Analysis of Ebh, a 1.1-Megadalton Cell Wall-Associated Fibronectin-Binding Protein of Staphylococcus aureus

Abstract

In order for Staphylococcus aureus to adhere to host extracellular matrix (ECM) substrates, it elicits a wide range of surface proteins. We have characterized a novel ϳ1.1-MDa protein in S. aureus, termed Ebh (for ECMbinding protein homologue), which has homology to other ECM-binding proteins. Ebh consists of several domains, including a large central region with 44 imperfect repeats of 126 amino acids. Expression analysis revealed ebh to be growth phase regulated and repressed by agr. A fragment of the central repeat region of Ebh was cloned, overexpressed, and used in ligand-binding studies to determine Ebh function. The recombinant protein was found to specifically bind human fibronectin. Ebh is produced during human infection since serum samples taken from patients with confirmed S. aureus infections were found to contain anti-Ebh antibodies. Localization studies revealed Ebh to be cell envelope associated and is proposed to form a specialized surface structure involved in cellular adhesion.Staphylococcus aureus is able to cause a wide range of different infections, such as endocarditis, arthritis, and septicemia (55). In order for S. aureus to colonize and disseminate through its host, the bacterium expresses an array of proteins which interact with molecules of the host extracellular matrix (ECM). These bacterial cell surface and extracellular proteins bind to a wide range of host proteins, such as fibronectin (Fn) (15,21,25,42), fibrinogen (Fg) (21,36,40,56), vitronectin (21), collagen (50), thrombospondin (18), bone sialoprotein (51), elastin (8), and von Willebrand factor (16), belying the ability of S. aureus to act as the etiological agent of a variety of pathologies. Fn is an adhesive glycoprotein found on the surface of mammalian cells and in serum (7). Previous studies have implicated staphylococcal Fn-binding proteins with adhesion to different cell types (1,23,37,38,47). Also, they bind Fn, which acts as an invasin, forming a bridge between S. aureus and an integrin on the surface of nonprofessional phagocytes (1,9,23,33,46,48). Most of the ligand-binding proteins that have previously been characterized are found associated with the cell wall and are known as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) (13). The specific interactions that these adhesins undergo with the ECM, coupled with the fact that they are found on the surface of the cell, means that they may be useful targets for prophylaxis or therapy, e.g., as vaccine components, as targets for passive immunotherapy, or for novel antiadhesive strategies.Recently, the genomes of S. aureus strains N315 and Mu50 have been published (31), and determination of the genomes of five additional strains has either been done or is nearing completion. These studies have revealed the presence of many uncharacterized putative surface proteins. The two largest genes, ebhA and ebhB, encode putative proteins of ca. 722 and 421 kDa, respectively. EbhA shows homology to the major adhesin of Streptococcus defectivus, Emb, a p...

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