Data from: Identification of the Minimal Cytolytic Unit for Streptolysin S and an Expansion of the Toxin Family

Background: Streptolysin 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. Results: In 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. Conclusions: The mutagenesis data described herein allowed us to define the minimal cytolytic unit of SLS as the NPH region. 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 is 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

Borrelia TOMM Alignment

BorB (A), borC (B) and borD (C) from Borrelia afzelii PKo (Bafz), B. spielmanii A14S (Bspi), and B. valaisiana VS116 (Bval) were aligned using ClustalW. Sites where PCR screening primers annealed are underlined. The primers used to detect borB/C/D were based on bvalB/C/D from B. valaisiana VS11

SLS like TOMM D ML Tree

A maximum-likelihood tree based on the D protein for the majority of known and putative producers of cytolytic TOMMs (as of December 2014)

TOMM D ML Tree

A maximum-likelihood tree of a representative sample of TOMMs based on the D protein from each cluster. Clades are color-coded based on the predicted class of TOMM natural product. NHLP, nitrile hydratase leader peptide; NHLP-Burk, NHLP from Burkholderia; PZN, plantazolicin; Balh, uncharacterized TOMM from Bacillus sp. Al Hakam; McB, microcin B from Gammaproteobacteria. SLS, streptolysin S; Bor TOMM, putative SLS-like cytolysin from Bbsl

MLSA Borrelia ML tree

A tree that shows the relationships of a number of Borrelia strains, including a subset of the Bbsl strains included in our PCR screen. A plus sign (+) next to the strain designation indicates that gene(s) from the Bor TOMM biosynthetic cluster (borB/borC/borD) was/were detected. WGS: whole genome sequence

MLSA Borrelia ML tree alignment

This is the alignment file for the tree that shows the relationships of a number of Borrelia strains, including a subset of the Bbsl strains included in our PCR screen. A plus sign (+) next to the strain designation indicates that gene(s) from the Bor TOMM biosynthetic cluster (borB/borC/borD) was/were detected. WGS: whole genome sequence

SLS like Precursor Alignment

Alignment using Clustal Omega [49] reveals that the SLS core region possesses a highly conserved N-terminus containing 9-10 contiguous heterocylizable residues (underlined), while the C-terminus is variable in terms of both identity of residues and length. The putative leader peptide cleavage sites are shown as carets. Residues of SagA from S. pyogenes are numbered

TOMM D Alignment

An alignment file for the maximum-likelihood tree of a representative sample of TOMMs based on the D protein from each cluster. Clades are color-coded based on the predicted class of TOMM natural product. NHLP, nitrile hydratase leader peptide; NHLP-Burk, NHLP from Burkholderia; PZN, plantazolicin; Balh, uncharacterized TOMM from Bacillus sp. Al Hakam; McB, microcin B from Gammaproteobacteria. SLS, streptolysin S; Bor TOMM, putative SLS-like cytolysin from Bbsl

SLS like TOMM D alignment

This is the alignment file for the maximum-likelihood tree based on the D protein for the majority of known and putative producers of cytolytic TOMMs (as of December 2014)