title: Characterization of the collagen-binding S-layer protein CbsA of Lactobacillus crispatus.
abstract: The cbsA gene of Lactobacillus crispatus strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The cbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other Lactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsA where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.
authors.affiliation: Division of General Microbiology, Department of Biosciences, FIN-00014 University of Helsinki, Finland.
authors.forename: J
authors.initials: J
authors.lastname: Sillanpää
authors.forename: B
authors.initials: B
authors.lastname: Martínez
authors.forename: J
authors.initials: J
authors.lastname: Antikainen
authors.forename: T
authors.initials: T
authors.lastname: Toba
authors.forename: N
authors.initials: N
authors.lastname: Kalkkinen
authors.forename: S
authors.initials: S
authors.lastname: Tankka
authors.forename: K
authors.initials: K
authors.lastname: Lounatmaa
authors.forename: J
authors.initials: J
authors.lastname: Keränen
authors.forename: M
authors.initials: M
authors.lastname: Höök
authors.forename: B
authors.initials: B
authors.lastname: Westerlund-Wikström
authors.forename: P H
authors.initials: PH
authors.lastname: Pouwels
authors.forename: T K
authors.initials: TK
authors.lastname: Korhonen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019677:Artificial Gene Fusion; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002645:Chickens; D003001:Cloning, Molecular; D003094:Collagen / Q000378:metabolism*; D005798:Genes, Bacterial; D007778:Lactobacillus / Q000737:chemistry / Q000235:genetics*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C055764:S-layer proteins; D003094:Collagen