RELATIVE CONTRIBUTIONS OF HYALURONIC-ACID CAPSULE AND M-PROTEIN TO VIRULENCE IN A MUCOID STRAIN OF THE GROUP-A STREPTOCOCCUS

The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsul ar mutant derived from the mucoid or highly encapsulated M protein typ e 18 group A streptococcal strain 282 indicated that loss of capsule e xpression was associated with decrease resistance to phagocytic killin g and with reduced virulence in mice. To study directly the relative c ontributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72 . Inactivation of emm18 was accomplished by integrational plasmid muta genesis, using the temperature-sensitive shuttle vector pJRS233 harbor ing a 5' DNA segment of emm18. As reported previously, wild-type strai n 282 was resistant to phagocytic killing in vitro, both in whole huma n blood and in 10% serum. The capsule mutant TX72 was highly susceptib le to phagocytic killing in 10% serum. The capsule mutant TX72 was hig hly susceptible to phagocytic killing in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- adn 80-fold for the c apsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the s train 282 capsule genes into the chromosome of a nonmucoid M1 strain r esulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results pro vide further evidence that the hyaluronic acid capsule confers resista nce tp phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocy tosis in 10% serum rather than in whole blood may be a more accurate r eflection of virulence in vivo of group A streptococci.