Gram-negative bacterial septicemia remains an important cause of morbidity and mortality in the United States. Gram-negative sepsis begins when bacterial membranes shed endotoxin (lipopolysacchadde, LPS)and engage signaling receptors on the surface of phagocytes and other LPS-sensitive cells. Although additional receptor components may yet be discovered, the names of the basic components of the LPS response machinery are now known. These include LPS-binding protein (LBP), CD14, Toll-like receptor (TLR) 4, MD-2 and one or more """"""""Toll, interleukin 1, resistance"""""""" (TIR)-domain containing adapter proteins. The hypothesis to be tested represents the current dogma: sepsis begins with LBP-mediated presentation of LPS to CD14. CD14 presents LPS to the TLR4/MD-2 complex. LPS binding to TLR4 results in receptor dimerization and recruitment of MyD88/Mal complexes resulting in the initiation of the NF-KB and IRF signal transduction pathways. These transcription factors drive cytokine production, resulting in septicemia. Many aspects of this dogma need considerable reassessment and refinement. For example, while preliminary data confirm that LPS directly binds to MD-2, and suggest that TLR4 is also bound, TLR4/MD-2 forms large multimers- much larger than dimmers- after being bound by LPS. In addition to MyD88 and Mal/TIRAP, at least two other adapter molecules, TRAM (which we recently discovered) and TRIF, are involved in the TLR4 pathway. We propose 4 specific aims: 1) To characterize the binding of LPS to TLR4 and MD-2. 2) To determine if the TLR4/MD-2 complex activates signal transduction by creating 'signa/osome' clusters.
The aim i s meant to precisely quantify receptor size and composition of the signalosome in LPS-stimulated cells. 3) To assess the roles of the five T/R domain containing adapter molecules in TLR4 signal transduction.
This aim combines aspects of biochemistry, molecular genetics and confocal microscopy to define the role of adapters in LPS stimulation. And finally, 4) to characterize the role of TRAM, a newly discovered adapter molecule, in LPS signal transduction by generating and characterizing a mouse with a targeted deletion in TRAM. Cells from this mouse will be tested for responses to LPS and other microbial products. In addition, we will challenge this knockout animal with Salmonella to determine if and hot TRAM expression contributes to host defense. ? ?
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