequently inoculated via filter feeding with 10⁶ colony-forming units (cfu) ml⁻¹ of V. vulnificus wild-type strains and phase variants, as well as strains with deletion mutations in genes related to CPS (Δ wza ), pili (Δ pilA ), flagella (Δ flaCDE/ Δ flaFBA ) and motility (Δ motAB ). All mutants were significantly reduced in their dissemination to oyster haemolymph as compared with wild type; however, recovery of mutants from gills and intestinal tissue was generally similar to wild type. Translucent and rugose inocula showed induction of high-frequency phase variation to the opaque encapsulated phenotype (100% and 72% respectively) during oyster infections that did not occur in strains recovered from seawater. Thus, multiple bacterial factors determine uptake of V. vulnificus in oysters, and phase variation during oyster infection is a likely mechanism for environmental survival and for induction of the more virulent phenotype.     

Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca2(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca2(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca2(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca2(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca2(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.     

Inhibitory effect of capsular antigen of Vibrio vulnificus on bactericidal activity of human serum

Opaque (Op) and translucent (Tr) colonial variants were isolated from Vibrio vulnificus strains. Op-type variants were more resistant than the isogenic Tr-type variants, but the survival rate of the Op-type variants varied with the strains. Antisera were prepared by immunizing rabbit with whole cells of Op and Tr variants of some strains, in which the difference of the sensitivity between Op and Tr cells was remarkable. Then agglutination tests with their living and heat-killed cells were carried out. The results suggested the presence of capsular antigen in Op cells and its absence in Tr cells, with the exception of the existence of a slight amount of capsular material in Tr variants of strain L-180. The thin capsular layer of Tr cells of strain L-180 was also demonstrated electron microscopically, but the layer was thinner than that of the isogenic Op cells. Results of determination of sugar content in the extracted capsular fraction also showed that Op to Tr transformation was due to loss of capsular antigen of the cells. These results confirmed the morphological studies previously reported which suggested the prevention of host defense system by the capsular material of the vibrio.     

Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood

The virulence factors of Vibrio vulnificus are not yet well understood. So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism. The present research was carried out in order to study the presence of some of these enzymes in 133 V. vulnificus strains isolated from 45 seafood samples. The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99·2% for caseinolytic protease, 96·9% for DNase, 65·4% for mucinase and 46·6% for elastase. None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells. In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59·4% of strains showed opaque morphology on BHI agar and 57·9% on nutrient agar, 10·5% presented translucent morphology on both agars and 30·1 and 31·6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively. None of the translucent colonies was virulent to mice. Therefore, opacity was a useful marker for potential virulence. Of 45 food samples contaminated with V. vulnificus , 29 (64·4%) presented strains lethal to adult mice.     

Thermal-death times of opaque and translucent morphotypes of Vibrio vulnificus.

Thermal-death times were determined for Vibrio vulnificus strains with different morphotypes. Opaque strains showed higher D values (times required to reduce the viable population of a given strain by 90%) than translucent strains. Z values (absolute values of the temperature required to reduce 1 log scale of D values) were also significantly higher in opaque morphotypes (2.4 to 2.5 degrees C) than in translucent ones (1.7 to 2.1 degrees C). These results indicate that the morphotype is related to the organism's susceptibility to heat.     

Factors influencing in vitro killing of bacteria by hemocytes of the eastern oyster (Crassostrea virginica).

A tetrazolium dye reduction assay was used to study factors governing the killing of bacteria by oyster hemocytes. In vitro tests were performed on bacterial strains by using hemocytes from oysters collected from the same location in winter and summer. Vibrio parahaemolyticus strains, altered in motility or colonial morphology (opaque and translucent), and Listeria monocytogenes mutants lacking catalase, superoxide dismutase, hemolysin, and phospholipase activities were examined in winter and summer. Vibrio vulnificus strains, opaque and translucent (with and without capsules), were examined only in summer. Among V. parahaemolyticus and L. monocytogenes, significantly (P < 0.05) higher levels of killing by hemocytes were observed in summer than in winter. L. monocytogenes was more resistant than V. parahaemolyticus or V. vulnificus to the bactericidal activity of hemocytes. In winter, both translucent strains of V. parahaemolyticus showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. In summer, only one of the V. parahaemolyticus translucent strains showed significantly (P < 0.05) higher susceptibility to killing by hemocytes than did the wild-type opaque strain. No significant differences (P > 0.05) in killing by hemocytes were observed between opaque (encapsulated) and translucent (nonencapsulated) pairs of V. vulnificus. Activities of 19 hydrolytic enzymes were measured in oyster hemolymph collected in winter and summer. Only one enzyme, esterase (C4), showed a seasonal difference in activity (higher in winter than in summer). These results suggest that differences existed between bacterial genera in their ability to evade killing by oyster hemocytes, that a trait(s) associated with the opaque phenotype may have enabled V. parahaemolyticus to evade killing by the oyster's cellular defense, and that bactericidal activity of hemocytes was greater in summer than in winter.     

