Bacterial infections are still a major cause of morbidity and mortality, especially in immunocompromised individuals. Peptidoglycan and lipopolysaccharide can reproduce most major signs and symptoms of infections with Gram-positive and Gram-negative bacteria, yet their molecular mechanism of action on host cells is virtually unknown. This laboratory has recently discovered one dominant peptidoglycan-binding protein on mouse lymphocytes and macrophages, and demonstrated that this protein is identical with the recently discovered lipopolysaccharide receptor. The overall goals of these studies are to unravel the molecular mechanisms of action of these bacterial constituents on host cells and to develop new treatments to prevent pathologic effects of bacterial infections. As a first step towards these goals, this project will test the hypothesis that peptidoglycan and lipopolysaccharide act on host cells through one specific cell surface receptor. This project has six specific aims: (i) To isolate this peptidoglycan-lipopolysaccharide receptor protein from mouse, sheep, and cow lymphocytes and macrophages; (ii) To obtain hamster monoclonal antibodies to this receptor protein; (iii) To prove that these monoclonal antibodies indeed bind to one peptidoglycan-lipopolysaccharide receptor, by demonstrating binding of these antibodies to this receptor on cells and in an isolated form, and by showing inhibition of ligand binding by monoclonal antibodies and inhibition of monoclonal antibody binding by the receptor ligands; (iv) To obtain further evidence for the function of the peptidoglycan-lipopolysaccharide receptor protein as a cell activating receptor by demonstrating agonistic or antagonistic nature of anti-receptor monoclonal antibodies for activation of macrophages and B cells, and modulation of cell surface expression and function of this receptor by these antibodies; (v) To characterize the fine specificity of this receptor by determining the structural requirements of peptidoglycan and lipopolysaccharide for the binding to this receptor, by studying competitive inhibition of ligand binding by a series of natural and synthetic peptidoglycan and lipopolysaccharide partial structures and analogs; (vi) To obtain highly purified peptidoglycan-lipopolysaccharide receptor protein by affinity chromatography with immobilized anti-receptor monoclonal antibodies. These studies will: (i) verify the hypothesis of receptor-mediated activation of lymphocytes and macrophages by two most important bacterial cell wall constituents; (ii) provide new tools to study novel mechanisms of signal transduction in leukocyte activation by bacterial cell wall components; (iii) enable future development of reagents that can prevent or reverse septic shock and other pathologic effects of bacterial products; and (iv) result in a discovery of a potentially clinically useful marker for various leukocyte subpopulations and hematologic neoplasms.
Showing the most recent 10 out of 29 publications
