Fluid accumulation in infant mice caused by Vibrio hollisae and its extracellular enterotoxin

Kothary, Mahendra H.; Richardson, S H

doi:10.1128/iai.55.3.626-630.1987

Cited by 37 publications

(10 citation statements)

References 16 publications

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“…and intraperitoneally (i.p.) with serial 10‐fold dilutions of protease A, respectively, as described previously [18,19]. For examining the virulence of V. parahaemolyticus no.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

Lee

Cheng

et al. 2002

FEMS Microbiology Letters

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A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

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“…and intraperitoneally (i.p.) with serial 10‐fold dilutions of protease A, respectively, as described previously [18,19]. For examining the virulence of V. parahaemolyticus no.…”

Section: Methodsmentioning

confidence: 99%

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

Lee

Cheng

et al. 2002

FEMS Microbiology Letters

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“…and intraperitoneally (i.p.) with serial 10-fold dilutions of protease A, respectively, as described previously [18,19]. For examining the virulence of V. parahaemolyticus no.…”

Section: Virulence In Micementioning

confidence: 99%

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

Lee¹

2002

FEMS Microbiology Letters

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A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl‐Sepharose CL‐4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS–PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40°C to 50°C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10‐phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N‐terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco‐2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat‐labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

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“…FA studies in suckling mice. The secretory response evoked by each of the isogenic strains was evaluated by using a sealed infant mouse model, as described by Kothary and Richardson (29). For the experiments involving unstarved mice, isolated colonies from each test strain were inoculated into 20 ml of L broth in a 250-ml flask and grown as described above to a viable cell concentration of 2 x 109 to 3 x 109 CFU/ml.…”

Section: Methodsmentioning

confidence: 99%

Role of Vibrio cholerae neuraminidase in the function of cholera toxin

et al. 1992

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Vibrio cholerae neuraminidase (NANase) is hypothesized to act synergistically with cholera toxin (CT) and increase the severity of a secretory response by increasing the binding and penetration of CT to enterocytes. To test this hypothesis, the NANase gene (nanH) from V. cholerae Ogawa 395 was first cloned and sequenced. Isogenic wild-type and NANase-V. cholerae 395 strains were then constructed by using suicide vectormediated mutagenesis. The influence of NANase on CT binding and penetration was examined in vitro by using culture ifitrates from these isogenic strains. Fluorescence due to binding of fluorescein-conjugated CT to C57BL/6 and C3H mouse fibroblasts exposed to NANase+ ifitrates increased five-and eightfold, respectively, relative to that with NANasefiltrates. In addition, NANase+ filtrates increased the short-circuit current measured in Ussing chambers 65% relative to that with NANase-filtrates, although this difference decreased as production of CT increased. The role of NANase in V. cholerae pathogenesis was examined in vivo by intragastric inoculation of the isogenic strains into CD1 suckling mice. No difference in fluid accumulation ratios was seen at doses of 104 to 108 CFU, but NANase+ strains produced 18% higher fluid accumulation ratios at 109 CFU than NANase-strains when inoculated into nonfasted suckling mice. It is concluded that NANase plays a subtle but significant role in the binding and uptake of CT by susceptible cells under defined conditions.

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Fluid accumulation in infant mice caused by Vibrio hollisae and its extracellular enterotoxin

Cited by 37 publications

References 16 publications

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

Role of Vibrio cholerae neuraminidase in the function of cholera toxin

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