REDUCED VIRULENCE OF GROUP-A STREPTOCOCCAL TN916 MUTANTS THAT DO NOT PRODUCE STREPTOLYSIN-S

Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly al l group A streptococci (GAS). SLS-deficient Tn916 insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps ( M serotypes 1 and 18, respectively), by transposon mutagenesis using T n916 donor strain Enterococcus faecalis CG110. Representative nonhemol ytic transconjugants SBNB5 and SB30-2 each harbored a single Tn916 ins ertion in identical loci. The insertion in SBNH5 was located in the pr omoter region of an open reading frame, designated sagA, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activi ties and the production of M protein remained the same in the nonhemol ytic mutants and the wild-type strains, as did the growth rates and ex oprotein profiles. Transconjugants were evaluated in an established mu rine model by injecting the organisms subcutaneously and monitoring th e mice for alterations in weight and the development of necrotic lesio ns. Animals infected with SBNH5, compared to those infected with MGAS1 66s, gained weight during the first 24 h (+1.15 versus -1.16 g; P < 0. 05) and had fewer necrotic lesions (0 versus 7; P = 0.0007). Animals i nfected with SB30-2, compared to those infected with T18Ps, also gaine d weight within the first 24 h (+0.54 versus -0.66 g; P < 0.05) and pr oduced fewer necrotic lesions (1 versus 8; P = 0.001). Revertants of t he mutants in which Tn916 had been excised regained the hemolytic phen otype and the virulence profile of the wild-type strains. This study d emonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn916 mutations, are markedly les s virulent than their isogenic parents.