Cellular streptolysin S-related hemolysins of group A Streptococcus C203S

Abstract

Group A streptococci strain C203S, grown in heart infusion broth with 0.3% maltose, produce two cellular hemolysins related to extracellular streptolysin S (SLS). Enzymatic lysis of the streptococci by group C streptococcal phageassociated lysin results in release of low titer, labile hemolysin, which can be stabilized by ribonucleic acid (RNA)-core (RNA preparation from yeast). This labile hemolysin can be detected only after the higher titer cellular streptolysin 0 is removed by erythrocyte membranes or inactivated by N-ethylmaleimide. The other cellular SLS-related hemolysin is released in a latent state (potential hemolysin) which can be activated.to high-titer hemolysin by sonication with RNA-core. The titer of such activated hemolysin depends upon the intensity of sonic energy, duration of sonication, and amount of RNA-core. RNA obtained from the streptococci is far less effective than RNA-core. When the cocci are disrupted by sonication or grinding, potential hemolysin and/or activated form may be released, depending upon the conditions employed. The potential hemolysin material is large and heterogeneous; activation appears to involve, in part, disaggregation or fragmentation. Labile hemolysin, potential hemolysin, and the activated form of potential hemolysin can all be converted to hemolysin having the same hemolytic and physical properties as RNA-core SLS, suggesting that all have the same hemolytic moiety. The presence of glucose in heart infusion broth prevents formation of both potential hemolysin and RNA-core SLS by log-phase cells, whereas addition of glucose to a culture in heart infusion broth with 0.3% maltose stops accumulation of potential hemolysin but does not affect continuation of RNA-core SLS release. These results suggest that potential hemolysin is a cellular precursor to RNA-core SLS.

Relationship of cellular potential hemolysin in group A streptococci to extracellular streptolysin S

“…When PH is activated in the presence of RNA-core, the resultant hemolysin has the same properties as RNA-core SLS. A hypothesis that PH is the cellular precursor to extracellular SLS was supported by prior results of experiments on the differing effects of glucose on the formation of PH and RNA-core SLS (5).…”

“…Group A streptococcus C203S contains several cellular hemolysins, two of which appear to be related to streptolysin S (SLS) (5). One is a very low-titer, active hemolysin, which is measurable only after removal or inactivation of streptolysin 0 (SLO) from the cell lysate.…”

“…Strains were grown in 100 ml of heart infusion (Difco)-0. 3% maltose media (5) to midlog phase (turbidity, 70 Klett-Summerson filter no. 520), harvested by centrifugation at 10,000 x g for 10 min, and washed with 0.85% saline.…”

“…(i) Cellular hemolysins. Washed streptococci suspended in 5 ml of lysing buffer (0.03 M NaPO4, pH 6.5; 0.85% NaCl; 10-2 M ethylenediaminetetraacetate; 10-3 M 2-mercaptoethanol) were lysed by incubation with excess phage-associated lysin (prepared as described previously) (5) at 28 C for 20 min. Debris was removed by centrifugation at 2,000 x g for 10 min.…”

“…PH was activated by sonication of part of the lysate plus RNA-core (0.3 mg/ml for 15 min; six 2.5-min periods of sonication interrupted for periods of cooling in ice) at 40 intensity using the large probe of the Brosonik IV (Bronwill). N-ethylmaleimide (NEM; 25 mg/ml) was mixed with unsonicated lysate for 10 min to inactivate SLO prior to assay of the remaining active hemolysin (5). This procedure was modified for the survey study (Table 1) in that PH was activated by sonication of whole cells plus RNA-core.…”