title: Identification of a collagen type I adhesin of Bacteroides fragilis.
abstract: Bacteroides fragilis is an opportunistic pathogen which can cause life threatening infections in humans and animals. The ability to adhere to components of the extracellular matrix, including collagen, is related to bacterial host colonisation. Collagen Far Western analysis of the B. fragilis outer membrane protein (OMP) fraction revealed the presence two collagen adhesin bands of ∼ 31 and ∼ 34 kDa. The collagen adhesins in the OMP fraction were separated and isolated by two-dimensional SDS-PAGE and also purified by collagen affinity chromatography. The collagen binding proteins isolated by both these independent methods were subjected to tandem mass spectroscopy for peptide identification and matched to a single hypothetical protein encoded by B. fragilis NCTC 9343 (BF0586), conserved in YCH46 (BF0662) and 638R (BF0633) and which is designated in this study as cbp1 (collagen binding protein). Functionality of the protein was confirmed by targeted insertional mutagenesis of the cbp1 gene in B. fragilis GSH18 which resulted in the specific loss of both the ∼ 31 kDa and the ∼ 34 kDa adhesin bands. Purified his-tagged Cbp1, expressed in a B. fragilis wild-type and a glycosylation deficient mutant, confirmed that the cbp1 gene encoded the observed collagen adhesin, and showed that the 34 kDa band represents a glycosylated version of the ∼ 31 kDa protein. Glycosylation did not appear to be required for binding collagen. This study is the first to report the presence of collagen type I adhesin proteins in B. fragilis and to functionally identify a gene encoding a collagen binding protein.
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Cape Town, RSA.
authors.forename: Bruna P G V
authors.initials: BP
authors.lastname: Galvão
authors.affiliation: Structural Biology Research Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, Western Cape, South Africa.
authors.forename: Brandon W
authors.initials: BW
authors.lastname: Weber
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Cape Town, RSA.
authors.forename: Mohamed S
authors.initials: MS
authors.lastname: Rafudeen
authors.affiliation: Departamento de Microbiologia Médica, UFRJ, Instituto de Microbiologia Prof. Paulo de Góes, Ilha do Fundão, Rio de Janeiro, Brazil; Universidade Federal do Rio de Janeiro - Polo Xerém, Duque de Caxias, Rio de Janeiro, Brazil.
authors.forename: Eliane O
authors.initials: EO
authors.lastname: Ferreira
authors.affiliation: Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, United Kingdom.
authors.forename: Sheila
authors.initials: S
authors.lastname: Patrick
authors.affiliation: Department of Molecular and Cell Biology, University of Cape Town, Cape Town, RSA.
authors.forename: Valerie R
authors.initials: VR
authors.lastname: Abratt
mesh_terms: D018829:Adhesins, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism; D001441:Bacteroides fragilis / Q000235:genetics / Q000378:metabolism*; D024042:Collagen Type I; D006031:Glycosylation; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D024042:Collagen Type I; D011993:Recombinant Fusion Proteins