Genetic Locus for Streptolysin S Production by Group A Streptococcus

Abstract

Group A streptococcus (GAS) is an important human pathogen that causes pharyngitis and invasive infections, including necrotizing fasciitis. Streptolysin S (SLS) is the cytolytic factor that creates the zone of betahemolysis surrounding GAS colonies grown on blood agar. We recently reported the discovery of a potential genetic determinant involved in SLS production, sagA, encoding a small peptide of 53 amino acids (S. D. Betschel, S. M. Borgia, N. L. Barg, D. E. Low, and J. C. De Azavedo, Infect. Immun. 66:1671-1679, 1998). Using transposon mutagenesis, chromosomal walking steps, and data from the GAS genome sequencing project (www .genome.ou.edu/strep.html), we have now identified a contiguous nine-gene locus (sagA to sagI) involved in SLS production. The sag locus is conserved among GAS strains regardless of M protein type. Targeted plasmid integrational mutagenesis of each gene in the sag operon resulted in an SLS-negative phenotype. Targeted integrations (i) upstream of the sagA promoter and (ii) downstream of a terminator sequence after sagI did not affect SLS production, establishing the functional boundaries of the operon. A rho-independent terminator sequence between sagA and sagB appears to regulate the amount of sagA transcript produced versus transcript for the entire operon. Reintroduction of the nine-gene sag locus on a plasmid vector restored SLS activity to the nonhemolytic sagA knockout mutant. Finally, heterologous expression of the intact sag operon conferred the SLS beta-hemolytic phenotype to the nonhemolytic Lactococcus lactis. We conclude that gene products of the GAS sag operon are both necessary and sufficient for SLS production. Sequence homologies of sag operon gene products suggest that SLS is related to the bacteriocin family of microbial toxins.Group A streptococcus (GAS), specifically Streptococcus pyogenes, is a common cause of pharyngitis, impetigo, and many other human respiratory tract and soft tissue infections. Recently, there has been a dramatic increase in reports of severe invasive GAS infections, including necrotizing fasciitis and toxic shock syndrome (44). Although GAS produces a wide array of virulence factors, those responsible for the rapid bacterial spread and tissue injury seen with invasive GAS infections are unknown.A hallmark feature of GAS in the clinical laboratory is the zone of beta-hemolysis observed surrounding colonies grown on the surface of blood agar media. The factor responsible for the beta-hemolytic phenotype is streptolysin S (SLS), the oxygen-stable cytolytic toxin of GAS. Despite detailed investigations over several decades, the exact chemical nature of SLS has remained a great mystery of GAS biology. SLS can exist in intracellular, cell-surface-bound, and extracellular forms (16), but attempts at purification are complicated by instability of the cytolytic activity in the absence of high-molecular-weight carriers, such as yeast RNA core or albumin (47).We have adopted a molecular genetic approach towards the identification of SLS and the study of...

Similarities between Complement-mediated and Streptolysin S-mediated Hemolysis

“…Based on genetic evidence, this polypeptide is predicted to contain a 17-amino acid leader sequence that would be processed to yield a 36-amino acid polypeptide (7,8). Studies of mutants and complementation experiments using nonhemolytic Lactococcus isolates support the conclusion that the polypeptide encoded by the pel/sagA gene is indeed a key constituent of streptococcal SLS (9).…”

“…Despite the functional property of hemolysis being easy to quantify the predicted protein composition of SLS from different isolates has varied widely (4 -6). Recent genetic studies have identified a locus in S. pyogenes that has been convincingly associated with encoding this oxygen-stable hemolysin (7)(8)(9). The predicted ORF encoding the hemolysin, pel/sagA, is present in group A, C, and G streptococci but not group B (10).…”

“…Recent genetic studies have identified the gene pel/sagA that is required for ␤-hemolysis in Streptococcus pyogenes (7)(8)(9). This gene is present in group A, C, and G but not group B streptococci (10), in which a distinct gene has been shown to encode the hemolysin (11,12).…”

Complete genome sequence of an M1 strain of Streptococcus pyogenes

“…The first is salivaricin A, a bacteriocin originally described in S. salivarius and that is present in 90% of S. pyogenes strains (36). The second is streptolysin S, a pore-forming hemolysin that has escaped identification for over 40 years (37). The genetic organizations of both of these operons resemble that of the lantibiotic nisin produced by L. lactis (38) and the cytolysin of Enterococcus faecalis (39).…”

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection

“…Integrational mutagenesis of ska and emm1 was performed essentially as previously described 20 . Internal fragments of the genes ska and emm1 were PCR amplified from GAS strain 5448 using specific primer pairs (Supplementary Table 2) and cloned by BamHI/XbaI digestion and T4 ligation into the temperature-sensitive plasmid pVE6007 21 .…”