Crystal structure of the Vibrio cholerae colonization factor TcpF and identification of a functional immunogenic site

Vibrio cholerae relies on two main virulence factors—toxin-coregulated pilus (TCP) and cholera toxin—to cause the gastrointestinal disease cholera. TCP is a type IV pilus that mediates bacterial autoagglutination and colonization of the intestine. TCP is encoded by the tcp operon, which also encodes TcpF, a protein of unknown function that is secreted by V. cholerae in a TCP-dependent manner. Although TcpF is not required for TCP biogenesis, a tcpF mutant has a colonization defect in the infant mouse cholera model that is as severe as a pilus mutant. Furthermore, TcpF antisera protect against V. cholerae infection. TcpF has no apparent sequence homology to any known protein. Here, we report the de novo X-ray crystal structure of TcpF and the identification of an epitope that is critical for its function as a colonization factor. A monoclonal antibody recognizing this epitope is protective against V. cholerae challenge and adds to the protection provided by an anti-TcpA antibody. These data suggest that TcpF has a novel function in V. cholerae colonization and define a region crucial for this function.     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

An intracellular replication niche for Vibrio cholerae in the amoeba Acanthamoeba castellanii

Vibrio cholerae is a human pathogen and the causative agent of cholera. The persistence of this bacterium in aquatic environments is a key epidemiological concern, as cholera is transmitted through contaminated water. Predatory protists, such as amoebae, are major regulators of bacterial populations in such environments. Therefore, we investigated the interaction between V. cholerae and the amoeba Acanthamoeba castellanii at the single-cell level. We observed that V. cholerae can resist intracellular killing. The non-digested bacteria were either released or, alternatively, established a replication niche within the contractile vacuole of A. castellanii. V. cholerae was maintained within this compartment even upon encystment. The pathogen ultimately returned to its aquatic habitat through lysis of A. castellanii, a process that was dependent on the production of extracellular polysaccharide by the pathogen. This study reinforces the concept that V. cholerae is a facultative intracellular bacterium and describes a new host–pathogen interaction.     

The Type II Secretion System Delivers Matrix Proteins for Biofilm Formation by Vibrio cholerae

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment. Here we demonstrate a new role in biofilm formation for the V. cholerae T2S system, since wild-type V. cholerae was found to secrete the biofilm matrix proteins RbmC, RbmA, and Bap1 into the culture supernatant, while an isogenic T2S mutant could not. In agreement with this finding, the level of biofilm formation in a static microtiter assay was diminished in T2S mutants. Moreover, inactivation of the T2S system in a rugose V. cholerae strain prevented the development of colony corrugation and pellicle formation at the air-liquid interface. In contrast, extracellular secretion of the exopolysaccharide VPS, an essential component of the biofilm matrix, remained unaffected in the T2S mutants. Our results indicate that the T2S system provides a mechanism for the delivery of extracellular matrix proteins known to be important for biofilm formation by V. cholerae. Because the T2S system contributes to the pathogenicity of V. cholerae by secreting proteins such as cholera toxin and biofilm matrix proteins, elucidation of the molecular mechanism of T2S has the potential to lead to the development of novel preventions and therapies.     

FATP4 missense and nonsense mutations cause similar features in Ichthyosis Prematurity Syndrome

Background

Vibrio cholerae O1 and V. cholerae O139 infect humans, causing the diarrheal and waterborne disease cholera, which is a worldwide health problem. V. cholerae and the free-living amoebae Acanthamoeba species are present in aquatic environments, including drinking water and it has shown that Acanthamoebae support bacterial growth and survival. Recently it has shown that Acanthamoeba species enhanced growth and survival of V. cholerae O1 and O139. Water samples from different cholera endemic areas in Sudan were collected with the aim to detect both V. cholerae and Acanthamoeba species from same natural water samples by polymerase chain reaction (PCR).

Findings

For the first time both V. cholerae and Acanthamoeba species were detected in same natural water samples collected from different cholera endemic areas in Sudan. 89% of detected V. cholerae was found with Acanthamoeba in same water samples.

Conclusions

The current findings disclose Acanthamoedae as a biological factor enhancing survival of V. cholerae in nature.     

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.     

