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.
Highlights
Bacteroides fragilis, a Gram negative, rod-shaped obligately anaerobic bacterium, is a commensal inhabitant of the human colon, assisting with digestion and the development of the host immune system [1]
The ability of the bacterium to interact with components of the extracellular matrix (ECM) is thought to play a major role in pathogenesis involving both adhesion to and degradation of these components during colonization and tissue invasion
We report the functional characterisation of a glycoprotein collagen adhesin from B. fragilis GSH18
Summary
Bacteroides fragilis, a Gram negative, rod-shaped obligately anaerobic bacterium, is a commensal inhabitant of the human colon, assisting with digestion and the development of the host immune system [1]. Under certain circumstances, it can become an opportunistic pathogen causing abscess formation, softtissue infections, and bacteraemia [2] Treatment of these lifethreatening infections is complicated by the emergence of antibiotic drug resistance in the clinical setting [3] and, alternative novel therapeutic targets, such as factors which enhance bacterial virulence, need to be investigated to assist in drug design. The host ECM is an intricate network of biologically active macromolecules underlying the epithelial and endothelial cells It surrounds connective tissue cells serving a structural function and participating in cellular adhesion, migration, proliferation and differentiation [4]. The ECM serves as a substrate for the attachment of microorganisms [5], a crucial first step in the infective process This attachment is mediated by microbial surface components which can recognise adhesive matrix molecules (MSCRAMMs) [5]. B. fragilis has been shown to interact with a number of these components including the glycoproteins laminin and fibronectin, and the proteoglycan fibrinogen [8,9,10,11]
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