A family of conserved toll-like receptors (TLRs) found on a variety of human cell types plays an important role in recognition of invading pathogens and in mounting an effective inflammatory immune response. Each these receptors binds a specific microbial ligand and triggers a signal transduction pathway leading to transcription of genes encoding pro-inflammatory cytokines, chemokines and variety of innate host-defense molecules . Activation of TLRs is critical for mobilization of the innate immune response, an early acting protective mechanism for the human host during many bacterial infections. Surprisingly, however, the same signaling pathways can be detrimental to the host and exacerbate the infection, causing extensive tissue destruction and bacterial dissemination by provoking excessive and damaging inflammation. This occurs in certain acute and chronic infections, including Pseudomonas aeruginosa and Burkholderia cepacia infections in cystic fibrosis as well as P. aeruginosa and B. pseudomallei infections in pneumonia and septicemia. One such TLR ligand is flagellin, a component of the bacterial flagellar motility apparatus, which is recognized by TLR5. In preliminary studies, this flagellin-TLR5 interaction was shown to be the major cause of Pseudomonas aeruginosa virulence, survival, and dissemination in a mouse pneumonia model. The overall objective of this research is to develop therapeutic agents that antagonize the pro-inflammatory signaling pathways resulting from the interaction between TLR5 and flagellin. In Phase I, a sensitive whole-cell assay will be applied to screen libraries of compounds for antagonists of flagellin/TLR5 interaction. Following confirmation of the hits from the high throughput screen, the active compounds will be characterized in secondary assays to identify those which are non-toxic and which specifically interfere with the flagellin-TLR5 signaling pathway. The most potent, specific, inhibitors will be tested in a P. aeruginosa infection model, in which lung colonization and dissemination into organs depends on the expression of flagellin by this organism. The results of this Phase I project will provide in vivo validated TLR5 antagonists for the development of therapeutics to reduce morbidity and mortality in bacterial sepsis and chronic infections. ? ? ?
