title: Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family.
abstract: 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 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.
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. emolloy@illinois.edu.
authors.forename: Evelyn M
authors.initials: EM
authors.lastname: Molloy
authors.affiliation: Division of Microbiology and Immunology, Department of Pathology, University of Utah Medical School, Salt Lake City, UT, 84112, USA. sherwood.casjens@path.utah.edu.
authors.forename: Sherwood R
authors.initials: SR
authors.lastname: Casjens
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. cox22@illinois.edu.|Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. cox22@illinois.edu.
authors.forename: Courtney L
authors.initials: CL
authors.lastname: Cox
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. maxson2@illinois.edu.
authors.forename: Tucker
authors.initials: T
authors.lastname: Maxson
authors.affiliation: School of Molecular and Cellular Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. nethri2@gmail.com.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Ethridge
authors.affiliation: Bavarian Health and Food Safety Authority, National Reference Centre for Borrelia, Oberschleissheim, Germany. gabriele.margos@lgl.bayern.de.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Margos
authors.affiliation: Bavarian Health and Food Safety Authority, National Reference Centre for Borrelia, Oberschleissheim, Germany. volker.fingerle@lgl.bayern.de.
authors.forename: Volker
authors.initials: V
authors.lastname: Fingerle
authors.affiliation: Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. douglasm@illinois.edu.|Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. douglasm@illinois.edu.|Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA. douglasm@illinois.edu.
authors.forename: Douglas A
authors.initials: DA
authors.lastname: Mitchell
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000633:toxicity*; D016000:Cluster Analysis; D004252:DNA Mutational Analysis; D006461:Hemolysis; D006801:Humans; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D013297:Streptococcus pyogenes / Q000378:metabolism; D013301:Streptolysins / Q000235:genetics* / Q000633:toxicity*
chemical_list: D001426:Bacterial Proteins; D013301:Streptolysins; C026050:streptolysin S