title: Sortase A substrate specificity in GBS pilus 2a cell wall anchoring.
abstract: Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(ΔN40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(ΔN40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.
authors: Novartis Vaccines and Diagnostics, Siena, Italy.
authors: Francesca
authors: F
authors: Necchi
authors: Vincenzo
authors: V
authors: Nardi-Dei
authors: Massimiliano
authors: M
authors: Biagini
authors: Michael
authors: M
authors: Assfalg
authors: Annalisa
authors: A
authors: Nuccitelli
authors: Roberta
authors: R
authors: Cozzi
authors: Nathalie
authors: N
authors: Norais
authors: John L
authors: JL
authors: Telford
authors: C Daniela
authors: CD
authors: Rinaudo
authors: Guido
authors: G
authors: Grandi
authors: Domenico
authors: D
authors: Maione
mesh_terms: D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D002473:Cell Wall / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D056148:Chromatography, Reverse-Phase; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010458:Peptidyl Transferases / Q000378:metabolism; D021382:Protein Sorting Signals; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013292:Streptococcus agalactiae / Q000166:cytology* / Q000201:enzymology*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D050505:Mutant Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D010458:Peptidyl Transferases; D006867:Hydrolases; D003546:Cysteine Endopeptidases