The effects of streptolysin S and cell-bound hemolytic factor were tested on tissue cultures of rat heart and kidney. Cytopathogenic changes were observed, characterized by swelling, cytoplasmic vacuolization and bleb formation and cells were disintegrated within 1–2 hours.
These changes were abolished by known inhibitors of streptolysin S.
In studies of the mechanism of lysis of red blood cells by washed streptococci with hemolytic activity (cell-bound hemolysin, CBH) no components released spontaneously by RBC or streptococci, or by interaction between these cells, could be found to induce the formation of soluble hemolysin by the streptococci. It was also found that separation of RBC from streptococci even by Millipore filter or a very thin layer of agar could prevent their hemolysis. By means of cellulose columns it was possible to separate RBC from streptococci after a short incubation. RBC thus separated from streptococci with which they had been incubated underwent hemolysis on subsequent incubation at 37°C. By varying the period of incubation prior to separation it was possible to demonstrate the transfer of increasing amounts of hemolysin from streptococci to RBC with increasing periods of incubation. A considerable part of this appeared to be at a constant rate. A theory is presented on the relationship between the streptococcal cell-bound hemolysin and the group of oxygen-stable streptococcal hemolysins, in terms of a transferable hemolytic moiety and binding sites for this moiety on the streptococcal cell, on various molecular species which can act as inducers of the oxygen-stable hemolysins, and on the RBC, with the affinity of the respective binding sites for the hemolytic moiety increasing in that order.
The relationship of the streptococcal hemolysin which is recognized on incubation of RBC with streptococcal cells (cell-bound hemolysin, CBH), to RNA hemolysin, a representative of oxygen-stable hemolysin (streptolysin S) has been studied. A number of similarities have been found in the conditions for optimal production of each of these hemolysins, a requirement for cysteine, Mg++, and glucose; maximal production by streptococci in the stationary phase; similar curves of pH-dependence. In both systems, the production of hemolysin was inhibited by certain antibiotics, by ultraviolet irradiation, and by sonic disruption and was absent in the same streptococcal mutant strain. The hemolytic activity of both systems was inhibited by lecithin, trypan blue, and papain. Similarities were also found in relative susceptibilities to the two hemolytic systems of erythrocytes of a number of animal species. These data support a suggestion advanced in an earlier study that a streptococcal hemolytic moiety, which can be induced by, and carried on, a number of diverse agents to comprise the group of oxygen-stable hemolysins, serves, in its original attachment to a component of the streptococcal cell, to produce the hemolytic effect recognized as the cell-bound hemolysin.
Cysteine and other sulfhydryl compounds markedly enhance the formation of streptolysin S by 4 strains of group A streptococci. In the presence of cysteine (0.001 M) 10,000-30,000 hemolytic units per ml were obtained from strain S 84 type 3 in comparison with 1,000-3,000 units in the absence of cysteine. The role of cysteine in formation of hemolysin is not clear but it probably acts as a donor of SH compounds essential for an unknown process, rather than as a reducing agent. The inhibition of hemolysin formation by 3 amino acid analogues phenylserine, phenylglycine and acetyl-pro-line is reversed to a large extent by sulfhydryl compounds. Thus 0.002 M of cysteine reverses to a large extent the inhibition of hemolysin formation caused by as much as 0.1 M of phenylserine.
The production of oxygen-stable hemolysin in growing and resting Group A streptococci has been induced by RNA, by detergents, and by mammalian blood serum proteins, in the presence of glucose, Mg(++), and cysteine. Of the serum proteins, albumin and alpha lipoprotein could act as inducers. In the case of both these serum proteins treatment with trypsin did not affect the capacity to induce hemolysin production, but removal of the bound lipids by alcohol-ether or chloroform-methanol destroyed this property. In comparisons of the conditions of production and of activity between the hemolysin produced by RNA on one hand and albumin and detergents on the other, some data indicated similarities among the hemolysins, and others, differences. The similarities included similar degrees of temperature dependence for production and equal degrees of inhibition by serum beta lipoprotein. Differences found among these hemolysins included differences between, the rate of production of the RNA hemolysin from that of albumin or detergent hemolysin by both resting and growing streptococci, and the failure of utilization of glucosamine as an energy source for the production of albumin hemolysin, in contrast with that of RNA hemolysin. The fact that the data have in some cases indicated similarities and in other cases differences among the hemolysins raises the question of whether these are different molecular species, or a single hemolysin synthesized by the streptococci via different pathways of metabolism, or complexes of a single hemolytic moiety with various molecular carriers.
The oxygen-stable streptococcal hemolysins, which can be induced by a number of diverse substances, have been studied. Differences among these hemolysins have been found in electrophoresis, chromatography, pH stability, and susceptibility to some organic solvents and to an enzyme, RNAase. These properties have in each case been found to characterize the inducing substances as well. In a number of instances it has been found possible to incubate one inducer with the hemolysin induced by another of these agents and then, after appropriate fractionation, to find hemolytic activity in the fraction containing the fresh inducer. These observations suggest that the oxygen-stable streptococcal hemolysins are constituted as carrier-hemolysin complexes, the carriers being the set of molecular species effective as inducers, and the prosthetic group being transferred from one carrier to another under appropriate conditions. After transfer of the hemolytic moiety from a hemolysin molecule which is susceptible to inactivation by a given agent or set of conditions to a carrier which is not itself significantly affected by this agent, the new, derived, hemolysin has been found not to be inactivated by the agent. The hemolysins of this group can thus be inactivated by enzymatic attack on the prosthetic group, or by hydrolysis or deformation of the postulated carrier molecule.
A heat stable factor (SF) in normal human serum which is cytotoxic to Landschutz ascites tumor cells has been described. The cytotoxic action requires the presence of human or rabbit complement but guinea pig complement is ineffective. The substance is found in the beta-globulin fraction and has characteristics similar to C′4 of human complement. It is selectively absorbed on human and mouse tumor cells and human placenta but not by normal human liver, kidney or heart. SF inhibits the action of heterologous antibody on ascites and HeLa cells.
We are indebted to Dr. A. Rimon, Marcus Memorial Blood Bank Institute, Jaffa, for the Cohn fractions, and to Dr. Esther Tenenbaum of this Department for a generous supply of HeLa cells and Chang cells.
