SummaryThe pathogen group A Streptococcus (GAS) produces a wide spectrum of infections including necrotizing fasciitis (NF). Streptolysin S (SLS) produces the hallmark b b b b -haemolytic phenotype produced by GAS. The nine-gene GAS locus ( sag A-sag I) resembling a bacteriocin biosynthetic operon is necessary and sufficient for SLS production. Using precise, inframe allelic exchange mutagenesis and single-gene complementation, we show sag A, sag B, sag C, sag D, sag E, sag F and sag G are each individually required for SLS production, and that sag E may further serve an immunity function. Limited site-directed mutagenesis of specific amino acids in the SagA prepropeptide supports the designation of SLS as a bacteriocin-like toxin. No significant pleotrophic effects of sag A deletion were observed on M protein, capsule or cysteine protease production. In a murine model of NF, the SLS-negative M1T1 GAS mutant was markedly diminished in its ability to produce necrotic skin ulcers and spread to the systemic circulation. The SLS toxin impaired phagocytic clearance and promoted epithelial cell cytotoxicity, the latter phenotype being enhanced by the effects of M protein and streptolysin O. We conclude that all genetic components of the sag operon are required for expression of functional SLS, an important virulence factor in the pathogenesis of invasive M1T1 GAS infection.

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Group A Streptococcus (GAS) is a Gram-positive bacterium associated with a variety of mucosal and invasive human infections. GAS systemic disease reflects the diverse abilities of this pathogen to avoid eradication by phagocytic defenses of the innate immune system. Here we review how GAS can avoid phagocyte engagement, inhibit complement and antibody functions required for opsonization, impair phagocytotic uptake mechanisms, promote phagocyte lysis or apoptosis, and resist specific effectors of phagocyte killing such as antimicrobial peptides and reactive oxygen species. Understanding the molecular basis of GAS phagocyte resistance may reveal novel therapeutic targets for treatment and prevention of invasive human infections.

Group A Streptococcus (GAS) is a leading human pathogen associated with a wide spectrum of mucosal and invasive infections. GAS expresses a large number of virulence determinants whose expression is under the control of several transcriptional regulatory networks. Here we performed the first mutational analysis of a genetic locus immediately upstream of the streptolysin S biosynthetic operon in several GAS genome sequences, including that of the M1T1 serotype, the leading isolates associated with serious invasive disease. The locus consists of a predicted RofA-like stand-alone transcriptional regulator (RALP3) and the largest open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large surface-anchored" protein). Comparative reverse transcription-PCR analysis of wildtype M1T1 GAS and an isogenic RALP3-deficient mutant identifies RALP3 as a global transcriptional regulator affecting expression of numerous virulence factor genes, including those for strong repression of the hyaluronic acid capsule and cysteine protease production. RALP3 contributed to GAS epithelial cell invasion and bloodstream survival. LSA was found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactions and GAS antimicrobial peptide sensitivity. Isogenic M1T1 GAS mutants lacking either RALP3 or LSA were attenuated in a murine model of systemic infection, indicating that this locus plays a role in the virulence potential of the organism.