Streptolysin S-like virulence factors: the continuing sagA

Abstract

Streptolysin S (SLS) is a potent cytolytic toxin and virulence factor that is produced by nearly all Streptococcus pyogenes strains. Despite a 100-year history of research on this toxin, it has only recently been established that SLS is just one of an extended family of post-translationally modified virulence factors (the SLS-like peptides) that are produced by some streptococci and other Gram-positive pathogens, such as Listeria monocytogenes and Clostridium botulinum. In this Review, we describe the identification, genetics, biochemistry and various functions of SLS. We also discuss the shared features of the virulence-associated SLS-like peptides, as well as their place within the rapidly expanding family of thiazole/oxazole-modified microcins (TOMMs).

Figure 1. Overview of the production, processing and export of SLS and microcin B17

The C-terminal core peptide of SagA (depicted in red; A), produced by GAS, and McbA (depicted in orange; B), produced by E. coli, serve as structural templates that undergo a series of post-translational tailoring reactions by the SagBCD and McbBCD biosynthetic complexes to form biologically active SLS (cytotoxin) and microcin B17 (DNA gyrase inhibitor), respectively. The N-terminal leader (depicted in black) is cleaved from the mature core peptide following modification, resulting in a mature peptide product. Also shown is the ‘leaky’ rho-independent terminator sequence between sagA and sagB, and mcbA and mcbB, that acts as a regulatory mechanism yielding a stoichiometric excess of structural gene transcripts versus a substoichiometric, catalytic amount of transcripts for the modification and transport machinery.

Figure 2. Post-translational modification of SagA via the combined activity of a cyclodehydratase (SagC) and dehydrogenase (SagB) from within a three protein complex (SagBCD)

SLS heterocycles are formed via two distinct steps: SagC, a zinc-tetrathiolate containing cyclodehydratase, removes water from cysteine, serine, and threonine residues in the peptide backbone to generate thiazoline and (methyl)-oxazoline rings. Subsequently, a flavin mononucleotide-dependent dehydrogenation reaction catalysed by SagB removes hydrogen to generate the aromatic thiazole and (methyl)-oxazole heterocycles. An example of each heterocyclizable residue is shown for illustrative purposes, with glycine included as a typical residue that is found N-terminal to cyclized residues to facilitate the orbital alignment required for cyclodehydration ^,,-.

Figure 3. Summary of the functions of SLS

The mechanisms via which SLS contributes to the virulence of GAS and other functions.

Figure 4. Top Panel: Amino acid sequence of the unmodified precursors of Staphylysin S (StsA), Listeriolysin S (LlsA), Streptolysin S (SagA) and Clostridolysin (BtsA)

The (predicted) leader regions are to the left and terminate in (putative) Gly-Gly/Ala-Gly leader cleavage sites (blue). Residues potentially involved in modification of the precursor peptides are indicated in red (cysteine), orange (serine), green (threonine) and blue (glycine). Based on the sequence of sagA and assuming cleavage after the Gly-Gly site, the molecular weight of modified SagA is estimated to be approximately 2.7 kDa. (although this does not preclude the possibility that mature SLS is an assemblage of modified SagA peptides). Bottom Panel: SLS-like gene clusters in S. aureus, L. monocytogenes, S. pyogenes and C. botulinum are depicted. Related genes are indicated by colour. A: structural gene; B: dehydrogenase; C: cyclodehydratase; D: “docking” protein; E/P: CaaX protease; G, H, I: ABC-type transporter components; Z, X, F: proteins of unknown function. In the case of RF122, the genomic map appears to be fragmented, giving multiple ORFs for several biosynthetic proteins.