DOI: 10.5061/dryad.d4863
title: Data from: Identification of the Minimal Cytolytic Unit for Streptolysin S and an Expansion of the Toxin Family

description: 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.
description: MLSA Borrelia ML
 treeA
 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.MLST
 tree_Doug_subm.nwkSLS like TOMM D
 alignmentThis 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).SLS-Like TOMM D
 Alignment.txtBorrelia TOMM
 AlignmentBorB (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
 VS116BorreliaTOMMAlignment.docxSLS like Precursor AlignmentAlignment 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.SLSlikePrecursorAlignment.docxSLS like TOMM D ML TreeA maximum-likelihood tree based on the D protein for the majority of known and putative producers of cytolytic TOMMs (as of December 2014).SLS-like TOMM D ML Tree.nwkTOMM D AlignmentAn 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.TOMM D ML TreeA 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 alignmentThis 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.MLSABorreliaMLtree.txt