Transcriptional regulation of the mannitol phosphotransferase system operon by the ferric uptake regulator (Fur) in Vibrio cholerae El Tor serogroup O1.

The phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS) allows bacteria to use various carbohydrates as energy resources including mannitol. The mannitol-specific PTS transporter in Vibrio cholerae is encoded by the mtlADR operon. Expression of the mtl operon has been shown to be strictly regulated by CRP, MtlS, and MtlR. In the present study, we investigated the regulation of mtlADR by the ferric uptake regulator (Fur). The results showed that Fur binds to the promoter-proximal DNA region of mtlADR to repress its transcription independent of iron, in mannitol-containing growth medium. The capacity for mannitol fermentation was significantly increased in Δfur relative to that of WT for normal and iron-replete growth media. The level of organic acids produced by Δfur was significantly enhanced relative to that produced by the WT strain in the normal and iron-replete media but not in an iron-starved medium. The results provided for a deeper understanding of the regulation of mtlADR in V. cholerae.

Vibrio cholerae Sialidase-Specific Immune Responses Are Associated with Protection against Cholera.

Cholera remains a major public health problem in resource-limited countries. Vaccination is an important strategy to prevent cholera, but currently available vaccines provide only 3 to 5 years of protection. Understanding immune responses to cholera antigens in naturally infected individuals may elucidate which of these are key to longer-term protection seen following infection. We recently identified Vibrio cholerae O1 sialidase, a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells, as immunogenic following infection in two recent high-throughput screens. Here, we present systemic, mucosal, and memory immune responses to sialidase in cholera index cases and evaluated whether systemic responses to sialidase correlated with protection using a cohort of household contacts. Overall, we found age-related differences in antisialidase immune response following cholera. Adults developed significant plasma anti-sialidase IgA, IgG, and IgM responses following infection, whereas older children (≥5 years) developed both IgG and IgM responses, and younger children only developed IgM responses. Neither older children nor younger children had a rise in IgA responses over the convalescent phase of infection (day 7/day 30). On evaluation of mucosal responses and memory B-cell responses to sialidase, we found adults developed IgA antibody-secreting cell (ASC) and memory B-cell responses. Finally, in household contacts, the presence of serum anti-sialidase IgA, IgG, and IgM antibodies at enrollment was associated with a decrease in the risk of subsequent infection. These data show cholera patients develop age-related immune responses against sialidase and suggest that immune responses that target sialidase may contribute to protective immunity against cholera.IMPORTANCE Cholera infection can result in severe dehydration that may lead to death within a short period of time if not treated immediately. Vaccination is an important strategy to prevent the disease. Oral cholera vaccines provide 3 to 5 years of protection, with 60% protective efficacy, while natural infection provides longer-term protection than vaccination. Understanding the immune responses after natural infection is important to better understand immune responses to antigens that mediate longer-term protection. Sialidase is a neuraminidase that facilitates binding of cholera toxin to intestinal epithelial cells. We show here that patients with cholera develop systemic, mucosal, and memory B-cell immune responses to the sialidase antigen of Vibrio cholerae O1 and that plasma responses targeting this antigen correlate with protection.

Catalases and PhoB/PhoR system independently contribute to oxidative stress resistance in Vibrio cholerae O1.

All cells are subjected to oxidative stress, a condition under which reactive oxygen species (ROS) production exceeds elimination. Bacterial defences against ROS include synthesis of antioxidant enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB and KatG, which contribute to ROS homeostasis. In this study, we analysed the mechanism behind katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, of classical and El Tor biotypes, respectively. Both strains express these genes, especially at stationary phase. However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to peroxide challenge than the classical strain, confirming a direct relationship between catalase activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961, katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase activity in N16961 and its phoB mutant cells was similar, independently of growth conditions, indicating that the PhoB/PhoR system is not required for katB and katG expression. However, N16961 cells from Pi-limited cultures were 50-100-fold more resistant to H2O2 challenge and accumulated less ROS than phoB mutant cells. Together, these findings suggest that, besides KatB and KatG, the PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also corroborate previous results from our and other groups, suggesting that the PhoB/PhoR system is fundamental for V. cholerae biology.

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

The current pandemic El Tor biotype of O1 Vibrio cholerae is resistant to polymyxins, whereas the previous pandemic strain of the classical biotype is polymyxin sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to polymyxins through the glycylation of lipopolysaccharide. Here, we present the mechanistic determination of initial steps in the AlmEFG pathway. We verify that AlmF is an aminoacyl carrier protein and identify AlmE as the enzyme required to activate AlmF as a functional carrier protein. A combination of structural information and activity assays was used to identify a pair of active site residues that are important for mediating AlmE glycine specificity. Overall, the structure of AlmE in complex with its glycyl-adenylate intermediate reveals that AlmE is related to Gram-positive d-alanine/d-alanyl carrier protein ligase, while the trio of proteins in the AlmEFG system forms a chemical pathway that resembles the division of labor in nonribosomal peptide synthetases.

