The Arcanobacterium pyogenes Collagen-Binding Protein, CbpA, Promotes Adhesion to Host Cells

Abstract

Arcanobacterium pyogenes is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys. A. pyogenes adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of A. pyogenes only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa A. pyogenes cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of A. pyogenes to adhere to HeLa and 3T6 cells, as a cbpA knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing cbpA on a multicopy plasmid. Furthermore, HIS-CbpA blocked A. pyogenes adhesion to HeLa or 3T6 cells in a dose-dependent manner. cbpA was only present in 48% of the A. pyogenes strains tested (n ‫؍‬ 75), and introduction of plasmid-encoded cbpA into a naturally cbpA-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9-and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of A. pyogenes, plays a role in the adhesion of this organism to host cells.

Functional and structural properties of CbpA, a collagen-binding protein from Arcanobacterium pyogenes

“…Cloning of recombinant CbpA, lacking the signal peptide (residues 28-1151), and the A domain of CbpA (residues 29-355) was performed as reported previously (Esmay et al, 2003).…”

“…Escherichia coli DH5aMCR harbouring plasmid pJGS591 (Esmay et al, 2003) was routinely grown in Luria broth or in Luria agar (Difco) containing 100 mg ampicillin ml 21 at 37 uC. Enterococcus faecium 395 cultures were grown in Brain Heart Infusion (BHI; Difco) at 37 uC.…”

“…CbpA is present in 48 % of A. pyogenes isolates; however, 100 % of turkey osteomyelitis isolates were cbpA + (Esmay et al, 2003), possibly implicating CbpA in the pathogenesis of osteomyelitis.…”

“…Economically significant disease includes liver abscessation in beef cattle, and pneumonia and arthritis in swine (Jost & Billington, 2005). A. pyogenes is armed with several factors that contribute to its pathogenesis, including a cholesterol-dependent cytolysin, pyolysin (Billington et al, 1997), several proteases (Schaufuss et al, 1989) and a number of adhesive mechanisms, including two cell-surface neuraminidases (Jost et al, 2002) and a collagen-binding protein, CbpA (Esmay et al, 2003).…”

“…In Gram-positive organisms, ECM binding is almost exclusively the property of a number of bacterial surface proteins designated microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (Foster & Höök, 1998). CbpA is a 121.9 kDa, surface-expressed protein that binds type I collagen, and is involved in mediating adherence to epithelial and fibroblast cell lines (Esmay et al, 2003). As reported for other MSCRAMMs (Foster & Höök, 1998), CbpA has a modular domain structure consisting of an Nterminal signal peptide, a non-repetitive A region, and four repeated units (B region), followed by a cell-wall anchor region, a transmembrane segment, and a short positively charged cytoplasmic tail.…”