title: Domains in the S-layer protein CbsA of Lactobacillus crispatus involved in adherence to collagens, laminin and lipoteichoic acids and in self-assembly.
abstract: The protein regions in the S-layer protein CbsA of Lactobacillus crispatus JCM 5810, needed for binding to collagens and laminin, anchoring to bacterial cell wall, as well as self-assembly, were mapped by deletion analysis of His-tagged peptides isolated from Escherichia coli and by heterologous expression on Lactobacillus casei. Mature CbsA is 410 amino acids long, and stepwise genetic truncation at both termini revealed that the region 32-271 carries the infor-mation for self-assembly of CbsA into a periodic structure. The lactobacillar S-layer proteins exhibit sequence variation in their assembly domain, but the border regions 30-34 and 269-274 in CbsA are conserved in valine-rich short sequences. Short deletions or substitutions at these regions affected the morphology of His-CbsA polymers, which varied from sheet-like to cylindrical tubular polymers, and further truncation beyond the DNA encoding residues 32 and 271 leads to a non-periodic aggregation. The self-assembly of the truncated peptides, as seen by electron microscopy, was correlated with their behaviour in a cross-linking study. The shorter peptides not forming a regular polymer were observed by the cross-linking study and mass spectrometry to form dimers, trimers and tetramers, whereas the other peptides were cross-linked to large multimers only. Binding of solubilized type I and IV collagens was observed with the His-CbsA peptides 1-274 and 31-287, but not with the smaller peptides regardless of their ability to form regular polymers. Strain JCM 5810 also adheres to immobilized laminin and, in order to analyse the possible laminin binding by CbsA, cbsA and its fragments were expressed on the surface of L. casei. Expression of the CbsA peptides 1-274, 1-287, 28-287 and 31-287 on L. casei conferred adhesiveness to both laminin and collagen immobilized on glass as well as to laminin- and collagen-containing regions in chicken colon and ileum. The C-terminal peptides 251-410 and 288-410 bound to L. crispatus JCM 5810 cells from which the S-layer had been depleted by chemical extraction, whereas no binding was seen with the His-CbsA peptides 1-250 or 1-269 or to cells with an intact S-layer. The His-CbsA peptides 251-410 and 288-410 bound to teichoic acids of several bacterial species. The results show that CbsA is an adhesive complex with an N-terminal assembly domain exhibiting affinity for pericellular tissue components and a cationic C-terminal domain binding to negatively charged cell wall components.
authors.affiliation: Department of Biosciences, University of Helsinki, Helsinki, Finland.
authors.forename: Jenni
authors.initials: J
authors.lastname: Antikainen
authors.forename: Lena
authors.initials: L
authors.lastname: Anton
authors.forename: Jouko
authors.initials: J
authors.lastname: Sillanpää
authors.forename: Timo K
authors.initials: TK
authors.lastname: Korhonen
mesh_terms: D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D017353:Gene Deletion; D007778:Lactobacillus / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007780:Lacticaseibacillus casei / Q000235:genetics / Q000378:metabolism; D007797:Laminin / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D013682:Teichoic Acids / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D007797:Laminin; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C055764:S-layer proteins; D013682:Teichoic Acids; C009900:lipoteichoic acid; D003094:Collagen