Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin

McCardell, Barbara A.

doi:10.1016/s0378-1097(99)00479-6

Cited by 3 publications

(7 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The molecular weight of the denatured and reduced VFH is much higher than the apparent molecular weight of the native protein (Ͻ10,000 [data not shown]) as determined by gel filtration using the method described by Andrews (1). Similar observations have been made for the El Tor and El Tor-like hemolysins of V. cholerae O1 and non-O1 strains that also exhibit a very low molecular weight by gel filtration but have a much higher molecular weight by SDS-PAGE (11,14,20,31). The pI of VFH is the same as that of the aberrant El Tor-like hemolysin of non-O1 V. cholerae (11) but is different from the values of 5.3 and 5.7 reported for the other El Tor-like hemolysins of non-O1 V. cholerae (11,20).…”

Section: Discussionsupporting

confidence: 79%

“…The molecular weight (63,000) of VFH is the same as that reported for the V. mimicus hemolysin (22) but is slightly higher than the 60,000 of the El Tor and El Tor-like hemolysins of V. cholerae (20,31) and the 48,000 and 52,000 of the aberrant El Tor hemolysin (8,11). The molecular weight of the denatured and reduced VFH is much higher than the apparent molecular weight of the native protein (Ͻ10,000 [data not shown]) as determined by gel filtration using the method described by Andrews (1).…”

Section: Discussionsupporting

confidence: 72%

“…This elution pattern is suggestive of an interaction between the gel and the protein molecule. Similar interactions between chromatographic gel matrices and proteins have been reported for hemolysins produced by V. cholerae O1 and non-O1 strains, Vibrio vulnificus, Vibrio mimicus, and Vibrio tubiashii (6,7,11,14,20,22,31). The extremely hydrophobic nature of the protein may also have been responsible for the loss of activity that was observed for the various purification methods (ammonium sulfate precipitation, ultrafiltration, dialysis, and lyophilization) we employed to isolate and concentrate VFH from the culture supernatant fluids.…”

Section: Discussionsupporting

confidence: 70%

“…The hemolytic spectrum of VFH is slightly different from those reported for the El Tor-like hemolysin of non-O1 V. cholerae (30) and the variant hemolysin of non-O1 V. cholerae (20). Erythrocytes from rabbit and guinea pig are the most sensitive and those from goose and chicken are the least sensitive to both the El Tor-like hemolysin of non-O1 V. cholerae and VFH.…”

Section: Discussioncontrasting

confidence: 69%

“…The results of immunoblot analysis (Fig. 5) also showed that the VFH reactive band has a slightly higher molecular weight (63,000) than do the major reactive bands of the El Tor and El Tor-like hemolysins, which have a molecular weight of 60,000 (20,31). VFH is heat labile and sensitive to proteases and is inhibited by the divalent cation Zn 2ϩ , which has been reported to inhibit the poreforming stage of the V. mimicus hemolysin (22).…”

Section: Discussionmentioning

confidence: 80%

See 4 more Smart Citations

Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis

et al. 2003

View full text Add to dashboard Cite

The halophilic bacterium Vibrio fluvialis is an enteric pathogen that produces an extracellular hemolysin. This hemolysin was purified to homogeneity by using sequential hydrophobic-interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. It has a molecular weight of 63,000 and an isoelectric point of 4.6, and its hemolytic activity is sensitive to heat, proteases, and preincubation with zinc ions. The hemolysin lyses erythrocytes of the eight different animal species that we tested, is cytotoxic against Chinese hamster ovary cells in tissue culture, and elicits fluid accumulation in suckling mice. Lysis of erythrocytes occurs by a temperature-dependent binding step followed by a temperature-and pH-dependent lytic step. Fourteen of the first 20 N-terminal amino acid residues (Val-Ser-Gly-Gly-Glu-Ala-Asn-Thr-LeuPro-His-Val-Ala-Phe-Tyr-Ile-Asn-Val-Asn-Arg) are identical to those of the El Tor hemolysin of Vibrio cholerae and the heat-labile hemolysin of Vibrio mimicus. This homology was further confirmed by PCR analysis using a 5 primer derived from the amino-terminal sequence of the hemolysin and a 3 primer derived from the El Tor hemolysin structural gene. The hemolysin also reacts with antibodies to the El Tor-like hemolysin of non-O1 V. cholerae.Vibrio fluvialis is a halophilic bacterial pathogen that has been isolated from humans with diarrhea and from river and estuarine water, marine mollusks, crustaceans, and fish. It has been implicated in outbreaks and sporadic cases of diarrhea, and gastrointestinal illness caused by this pathogen is usually associated with consumption of raw or improperly cooked seafood. In the United States, recent data show that it was responsible for 82 of the 1,584 Vibrio infections reported to the Centers for Disease Control and Prevention since 1997 (M. C. Evans, P. M. Griffin, and R. V. Tauxe, CDC report: Vibrio surveillance system, summary data, 1997-2000 [http://www.cdc.gov /ncidod/dbmd/diseaseinfo/files/CSTE_Vibrio_2000.pdf]). Clinical symptoms of gastroenteritis caused by V. fluvialis and Vibrio cholerae are very similar, and therefore the ability of V. fluvialis to cause diarrhea has been examined in various animal models originally designed for V. cholerae. V. fluvialis elicits intestinal fluid when fed to suckling mice (18, 23) and in rabbit ileal loops (5, 25). It produces a toxin that elongates Chinese hamster ovary (CHO) cells, a nonhemolytic CHO cell-killing cytotoxin, a protease, and a cytolysin active against rabbit erythrocytes (5, 17). Lockwood et al. (18) showed that partially purified preparations of the elongation factor, protease, and cytolysin/hemolysin induced fluid accumulation in suckling mice. However, it is not clear what roles these products play in diarrheal disease since none of them have been purified. In their research with the V. fluvialis hemolysin (VFH), Rahim and Aziz (26) reported that of the six different growth media tested, brain heart infusion broth yielded maximal amounts of hemolysin. In order to ha...