[Membrane-bound proteases of ompT+ and ompT- Vibrio cholerae strains].

AIM: Detection ofproteases in outer membranes (OM) of ompT+ and ompT- Vibrio cholerae strains of O1 and O139 serogroups. MATERIALS AND METHODS: Specific sterile preparations of OM were obtained by lysis of live V. cholerae cells by 4.5 M urea solution with subsequent differential centrifugation and treatment by nucleases. Extraction of OM proteins previously treated by sodium sarcosinate was carried out by Triton X-100 in the presence of EDTA. Protease and polypeptide spectra were studied in substrate and SDS electrophoresis. Sensitivity of proteases to inhibitors was determined in diffusion test in agarose gel containing substrate by using soy trypsin inhibitor (STI) and phenylmethylsulfonyl fluoride (PMSF). The presence of ompT was determined in PCR by using specific primers. RESULTS: According to PCR data 13 Vibrio cholerae O1 strains and 3 V. cholerae O139 strains isolated from clinical material as well as 22 V. cholerae O1 strains isolated from environmental objects contained ompT gene. 2 V. cholerae O1 human isolated strains, 9 V. cholerae O1 strains and 2 V. cholerae O139 strains isolated from the environment did not have ompT gene. By using SDS- and enzyme-electrophoresis in polyacrylamide gel quantitative and qualitative differences in composition of polypeptides and proteases of OM ompT+ and ompT- V. cholerae strains that hydrolyze gelatin, casein and protamine sulfate were detected. Inhibition of OM by STI and PMSF resulted in a decrease of their proteolytic activity. CONCLUSION: In preparations and extracts of ompT+ and ompT- V. cholerae OM up to 3 proteases some of which may be related to ompT-like were detected.

Role of polyphosphate kinase gene (ppk) for survival of Vibrio cholerae O1 in surface water of Bangladesh.

Polyphosphate provides a substitute for ATP and energy source when phosphorus is a limiting resource in nature. The present study focuses on the role ofpolyphosphate for the survival of Vibrio cholerae in the aquatic habitats as an autochthonous bacterium. The survival advantages of polyphosphate of V. cholerae O1 having (wild type) and lacking (mutant) polyphosphate kinase (ppk) gene in surface water and with Anabaena variabilis were compared by cultural, Direct Fluorescent Antibody (DFA) and polymerase chain reaction methods in natural water microcosms. The microcosm's water was prepared by filtering and physicochemical parameters were also investigated by standard methods. The results revealed that both fresh and saline water, the wild type strain enhanced survival in cultural conditioned than ppk mutant strain. However, Fluorescent Antibody Direct Viable Counts (FADVC) and Polymerase Chain Reaction (PCR) results noted both strains have the equal survival strategy in viable but nonculturable state (VNC). In conclusion, it could be hypothesized that the polyphosphate inclusion body might keep cultivable and survivable at low phosphate natural environment of the aquatic bacterium.

Third-generation cephalosporin-resistant Vibrio cholerae, India.

Vibrio cholerae resistance to third-generation cephalosporins is rarely reported. We detected a strain that was negative for extended-spectrum ß-lactamase and positive for the AmpC disk test, modified Hodge test, and EDTA disk synergy test and harbored the blaDHA-1 and blaNDM-1 genes. The antimicrobial drug susceptibility profile of V. cholerae should be monitored.

Genetic characterization of multidrug-resistant, extended-spectrum- ß-lactamase-producing Vibrio cholerae O1 outbreak strains, Mpumalanga, South Africa, 2008.

Thirty-one antimicrobial-resistant, extended-spectrum-ß-lactamase-producing strains of Vibrio cholerae O1 serotype Ogawa associated with an outbreak of cholera in South Africa (2008) were investigated. Ten selected cholera strains were PCR positive for the SXT element, harbored mutations in the quinolone resistance-determining regions of GyrA (Ser83-Ile) and ParC (Ser85-Leu), and produced TEM-63 ß-lactamase.

Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model.

BACKGROUND: Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL). METHODOLOGY/PRINCIPAL FINDINGS: We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/-0.3 n = 3), CHA6.8 (FA ratio 1.08+/-0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/-0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/-0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/-0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/-0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure. CONCLUSION: Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

[Detection of lipase in Vibrio cholerae serogroup O1 in reaction of volumetric agglomeration].

