Role of exopolysaccharide, the rugose phenotype and VpsR in the pathogenesis of epidemic Vibrio cholerae

Vibrio cholerae, the causative agent of cholera can produce an exopolysaccharide (EPS). Some strains can also phenotypically switch from a smooth to a 'rugose' phenotype characterized by small wrinkled colonies, overproduction of EPS, increased biofilm formation in vitro and increased resistance to various stressful conditions. High frequency switching to the rugose phenotype is more common in epidemic strains than in non-pathogenic strains, suggesting EPS production and the rugose phenotype are important in cholera epidemiology. VpsR up-regulates Vibrio polysaccharide (VPS) genes and the synthesis of extracellular EPS (VPS). However, the function of VPS, the rugose phenotype and VpsR in pathogenesis is not well understood. We report that rugose strains of both classical and El Tor biotypes of epidemic V. cholerae are defective in the in vitro production of extracellular collagenase activity. In vivo studies in rabbit ileal loops suggest that VpsR mutants are attenuated in reactogenicity. Intestinal colonization studies in infant mice suggest that VPS production, the rugose phenotype and VpsR have a role in pathogenesis. Our results indicate that regulated VPS production is important for promoting in vivo biofilm formation and pathogenesis. Additionally, VpsR might regulate genes with roles in virulence. Rugose strains appear to be a subpopulation of cells that might act as a 'helper' phenotype promoting the pathogenesis of certain strains. Our studies provide new insight into the potential role of VPS, the rugose phenotype and VpsR in the pathogenesis of epidemic V. cholerae.     

VpsR, a Member of the Response Regulators of the Two-Component Regulatory Systems, Is Required for Expression of vps Biosynthesis Genes and EPS(ETr)-Associated Phenotypes in Vibrio cholerae O1 El Tor

The rugose colonial variant of Vibrio cholerae O1 El Tor produces an exopolysaccharide (EPS(ETr)) that enables the organism to form a biofilm and to resist oxidative stress and the bactericidal action of chlorine. Transposon mutagenesis of the rugose variant led to the identification of vpsR, which codes for a homologue of the NtrC subclass of response regulators. Targeted disruption of vpsR in the rugose colony genetic background yielded a nonreverting smooth-colony morphotype that produced no detectable EPS(ETr) and did not form an architecturally mature biofilm. Analysis of two genes, vpsA and vpsL, within the vps cluster of EPS(ETr) biosynthesis genes revealed that their expression is induced above basal levels in the rugose variant, compared to the smooth colonial variant, and requires vpsR. These results show that VpsR functions as a positive regulator of vpsA and vpsL and thus acts to positively regulate EPS(ETr) production and biofilm formation.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30°C form rugose colonies, while the same cells grown at 37°C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30°C produced more biofilm material than rugose colonies grown at 37°C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

The rbmBCDEF gene cluster modulates development of rugose colony morphology and biofilm formation in Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, can undergo phenotypic variation generating rugose and smooth variants. The rugose variant forms corrugated colonies and well-developed biofilms and exhibits increased levels of resistance to several environmental stresses. Many of these phenotypes are mediated in part by increased expression of the vps genes, which are organized into vps-I and vps-II coding regions, separated by an intergenic region. In this study, we generated in-frame deletions of the five genes located in the vps intergenic region, termed rbmB to -F (rugosity and biofilm structure modulators B to F) in the rugose genetic background, and characterized the mutants for rugose colony development and biofilm formation. Deletion of rbmB, which encodes a protein with low sequence similarity to polysaccharide hydrolases, resulted in an increase in colony corrugation and accumulation of exopolysaccharides relative to the rugose variant. RbmC and its homolog Bap1 are predicted to encode proteins with carbohydrate-binding domains. The colonies of the rbmC bap1 double deletion mutant and bap1 single deletion mutant exhibited a decrease in colony corrugation. Furthermore, the rbmC bap1 double deletion mutant was unable to form biofilms at the air-liquid interface after 2 days, while the biofilms formed on solid surfaces detached readily. Although the colony morphology of rbmDEF mutants was similar to that of the rugose variant, their biofilm structure and cell aggregation phenotypes were different than those of the rugose variant. Taken together, these results indicate that vps intergenic region genes encode proteins that are involved in biofilm matrix production and maintenance of biofilm structure and stability.     

