Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family

Abstract

BackgroundStreptolysin S (SLS) is a cytolytic virulence factor produced by the human pathogen Streptococcus pyogenes and other Streptococcus species. Related “SLS-like” toxins have been characterized in select strains of Clostridium and Listeria, with homologous clusters bioinformatically identified in a variety of other species. SLS is a member of the thiazole/oxazole-modified microcin (TOMM) family of natural products. The structure of SLS has yet to be deciphered and many questions remain regarding its structure-activity relationships.ResultsIn this work, we assessed the hemolytic activity of a series of C-terminally truncated SLS peptides expressed in SLS-deficient S. pyogenes. Our data indicate that while the N-terminal poly-heterocyclizable (NPH) region of SLS substantially contributes to its bioactivity, the variable C-terminal region of the toxin is largely dispensable. Through genome mining we identified additional SLS-like clusters in diverse Firmicutes, Spirochaetes and Actinobacteria. Among the Spirochaete clusters, naturally truncated SLS-like precursors were found in the genomes of three Lyme disease-causing Borrelia burgdorferi sensu lato (Bbsl) strains. Although unable to restore hemolysis in SLS-deficient S. pyogenes, a Bbsl SLS-like precursor peptide was converted to a cytolysin using purified SLS biosynthetic enzymes. A PCR-based screen demonstrated that SLS-like clusters are substantially more prevalent in Bbsl than inferred from publicly available genome sequences.ConclusionsThe mutagenesis data described herein indicate that the minimal cytolytic unit of SLS encompasses the NPH region of the core peptide. Interestingly, this region is found in all characterized TOMM cytolysins, as well as the novel putative TOMM cytolysins we discovered. We propose that this conserved region represents the defining feature of the SLS-like TOMM family. We demonstrate the cytolytic potential of a Bbsl SLS-like precursor peptide, which has a core region of similar length to the SLS minimal cytolytic unit, when modified with purified SLS biosynthetic enzymes. As such, we speculate that some Borrelia have the potential to produce a TOMM cytolysin, although the biological significance of this finding remains to be determined. In addition to providing new insight into the structure-activity relationships of SLS, this study greatly expands the cytolysin group of TOMMs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0464-y) contains supplementary material, which is available to authorized users.

Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic CellsIn Vivo

“…SLS is a potent membrane-damaging agent and a major virulence factor contributing to GAS infection through rapid destruction of eukaryotic cells and tissue damage (6, 9–11, 15, 18–20). It has been proposed that SLS-like virulence factors from other Gram-positive pathogens also behave as potent cytotoxins (6, 14). Interestingly, functional experimental data of LLS activity on eukaryotic cells are scarce (3), despite the fact that LLS is almost exclusively detected within lineage I strains (the most frequent lineage among L. monocytogenes clinical isolates) and that it has been related to the L. monocytogenes infectious potential in epidemiological and comparative genomic studies (21, 22).…”

“…It has been suggested that given the genetic similarities of the TOMM operons in Gram-positive pathogens, it is likely that these TOMMs contribute to the pathogenic potential of each bacterial producer, similarly to SLS for S. pyogenes (6, 14). However, it is currently unknown if all SLS-like molecules of bacterial pathogens are cytotoxins, which play a role in tissue injury and contribute to virulence by targeting eukaryotic cells.…”

Heterogeneity of Streptococcus anginosus ß‐hemolysis in relation to CRISPR/Cas

“…We designated this constellation as sag genotype I and the gene cluster carrying a duplication of the sagA genes as genotype II. To the best of our knowledge, the sag genotype II is unique to S. anginosus and has so far not been found in other streptococcal species or in any of the Gram-positive bacteria such as listeria that harbor a SLS homologous gene cluster (Molloy et al, 2015).…”

“…We designated this constellation as sag genotype I and the gene cluster carrying a duplication of the sagA genes as genotype II. To the best of our knowledge, the sag genotype II is unique to S. anginosus and has so far not been found in other streptococcal species or in any of the Gram‐positive bacteria such as listeria that harbor a SLS homologous gene cluster (Molloy et al, ). Nevertheless, in the ß‐hemolytic strains we analyzed, genotype I was more prevalent, matching the SLS gene set up typically found in other streptococci that have thus far been shown to harbor SLS genes (Molloy et al, ).…”

Bacteriocins of Listeria monocytogenes and Their Potential as a Virulence Factor

“…LLS is a bacteriocin that possesses post-translationally modified amino acids and belongs to a subgroup of the family of thiazole/oxazole-modified microcins (TOMMs), which have been identified in various Gram-positive pathogens including Streptococcus pyogenes (SLS), Staphylococcus aureus (staphylolysin S; STS), and Clostridium botulinum (botulysin S or clostridiolysin S; BTS) [22][23][24][25][26][27][28]. Among these related bacteriocins, LLS displays the highest similarity to STS in the organization of the gene cluster and amino acid sequence of the encoded proteins, whereas SLS more closely corresponds to BTS [22][23][24][25]29].…”