Study showed that El-Tor strains of V. cholerae isolated from different sources produce lipase for hemolysis after cultivation during 24 h on meat-peptone broth independently from their toxigenic and hemolytic abilities. Study of 3- and 4-hours broth cultures of vibrios revealed possibility to differentiate between hemolytic nontoxigenic strains and toxigenic nonhemolytic ones. Using antilipaze diagnostic kit it was possible to differentiate El-Tor vibrios from vibrios of classic biovar basing on lipase production 24 h after cultivation on meat-peptone broth that was evident in El-Tor vibrios but not in classic biovar strains.

Comparative study of different methods for detection of toxic and other enzymatic factors in Vibrio cholerae strains.

The purpose of this work was to characterize the toxin profile and the presence of other virulence factors involved in the pathogenesis and biology of 13 V. cholerae O1 (11 clinical cases and 2 waters) and 6 V. cholerae non O1 strains (4 clinical cases and 2 waters) using genetic (PCR), immunological (RPLA), biochemical (NAD degradation, haemolysis, Kanagawa phenomenon, caseinase, lecithinase, mucinase, amylase, esculine hydrolysis) and cell culture (Vero E6, HEp-2) assays. The results indicated a concordance between PCR-RPLA (84%), PCR-NAD (73%) and RPLA-NAD (84%) methods. The sensitivity of RPLA and NAD degradation methods were comparable to PCR in detecting CT in Vibrio cholerae O1 strains. Although NAD degradation method was not exclusively specific for the CT detection, it proved its usefulness in screening certain virulent, CT-negative clones of V. cholerae. The cytotoxic effect on Vero E6 cells, enzyme production (Kanagawa haemolysins, lecithinase, caseinase, esculine hydrolysis) as well as adherence ability on inert substrate proved to be much more constant in V. cholerae non O1 (CT- negative) than in V. cholerae O1 (CT-positive). All V. cholerae non O1 strains isolated in diarrheal cases were Kanagawa positive. This complex of virulence factors detected in V. cholerae non O1 strains could probably contribute during interepidemic periods to human-to-human transmission and to greater resistance as compared to O1 strains in the environment.

Factors associated with virulence and survival in environmental and clinical isolates of Vibrio cholerae O1 and non O1 in Romania.

Four hundred ninety seven strains of Vibrio cholerae selected from isolates in Romania in the last decade 1990-1999 were investigated for antibiotic resistance and for classical and putative virulence factors. V. cholerae O1 strains predominated in clinical cases and non O1 strains in the environment, excepting in 1992 when non O1 strains were frequent in clinical and environmental sources. V. cholerae O1 strains previously susceptible to tetracycline acquired clinically significant resistance to this drug during 1993-1994, but this trend was reversed in 1995, following the introduction of nalidixic acid in cholera treatment in 1994. V. cholerae O1 and non O1 clinical isolates acquired simultaneous resistance to the vibriostatic agent O/129 and cotrimoxazole during 1994-1995. High levels of intrinsic resistance to multiple antibiotics were exhibited by all strains examined. The presence of cholera toxin (CT) was concentrated in clinical V. cholerae O1 strains and was substituted in clinical non O1 strains by four putative virulence markers (Kanagawa haemolysin, slime, lipase, and colonial opacity). Colonial opacity (30%) was present only in clinical isolates of V. cholerae non O1. Pigmentogenesis (11.7%) has present only in environmental sources. Antibioresistance profiles differ for V. cholerae O1 and non O1 strains with respect to their source of isolation. This aspect may imply a role in virulence and survival of V. cholerae in the natural environment where they may serve as a reservoir of virulence and multiple drug resistance genes.

Involvement of the hap gene (mucinase) in the survival of Vibrio cholerae O1 in association with the blue-green alga, Anabaena sp.

Mucinase is a soluble haemagglutinin protease, which may be important for the survival of Vibrio cholerae in association with mucilaginous blue-green algae (cyanobacteria). A comparative survival study was carried out with an Anabaena sp. and a wild-type V. cholerae O1 strain hap+ gene (haemagglutinin-protease), together with its isogenic mutant hap (hap-deleted gene). A simple spread plate technique was followed to count culturable V. cholerae O1 on taurocholate tellurite gelatin agar plate. The fluorescent antibody technique of Kogure et al. (1979) was used for the microscopical viable count of V. cholerae O1. Polymerase chain reaction (PCR) and Southern blot hybridization were carried out to detect a lower number of viable but nonculturable (VBNC) V. cholerae O1 from the laboratory-based experiments. The wild and mutant V. cholerae O1 strains survived in culturable form for 22 and 10 days. respectively, in association with the Anabaena sp., with the difference being statistically significant (P < 0.01). The fluorescent antibody technique, PCR, and hybridization results also showed that the wild strain survived better in the VBNC state than did the mutant VBNC strain in association with an Anabaena sp. These results indicate that the enzyme mucinase may play an important role in the association and long-term survival of V. cholerae O1 with a mucilaginous blue-green alga, Anabaena sp.