Inactivation of the biological activities of the thermostable direct hemolysin of Vibrio parahaemolyticus by ganglioside Gt1

Abstract

Biological activities of the thermostable direct hemolysin produced by Vibrio parahaemolyticus, such as its hemolytic activity and lethal activity, were inhibited by neuraminidase-sensitive gangliosides, of which GT1 ganglioside was the most inhibitory. Neuraminidase-resistant gangliosides did not affect the activities of the hemolysin. Results showed that horse erythrocytes, which are resistant to the hemolysin, do not contain the neuraminidase-sensitive gangliosides GT, and GD1a. Therefore, we propose that neuraminidase-sensitive gangliosides, and especially GT1 ganglioside, may be the receptor sites on the membranes for the thermostable direct hemolysin of V. parahaemolyticus.In a previous paper, we reported that the hemolytic activity of the thermostable direct hemolysin of Vibrio parahaemolyticus was inhibited by a ganglioside mixture and that GM, ganglioside was not related to this inhibitory effect (14). To determine the tissue receptor for the thermostable direct hemolysin, we studied the inhibitory effects of various ganglioside components on biological activities of the thermostable direct hemolysin and found that neuraminidase-sensitive gangliosides, and especially GTI ganglioside, inhibited these biological activities. MATERIALS AND METHODSPurification of thermostable direct hemolysin. The thermostable direct hemolysin was isolated from culture filtrates of a Kanagawa phenomenonpositive strain, V. parahaemolyticus WP-1, and purified by successive column chromatographies on diethylaminoethyl-cellulose, hydroxylapatite, and Sephadex G-200, as described previously (7). The purified preparation of thermostable direct hemolysin contained a single protein with a molecular weight of about 42,000 (7).Assay of hemolytic activity. Hemolytic activity was assayed as described previously (7,13). The standard reaction mixture (2.5 ml) contained 10 mM tris(hydroxymethyl)aminomethane (Tris) -hydrochloride buffer (pH 7.2), 10 mM CaCl12, 0.9% NaCl, the thermostable direct hemolysin, and 1.25 ml of a 1% suspension of human erythrocytes (about 8.3 x 107 cells/ml). The reaction mixture was incubated at 37 C for 30 min and then centrifuged at 3,000 rpm for 5 min. The hemolytic activity was determined by measuring the absorbance of the resulting supernatant fluid at 540 nm.Assay of lethal toxicity. Lethal toxicity was assayed by injecting the thermostable direct hemolysin intravenously into 4-to 6-week-old mice (ddO strain) and measuring the survival time of the animals.Gangliosides. The ganglioside mixture used was type II from Sigma Chemical Co. Gangliosides GTI; GDIa, GM,, and GM2 (8). were obtained from Supelco Inc. GM, and GM2 were treated with V. cholerae neuraminidase (Calbiochem, B grade), as described below, before use. Treatment of gangliosides with neuraminidase.Ganglioside was incubated with V. cholerae neuraminidase (Calbiochem, B grade) in 0.2 ml of 0.01 M Tris-hydrochloride buffer (pH 7.2) for 48 h at 37 C. Then, the reaction mixture was dried in a rotary evaporator and the ganglioside was extracted into a mi...

Purification and characterization of a putative virulence factor, serine protease, fromVibrio parahaemolyticus

“…3). In addition, it is reported that the hemolytic activity of TDH was blocked after incubation with ganglioside G T1 and G D1a , which are usually located on the cell membrane and play the role of cell receptors [21]. Ganglioside G M1 is identified as the receptor for cholera toxin [23].…”

“…For example, G M1 has been characterized as a cholera toxin receptor, and can block the activity of that toxin [23]. TDH activity was inactivated by treatment with G T1 and G D1a but not by G M2 [21]. Nevertheless, G T1 and G D1a did not neutralize the CT of protease A.…”

SEPARATION AND CHARACTERIZATION OF ENTEROTOXIN AND TWO HAEMOLYSINS FROM AEROMONAS HYDROPHILA

“…The V. cholerue E 1 Tor haemolysin was likewise inhibited by this ganglioside mixture (1 5). The cell receptor for Y. parahuemolyticus haemolysin was earlier identified as ganglioside (29). Several physico-chemical properties are shared by the 6-haemolysin and the vibrio haemolysin, and the effects on tissue cultures are also similar (20,22,30).…”

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus

“…3). In addition, it is reported that the hemolytic activity of TDH was blocked after incubation with ganglioside G T1 and G D1a , which are usually located on the cell membrane and play the role of cell receptors [21]. Ganglioside G M1 is identi¢ed as the receptor for cholera toxin [23].…”