Production, Purification, and Composition of Staphylococcal α Toxin

Abstract

COULTER, JOHN R. (Institute of Medical and Veterinary Science, Adelaide, Australia); Production, purification, and composition of staphyloccocal a toxin. J. Bacteriol. 92:1655-1662. 1966-Pure staphylococcal a toxin has been prepared in quantities suitable for chemical, biological, and clinical characterization. Purification was achieved by acid-methanol precipitation, chromatography on G100 Sephadex, and electrophoresis in G100 Sephadex. We recovered 25% of the crude toxin in pure form, a yield of 12 mg/liter of crude culture supernatant fluid. The pure material gave a single line on gel diffusion and on immunoelectrophoresis and gave a single symmetrical peak in the ultracentrifuge. The a toxin was highly unstable, with a halflife of 3 days at 0 C (pH 7.8); solutions of it could not be frozen, and we found no method to stabilize it. On standing, a thready precipitate appeared; it was inactive against rabbit red cells, was not lethal to rabbits, but was able to elicit specific anti-a antibody production in the rabbit. There is evidence that a toxin is an associating molecule, with a mean sedimentation coefficient of approximately 3.0 and a molecular weight of approximately 30,000. The lowest molecular weight, found by equilibrium ultracentrifugation, was 21,200 400. The amino acid composition was determined, and the high positive charge was explained by the presence of lysine, arginine, and histidine, and by amination of the aspartic and glutamic acid residues. Histidine and arginine were shown to be N-terminal amino acids, a fact which suggests the presence of two polypeptide chains. No carbohydrate was present. The ultraviolet absorption spectrum showed a maximum at 274.5 m,, a minimum at 251.5 m,u, and a shoulder at 292 m,u. The toxin was without proteolytic or phospholipase activity, and its highly specific action on cell membranes still remains unexplained.

Electron Microscopic Localization of Alpha Toxin Within the Staphylococcal Cell by Ferritin-Labeled Antibody

“…Examination in the electron microscope confirmed the characteristic shape of the ferritin particles (Fig. 2,3,4).…”

“…Anti-alpha gamma globulin. Several rabbits were injected with purified alpha toxin (3). The toxin was allowed to aggregate by standing at 4 C for several weeks, and the aggregate was suspended in distilled water.…”

Cellular streptolysin S-related hemolysins of group A Streptococcus C203S

“…A tendency to aggregate or associate with cellular material is apparently characteristic of several bacterial extracellular hemolysins or cytolytic toxins (11). Staphylococcal alphatoxin aggregates during purification to a larger, but still soluble, particle and can also polymerize to a water-insoluble protein (4,15,16). The F and S components of staphylococcal leukocidin both polymerize in low-ionic-strength solutions (48).…”

Cellular location of alpha-hemolysin in Staphylococcus aureus

“…McNiven and Arbuthnott reported that less than 1% of the total hemolytic activity in Staphylococcus aureus strain Wood 46 is cell associated (11), suggesting that alphahemolysin is secreted rapidly without lag after synthesis. Recently, Coulter reported that alpha-hemolysin, as detected by ferritin-labeled antisera, is associated with the inner surface of the membrane (5). Using established protoplast fractionation techniques (14), we found that the cellular localization of alpha-hemolysin was almost exclusively in the periplasmic fraction of several strains of S. aureus.…”

“…Extracellular staphylococcal alpha-hemolysin has been purified and characterized by many investigators (3,5,9). In contrast, information pertaining to the location and characteristics of cellular alpha-hemolysin is almost nonexistent.…”