Novel insights into Haemagglutinin Protease (HAP) gene regulation in Vibrio cholerae

Quorum sensing is the phenomenon, whereby bacteria use signal molecules to communicate with each other. For example, to establish a successful infection, pathogenic bacteria become virulent only when they reach a certain local concentration in their host. Bassler and others have highlighted the surprising observation that quorum sensing seems to repress Vibrio cholerae virulence factor expression (e.g. cholera toxin), in contrast to what has been observed for virulence gene expression in other bacteria. Here, I present a novel insight that may clarify the way V. cholerae quorum‐sensing signals regulate its genes. Chironomids (Diptera; Chironomidae), which occur worldwide and are frequently the insect found most abundantly in fresh water bodies, are natural reservoirs of V. cholerae. Quorum‐sensing signals in V. cholerae up‐regulate the production of an extracellular enzyme, haemagglutinin protease (HAP), which degrades chironomid egg masses and prevents the eggs from hatching. HAP, therefore, is a virulence factor against chironomids. Indeed, in a survey carried out over the course of a year, V. cholerae and chironomids showed a pattern that mirrored the dynamics of predator‐prey populations. Globally, the numbers of chironomids are much larger than those of humans, so quorum‐sensing signals of V. cholerae and HAP gene regulation should be understood with regard to their role in chironomids rather than humans. Further research is needed to understand the role of cholera toxin in the environmental existence of V. cholerae.     

H-ras oncogene-transformed human bronchial epithelial cells (TBE-1) secrete a single metalloprotease capable of degrading basement membrane collagen

H-ras-transformed human bronchial epithelial cells (TBE-1) secrete a single major extracellular matrix metalloprotease which is not found in the normal parental cells. The enzyme is secreted in a latent form of 72 kDa, which can be activated to catalyze the cleavage of the basement membrane macromolecule type IV collagen. The substrates in their order of preference are: gelatin, type IV collagen, type V collagen, fibronectin, and type VII collagen; but the enzyme does not cleave the interstitial collagens or laminin. This protease is identical to gelatinase isolated from normal human skin explants, normal human skin fibroblasts, and SV40-transformed human lung fibroblasts. Based on its ability to initiate the degradation of type IV collagen in a pepsin-resistant portion of the molecule, it will be referred to as type IV collagenase. This enzyme is most likely the human analog of type IV collagenase detected in several rodent tumors, which has the same molecular mass and has been linked to their metastatic potential. Type IV collagenase consists of three domains. Two of them, the amino-terminal domain and the carboxyl-terminal domain, are homologous to interstitial collagenase and human and rat stromelysin. The middle domain, of 175 residues, is organized into three 58-residue head-to-tail repeats which are homologous to the type II motif of the collagen-binding domain of fibronectin. Type IV collagenase represents the third member of a newly recognized gene family coding for secreted extracellular matrix metalloproteases, which includes interstitial fibroblast collagenase and stromelysin.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

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.     

Termination of RNA by nucleotides of 9-beta-D-xylofuranosyladenine

The protease susceptibilities of recently identified cartilage collagens HMW, 1α, 2α, and 3α were investigated. Mammlian skin collagenase cleaved the 3α chain under conditions where HMW, 1α and 2α were not degraded. A tumor cell derived type V collagenolytic metalloproteinase degraded HMW, 1α and 2α, but not 3α. Plasmin or leucocyte elastase failed to significantly degrade any of the cartilage collagens when digestion was performed at 25°C (15 hours, enzyme to substrate ratio 1:100). At 36°C but not 33°C α thrombin degraded HMW, 1α and 2α, with little or no degradation of 3α. This pattern of protease susceptibility for HMW, 1α and 2α is therefore similar to type V collagen. The cleavage of 3α by skin collagenase but not by elastase is similar to type II collagen. These results suggest that HMW, 1α and 2α are part of the type V collagen family.     

Vibrio cholerae Hemagglutinin/Protease Degrades Chironomid Egg Masses

Cholera is a severe diarrheal disease caused by specific serogroups of Vibrio cholerae that are pathogenic to humans. The disease does not persist in a chronic state in humans or animals. The pathogen is naturally present as a free-living organism in the environment. Recently, it was suggested that egg masses of the nonbiting midge Chironomus sp. (Diptera) harbor and serve as a nutritive source for V. cholerae, thereby providing a natural reservoir for the organism. Here we report that V. cholerae O9, O1, and O139 supernatants lysed the gelatinous matrix of the chironomid egg mass and inhibited eggs from hatching. The extracellular factor responsible for the degradation of chironomid egg masses (egg mass degrading factor) was purified from V. cholerae O9 and O139 and was identified as the major secreted hemagglutinin/protease (HA/P) of V. cholerae. The substrate in the egg mass was characterized as a glycoprotein. These findings show that HA/P plays an important role in the interaction of V. cholerae and chironomid egg masses.     

