This project is a strategic component of our long-range research goals, which are to identify molecular mechanisms responsible for the contribution of toxic bacterial cell wall components to septicemia, and to use this knowledge to develop efficacious treatments. The project has four specific aims designed to test our central hypothesis that PGN induces hetero-dimerization of Toll like receptor (TLR) 2 and TLR6, leading to cellular activation. The proposed role of the cluster differentiation antigen CD 14 is to enhance cellular responsiveness to PGN by immobilizing it on the cell surface through interactions with multiple CD14 receptors. Furthermore, the available data suggest that CD14 recognizes the carbohydrate moiety of PGN, whereas TLR2 and TLR6 recognize the peptide portion. To test these hypotheses, a series of partial structures of PGN will be synthesized, which will be attached to synthetic polymers and well-defined multivalent scaffold to give a range of poly- and multivalent ligands. These ligands will be tested for agonist activity in human monocytic cells using as primary read-outs production of TNFalpha protein, activation of NF-kappaB and MAP kinases, and expression of TNFalpha mRNA. To determine the ligand specificities of the receptors, parallel studies will be performed with human cells transfected with CD14, TLR2, TLR6, and combinations of the three receptors. Ligands that bind to the cells yet lack agonist activity will be evaluated for their ability to antagonize the proinflammatory effects of PGN. The binding affinities of the synthetic ligands will be determined by measuring their ability to prevent binding of radiolabeled reference compounds in a cell-based assay. Correlations between structures of individual multivalent ligands, their relative binding affinities for CD14, TLR2, and TLR6 and agonist or (partial) antagonist properties will permit identification of the ligand requirements (part structure, valency and linker) of the individual receptors, demonstrate the importance of multi-valency for high avidity binding, and establish how the different receptors cooperate to induce cell signaling. Finally, confocal microscopy and fluorescence resonance energy transfer (FRET) studies will be employed to determine whether PGN and the multivalent ligands induce clustering of the relevant receptors. The results of the proposed studies will provide a scientific foundation for the development of therapeutic strategies to increase the survival rates of patients with Gram-positive septicemia.
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