Phospholipase Activity of the Delta Hemolysin of Staphylococcus aureus

Abstract

The alpha, beta, and delta hemolysins of Staphylococcus aureus are implicated in the pathogenicity of this microorganism for man and animals. However, only the mode of action of the beta hemolysin is known with certainty. This enzyme hydrolyzes sphingomyelin, a common constituent of cellular membranes, resulting in the liberation of water-soluble phosphorylcholine and waterinsoluble K-acyl sphingosine ( 1,2). In the present experiments, we have shown that two highly purified preparations of the delta hemolysin liberate organic phosphorus from phospholipid extracts of mammalian erythrocytes. Of several phospholipids investigated as substrates, phosphatidylinositol was the most susceptible to degradation by the delta hemolysin.Materials and Methods. Crude delta hemolysin from the Newman and E-delta strains of S. aureusl was prepared in stainless steel trays of Dolman-Wilson agar (3) overlain with sterile cellophane and covered with aluminum foil. The cellophane surface was inoculated with 2 ml of a saline suspension of a 24-hour agar slope culture of the organisms, which was spread with a glass rod. After 24-hours incubation at 37OC in a sealed plexiglass tank in an atmosphere of 25% C 0 2 in air, 0.01 A1 phosphate buffered saline at pH 7.0 was added to the trays. The growth was taken up in the buffer, pooled, and centrifuged at 11,OOOg for 30 min. The supernatant fluid containing the hemolysin was stored at -20°C.Crude delta hemolysin prepared in this manner was dialyzed against 0.05 M acetate buffer a t pH 4.0 for 48 hours. The resulting ~~ * Supported by the Medical Research Council.precipitate which contains the active material was collected by centrifugation and dissolved in 0.05 M tris (hydroxymethyl) aminomethane (Tris) buffer a t pH 9.0 to effect solution. Further dialysis of the preparation was then carried out against 0.1 M phosphate buffer a t pH 7.0 for 48 hours. I t was centrifuged and the supernatant fluid was added to hydroxylapatite (4) in the amount of 10 m1/0.2 gm of dry weight of adsorbent. Elution of activity was accomplished after 1-hour standing a t 4°C by addition of 2 M NaCl in buffer to the adsorbent, followed by centrifugat ion.Further purification of the delta hemolysin was achieved on a column of diethylaminoethyl (DEAE) cellulose equilibrated with 0.02 M phosphate buffer a t p H 7.0. The hemolysin was eluted from the column against a linear gradient of NaCl (W.5 M ) in phosphate buffer prepared in a Varigrad.2 About 40 mg of protein was applied to the column and 10 ml fractions were collected, the active material appearing in the first of several peaks eluted.A 30-fold increase in specific activity was achieved by this method when crude hemolysin was compared to that eluted from the DEAE column. One strong line of precipitation was observed in Ouchterlony plates when purified hemolysin was incubated with a rabbit-produced antiserum to crude material. However, Jackson and Little ( 5 ) and Gladstone and Yoshida (6) have observed that protein components of normal serum strongly inhibit hem...

Purity of Staphylococcal -haemolysin obtained by three different procedures

“…This study shows that we were unable to obtain purified 6-haemolysin by employing some of the methods reportedly capable of yielding such preparations (Wiseman and Caird, 1968;Kreger et al, 1971;Kantor et al, 1972). In fact, with one procedure our final product did not contain demonstrable 6-haemolysin when tested by the electrophoretic localisation technique.…”

“…Our choice of the 146P strain of S. aureus instead of the Wood 46M strain used by Kreger et al (1971) and Kantor et al (1972), and the strain E-delta used by Wiseman and Caird (1968), was based on studies that we previously conducted (Ali and Haque, 1974) wherein 13 selected strains of S. aurew were compared for their ability to produce various haemolysins and other biologically active extracellular products. Of the 13 strains, the two best suited for the production and purification of 6-haemolysin were the 146P and the Wood 46M strains.…”

“…The methods of purification as described by Wiseman and Caird (1968) Purity tests. The purity of the various preparations of 6-haemolysin was tested by (a) determining the presence of biological activities known to be due to products other than the 8-haemolysin, and (b) the electrophoretic localisation technique.…”

“…At present, three methods (Wiseman and Caird, 1968 ; Kreger, Zabovetzky and Bernheimer, 1971 ;Kantor, Temples and Shaw, 1972) have been reported for purifying 6-haemolysin. In this investigation we tried to compare these methods and to test the purity of the 8-haemolysin preparations so obtained.…”

Assay of Staphylococcal -Haemolysin with Fish Erythrocytes

“…Current evidence suggests that the action of 8-haemolysin resembles that of certain surface-active agents, namely Triton X-100 and melittin (Bernheimer, 1974;Freer and Arbuthnott, 1976) in producing large " functional holes " in the erythrocyte membrane (Thelestam and Mollby, 1975), and fish erythrocytes may have a specific receptor that facilitates the interaction of 6-haemolysin with the hydrophobic region of the membrane. Recent reports Finean, 1976 and1977) of a phosphatidylinositol-specific phospholipase C, distinct from 6-haemolysin, may explain previous suggestions that 8-haemolysin is a phospholipase (Wiseman and Caird, 1968;Wiseman, 1975).…”