Identification of a collagen type I adhesin of Bacteroides fragilis

Bruna P G V Galvão; Brandon W Weber; Mohamed S Rafudeen; Eliane O Ferreira; Sheila Patrick; Valerie R Abratt

doi:10.1371/journal.pone.0091141

Identification of a collagen type I adhesin of Bacteroides fragilis

PLoS One. 2014 Mar 11;9(3):e91141. doi: 10.1371/journal.pone.0091141. eCollection 2014.

Authors

Bruna P G V Galvão¹, Brandon W Weber², Mohamed S Rafudeen¹, Eliane O Ferreira³, Sheila Patrick⁴, Valerie R Abratt¹

Affiliations

¹ Department of Molecular and Cell Biology, University of Cape Town, Cape Town, RSA.
² Structural Biology Research Unit, Division of Medical Biochemistry, Department of Clinical Laboratory Sciences, University of Cape Town, Observatory, Western Cape, South Africa.
³ 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.
⁴ Centre for Infection and Immunity, School of Medicine, Dentistry and Biomedical Sciences, Queen's University Belfast, Belfast, United Kingdom.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adhesins, Bacterial / chemistry
Adhesins, Bacterial / genetics
Adhesins, Bacterial / metabolism*
Amino Acid Sequence
Bacterial Adhesion / genetics
Bacterial Outer Membrane Proteins / genetics
Bacterial Outer Membrane Proteins / metabolism
Bacteroides fragilis / genetics
Bacteroides fragilis / metabolism*
Collagen Type I
Glycosylation
Molecular Sequence Data
Mutation
Protein Binding
Recombinant Fusion Proteins / chemistry
Recombinant Fusion Proteins / metabolism

Substances

Adhesins, Bacterial
Bacterial Outer Membrane Proteins
Collagen Type I
Recombinant Fusion Proteins

Grants and funding

This study was supported by grants from the Medical Research Council of South Africa and the South African National Research Foundation (NRF). BPGVG acknowledges financial support from NRF Africa and Innovation Scholarship programs, University of Cape Town Research Scholarship and the Claude Leon Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.