title: Evidence for the Sialylation of PilA, the PI-2a Pilus-Associated Adhesin of Streptococcus agalactiae Strain NEM316.
abstract: Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Eric
authors.initials: E
authors.lastname: Morello
authors.affiliation: Institut Pasteur, Imagopole, Ultrastructural Microscopy Platform, Paris, France.
authors.forename: Adeline
authors.initials: A
authors.lastname: Mallet
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Yoan
authors.initials: Y
authors.lastname: Konto-Ghiorghi
authors.affiliation: Institut Pasteur, Spectrométrie de Masse Structurale et Protéomique, Paris, France; INRA UMR 1319, MICALIS, Jouy-en-Josas, France.
authors.forename: Thibault
authors.initials: T
authors.lastname: Chaze
authors.affiliation: INRA UMR 1319, MICALIS, Jouy-en-Josas, France.
authors.forename: Michel-Yves
authors.initials: MY
authors.lastname: Mistou
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Giulia
authors.initials: G
authors.lastname: Oliva
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Liliana
authors.initials: L
authors.lastname: Oliveira
authors.affiliation: Université Grenoble Alpes, Institut de Biologie Structurale (IBS), Grenoble, France.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Di Guilmi
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Trieu-Cuot
authors.affiliation: Institut Pasteur, Unité des Bactéries Pathogènes à Gram positif, Paris, France; Centre National de la Recherche Scientifique (CNRS ERL 3526), Paris, France.
authors.forename: Shaynoor
authors.initials: S
authors.lastname: Dramsi
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001216:Asparagine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010861:Fimbriae, Bacterial / Q000378:metabolism / Q000648:ultrastructure; D005964:Glucosyltransferases / Q000378:metabolism; D008958:Models, Molecular; D019158:N-Acetylneuraminic Acid / Q000378:metabolism*; D030781:Organisms, Genetically Modified; D037121:Plant Lectins / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054788:Ribosome Inactivating Proteins / Q000378:metabolism; D013292:Streptococcus agalactiae / Q000235:genetics / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D037121:Plant Lectins; D021122:Protein Subunits; C050600:Sambucus nigra lectins; D036781:Fimbriae Proteins; D001216:Asparagine; D005964:Glucosyltransferases; C410788:N-glycosyltransferase, Nocardia aerocolonigenes; D054788:Ribosome Inactivating Proteins; D019158:N-Acetylneuraminic Acid