Staphylococcal Toxins

BERNHEIMER, ALAN W. (New York University School of Medicine, New York, N.Y.). Disruption of wall-less bacteria by streptococcal and staphylococcal toxins. J. Bacteriol. 91:1677-1680. 1966.-Earlier studies demonstrating that staphylococcal a-toxin and streptolysin S have the capacity to lyse protoplasts and spheroplasts of certain species of bacteria and Mycoplasma have been extended to encompass additional kinds of wall-less bacteria including L forms. It is suggested that sensitivity to staphylococcal a-toxin and streptolysin S may be explicable in terms of specific phospholipid composition of cell membranes, whereas sensitivity to streptolysin 0 is dependent upon the presence in cell membranes of cholesterol. The results suggest that streptolysin 0 might prove to be a useful reagent for differentiating L forms from parasitic pleuropneumonia-like organisms. It has been demonstrated that protoplasts and spheroplasts of certain bacterial species are lysed by staphylococcal a-toxin and by streptolysin S, and that they are not lysed by streptolysin 0 (2). In contrast, bacteria having unimpaired cell walls were not affected by any of the toxins. It follows from these and other observations that the cell membrane is the site of action of the toxins, and it seems probable, though as yet unproved, that one or more membrane lipids function as specific receptors (or substrates?) for the first two toxins mentioned. Present knowledge (6), though incomplete, indicates that there are substantial differences in the lipid composition of membranes of different bacterial species. It is reasonable to predict that a correlation might exist between membrane lipid composition and sensitivity to a particular toxin, as, in fact, has been established for streptolysin 0, which is active only on cells whose membranes contain cholesterol (1, 2). We present here data on the toxin sensitivity of additional species of wall-less bacteria with the view that the findings may eventually become interpretable in terms of specific lipid composition of membranes. In addition to protoplasts and spheroplasts, it was considered of interest to examine bacterial L forms for sensitivity to toxins to learn how they compare with pleuropneumonia-like organisms and with protoplasts and spheroplasts. MATERIALS AND METHODS Criterion of lysis. Susceptibility of test organisms to toxins was estimated by following the optical density of mixtures in a Cary recording spectrophotometer at 35 C. The conditions were the same as described earlier (2). Toxins. Staphylococcal a-toxin, streptolysin S, and streptolysin 0 were the same as used before (2). Protoplasts and spheroplasts. Protoplasts of Streptococcus pyogenes, type 19, strain GL8 were prepared with phage-associated lysin in the same way as for the C203S strain in the earlier study. Protoplasts of S. faecalis ATCC 9790 were prepared according to Shockman and Slade (10), by use of 1 mg of lysozyme per ml for 16 hr at 37 C. The protoplasts were centrifuged and suspended in 0.5 M sucrose in 0.05 M phosphate, pH 6.6, containing 0.01 M MgCl2. Protoplasts of Staphylococcus aureus Wood 46 were prepared with lysostaphin (9). Cocci from 25 ml of Neopeptone (Difco) meat-infusion broth that had been inoculated with 0.02 volume of culture and incubated at 37 C for 5 hr were washed in 0.3 volume of 0.15 M NaCl solution. They were suspended in 0.3 volume of 0.6M sucrose, 0.15M NaCl, and 0.05 M tris(hydroxymethyl)aminomethane (Tris), pH 7.5, containing 2 units of lysostaphin per ml. After 10 min at 37 C, the mixture was centrifuged and the protoplasts were suspended in 0.3 volume of the same solution, but lacking lysostaphin. Protoplasts of Micrococcus lysodeikticus were prepared from cocci grown in peptone-water-yeast extract medium (8) that had been inoculated with 0.1 volume of culture and incubated on a shaker at 30 C for 16 hr. The cocci from 40 ml were washed in 1677 on N ovem er 6, 2017 by gest http/jb.asm .rg/ D ow nladed fom