The effect of iron on the toxigenicity of Vibrio cholerae.
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In vitro and in vivo studies were conducted to assess the response of cholera toxin (CT) production to increasing iron concentrations in an aquatic environment. Production of CT by seven of eight Vibrio cholerae strains tested, including the Bengal strain (O139), was significantly enhanced in the presence of iron concentrations of 1.0 and 10 g/L. The exception (El Tor Ogawa) had a significant CT response only in the presence of 10 g of iron/L. Enhancement of CT production also occurred at iron concentrations less than 1.0 g/L, but not to a statistically significant degree. The high iron concentrations, which in this study were found to stimulate CT production, have been described by others in association with sediments, water plants, and chitinous fauna. Other investigators have shown a predilection by V. cholerae to attach to these sites in the aquatic environment. The importance of excess in vivo iron with respect to the pathogenicity of several gram-negative bacilli is well recognized. However, the possible impact of environmental iron on the in vitro toxigenicity of a microorganism, in this case V. cholerae in its aquatic environment, is to the best of our knowledge a new finding with important epidemiologic implications. These findings, coupled with the fact that iron concentration is considerably enhanced in industrially polluted waters and sediments, may reflect a causal link between the concurrent global upsurge of industrialization and pandemic occurrence of cholera during the latter half of the 20th century. Enhanced toxigenicity may also cause clinical disease following ingestion of lower than usual infective doses of cholera vibrios, thereby increasing the incidence of symptomatic cases and, possibly, of severe cases. (+info)
Cross-reaction between a strain of Vibrio mimicus and V. cholerae O139 Bengal.
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Of 200 isolates of Vibrio mimicus screened, one from water (N-57) agglutinated with V. cholerae O139 polyclonal antiserum (absorbed with a rough strain of V. cholerae only) and not with O139 polyclonal diagnostic antiserum (absorbed with the rough strain and V. cholerae O22 and O155). The antigenic relationship between V. cholerae 0139 and N-57 is of a,b-a,c type, where a is the common antigenic epitope and b and c are unique epitopes. Strain N-57 was assigned to a new serogroup of V. cholerae O194. It gave negative results in a monoclonal antibody-based rapid test and a PCR test specific for V. cholerae O139. It did not possess the ctx gene or produce cholera toxin. Antiserum to strain N-57 cross-protected infant mice against cholera on challenge with V. cholerae O139. Structural studies of the surface polysaccharides and studies of the rfb genes will shed more light on the extent of relatedness between V. mimicus N-57 and V. cholerae O139. (+info)
Cloning and sequencing of the genes downstream of the wbf gene cluster of Vibrio cholerae serogroup O139 and analysis of the junction genes in other serogroups.
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The DNA sequence of the O-antigen biosynthesis cluster (wbf) of a recently emergent pathogen, Vibrio cholerae serogroup O139, has been determined. Here we report the sequence of the genes downstream of the O139 wbfX gene and analysis of the genes flanking the wbf gene cluster in other serogroups. The gene downstream of wbfX, designated rjg (right junction gene), is predicted to be not required for O-antigen biosynthesis but appears to be a hot spot for DNA rearrangements. Several variants of the rjg gene (three different insertions and a deletion) have been found in other serogroups. DNA dot blot analysis of 106 V. cholerae strains showed the presence of the left and right junction genes, gmhD and rjg, respectively, in all strains. Further, these genes mapped to a single I-CeuI fragment in all 21 strains analyzed by pulsed-field gel electrophoresis, indicating a close linkage. The insertion sequence element IS1358, found in both O1 and O139 wb* regions, is present in 61% of the strains tested; interestingly, where present, it is predominantly linked to the wb* region. These results indicated a cassette-like organization of the wb* region, with the conserved genes (gmhD and rjg) flanking the divergent, serogroup-specific wb* genes and IS1358. A similar organization of the wb* region in other serogroups raises the possibility of the emergence of new pathogens by homologous recombination via the junction genes. (+info)
Differential transcription of the tcpPH operon confers biotype-specific control of the Vibrio cholerae ToxR virulence regulon.
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Epidemic strains of Vibrio cholerae O1 are divided into two biotypes, classical and El Tor. In both biotypes, regulation of virulence gene expression depends on a cascade in which ToxR activates expression of ToxT, and ToxT activates expression of cholera toxin and other virulence genes. In the classical biotype, maximal expression of this ToxR regulon in vitro occurs at 30 degrees C at pH 6.5 (ToxR-inducing conditions), whereas in the El Tor biotype, production of these virulence genes only occurs under very limited conditions and not in response to temperature and pH; this difference between biotypes is mediated at the level of toxT transcription. In the classical biotype, two other proteins, TcpP and TcpH, are needed for maximal toxT transcription. Transcription of tcpPH in the classical biotype is regulated by pH and temperature independently of ToxR or ToxT, suggesting that TcpP and TcpH couple environmental signals to transcription of toxT. In this study, we show a near absence of tcpPH message in the El Tor biotype under ToxR-inducing conditions of temperature and pH. However, once expressed, El Tor TcpP and TcpH appear to be as effective as classical TcpP and TcpH in activating toxT transcription. These results suggest that differences in regulation of virulence gene expression between the biotypes of V. cholerae primarily result from differences in expression of tcpPH message in response to environmental signals. We present an updated model for control of the ToxR virulence regulon in V. cholerae. (+info)
Purification and characterization of novel toxin produced by Vibrio cholerae O1.
