Grant: Discovery Grants Program - Individual

Mechanism of Pseudomonas aeruginosa infection remains incompletely defined. We showed that Toll-like receptor (TLR) signaling pathways (the MyD88-IRAK pathway and the TRIF-IRF3/7 pathway) are activated by P. aeruginosa. Activation signals are subsequently inhibited by the negative signals that allow host cells to return to the basal resting condition. Thus, TLR-mediated activation and subsequent inhibition are highly ordered, sequential molecular events. However, little is known on how activated TLR signals are down-regulated in P. aeruginosa infection.******Irreversible protein degradation and reversible phosphorylation are two fundamental processes in controlling signal transduction. Autophagy is an intracellular degradation process. Many of the signaling components in the TLR signaling pathways are regulated by protein degradation. Thus, it is possible that P. aeruginosa-triggered autophagy serves as a negative regulator to down regulate TLR signaling. A role of protein phosphatase in P. aeruginosa infection is not known. We found that PTP1B deficiency led to enhanced TLR signaling in P. aeruginosa infection. Here, we propose to examine two possible mechanisms in the down-regulation of TLR signaling, the autophagy-mediated protein degradation and protein tyrosine phosphatase 1B (PTP1B)-mediated protein dephosphorylation. ******1. To examine whether autophagy modulates the MyD88-IRAK4 and TRIF-IRF3/7 pathways in response to P. aeruginosa infection. Although, a role of TLRs and TLR signaling molecules MyD88 and TRIF have been associated in triggering autophagy, little is known whether autophagy serves as a feedback regulatory mechanism in controlling TLR signaling. To test this hypothesis, autophagy will be up-regulated by various stimuli or down-regulated by chemical inhibitors or genetic approaches. Activation of TLR signaling pathways and degradation of various components of the MyD88 and TRIF pathways will be determined. Association of TLR signaling molecule with autophagy markers will be examined. Activation or degradation of TLR pathways and association with autophagy markers will be compared between cells treated with autophagy promoters or inhibitors and cells without treatment. ***2. To examine a role of PTP1B in the regulation of P. aeruginosa-induced activation of the MyD88-IRAK4 and TRIF-IRF3/7 pathways. PTP1B-deficient macrophages and mast cells and wild-type controls will be stimulated by P. aeruginosa infection. Activation of the MyD88-IRAK4 and TRIF-IRF3/7 pathways will be examined by various biochemical assays and compared between PTP1B-deficient and wild-type cells. Direct physical interaction between PTP1B and components of TLR signaling pathway will be examined by immunoprecipitation assay. *** These studies will provide novel information on the regulation of TLR signaling in P. aeruginosa infection. ***********