Purification and characterization of an extracellular cytolysin produced by Vibrio vulnificus

Abstract

An extracellular cytolytic toxin produced by the halophilic bacterium V. vulnificus was isolated free of detectable contamination with medium constituents and other bacterial products by sequential ammonium sulfate precipitation, gel filtration with Sephadex G-75, hydrophobic interaction chromatography with phenyl-Sepharose CL-4B, and isoelectric focusing in an ethylene glycol density gradient. The cytolysin is a heat-labile, hydrophobic protein that is inhibited by large amounts of cholesterol, is partially inactivated by proteases and trypan blue, has a MW (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by amino acid analysis) of .apprx. 56,000, and has an isoelectric point of .apprx. 7.1. The first 10 amino-terminal amino acid residues of the cytolysin are Gln-Glu-Tyr-Val-Pro-Ile-Val-Glu-Lys-Pro. Lysis of mouse erythrocytes by the purified cytolysin is a multi-hit, at least 2-step process consisting of a temperature-independent, toxin-binding step, followed by a temperature-dependent, membrane-perturbation step(s). In addition to possessing cytolytic activity against erythrocytes from 17 animal species and against Chinese hamster ovary cells in tissue culture, the purified cytolysin preparation was lethal for mice (.apprx. 3 .mu.g/kg, i.v. LD50) and had vascular permeability factor activity in guinea pig skin.