Identification and characterization of a locus putatively involved in colanic acid biosynthesis in Vibrio alginolyticus ZJ-51

Colanic acid (CA) is a group I extracellular polysaccharide (EPS) that contributes to resistance against adverse environments in many members of the Enterobacteriaceae. In the present study, a genetic locus EPS_C putatively involved in CA biosynthesis was identified in Vibrio alginolyticus ZJ-51, which undergoes colony morphology variation between translucent/smooth (ZJ-T) and opaque/rugose (ZJ-O). EPS_C in ZJ-T carries 21 ORFs and resembles the CA cluster of Escherichia coli K-12. The deletion of EPS_C led to decreased EPS and biofilm formation in both genetic backgrounds but no alternation of lipopolysaccharide. The loss of this locus also changed the colony morphology of ZJ-O on the 2216E plate and reduced the motility of ZJ-T. Compared with ZJ-T, ZJ-O lacks a 10-kb fragment (eps^T) in EPS_C containing homologs of wecA, wzx and wzy that are essential for O-antigen synthesis. However, the deletion or overexpression of eps^T resulted in no change of colony morphology, biofilm formation or EPS production. This study reported at the first time a genetic locus EPS_C that may be involved in colanic acid synthesis in V. alginolyticus ZJ-51, and found that it was related to EPS biosynthesis, biofilm formation, colony morphology and motility, which may shed light on the environmental adaptation of the vibrios.     

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.     

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.     

Rugosity in Grimontia hollisae

Grimontia hollisae, formerly Vibrio hollisae, produces both smooth and rugose colonial variants. The rugose colony phenotype is characterized by wrinkled colonies producing copious amounts of exopolysaccharide. Cells from a rugose colony grown at 30 degrees C form rugose colonies, while the same cells grown at 37 degrees C form smooth colonies, which are characterized by a nonwrinkled, uncrannied appearance. Stress response studies revealed that after exposure to bleach for 30 min, rugose survivors outnumbered smooth survivors. Light scatter information obtained by flow cytometry indicated that rugose cells clumped into clusters of three or more cells (average, five cells) and formed two major clusters, while smooth cells formed only one cluster of single cells or doublets. Fluorescent lectin-binding flow cytometry studies revealed that the percentages of rugose cells that bound either wheat germ agglutinin (WGA) or Galanthus nivalis lectin (GNL) were greater than the percentages of smooth cells that bound the same lectins (WGA, 35% versus 3.5%; GNL, 67% versus 0.21%). These results indicate that the rugose exopolysaccharide consists partially of N-acetylglucosamine and mannose. Rugose colonies produced significantly more biofilm material than did smooth colonies, and rugose colonies grown at 30 degrees C produced more biofilm material than rugose colonies grown at 37 degrees C. Ultrastructurally, rugose colonies show regional cellular differentiation, with apical and lateral colonial regions containing cells embedded in a matrix stained by Alcian Blue. The cells touching the agar surface are packed tightly together in a palisade-like manner. The central region of the colony contains irregularly arranged, fluid-filled spaces and loosely packed chains or arrays of coccoid and vibrioid cells. Smooth colonies, in contrast, are flattened, composed of vibrioid cells, and lack distinct regional cellular differences. Results from suckling mouse studies showed that both orally fed rugose and smooth variants elicited significant, but similar, amounts of fluid accumulated in the stomach and intestines. These observations comprise the first report of expression and characterization of rugosity by G. hollisae and raise the possibility that expression of rugose exopolysaccharide in this organism is regulated at least in part by growth temperature.     

Roles of the Structural Symbiosis Polysaccharide (syp) Genes in Host Colonization,Biofilm Formation,and Polysaccharide Biosynthesis in Vibrio fischeri

The symbiosis polysaccharide locus, syp, is required for Vibrio fischeri to form a symbiotic association with the squid Euprymna scolopes. It is also required for biofilm formation induced by the unlinked regulator RscS. The syp locus includes 18 genes that can be classified into four groups based on putative function: 4 genes encode putative regulators, 6 encode glycosyltransferases, 2 encode export proteins, and the remaining 6 encode proteins with other functions, including polysaccharide modification. To understand the roles of each of the 14 structural syp genes in colonization and biofilm formation, we generated nonpolar in-frame deletions of each gene. All of the deletion mutants exhibited defects in their ability to colonize juvenile squid, although the impact of the loss of SypB or SypI was modest. Consistent with their requirement for colonization, most of the structural genes were also required for RscS-induced biofilm formation. In particular, the production of wrinkled colonies, pellicles, and the matrix on the colony surface was eliminated or severely decreased in all mutants except for the sypB and sypI mutants; in contrast, only a subset of genes appeared to play a role in attachment to glass. Finally, immunoblotting data suggested that the structural Syp proteins are involved in polysaccharide production and/or export. These results provide important insights into the requirements for the syp genes under different environmental conditions and thus lay the groundwork for a more complete understanding of the matrix produced by V. fischeri to enhance cell-cell interactions and promote symbiotic colonization.     