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Vibrio cholerae WO7 (serogroup O1) isolated from patients with diarrhea produces an extracellular toxin despite the absence of ctx, zot, and ace genes from its genome. The toxin elongates Chinese hamster ovary cells, produces fluid accumulation in ligated rabbit ileal loops, and agglutinates freshly isolated rabbit erythrocytes. Maximal production of this toxin (WO7 toxin) was seen in AKI medium with the pH adjusted to 8.5 at 37 degrees C under shaking conditions. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, affinity chromatography using a fetuin-Sepharose CL-4B column, and gel filtration chromatography, which increased the specific activity of the toxin by 1.6 x 10(6)-fold. The toxin is heat labile and sensitive to proteases and has a subunit structure consisting of two subunits with molecular masses of about 58 and 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Agglutination of GM1-coated sheep erythrocytes by toxin suggests that GM1 might be the physiologic receptor for WO7 toxin on the enterocytes. An immunodiffusion test between the antiserum raised against the purified WO7 toxin and the purified toxin gave a well-defined precipitation band. In the immunoblot assay, two bands were observed in the 58- and 40-kDa region. At the same time, antiserum against WO7 toxin failed to show any cross-reactivity with cholera toxin or Escherichia coli heat-labile toxin (LT1) in an immunodiffusion test or immunoblot assay. The enterotoxic activity of WO7 toxin could be inhibited by antiserum against purified WO7 toxin. Our results indicate that WO7 toxin is structurally and functionally distinct from other cholera toxins and that the enterotoxic activities expressed by WO7 toxin appear to contribute to the pathogenesis of disease associated with V. cholerae O1 strains. (+info)
An improved method for detecting faecal Vibrio cholerae by PCR of the toxin A gene.
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A method for removing inhibitor(s) of the PCR assay for the direct detection of cholera toxin A gene (ctxA) in human faeces is described. Inhibitors of the PCR were removed by centrifugation and the activity of the remaining inhibitors by dilution. Based on these data, a protocol was developed for pre-treatment of stool specimens for PCR assay, and a simple and rapid protocol was constructed for the diagnostic detection of the ctxA genes in stool specimens in combination with single band detection on gel electrophoresis, dot-blot hybridisation and enrichment culture. This protocol was applied to clinical specimens and showed that the PCR method gave 100% agreement with established culture methods for the detection of cholera toxin-producing Vibrio cholerae O1. This protocol was considered to be useful because of its simplicity and the rapidity of diagnosis. (+info)
Effect of small bowel bacterial overgrowth on the immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR.
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Several live oral vaccines (polio, bovine rotavirus, CVD 103-HgR cholera) are less immunogenic in developing than in industrialized countries. It was hypothesized that proximal small bowel bacterial overgrowth (common in children in less developed countries but rare in industrialized settings) diminishes the vibriocidal antibody response to CVD 103-HgR. In total, 202 fasting Santiago schoolchildren aged 5-9 years had lactulose breath H2 tests to detect proximal small bowel bacteria 1 day before ingesting CVD 103-HgR. Florid small bowel overgrowth was observed in 10 (5.6%) of 178 analyzable children. In children with florid overgrowth, vibriocidal seroconversion differed little from other children (60% vs. 67%), but the geometric mean titer was lower (160 vs. 368; P=.25). By logistic regression, increased peak breath H2 at small bowel time points was associated with diminished seroconversion (P=.04), as was the interaction of H2 value and weight (children >25 kg had lower seroconversion rates among subjects with heaviest overgrowth). (+info)
A role for the PhoBR regulatory system homologue in the Vibrio cholerae phosphate-limitation response and intestinal colonization.
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To survive and multiply in different environments, Vibrio cholerae has to coordinately regulate the expression of genes involved in adaptive responses. In many pathogens, adaptive responses, including pathogenic responses, are regulated by two-component regulator (TCR) systems. It is likely that members of a TCR family play a role in the regulation of processes involved in intestinal colonization, and therefore pathogenesis, in V. cholerae. We have identified and characterized a TCR system of V. cholerae: this system is a homologue of Escherichia coli PhoBR. The presence of a putative Pho box suggests that the V. cholerae phoBR operon is regulated by inorganic phosphate levels. The phoR and phoB genes are organized the same way as in E. coli. Mutation of the V. cholerae phoB gene affected the expression of the putative Pho regulon, including PhoA, but did not affect the production of cholera toxin. V. cholerae phoB mutants are less able to colonize rabbit intestine than wild-type V. cholerae. The addition of inorganic phosphate at a high concentration to the inoculum only partially restored the ability of the mutants to colonize the intestine, suggesting that the V. cholerae Pho regulon in vivo may not be regulated by inorganic phosphate levels alone. (+info)