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Infection and Immunity, June 2003, p. 3213-3220, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3213-3220.2003

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

Mahendra H. Kothary,1* Heather Lowman,2 Barbara A. McCardell,1 and Ben D. Tall2

Division of Virulence Assessment, Center for Food Safety and Applied Nutrition, Food and Drug Administration, Laurel, Maryland 20708,1 Division of Microbiological Studies, Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 207402

Received 5 November 2002/ Returned for modification 29 January 2003/ Accepted 14 March 2003

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-Leu-Pro-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.

* Corresponding author. Mailing address: Division of Virulence Assessment (HFS-025), Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Rd., Laurel, MD 20708. Phone: (301) 827-8616. Fax: (301) 827-8615. E-mail: mkothary{at}cfsan.fda.gov.

Editor: A. D. O'Brien

Infection and Immunity, June 2003, p. 3213-3220, Vol. 71, No. 6
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.6.3213-3220.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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