Highly Dynamic Genomic Loci Drive the Synthesis of Two Types of Capsular or Secreted Polysaccharides within the Mycoplasma mycoides Cluster

Mycoplasmas of the Mycoplasma mycoides cluster are all ruminant pathogens. Mycoplasma mycoides subsp. mycoides is responsible for contagious bovine pleuropneumonia and is known to produce capsular polysaccharide (CPS) and exopolysaccharide (EPS). Previous studies have strongly suggested a role for Mycoplasma mycoides subsp. mycoides polysaccharides in pathogenicity. Mycoplasma mycoides subsp. mycoides-secreted EPS was recently characterized as a β(1→6)-galactofuranose homopolymer (galactan) identical to the capsular product. Here, we extended the characterization of secreted polysaccharides to all other members of the M. mycoides cluster: M. capricolum subsp. capripneumoniae, M. capricolum subsp. capricolum, M. leachii, and M. mycoides subsp. capri (including the LC and Capri serovars). Extracted EPS was characterized by nuclear magnetic resonance, resulting in the identification of a homopolymer of β(1→2)-glucopyranose (glucan) in M. capricolum subsp. capripneumoniae and M. leachii. Monoclonal antibodies specific for this glucan and for the Mycoplasma mycoides subsp. mycoides-secreted galactan were used to detect the two polysaccharides. While M. mycoides subsp. capri strains of serovar LC produced only capsular galactan, no polysaccharide could be detected in strains of serovar Capri. All strains of M. capricolum subsp. capripneumoniae and M. leachii produced glucan CPS and EPS, whereas glucan production and localization varied among M. capricolum subsp. capricolum strains. Genes associated with polysaccharide synthesis and forming a biosynthetic pathway were predicted in all cluster members. These genes were organized in clusters within two loci representing genetic variability hot spots. Phylogenetic analysis showed that some of these genes, notably galE and glf, were acquired via horizontal gene transfer. These findings call for a reassessment of the specificity of the serological tests based on mycoplasma polysaccharides.     

High-frequency rugose exopolysaccharide production by Vibrio cholerae

Vibrio cholerae can shift to a "rugose" phenotype, thereby producing copious exopolysaccharide (EPS), which promotes its environmental survival and persistence. We report conditions that promote high-frequency rugose EPS production (HFRP), whereby cells switch at high frequency (up to 80%) to rugose EPS production. HFRP appeared to be more common in clinical strains, as HFRP was found in 6 of 19 clinical strains (32%) (including classical, El Tor, and non-O1 strains) but in only 1 of 16 environmental strains (6%). Differences were found between strains in rugose colony morphology, conditions promoting HFRP, the frequency of rugose-to-smooth (R-S) cell reversion, and biofilm formation. We propose that rugose EPS and HFRP provide an evolutionary and adaptive advantage to specific epidemic V. cholerae strains for increased persistence in the environment.     

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.     

High-Frequency Rugose Exopolysaccharide Production by Vibrio cholerae Strains Isolated in Haiti

In October, 2010, epidemic cholera was reported for the first time in Haiti in over 100 years. Establishment of cholera endemicity in Haiti will be dependent in large part on the continued presence of toxigenic V. cholerae O1 in aquatic reservoirs. The rugose phenotype of V. cholerae, characterized by exopolysaccharide production that confers resistance to environmental stress, is a potential contributor to environmental persistence. Using a microbiologic medium promoting high-frequency conversion of smooth to rugose (S–R) phenotype, 80 (46.5%) of 172 V. cholerae strains isolated from clinical and environmental sources in Haiti were able to convert to a rugose phenotype. Toxigenic V. cholerae O1 strains isolated at the beginning of the epidemic (2010) were significantly less likely to shift to a rugose phenotype than clinical strains isolated in 2012/2013, or environmental strains. Frequency of rugose conversion was influenced by incubation temperature and time. Appearance of the biofilm produced by a Haitian clinical rugose strain (altered biotype El Tor HC16R) differed from that of a typical El Tor rugose strain (N16961R) by confocal microscopy. On whole-genome SNP analysis, there was no phylogenetic clustering of strains showing an ability to shift to a rugose phenotype. Our data confirm the ability of Haitian clinical (and environmental) strains to shift to a protective rugose phenotype, and suggest that factors such as temperature influence the frequency of transition to this 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.