title: Roles of minor pilin subunits Spy0125 and Spy0130 in the serotype M1 Streptococcus pyogenes strain SF370.
abstract: Adhesive pili on the surface of the serotype M1 Streptococcus pyogenes strain SF370 are composed of a major backbone subunit (Spy0128) and two minor subunits (Spy0125 and Spy0130), joined covalently by a pilin polymerase (Spy0129). Previous studies using recombinant proteins showed that both minor subunits bind to human pharyngeal (Detroit) cells (A. G. Manetti et al., Mol. Microbiol. 64:968-983, 2007), suggesting both may act as pilus-presented adhesins. While confirming these binding properties, studies described here indicate that Spy0125 is the pilus-presented adhesin and that Spy0130 has a distinct role as a wall linker. Pili were localized predominantly to cell wall fractions of the wild-type S. pyogenes parent strain and a spy0125 deletion mutant. In contrast, they were found almost exclusively in culture supernatants in both spy0130 and srtA deletion mutants, indicating that the housekeeping sortase (SrtA) attaches pili to the cell wall by using Spy0130 as a linker protein. Adhesion assays with antisera specific for individual subunits showed that only anti-rSpy0125 serum inhibited adhesion of wild-type S. pyogenes to human keratinocytes and tonsil epithelium to a significant extent. Spy0125 was localized to the tip of pili, based on a combination of mutant analysis and liquid chromatography-tandem mass spectrometry analysis of purified pili. Assays comparing parent and mutant strains confirmed its role as the adhesin. Unexpectedly, apparent spontaneous cleavage of a labile, proline-rich (8 of 14 residues) sequence separating the N-terminal approximately 1/3 and C-terminal approximately 2/3 of Spy0125 leads to loss of the N-terminal region, but analysis of internal spy0125 deletion mutants confirmed that this has no significant effect on adhesion.
authors.affiliation: Institute for Cell and Molecular Biosciences and Institute for Cellular Medicine, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom.
authors.forename: Wendy D
authors.initials: WD
authors.lastname: Smith
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Pointon
authors.forename: Emily
authors.initials: E
authors.lastname: Abbot
authors.forename: Hae Joo
authors.initials: HJ
authors.lastname: Kang
authors.forename: Edward N
authors.initials: EN
authors.lastname: Baker
authors.forename: Barry H
authors.initials: BH
authors.lastname: Hirst
authors.forename: Janet A
authors.initials: JA
authors.lastname: Wilson
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Banfield
authors.forename: Michael A
authors.initials: MA
authors.lastname: Kehoe
mesh_terms: D001422:Bacterial Adhesion / Q000235:genetics / Q000502:physiology; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D036781:Fimbriae Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D013297:Streptococcus pyogenes / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D036781:Fimbriae Proteins