Reduced Virulence of Group A Streptococcal Tn
916
Mutants That Do Not Produce Streptolysin S
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abstract
ABSTRACT
Streptolysin S (SLS) is a potent cytolytic toxin produced by nearly all group A streptococci (GAS). SLS-deficient Tn
916
insertional mutants were generated from two clinical isolates of GAS, MGAS166s and T18Ps (M serotypes 1 and 18, respectively), by transposon mutagenesis using Tn
916
donor strain
Enterococcus faecalis
CG110. Representative nonhemolytic transconjugants SBNH5 and SB30-2 each harbored a single Tn
916
insertion in identical loci. The insertion in SBNH5 was located in the promoter region of an open reading frame, designated
sagA
, rendering it transcriptionally inactive. Protease, streptolysin O, and DNase activities and the production of M protein remained the same in the nonhemolytic mutants and the wild-type strains, as did the growth rates and exoprotein profiles. Transconjugants were evaluated in an established murine model by injecting the organisms subcutaneously and monitoring the mice for alterations in weight and the development of necrotic lesions. Animals infected with SBNH5, compared to those infected with MGAS166s, 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 infected with SB30-2, compared to those infected with T18Ps, also gained weight within the first 24 h (+0.54 versus −0.66 g;
P
< 0.05) and produced fewer necrotic lesions (1 versus 8;
P
= 0.001). Revertants of the mutants in which Tn
916
had been excised regained the hemolytic phenotype and the virulence profile of the wild-type strains. This study demonstrates that SLS-deficient mutants of GAS, belonging to different M serotypes and containing identical Tn
916
mutations, are markedly less virulent than their isogenic parents.
