Rabbits injected intravenously with extracellular products ("toxins") of group A streptococci develop myocardial, muscular, and hepatic lesions. When such animals are then challenged with fluorochrome-labeled group A streptococci or with titanium oxide particles the labeled bacteria or particles localize within phagocytic cells in the tissue lesions caused by the toxins. Similarly, labeled streptococci or titanium oxide particles will also localize within phagocytic cells in skin lesions of guinea pigs that develop delayed hypersensitivity to tuberculin or to bovine gamma globulin. It is proposed that a combined mechanism of injury and localization of bacteria in damaged tissues may be responsible for poststreptococcal sequelae or other chronic inflammatory diseases.

Studies on the host parasite relationship in experimental streptococcal infections have been greatly aided by fluorescent antibody techniques (1). By such methods it is possible to follow the localization and persistence of group A streptococcal antigen in cells and tissues of laboratory animals (2-6). To detect such antigens by fluorescent techniques, frozen sections are often employed and the antisera to streptococcal antigens must be repeatedly absorbed with organ powders to remove nonspecific staining. The present communication describes a simple method for applying a label of fluorescein isothiocyanate (FITC) to living group A streptococci, streptococcal cell-wall fragments,mucopeptides, and streptococcal Lforms. Such labeled streptococci and products were found to persist for long periods of time in inflammatory sites in the muscle and heart of mice and rabbits.

Extracellular antigens have been isolated from Streptococcus mitis, Streptococcus salivarius and group A streptococcal cultures grown in a synthetic medium. Analysis of the antigens was performed by immunoelectrophoretic and double diffusion techniques using rabbit immune sera. S. mitis cultures produced 10 antigens, S. salivarius six antigens and group A streptococcus 12 antigens, when tested with their corresponding antisera. S. mitis and S. salivarius antigens had only one common antigen when tested with antisera to both antigen pools. No cross reaction was found between the exo-antigens of group A and viridans streptococci. While the extracellular antigen pool of group A streptococci contained ribonuclease, deoxyribonuclease, hyaluronidase, diphosphopyridine-nucleotidase, streptokinase and streptolysin 0, that of S. mitis contained only ribonuclease and that of S. salivarius contained hyaluronidase and collagenase. Each of the three streptococcal antigen pools contained a hemosensitizing factor which sensitized mammalian cells to passive immune kill. Sonicates produced from the mitis, salivarius and group A streptococcus contained six antigens, most of which cross reacted with each other. S. mitis sonicates were separated into six fractions by ion exchange chromatography on ECTEOLA cellulose, and into three major fractions following gel-filtration on Sephadex 0-200 columns. Rabbits injected i.v. with sonicates derived from S. mitis developed cardiac and hepatic lesions which, in some cases, were accompanied by a steep rise in serum glutamic oxalacetic transaminase, sorbitol dehydrogenase and total lipids. The relationship of tissue damage to enzyme rise is discussed in relation to the possible early diagnosis of tissue damage following streptococcal infection.

Rabbits injected with streptococcal extracellular protein (SEP) developed degenerative and infiltrative lesions in the heart and liver, with coagulation necrosis and multinucleated giant cells in the latter organ. The majority of these rabbits also showed elevated levels of glutamic-oxalacetic transaminase, or of sorbitol dehydrogenase, and all showed elevated serum lipids. These biochemical indications of cell injury could be found within 2 to 4 hours after a first injection of SEP. No such biochemical or pathologic effects were found following an injection of heated SEP or a commercially available streptokinase-streptodornase preparation from group C streptococcal culture. In a preliminary fractionation of SEP all the activity was found in a fraction not adsorbed to DEAE cellulose at pH 7.4 (0.05 M P04). The active fraction contained at least five antigens and four of the known streptococcal enzymes. Injection of extracts of sonically disrupted streptococci produced similar biochemical and pathologic changes. There was some cross-reacting material between the sonicates and anti-SEP serum.

Intraartikuläre Injektion von Streptolysin-S-freien extrazellulären Produkten der Streptokokken Gruppe A verursacht eine zunächst akute, in der Folge aber subkutane Synovitis. Die Veränderungen gleichen denjenigen nach Injektion von Streptolollensonikaten, so dass angenommen wird, ausser Streptolysin S bedingen auch andere streptokokkale Faktoren eine Arthritis.

Many studies have been made of tissue alterations due to infections with Group A streptococci in laboratory animals. Cardiac lesions characterized by muscle necrosis, myocarditis, and giant-cell formation have been reported in a variety of laboratory animals following injections of living Group A streptococci and some of their products.1l Some of these lesions were described as quite similar to the Aschoff bodies of rheumatic carditis.4 The mechanism of formation of these cardiac lesions has been attributed to toxic effects of streptococcal products,6 7 to immune response to streptococcal components (hypersensitivity or autoimmunity) ,10 or to combinations of these processes. Among these studies, the pharyngeal cavity of animals was used as the portal of entry of streptococci only in that of Glaser et al.,11 who found cardiac lesions within 72 hr. of a single intratonsillar injection of virulent Group A streptococci. These lesions were focal, and showed muscle necrosis, infiltrations of mononuclear cells, and occasional giant cells. No streptococci could be isolated from such lesions, and it was concluded that the lesions were probably not caused by an immunologic process."' The present report describes the induction of lesions in cardiac and other tissues of rabbits following injection, by intratonsillar and other routes, of streptococcal extracellular products (SEP) obtained from Group A streptococci grown in steady-state culture. These lesions occured soon after single injections of these materials and probably reflect direct toxic effect on the cells of these tissues.

Various streptococcal species produce an haemosensitizing factor during the logarithmic phase of growth. A variety of mammalian cells sensitized with this factor become agglutinated following the addition of antistreptoccocal serum and also undergo cytopathic changes in the presence of complement. The haemosensitizing factor is thermostable and is unaltered by trypsin, papain, chymotrypsin, lipases or ribonucleases. Attempts to destroy the binding sites on the cell membrane by treatment with phospholipase C from Clostridium welchii or by neuraminidase failed. Treatment with trypsin or papain on the other hand markedly increased the binding capacity of red blood cell for the haemosensitizing factor. Studies on the nature of the binding sites on the erythrocyte membrane of the haemosensitizing factor suggest that cholesterol and phospholipids constitute some of the binding sites for this factor.

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.

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.

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.

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 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.

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.