title: Noncanonical sortase-mediated assembly of pilus type 2b in group B Streptococcus.
abstract: Group B Streptococcus (GBS) expresses 3 structurally distinct pilus types (1, 2a, and 2b) identified as important virulence factors and vaccine targets. These pili are heterotrimeric polymers, covalently assembled on the cell wall by sortase (Srt) enzymes. We investigated the pilus-2b biogenesis mechanism by using a multidisciplinary approach integrating genetic, biochemical, and structural studies to dissect the role of the 2 pilus-2b-associated Srts. We show that only 1 sortase (SrtC1-2b) is responsible for pilus protein polymerization, whereas the second one (Srt2-2b) does not act as a pilin polymerase, but similarly to the housekeeping class A Srt (SrtA), it is involved in cell-wall pilus anchoring by targeting the minor ancillary subunit. Based on its function and sequence features, Srt2-2b does not belong to class C Srts (SrtCs), nor is it a canonical member of any other known family of Srts. We also report the crystal structure of SrtC1-2b at 1.9 Å resolution. The overall fold resembles the typical structure of SrtCs except for the N-terminal lid region that appears in an open conformation displaced from the active site. Our findings reveal that GBS pilus type 2b biogenesis differs significantly from the current model of pilus assembly in gram-positive pathogens.
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Maddalena
authors.initials: M
authors.lastname: Lazzarin
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Cozzi
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Enrico
authors.initials: E
authors.lastname: Malito
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Manuele
authors.initials: M
authors.lastname: Martinelli
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Mariapina
authors.initials: M
authors.lastname: D'Onofrio
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Domenico
authors.initials: D
authors.lastname: Maione
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy.
authors.forename: Immaculada
authors.initials: I
authors.lastname: Margarit
authors.affiliation: *Novartis Vaccines and Diagnostics, GlaxoSmithKline, Siena, Italy; and Nuclear Magnetic Resonance Laboratory, Department of Biotechnology, University of Verona, Verona, Italy daniela.rinaudo@novartis.com.
authors.forename: C Daniela
authors.initials: CD
authors.lastname: Rinaudo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000201:enzymology* / Q000235:genetics; D017434:Protein Structure, Tertiary; D013292:Streptococcus agalactiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases
keywords: FRET assay; ancillary protein; backbone protein; crystal structure