title: Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.
abstract: Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells.
authors.affiliation: INRA, UMR1319 Micalis, F-78350 Jouy-en-Josas, France;
authors.forename: Mickael
authors.initials: M
authors.lastname: Meyrand
authors.forename: Alain
authors.initials: A
authors.lastname: Guillot
authors.forename: Mélodie
authors.initials: M
authors.lastname: Goin
authors.forename: Sylviane
authors.initials: S
authors.lastname: Furlan
authors.forename: Julija
authors.initials: J
authors.lastname: Armalyte
authors.forename: Saulius
authors.initials: S
authors.lastname: Kulakauskas
authors.forename: Naima G
authors.initials: NG
authors.lastname: Cortes-Perez
authors.forename: Ginette
authors.initials: G
authors.lastname: Thomas
authors.forename: Sophie
authors.initials: S
authors.lastname: Chat
authors.forename: Christine
authors.initials: C
authors.lastname: Péchoux
authors.forename: Vincent
authors.initials: V
authors.lastname: Dupres
authors.forename: Pascal
authors.initials: P
authors.lastname: Hols
authors.forename: Yves F
authors.initials: YF
authors.lastname: Dufrêne
authors.forename: Germain
authors.initials: G
authors.lastname: Trugnan
authors.forename: Marie-Pierre
authors.initials: MP
authors.lastname: Chapot-Chartier
mesh_terms: D018829:Adhesins, Bacterial / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases / Q000235:genetics / Q000378:metabolism; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D018938:Caco-2 Cells; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D036781:Fimbriae Proteins / Q000235:genetics* / Q000378:metabolism; D010861:Fimbriae, Bacterial / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007422:Intestines / Q000166:cytology / Q000382:microbiology; D013294:Lactococcus lactis / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D008854:Microscopy, Electron; D058977:Molecular Sequence Annotation; D008969:Molecular Sequence Data; D005810:Multigene Family; D010446:Peptide Fragments / Q000032:analysis; D010957:Plasmids; D019936:Probiotics / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000235:genetics* / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
chemical_list: D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D010446:Peptide Fragments; D020543:Proteome; C509822:sortase C; D036781:Fimbriae Proteins; D019881:Aminoacyltransferases; D014357:Trypsin; D003546:Cysteine Endopeptidases