It is widely believed that the virulence or pathogenicity of many bacterial species, particularly among the Gram-positive cocci, is due in large part to their ability to resist destruction by host phagocytes. The group A streptococci possess two elements that have been implicated in virulence, namely the M substance and the capsule; and of these the M substance is thought to be the more important (1). Nevertheless, there are certain observations which suggest that there may be other factors involved in streptococcal virulence. For example, strains freshly isolated from acute human streptococcal infections may be of low virulence for mice, even though they contain abundant M substance and produce good capsules. Serial intraperitoneal passage of such a strain in mice often causes its mouse virulence to be increased many fold. This increase is associated with an increase in the M substance, when the strain has relatively little M substance to begin with, but the problem of why unpassed strains with abundant M and well developed capsules should be relatively avirulent for mice, continues unsolved. Speculation has been offered that there may be other as yet unrecognized structural elements in the streptococcus involved in virulence. It has been suggested also that certain undefined metabolic activities of the cocci may play a role in virulence, and this idea is supported by the practice of using young, actively proliferating and actively metabolizing cocci in virulence and phagocytosis studies. No specific indication has been offered of the nature of such metabolic functions other than those concerned with the synthesis of M and the capsule.In 1898 Marchand (2) published a classic work showing that streptococci retained their ability to resist ingestion by phagocytes after they had been killed by heat or several chemical agents. This work has attracted little notice, although Todd (3) in 1927 and Hare (4) in 1931 each published similar views without giving experimental data.This seemed to us an important observation that deserved reinvestigation. If the factors in the streptococcal cell that enable it to resist phagocytosis sur-15 on

The immunoelectrophoretic characteristics of the known cellular antigens of serotype 17, Group A streptococci have been presented. These include C, M, T, and polyglycerophosphate. In addition, three hitherto undescribed antigens of serotype 17 have been encountered. The F antigen occurs in most serotype 17 strains, has a faster electrophoretic mobility than M, appears in acid, distilled water, and other extracts of harvested cells, is released from the cells in large quantity into the culture medium during growth when the pH is maintained at 7.3 or over, is probably protein in nature, and may play a minor role in mouse virulence of serotype 17 strains. Its antibodies do not confer bactericidal power on human blood. The E antigen is serotype-specific and is closely associated with the M antigen. The suggestion is made here that E is a part of the M molecule, acquiring independent electrophoretic mobility when separated from the rest of the M molecule by acid hydrolysis and carrying an antigenic determinant serologically distinct from the determinant on the rest of the M molecule. E can be recognized only by immunoelectrophoresis. Its role, if any, in virulence has not been established. The third antigen, E4, is a non-serotype-specific antigen found in most serological groups and types of hemolytic streptococci. It is serologically related to polyglycerophosphate, but its chemical nature has not been determined. It appears to be unrelated to virulence.

The fate of non-virulent group A streptococci phagocytized in vitro has been investigated by destroying the phagocyte with electric current and observing whether the liberated cocci multiply. Human and mouse peripheral blood neutrophils quickly injure ingested cocci, the time required to produce 50 per cent non-survival of chains being 8 and 6¾ minutes, respectively.

The type classification of Group A streptococci depends upon the M proteins, which are imrnunologically different for each type. These antigens are of special importance among the several well studied antigens of this group of streptococci because of their dose association with virulence. They are demonstrable by precipitin tests, and antibodies to them react type-specifically in active and passive animal protection tests and in ingestion and bactericidal tests. There have been described, up to the present time, some 45 types of streptococci, and in each type the determinant has been a single M antigen characteristic of the type.The work reported here shows that most Type 14 streptococci possess two M antigens, the presently recognized M antigen of Type 14 and another M designated here Type 51. A few strains have been encountered possessing the Type 51 M antigen without the Type 14 M antigen and one strain possessed the Type 14 M antigen alone. Mthough it has previously been shown that Group A streptococci may possess more than one T antigen (1), the present work is the first demonstration that strains may have two distinct M antigens. Materials and MahodsStreptococcal Strains.raThe principle strains used in this work are listed in Table I. In addition to these, 34 strains obtained from many sources and covering a wide geographical distribution were tested for their M antigen composition. All strains were preserved by freezing and drying. For routine work strains were grown in neopeptone--sheep blood broth and maintained in the refrigerator at approximately 4°C. To roa~ntaln cultures capable of vigorous multiplication in normal human blood, strains were recovered approximately once a month from the dried state, or as an alternative method, stock blood broth cultures were sometimes given a single intraperitoneal mouse passage.Precipitin tests were performed by the capillary precipitin method using crude acid extracts (2) or M extracts (3).Antlsera were prepared by injecting rabbits three times weekly with heat-killed bacterial suspensions, and collecting serum when adequate antibody content had been achieved. Sera were absorbed with packed hcat-ldlled cells in the desired proportions. We are indebted to the Commtmicable Disease Center, Chamblee, Georgia for absorbed typing sera for many streptococcal types and to Dr. Rebecca C. Lancefield for a generous supply of antiserum prepared against strain Type 14146.

Strains of four streptococcal types, 33, 41, 43, 52, and a nontypable strain, Ross, cross-reacted in precipitin and bactericidal tests. The homologous reactions, which determined the type, afforded the major protection and developed promptly and regularly in the serum of rabbits during immunization. The associated cross-reactions, on the other hand, appeared in the serum of certain rabbits only, were often not as strong as the associated homologous reactions, and required for their presence a longer period of immunization than the homologous reactions. Agar gel analysis of the homologous precipitin reactions revealed, as would be expected, reactions of serological identity, while those cross-reactions which were strong enough to test in this way formed bands of precipitate which joined with spur formation on the side of the homologous reaction. These experiments and others referred to in the text suggest that cross-protection, as demonstrated in bactericidal tests, is sufficiently widespread to be a factor in streptococcal immunity, if a corresponding protection occurs in vivo. Thus, streptococcal infection with one of the cross-reacting strains might confer, in addition to strong homologous protection, a certain amount of cross-protection.