Gram-negative bacterial sepsis is a medical catastrophe, claiming the lives of 35-40 percent of affected patients. There has been little progress in improving the poor outcome of sepsis, due to our inadequate understanding of the basic pathophysiology of the syndrome. Sepsis results from a cascade of life-threatening host responses beginning with the interaction of bacterial lipopolysaccharide (LPS) and the LPS-receptor complex on effector cells. CD14 appears to be the LPS-binding component of this complex. The exact components of the complex, and the mechanism of signal transduction, are uncertain. Two Toll-like receptors (TLRs), TLR2 and TLR4, function as LPS signal transducers. Expression of TLR4, but not TLR2, is obligatory for sensitive responses to LPS. TLR2 functions as a pattern recognition molecule for bacterial products (including LPS) from multiple types of bacteria. LPS-receptor specificity resides in TLR4: expression of TLR4 defines a highly specific pharmacology to lipid A and its analogs. The central hypothesis of this proposal is that LPS is recognized by a multimeric receptor consisting of CD14 and of a complex of TLRs and accessory molecules. TLR4 is the predominant LPS receptor in most cells; TLR2 may be important for sCD14/LPS recognition. Upon ligand binding, TLRs multimerize and engage signaling molecules similar or identical to those used by the IL-1 receptor. LPS antagonists may function by preventing TLR multimerization. We propose three Aims to test this hypothesis. 1. To analyze available known TLRs (including TLRs 1, 3, 5, 6 and 7) and chimeric TLR constructs as potential LPS receptor components. To identify regions of TLR4 that are necessary for specific ligand recognition. To determine if epitope-tagged TLRs multimerize, and if multimerization is inhibited by LPS antagonists. 2. To use [32P]-lipid IVa to analyze lipid A binding to TLRs, especially TLR4. 3. To identify new genes in the LPS signal transduction pathway using somatic cell mutagenesis and complementation analysis approaches. The discovery of Toll-like receptors as mediators of signal transduction for both Gram-positive and Gram-negative bacteria suggests that there are common signaling elements that can be discovered and targeted for the rational design of novel therapeutic agents for sepsis, an thus reduce the appalling morbidity and mortality associated with this disease.
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