Effects of Shear on Specific Adhesion Receptor Expression and Binding in the Bact

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DESCRIPTION (provided by applicant): Bacterial specific adhesion to biological (host tissue) and synthetic substrata (e.g., biomedical devices) through receptor:ligand interactions with adsorbed molecules (e.g., blood plasma proteins, carbohydrates, and glycoconjugates) is a critical first step in a cascade of processes leading to biofilm formation; host tissue invasion, virulence and infection; and potentially death. Nosocomial infections are the fourth leading cause of death in the U.S. with >2 million cases annually (or ~10% of American hospital patients). About 60-70% of all such infections are associated with an implanted medical device causing >$4.5 billion medical costs in 2002 and ~99,000 deaths annually. One of the first steps in biofilm formation is the specific adhesion of bacterial cells via `adhesins' (or receptors) to ligand molecules present on the target surface. Prevention of this initial adherence binding could potentially abrogate biofilm formation and any subsequent infection. Development of small molecule therapies or vaccines to prevent specific adhesion to biomedical devices or, conversely, the fabrication of "lab-on-a-chip" arrays designed to promote adhesion and identify specific pathogens will require detailed information on bacterial specific binding events, under pertinent hydrodynamic conditions. Attempts to identify binding epitopes on both bacterial receptors and their immobilized ligands, in vitro, are complicated by (1) the inability to "present" the ligand in a defined and consistent orientation and (2) lack of detailed kinetics for adhesion receptor expression, as a function of cell growth and ambient hydrodynamic conditions, Based on prior work, we hypothesize that to precisely quantify bacterial specific adhesion will require (1) controlling the orientation and surface density of the binding ligand; (2) determination of the number, affinity and avidity of bacterial adhesion receptors; and (3) characterization of the binding interactions of the receptor:ligand pair - all under pertinent conditions of growth and fluid shear. Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. PUBLIC HEALTH RELEVANCE: Our ultimate goal is to develop a general protocol that will define bacterial adhesion receptor:ligand interactions, in their native states as a function of fluid shear - for both pure and mixed culture biofilms. With NIH support, we will develop this protocol using the model pure culture system of Staphylococcus epidermidis (SE) binding to immobilized fibronectin (FN). Our specific aims in this two-year project will be: 1. Quantify, as a function of prevailing fluid shear, the specific binding of SE strains via fibronectin binding receptors (FNBR) to FN immobilized in a controlled orientation and known surface density. 2. Quantify the kinetics of FNBR adhesin receptor expression on SE surfaces, as function of bacterial growth condition and prevailing fluid shear; both as planktonic and adherent cells.