Viability of Vibrio vulnificus in Association with Hemocytes of the American Oyster (Crassostrea virginica)

Certain indigenous estuarine bacteria, such as Vibrio vulnificus, may cause opportunistic human infections after consumption of raw oysters or exposure of tissues to seawater. V. vulnificus is known to be closely associated with oyster (Crassostrea virginica) tissues and is not removed by controlled purification methods, such as UV light-assisted depuration. In fact, when live shellfish are subjected to controlled purification, the number of V. vulnificus cells can markedly increase. A review of previous studies showed that few workers have examined mechanisms in oysters which may influence the persistence of V. vulnificus in shellfish, such as the fate of V. vulnificus following phagocytosis by molluscan hemocytes. The objectives of this study were to define the intracellular viability and extracellular viability of V. vulnificus during the phagocytic process and to study the release of specific lysosomal enzymes. The viability of a virulent estuarine V. vulnificus isolate with opaque morphology was compared with the viability of a translucent, nonvirulent form, the viability of Vibrio cholerae, and the viability of Escherichia coli in phagocytosis experiments. Our results showed that the levels of phagocytosis and bactericidal degradation of the opaque V. vulnificus isolate were less than the levels of phagocytosis and bactericial degradation of the translucent morphotype. These findings indicate that encapsulation may contribute to resistance to ingestion and degradation by hemocytes. The rates of intracellular death of V. cholerae and E. coli exceeded the rate of intracellular death of the opaque V. vulnificus isolate, even though the ingestion or uptake rates did not differ significantly. The levels of lysozyme activity and acid phosphatase activity were not significantly different in hemocyte monolayers inoculated with V. vulnificus.     

A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans

Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.     

High-frequency phase variation of Vibrio vulnificus 1003: isolation and characterization of a rugose phenotypic variant

The marine bacterium Vibrio vulnificus is a human pathogen that can spontaneously switch between virulent opaque and avirulent translucent phenotypes. Here, we document an additional form, the rugose variant, which produces copious biofilms and which may contribute both to pathogenicity of V. vulnificus and to its survival under adverse environmental conditions.     

Candida albicans MTLalpha tup1Delta mutants can reversibly switch to mating-competent, filamentous growth forms

Candida albicans strains that are homozygous at the mating type locus (MTLa or MTLalpha) can spontaneously switch from the normal round-to-oval yeast cell morphology to an elongated, so-called opaque cell form that can mate with opaque cells of the opposite mating type. In response to environmental signals, C. albicans also undergoes a transition from yeast to filamentous growth, which is negatively regulated by the general repressor Tup1p. Therefore, C. albicans mutants in which the TUP1 gene is inactivated grow constitutively in the filamentous form. We found that tup1Delta mutants of the MTLalpha strain WO-1 are still able to undergo phenotypic switching. Although the mutants had lost the capacity to grow in the normal yeast (white) or opaque forms, they could still reversibly switch between four different cell and colony phenotypes (designated as fuzzy, frizzy, wrinkled and smooth) at a frequency of about 10(-3) to 10(-4). Deletion of TUP1 resulted in deregulated expression of phase-specific genes. While the white-specific WH11 gene was constitutively expressed in all four cell types, the opaque-specific SAP1 gene remained repressed and the opaque-specific OP4 gene was weakly induced in all phase variants. In spite of the loss of white- and opaque-specific cell morphology and gene expression, the tup1Delta mutants retained an important characteristic of their wild-type parent, the ability to switch to a mating-competent form. The three filamentous phase variants (fuzzy, frizzy and wrinkled) all were able to mate and produce recombinant progeny with opaque cells of an MTLa strain at frequencies that were somewhat lower than those of normal opaque cells, whereas the smooth phase variant was unable to do so. Therefore, although deletion of TUP1 in C. albicans MTLalpha cells affects cellular morphology and gene expression patterns, the mutants can still reversibly switch between mating-competent and -incompetent cell types and mate with a partner of the opposite mating type.     

Genetic Analysis and Prevalence Studies of the brp Exopolysaccharide Locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus.     

Cyclic-di-GMP regulates extracellular polysaccharide production, biofilm formation, and rugose colony development by Vibrio vulnificus

Vibrio vulnificus is a human and animal pathogen that carries the highest death rate of any food-borne disease agent. It colonizes shellfish and forms biofilms on the surfaces of plankton, algae, fish, and eels. Greater understanding of biofilm formation by the organism could provide insight into approaches to decrease its load in filter feeders and on biotic surfaces and control the occurrence of invasive disease. The capsular polysaccharide (CPS), although essential for virulence, is not required for biofilm formation under the conditions used here. In other bacteria, increased biofilm formation often correlates with increased exopolysaccharide (EPS) production. We exploited the translucent phenotype of acapsular mutants to screen a V. vulnificus genomic library and identify genes that imparted an opaque phenotype to both CPS biosynthesis and transport mutants. One of these encoded a diguanylate cyclase (DGC), an enzyme that synthesizes bis-(3'-5')-cyclic-di-GMP (c-di-GMP). This prompted us to use this DGC, DcpA, to examine the effect of elevated c-di-GMP levels on several developmental pathways in V. vulnificus. Increased c-di-GMP levels induced the production of an EPS that was distinct from the CPS and dramatically enhanced biofilm formation and rugosity in a CPS-independent manner. However, the EPS could not compensate for the loss of CPS production that is required for virulence. In contrast to V. cholerae, motility and virulence appeared unaffected by elevated levels of c-di-GMP.