Molecular Characterization of the Multiple Interactions of SpsD, a Surface Protein from Staphylococcus pseudintermedius, with Host Extracellular Matrix Proteins

Abstract

Staphylococcus pseudintermedius, a commensal and pathogen of dogs and occasionally of humans, expresses surface proteins potentially involved in host colonization and pathogenesis. Here, we describe the cloning and characterization of SpsD, a surface protein of S. pseudintermedius reported as interacting with extracellular matrix proteins and corneocytes. A ligand screen and Western immunoblotting revealed that the N-terminal A domain of SpsD bound fibrinogen, fibronectin, elastin and cytokeratin 10. SpsD also interfered with thrombin-induced fibrinogen coagulation and blocked ADP-induced platelet aggregation. The binding site for SpsD was mapped to residues 395–411 in the fibrinogen γ-chain, while binding sites in fibronectin were localized to the N- and C-terminal regions. SpsD also bound to glycine- and serine-rich omega loops within the C-terminal tail region of cytokeratin 10. Ligand binding studies using SpsD variants lacking the C-terminal segment or containing an amino-acid substitution in the putative ligand binding site provided insights into interaction mechanism of SpsD with the different ligands. Together these data demonstrate the multi-ligand binding properties of SpsD and illustrate some interesting differences in the variety of ligands bound by SpsD and related proteins from S. aureus.

Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius

“…The rabbit polyclonal antibody against the ␣ 5 chain of the ␣ 5 ␤ 1 integrin and the mouse monoclonal antibodies BV7 against the human ␤ 1 chain and B212 against the human ␤ 3 chain of the integrin ␣ v ␤ 3 were a generous gift from G. Tarone (University of Turin, Italy). Mouse polyclonal antibodies against region A of SpsD and SpsL were prepared as previously reported (16).…”

“…SpsD and SpsL mediate bacterial adherence to fibrinogen (15) and fibronectin (Fn) (15), while SpsD also binds to cytokeratin 10 and elastin (16). The binding site in fibrinogen for SpsD was mapped to residues 395 to 411 in the ␥-chain, while a binding site for SpsD in Fn was localized to the N-terminal region.…”

The Staphylococcal Biofilm: Adhesins, Regulation, and Host Response

“…One example is S. pseudintermedius, a commensal of dogs that is implicated in canine opportunistic infection (309). Biofinformatic and proteomic analyses have been employed to study the cell wall adhesins of S. pseudintermedius , leading to further study of two proteins (SpsD and SpsL) to which canines have antibodies, indicating their expression in vivo (310, 311). …”

Fibronectin and Its Role in Human Infective Diseases

“…SpsD (S. pseudintermedius surface protein D) and SpsL (S. pseudintermedius surface protein L) are examples of MSCRAMMs that mediate the binding of S. pseudintermedius to fibrinogen and Fn. Domain binding to fibrinogen is located to the N-terminal region in both the proteins [51,52], while the Fn-binding is mediated by a segment that maps to the C-terminus of the proteins ( Figure 2B,C) [53]. Both SpsD and SpsL play an important role in the internalization of S. pseudintermedius by host cells and, as reported for FnBPA, the process involves Fn and integrin α 5 β 1 [53].…”