Role of Zooplankton Diversity in Vibrio cholerae Population Dynamics and in the Incidence of Cholera in the Bangladesh Sundarbans

Vibrio cholerae, a bacterium autochthonous to the aquatic environment, is the causative agent of cholera, a severe watery, life-threatening diarrheal disease occurring predominantly in developing countries. V. cholerae, including both serogroups O1 and O139, is found in association with crustacean zooplankton, mainly copepods, and notably in ponds, rivers, and estuarine systems globally. The incidence of cholera and occurrence of pathogenic V. cholerae strains with zooplankton were studied in two areas of Bangladesh: Bakerganj and Mathbaria. Chitinous zooplankton communities of several bodies of water were analyzed in order to understand the interaction of the zooplankton population composition with the population dynamics of pathogenic V. cholerae and incidence of cholera. Two dominant zooplankton groups were found to be consistently associated with detection of V. cholerae and/or occurrence of cholera cases, namely, rotifers and cladocerans, in addition to copepods. Local differences indicate there are subtle ecological factors that can influence interactions between V. cholerae, its plankton hosts, and the incidence of cholera.     

Association of Vibrio cholerae O1 El Tor and O139 Bengal with the Copepods Acartia tonsa and Eurytemora affinis

The association of Vibrio cholerae with zooplankton has been suggested as an important factor in transmission of human epidemic cholera, and the ability to colonize zooplankton surfaces may play a role in the temporal variation and predominance of the two different serogroups (V. cholerae O1 El Tor and O139) in the aquatic environment. To date, interactions between specific serogroups and species of plankton remain poorly understood. Laboratory microcosm experiments were carried out to compare quantitatively the colonization of two copepod species, Acartia tonsa and Eurytemora affinis, by each of the epidemic serogroups. V. cholerae O1 consistently achieved higher abundances than V. cholerae O139 in colonizing adults of each copepod species as well as the multiple life stages of E. affinis. This difference in colonization may be significant in the general predominance of V. cholerae O1 in cholera epidemics in rural Bangladesh where water supplies are taken directly from the environment.     

Enzyme-antibody histochemistry

Summary Different types of distinct molecular forms of collagen are components of the extracellular matrix in most tissues. The common types can usually be detected by immunohistochemical methods but others may escape detection for lack of specific antisera. However, all these collagens are substrates for the collagenase of Clostridium histolyticum. In this report we describe a method that allows the visualization of collagens, collectively, in a tissue preparation. The method is based on the affinity between clostridial collagenase and collagen on one hand, and collagenase and its antibody on the other. Under the conditions of low temperature used in the procedure, collagenase binds to collagen, but digestion does not occur. Subequent reaction of the bound collagenase with the specific collagenase antibody is followed by reaction with a tagged anti-IgG reagent. This allows the visualization of the enzyme-substrate complex.The procedure is illustrated in sections of the heart and the aorta, as well as in the isolated cardiomyocytes and the collagen distribution is verified using collagens type I and IV specific antibodies. In all instances the collagenase staining pattern includes all structural features seen individually with the type specific anticollagen antibodies.Abbreviations BSA Bovine serum albumin - PBS phosphate buffored saline     

Susceptibility to Vibrio cholerae infection in a cohort of household contacts of patients with cholera in Bangladesh

Background

Despite recent progress in understanding the molecular basis of Vibrio cholerae pathogenesis, there is relatively little knowledge of the factors that determine the variability in human susceptibility to V. cholerae infection.

Methods and Findings

We performed an observational study of a cohort of household contacts of cholera patients in Bangladesh, and compared the baseline characteristics of household members who went on to develop culture-positive V. cholerae infection with individuals who did not develop infection. Although the vibriocidal antibody is the only previously described immunologic marker associated with protection from V. cholerae infection, we found that levels of serum IgA specific to three V. cholerae antigens—the B subunit of cholera toxin, lipopolysaccharide, and TcpA, the major component of the toxin–co-regulated pilus—also predicted protection in household contacts of patients infected with V. cholerae O1, the current predominant cause of cholera. Circulating IgA antibodies to TcpA were also associated with protection from V. cholerae O139 infection. In contrast, there was no association between serum IgG antibodies specific to these three antigens and protection from infection with either serogroup. We also found evidence that host genetic characteristics and serum retinol levels modify susceptibility to V. cholerae infection.

Conclusions

Our observation that levels of serum IgA (but not serum IgG) directed at certain V. cholerae antigens are associated with protection from infection underscores the need to better understand anti–V. cholerae immunity at the mucosal surface. Furthermore, our data suggest that susceptibility to V. cholerae infection is determined by a combination of immunologic, nutritional, and genetic characteristics; additional factors that influence susceptibility to cholera remain unidentified.     

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand

Background

Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.

Methods/Findings

A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.

Conclusions

MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.