Further observations on the mode of action of the alpha toxin of Staphylococcus aureus "Wood-46"

Journal of Medical Microbiology

1974

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PLATE XIXAT T E M P,T s by several investigators have failed to demonstrate the immunogenicity of the 6-haemolysin of StaphyZococcus aureus. Gladstone and Yoshida (1967) did not convincingly demonstrate that 8-lysin was immunogenic and they showed that inhibitors of its haemolytic activity were present in serum. Recent evidence suggests that these serum inhibitors are lipoproteins (Donahue, 1969;Kantor, Temples and Shaw, 1972).In this study, the immunogenicity of 8-lysin has been demonstrated by the use of purified antibody preparations. MATERIALS AND METHODSPreparation of 6-lysin The strain employed, methods of production and purification of 6-lysin were those of Caird and Wiseman (1970). Purified S-lysin was also kindly sent to us by Dr Arnold Kreger. Haemolysin titrations were performed as described by Wiseman and Caird (1972). Buffers were prepared according to Gomori (1955) and, where required, 0 . 0 1~ phosphate buffer at pH 7 was supplemented with 0.85% NaCl. Nitrogen content was assayed by the microKjeldahl technique as described by Markham (1942) and an estimate of protein content was derived from the assay value multiplied by 6.25.Preparation of u-, ,8-and 7-lysins u-and y-Haemolysins were prepared and purified as described elsewhere (Fackrell and Wiseman, 1974; Wiseman, Caird and Fackrell, 1975). Purified ,!I-lysin was used in one immunodiffusion study and was prepared by a modification of the method of Wiseman and Caird (1967) in which the haemolysin was passed through Sephadex G-75 equilibrated with Hallander's (1963) buffer. The active lysin was pooled when removed from the column, dialysed against distilled water for 48 hours, centrifuged and the supernatant fluid lyophilised.Serological procedures Immunodi'usion, immunoelectrophoresis and quantitative precipitin methods were those of Campbell et al. (1970). The blood agarose required in some immunodiffusion tests was prepared by the addition of 1.0 ml of packed, washed human group-0 RBC to 100 ml of a 1 % (w/v) solution of Difco agarose in phosphate-buffered saline held at 50°C.Production of antisera. Antibodies to the haemolysins were prepared in 2-month-old New Zealand white rabbits purchased from the Canadian Research Animal Farm, Bradford,

Section: Methodsmentioning

confidence: 99%

Immunogenicity of the -Haemolysin of Staphylococcus Aureus

Journal of Medical Microbiology

1974

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“…Haemolytic titrations of gamma lysin were performed with human red cells as described by Wiseman & Caird (1972). Human blood (Rh+, group 0) obtained from the Canadian Red Cross in Winnipeg was stored in acid-citrate-dextrose solution as supplied.…”

Section: Methodsmentioning

confidence: 99%

“…Proteolytic activity of cultures in some experiments was determined as described by Wiseman & Caird (1972).…”

Section: Methodsmentioning

confidence: 99%

Production and Purification of the Gamma Haemolysin of Staphylococcus aureus 'Smith 5R'

Journal of General Microbiology

1976

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S U M M A R YThe gamma haemolysin of Staphylococcus aureus 'Smith 5~' was produced on Dolman-Wilson agar overlain with cellophane. Maximal yields of crude lysin with titres of 2000 to 4000 haemolytic unitslml were obtained within 24 h at 37 "C in 10 % (v/v) C02 in air, on medium adjusted to pH 7.0.The crude lysin was purified 2700-fold (with 75 % recovery) by ultrafiltration, gel filtration and ammonium sulphate fractionation. The specific activity of the lysin was 106 haemolytic unitslmg protein after the dialysed active precipitate was extracted with NaCl and reprecipitated with ammonium sulphate. purified gamma lysin was homogeneous by disc electrophoresis and immunoelectrophoresis.

“…Both the proteolytic and haemolytic activities of activated toxin have been shown to be neutralised by a-antitoxin (Wiseman and Caird, 1972). It has also been suggested that this a-'' protoxin " is activated by erythrocyte proteases and that the level of protease activity in red cells of various species explains their differing sensitivity to the toxin.…”

mentioning

confidence: 99%

“…Haemolytic activity was measured according to the method outlined by Wiseman and Caird (1972). Proteolytic activity was assayed according to the technique described by Hummel (1959) in which p-toluenesulphonyl-L-arginine methylester (TAME) was used as a substrate.…”

mentioning

confidence: 99%

Trypsin-Mediated Activation of the -Haemolysin of Staphylococcus Aureus

Caird

Journal of Medical Microbiology

1975

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PLATE IRECENT work in this laboratory indicated that the a-toxin of Staphylococcus aureus strain Wood 46 is produced by the organisms as an inactive proteolytic enzyme (Wiseman and Caird, 1970). Both the proteolytic and haemolytic activities of activated toxin have been shown to be neutralised by a-antitoxin (Wiseman and Caird, 1972). It has also been suggested that this a-'' protoxin " is activated by erythrocyte proteases and that the level of protease activity in red cells of various species explains their differing sensitivity to the toxin. In our study of this phenomenon, we were interested to know whether other proteolytic enzymes could serve as activators and our attention was thus directed toward trypsin. MATERIALS AND METHODSProduction and purification of a-haemolysin. Crude protoxin was prepared from the Wood-46 strain of S. aureus by a method similar to that given by Gow and Robinson (1969) except that air rather than pure oxygen was used in conjunction with carbon dioxide to provide the gaseous environment during growth.Gel filtration and ion-exchange chromatographic procedures were as follows. A K50 x 100-cm column containing Sephadex G-75 (Pharmacia, Montreal, Canada) was set up according to the manufacturer's instructions and equilibrated with Hallander's buffer (Hallander, 1963). Haemolysin was applied to the column in 5-ml volumes and 15-ml fractions were collected by upward elution.Ion-exchange chromatography was performed on a column of carboxymethylcellulose (CMC) according to the method of Robinson, Thatcher and Montford (1960). Haemolysin samples were dialysed against distilled water for 24 h, centrifuged and then dialysed against 0 . 0 5 6~ sodium-phosphate buffer, p H 6.0. Five-ml volumes of haemolysin were applied to a column equilibrated with the same buffer. The haemolysin was eluted in 15-ml fractions from the column by the gradient described by Robinson et al. (1960).Ammonium-sulphate fractionation of partially purified haemolysin was performed in which haemolysin was divided into eight fractions, to each of which the salt was added in concentrations between 0 and 100% saturation. The precipitates, held overnight at 4"C, were centrifuged, resuspended and dialysed against phosphate-buffered saline.The purification procedure was the method developed in this laboratory (Fackrell, 1973). The technique of methanol precipitation of a-haemolysin, described by Wittler and Pillemer (1948), was re-examined. Crude haemolysin adjusted to pH 4.0 was mixed with methanol at -20°C to a final concentration of 35% (v/v) solvent and the precipitate after harvesting was dialysed against phosphate-buffered saline. As a second step, methanol-precipitated ~~