Purification and Characterization of Cytotoxin Produced by a Clinical Isolate of Vibrio cholerae O54 TV113

• Published in: Applied biochemistry and biotechnology Vol. 167; no. 4; pp. 809 - 823
• Main Authors: Isac, Sree Renjini, Nair, Gopinath Balakrish, Singh, Durg Vijai
• Format: Journal Article
• Language: English
• Published: New York Springer-Verlag 01.06.2012; Springer; Springer Nature B.V
• Subjects: Ammonium; Animals; Anion exchange; Bacteria; Biochemistry; Biological and medical sciences; Biotechnology; Chemical Phenomena; Chemistry; Chemistry and Materials Science; CHO Cells; Chromatography; Cricetinae; Cricetulus; Cytotoxins - biosynthesis; Cytotoxins - chemistry; Cytotoxins - isolation & purification; Cytotoxins - toxicity; Fundamental and applied biological sciences. Psychology; Hemorrhage - chemically induced; Hemorrhage - pathology; Humans; Ileum - drug effects; Ileum - pathology; Microbiology; Rabbits; Sulfates; Toxins; Ultrafiltration; Vibrio cholerae; Vibrio cholerae - isolation & purification; Vibrio cholerae - metabolism; Characterization; SDS-PAGE; Purification; Cytotoxin; O54 TV113; Chromatography; Anionic surfactant; Vibrio cholerae; Bacteria; Vibrionaceae; O54 TV 113; Clinical isolate; Lauryl sulfate
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-012-9718-4

Vibrio cholerae O54 TV113 isolated from a diarrheal patient produces an extracellular cytotoxin that caused alteration in the morphology of Chinese hamster ovary cells manifested as cell shrinkage with intact cell boundaries and finally causing cell death. Syncase medium supplemented with lincomycin (50 μg/ml), pH 7.2, and 18 h incubation with shaking at 37 °C supported optimal cytotoxin production. We isolated and purified this cytotoxin to homogeneity by ultrafiltration, 40–80 % ammonium sulfate precipitation, gradient–anion exchange chromatography, stepwise-anion exchange chromatography, and size exclusion chromatography increasing the specific activity by 866-fold. The cytotoxin is heat-labile, sensitive to protease and papain, and has a molecular weight of 64 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and enterotoxic activity in rabbit ileal loop assay. Both cytotoxic and enterotoxic activity could be inhibited or neutralized by antiserum raised against purified cytotoxin but not by preimmune serum. Immunodiffusion test between purified cytotoxin and its antiserum gave a single well-defined precipitin band showing reaction of complete identity and a well-defined single band in an immunoblot assay. This study thus indicate that the cytotoxin expressed by strain TV113 has both cytotoxic and enterotoxic activity and appears to contribute in pathogenesis of non-O1, non-O139 strains.