show abstract

Section: Discussionsupporting

confidence: 79%

Section: Discussionsupporting

confidence: 72%

Section: Discussionsupporting

confidence: 70%

Section: Discussioncontrasting

confidence: 69%

Section: Discussionmentioning

confidence: 80%

See 3 more Smart Citations

Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis

et al. 2003

View full text Add to dashboard Cite

show abstract

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced byVibrio tubiashii

Kothary¹,

Delston

Curtis

et al. 2001

Appl Environ Microbiol

View full text Add to dashboard Cite

An extracellular cytolysin from Vibrio tubiashii was purified by sequential hydrophobic interaction chromatography with phenyl-Sepharose CL-4B and gel filtration with Sephacryl S-200. This protein is sensitive to heat and proteases, is inhibited by cholesterol, and has a molecular weight of 59,000 and an isoelectric point of 5.3. In addition to lysing various erythrocytes, it is cytolytic and/or cytotoxic to Chinese hamster ovary cells, Caco-2 cells, and Atlantic menhaden liver cells in tissue culture. Lysis of erythrocytes occurs by a multihit process that is dependent on temperature and pH. Twelve of the first 17 N-terminal amino acid residues (Asp-AspTyr-Val-Pro-Val-Val-Glu-Lys-Val-Tyr-Tyr-Ile-Thr-Ser-Ser-Lys) are identical to those of the Vibrio vulnificus cytolysin.Vibrio tubiashii is a marine organism that causes bacillary necrosis in larval and juvenile bivalve mollusks (24,25). The disease is characterized by a rapid onset of symptoms, such as a generalized reduction in larval motility, an increase in larval quiescence, and extensive soft tissue necrosis. The pathogen has been isolated from hard clam larvae, juvenile hard clams, and Eastern oyster spat and larvae (7,12,25). V. tubiashii has been isolated from diseased mollusks in the United States and United Kingdom and has been associated with red tides caused by Mesodinum rubrum along the northwest coast of Spain (12,23,24,25). In spite of the economic importance of V. tubiashii in the cultivation of bivalve mollusks, nothing is known about the virulence mechanisms of this pathogen. Romalde et al. (23) reported that culture supernatants of the pathogen exhibited cytotoxicity towards fathead minnow peduncle cells and mouse lung fibroblasts in tissue culture. We describe the purification and properties of a cytolysin that lyses various types of erythrocytes and Chinese hamster ovary (CHO) cells and is cytotoxic to human intestinal (Caco-2) cells and fish (Atlantic menhaden liver [AML]) cells in tissue culture.Cytolysin production and purification. Two V. tubiashii strains (ATCC 19105 and ATCC 19109) were obtained from the American Type Culture Collection (Manassas, Va). Both strains were confirmed to be V. tubiashii using biochemical tests and were stored at Ϫ70°C. The ATCC 19105 frozen culture was rapidly thawed and inoculated onto two plates containing Trypticase soy agar (BBL, Cockeysville, Md.) supplemented with 1% NaCl. The plates were incubated at 30°C for 16 to 18 h, and the bacteria were harvested in 5 ml of Casamino Acids-yeast extract broth (3% Casamino Acids, 0.4% yeast extract, 0.05% K 2 HPO 4 [pH 7.4] supplemented with 1% NaCl). A 2-liter flask containing 500 ml of Casamino Acids-yeast extract broth was inoculated with the seed culture suspension (25 optical density units at 650 nm; ca. 10 10 CFU), and the culture was incubated for 7 h at 37°C on a rotary shaker at 100 rpm. Culture supernatant fluids (stage 1) were recovered by centrifugation at 16,000 ϫ g (20 min). Disodium hydrogen phosphate and sodium chloride were dissolved in the sta...

show abstract

Overexpression, Purification, Characterization, and Pathogenicity of Vibrio harveyi Hemolysin VHH

et al. 2006

View full text Add to dashboard Cite

show abstract

Two-step purification and partial characterization of a variant of the Vibrio cholerae non-O1 hemolysin

Cited by 3 publications

References 16 publications

Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis

Purification and Characterization of Enterotoxigenic El Tor-Like Hemolysin Produced byVibrio fluvialis

Purification and Characterization of a Vulnificolysin-Like Cytolysin Produced byVibrio tubiashii

Overexpression, Purification, Characterization, and Pathogenicity of Vibrio harveyi Hemolysin VHH

Contact Info

Product

Resources

About