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 beta-hemolysis 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.

FIG. 1

Map of the sag genetic locus required for SLS production by GAS. Loci targeted for mutagenesis are also shown.

FIG. 2

Amino acid sequence similarity between SagA and McbA of E. coli.

FIG. 3

Southern blot analysis showing the conservation of the sag locus among GAS isolates from a variety of emm (M protein) genotypes. Blot was probed with a digoxigenin-labeled PCR amplicon encompassing sagD to sagF.

FIG. 4

SLS phenotype following targeted knockouts of sag locus genes.

FIG. 5

(a) RT-PCR analysis of total RNA from NZ131. Transcript of predicted size is identified using a sagA forward primer and a sagB reverse primer, sagB forward primer and sagC reverse primer, etc. (b) RT-PCR analysis of total RNA from SLS-negative mutants NZ131:sagA.KO and NZ131:sagB.KO, demonstrating presence of transcript extending from the 3′ end of the integrative vector (T7) to the next gene in the operon.

FIG. 6

Restoration of SLS-activity to the nonhemolytic sagA allelic exchange mutant by reintroduction of the nine-gene sag operon on a plasmid vector.

FIG. 7

Plasmid pSagLocus confers beta-hemolytic phenotype to L. lactis.