Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Wed, 02 Mar 2016 17:00:08 Functional and structural properties of CbpA, a collagen-binding protein from Arcanobacterium pyogenes Giampiero Pietrocola, 1 Viviana Valtulina, 1 Simonetta Rindi, 1 B. Helen Jost 2 and Pietro Speziale 1 Correspondence P. Speziale pspeziale@unipv.it 1 University of Pavia, Department of Biochemistry, Viale Taramelli 3/B, 27100 Pavia, Italy 2 Department of Veterinary Science and Microbiology, The University of Arizona, Tucson, AZ 85721, USA Received 24 April 2007 Revised 26 June 2007 Accepted 26 June 2007 Arcanobacterium pyogenes, an opportunistic pathogen of economically important food animals, is the causative agent of liver abscesses in feedlot cattle, osteomyelitis in turkeys, and pneumonia and arthritis in pigs. Previous studies identified the first A. pyogenes adhesin, CbpA, a protein located on the bacterial surface which has the ability to bind collagen and promotes adhesion to the host cells. The protein has an N-terminal ligand-binding region (region A) and a C-terminal repetitive domain (region B). In this study we found that CbpA bound to almost all the collagen types tested but not to other proteins, and it displayed a propensity to interact with several collagenous peptides derived by CNBr cleavage of type I and II collagens. The K D values of CbpA for type I and II collagens and collagen peptides determined by solid-phase binding assay and intrinsic tryptophan fluorescence were in the range of 1–15 nM. It was also found that CbpA and its A region bound fibronectin, and that collagen and fibronectin interacted with distinct subsites. Anti-CbpA antibodies were effective at inhibiting both binding of isolated CbpA and bacterial adhesion to immobilized collagen, suggesting that CbpA is a functional collagen-binding adhesin. Analysis of the immunological cross-reactivity of CbpA with antibodies against other bacterial collagen-binding proteins indicated that CbpA is immunologically related to ACE from Enterococcus faecalis but not to CNA from Staphylococcus aureus or Acm from Enterococcus faecium. Far-UV and near-UV circular dichroism spectra showed that full-length CbpA and its region A are mainly composed of b-sheet with only a minor a-helical component and that both the proteins have a well-defined tertiary structure. INTRODUCTION Arcanobacterium pyogenes is a ubiquitous inhabitant of the mucous membranes of cattle, swine and many other animal species (Jost & Billington, 2005). As an opportunistic pathogen, A. pyogenes can cause a variety of suppurative diseases in animals compromised by previous microbial infection or trauma. 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 patho- genesis, including a cholesterol-dependent cytolysin, pyoly- sin (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). Adhesion to the host is an important first step in bacterial colonization and disease pathogenesis. Bacteria target a wide variety of host molecules, but a common mechanism used by a number of pathogens is binding to components of the host extracellular matrix (ECM). 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 & Ho ¨o ¨k, 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 & Ho ¨o ¨k, 1998), CbpA has a modular domain structure consisting of an N- terminal 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 Abbreviations: CB peptide, collagen fragment generated by CNBr cleavage; CD, circular dichroism; ECM, extracellular matrix; ITF, intrinsic tryptophan fluorescence; MSCRAMM, microbial surface component recognizing adhesive matrix molecules. Microbiology (2007), 153, 3380–3389 DOI 10.1099/mic.0.2007/009100-0 3380 2007/009100 G 2007 SGM Printed in Great Britain