Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease

Mitra, Rupak

doi:10.1016/s0378-1097(98)00489-3

Cited by 4 publications

(14 citation statements)

References 32 publications

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“…The presence of CT in toxigenic V. cholerae strains may obliterate the cell vacuolating effect, which may have led to the conclusion that toxigenic V. cholerae strains do not produce cell vacuolating factor but, interestingly, CO853, a nontoxigenic O139 strain, induced cell vacuolation. V. cholerae clinical isolates have also been reported to produce a cytotoxin (cell-rounding factor) which induces rounding of cultured HeLa cell line (17,22). The exact mechanism of how the El Tor or hemolysin induces vacuolation was not studied here, but it can be inferred that intoxication of cells with mature hemolysin after the action of cell protease on the prohemolysin moiety leads to the dramatic vacuolation in the cell cytoplasm.…”

Section: Discussionmentioning

confidence: 99%

“…A freshly prepared concentrated culture supernatant of V. cholerae strain CO848b was incubated with 2.5 U of insoluble proteinase K-bead suspension at 37°C for 1 h. As a control, the supernatant was incubated under the same conditions without added protease. After incubation, the beads were elim-inated by centrifugation at 3,000 ϫ g for 1 min, and the supernatants were tested on nonconfluent HeLa cells after serial dilution as described earlier (17).…”

Section: Methodsmentioning

confidence: 99%

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Cell Vacuolation, a Manifestation of the El Tor Hemolysin of Vibrio cholerae

et al. 2000

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Culture supernatants of nontoxigenic nonepidemic clinical strains of Vibrio cholerae belonging to diverse serogroups were found to induce vacuolation of nonconfluent HeLa cells. The vacuoles became prominent 18 h after introduction of culture supernatant, and vacuolated cells survived for 48 h and then died. Only a fraction of the vacuolated cells took up neutral red dye, implying that there were differences in the vacuolar microenvironment. Further tests showed that the factor responsible for vacuolation was heat labile and proteinaceous. Vacuolating activity was completely neutralized by antibody to hemolysin of V. cholerae but not by antibody to vacuolating cytotoxin of Helicobacter pylori. Partial purification of the vacuolating factor led to elution of fractions, which showed both hemolytic and vacuolating activity. PCR amplification and cloning of the hemolysin structural gene (hlyA) into Escherichia coli DH5␣ led to isolation of clones producing cell vacuolating factor in a cell-associated form. Further, a null insertion mutation in the hlyA gene of a high-vacuolatingfactor-producing strain led to complete abolition of both cell vacuolating and hemolytic activities. These analyses establish vacuolation as a potentially important but previously unrecognized property of V. cholerae El Tor hemolysin.Much of the virulence of bacterial pathogens is initiated by secreted factors that induce specific biochemical changes in host tissue, culminating in pathology and disease. Cholera toxin (CT), the potent secretory toxin produced by toxigenic strains of Vibrio cholerae, is critically involved in key features of the disease cholera. Other putative factors, including zonula occludens toxin (Zot), accessory cholera enterotoxin (Ace), El Tor hemolysin, Shiga-like toxin, and heat-stable toxin, are reportedly produced by virulent strains of V. cholerae and are thought to contribute to the disease process (12). Despite attenuation of several virulence genes in recombinant candidate vaccine strains of V. cholerae, a safe and efficacious vaccine still eludes us. The residual diarrhea caused by genetically attenuated live oral vaccine strains of V. cholerae O1 prompted us to look for new factors secreted by V. cholerae.One means of assessing the toxicity of the secreted products of bacterial pathogens is to study the effect of culture supernatants on eukaryotic cell lines (12). In this context, we have previously reported that cell-free culture supernatants of V. cholerae strains induced morphological changes, including elongation and rounding in eukaryotic cells (18,21,23). We initiated the present study to examine the mechanism as to how nontoxigenic nonepidemic V. cholerae strains are able to cause a disease that resembles cholera in absence of already known virulence determinants, especially CT, found in their toxigenic epidemic causing counterparts. While attempting this, we observed that some clinical strains of V. cholerae induce vacuolation on HeLa cells, which seemed reminiscent of that induced by VacA cytotoxin of...

