Title

Quantification of Staphylococcus epidermidis specific adhesion to ligand-modified substrata controlling fibronectin orientation using immobilized antibodies

Date of Completion

January 2005

Keywords

Biology, Microbiology|Engineering, Biomedical|Health Sciences, Immunology

Degree

Ph.D.

Abstract

Specific bacterial adhesion to implanted medical devices occurs through receptor:ligand interactions with adsorbed proteins (e.g., fibronectin and fibrinogen), a critical first step in a cascade of processes leading to biofilm formation, virulence and infection, and potentially death. Several bacterial species, including Staphylococcus aureus and Staphylococcus epidermidis (SE) are known to express cell receptors that bind specifically to surface immobilized or extracellular matrix ligands, such as the protein fibronectin (FN). Yet, few existing studies have definitively examined the effect of protein surface orientation on bacterial adhesion. We report here a substratum modification protocol that controls the specific orientation of FN molecules on a surface, at known levels of surface coverage. Monoclonal antibodies (Mabs), specific to either the COOH-terminus or NH3-terminus of FN, were either immobilized to polystyrene by adsorption or were conjugated to biotin then immobilized to streptavidin-coated glass substrata. Specific orientation of the bound FN molecules is verified using the same Mabs in an ELISA. Modified glass surfaces were further characterized by XPS and AFM. Bacterial adhesion of Staphylococcus epidermidis (SE) to FN bound by either its C-terminus or its NH3-terminus was quantified in batch static adhesion assays. Results indicate an increase in SE adhesion to FN-coated surfaces when the FN is bound by its C-terminus (NH3-terminus free), indicating SE receptor specific adhesion to the FN NH3-terminus. These studies demonstrate that anti-fibronectin monoclonal antibodies can be used to specifically bind and orient fibronectin on a surface. In addition, adhesion of SE to these model substrata can be controlled by the orientation of an adhesion ligand. ^