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

Abstract

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...

The Photobacterium damselae subsp. damselae Hemolysins Damselysin and HlyA Are Encoded within a New Virulence Plasmid

“…damselae supernatants (27). These observations suggested that other hemolysins in addition to Dly might be produced by this subspecies.…”

Toxicity of Photobacterium damselae subsp. damselae strains isolated from new cultured marine fish

“…Dis Aquat Org 92: [31][32][33][34][35][36][37][38][39][40] 2010 brane, has been described (Kreger 1984, Kothary & Kreger 1985, Kreger et al 1987. In addition, a relationship between the degree of virulence and the hemolytic activity has been demonstrated in P. damselae subsp.…”

“…In the case of P. damselae subsp. damselae only an extracellular cytolytic toxin, named damselysin, which is a phospholipase-D active against the sphingomyelin of the sheep erythrocyte mem-brane, has been described (Kreger 1984, Kothary & Kreger 1985, Kreger et al 1987. In addition, a relationship between the degree of virulence and the hemolytic activity has been demonstrated in P. damselae subsp.…”

Phobalysin, a Small β-Pore-Forming Toxin of Photobacterium damselae subsp. damselae

“…Dly is a phospholipase D that until recently was the only known hemolysin of P. damselae subsp. damselae (10)(11)(12), whereas HlyA pl is a putative pore-forming toxin (PFT) (9). A third, chromosomally encoded hemolysin, HlyA ch , present in all hemolytic strains, is 92% identical to HlyA pl (13), and it makes a minor contribution to hemolysis and virulence (14).…”