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cell Vacuolation, a Manifestation of the El Tor Hemolysin of Vibrio cholerae

et al. 2000

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“…A cell-rounding factor called NMDCY (non-membrane-damaging cytotoxin) is produced by clinical strains of V. cholerae Non-O1, usually in association with sporadic cases of gastroenteritis (Saha et al 1996). This extracellular factor causes cell-rounding of the Chinese hamster ovary (CHO), and of Int 407 (human intestinal) and HeLa (human non-intestinal) cell lines without cell-membrane disruption and without cell death (Saha et al 1996, Mitra et al 1998, Basu et al 1999. NMDCY toxin-treated cells exibited important changes in morphological and cytoskeletal structures.…”

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confidence: 99%

Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Choquet¹,

Soudant²,

Lambert³

et al. 2003

Dis. Aquat. Org.

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Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated. KEY WORDS: Cytotoxicity · Flow cytometry · Vibrio tapetis · Ruditapes philippinarum · Bivalve hemocytes · Pathogenicity Resale or republication not permitted without written consent of the publisherDis Aquat Org 57: [109][110][111][112][113][114][115][116] 2003 external defense factors, such as phagocytosis by hemocytes in the pallial fluid as well as the epithelium.However, mechanisms of interactions between Vibrio tapetis and clam hemocytes remain poorly understood. Measurement of cytotoxicity of V. tapetis against hemocytes could be a good indicator of the virulence properties of this bacterium. In the literature, 2 assays using bacteria-hemocyte interaction have already been described: one was direct and employed microscopic observation, the other was indirect and used a chemiluminescence assay. The first used a time-lapse video recording to determine the toxic effect of V. anguillarum and V. alginolyticus on mussel Mytilus edulis hemocytes (Lane & Birkbeck 1999). These 2 Vibrio species cause cell-rounding of the hemocytes. The most toxic bacterium, V. anguillarum, was able to induce rounding in 50% of the hemocytes at a ratio of only 1 bacterium per hemocyte. Differences in cellrounding percentages permitted quantification of cytotoxicity. The second method was used to examine the effects of V. pectenicida and of its cytoplasmic extract on the respiratory burst of Pecten maximus hemocytes , Lambert et al. 2001). This indirect method used chemiluminescence to quantify production of reactive oxygen intermediates (ROIs). V. pectenicida, and in particular the cytoplasm extract, were responsible for the inhibition of chemiluminescence activity in oyster and scallop hemocytes.To better understand the cellular effects of Vibrio tapetis toward hemocytes, the present study was designed first to develop a cytotoxicity test using flow cytometry, and second to compare the pathogenicity of different V. tapetis strains on clam and oyster hemocytes. MATERIALS AND METHODSBacteria. The bacteria used in this study were generally isolated from BRD-diseased clams and asymptomatic cockles at ...

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“…NMDCY also elicits impressive enterotoxic activity in the rabbit ileal loop assay [7,8]. NMDCY further elicited a concentration-dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on an Ussing chamber [8]. A signi¢cant serum globulin G response against NMDCY was elicited by patients infected with V. cholerae O139 [8].…”

Section: Introductionmentioning

confidence: 99%

“…NMDCY further elicited a concentration-dependent increase in short circuit current and tissue conductance of rabbit ileal mucosa mounted on an Ussing chamber [8]. A signi¢cant serum globulin G response against NMDCY was elicited by patients infected with V. cholerae O139 [8]. Monoclonal and polyclonal antibodies raised against this cytotoxic enterotoxin inhibited the cell rounding e¡ect on HeLa cells and also neutralized the biological activity of puri¢ed NMDCY in the rabbit ileal loop assay [9].…”

Section: Introductionmentioning

confidence: 99%

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on Int 407 and HeLa cells

Basu

1999

FEMS Microbiology Letters

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Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery. ß

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Characterization of non-membrane-damaging cytotoxin of non-toxigenic Vibrio cholerae O1 and its relevance to disease

Cited by 4 publications

References 32 publications

Cell Vacuolation, a Manifestation of the El Tor Hemolysin of Vibrio cholerae

Cell Vacuolation, a Manifestation of the El Tor Hemolysin of Vibrio cholerae

Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on Int 407 and HeLa cells

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