Mechanisms and regulation of Toll-like receptor signaling in Pseudomonas aeruginosa infection

铜绿假单胞菌感染的机制仍不完全确定。我们发现Toll样受体（TLR）信号通路（MyD 88-IRAK通路和TRIF-IRF 3/7通路）被铜绿假单胞菌激活。激活信号随后被允许宿主细胞返回到基础静息状态的负信号抑制。因此，TLR介导的激活和随后的抑制是高度有序的连续分子事件。然而，关于铜绿假单胞菌感染中激活的TLR信号如何下调，人们知之甚少。 蛋白质的不可逆降解和可逆磷酸化是控制信号转导的两个基本过程。自噬是细胞内的降解过程。TLR信号通路中的许多信号组分受蛋白质降解调节。因此，铜绿假单胞菌触发的自噬可能作为负调节剂下调TLR信号传导。蛋白磷酸酶在铜绿假单胞菌感染中的作用尚不清楚。我们发现PTP 1B缺陷导致铜绿假单胞菌感染中TLR信号增强。在这里，我们提出了两种可能的机制，在TLR信号的下调，自噬介导的蛋白质降解和蛋白酪氨酸磷酸酶1B（PTP 1B）介导的蛋白质去磷酸化。 1.检查自噬是否调节响应铜绿假单胞菌感染的MyD 88-IRAK 4和TRIF-IRF 3/7通路。虽然TLR和TLR信号分子MyD 88和TRIF的作用与触发自噬有关，但很少有人知道自噬是否作为控制TLR信号传导的反馈调节机制。为了验证这一假设，自噬将被各种刺激上调或被化学抑制剂或遗传方法下调。将确定TLR信号传导途径的激活以及MyD 88和TRIF途径的各种组分的降解。TLR信号分子与自噬标志物的关联将被检查。TLR途径的活化或降解以及与自噬标志物的关联将在用自噬促进剂或抑制剂处理的细胞与未经处理的细胞之间进行比较。 2.研究PTP 1B在铜绿假单胞菌诱导的MyD 88-IRAK 4和TRIF-IRF 3/7通路激活的调节中的作用。将通过铜绿假单胞菌感染刺激PTP 1B缺陷型巨噬细胞和肥大细胞以及野生型对照。MyD 88-IRAK 4和TRIF-IRF 3/7通路的激活将通过各种生物化学测定进行检查，并在PTP 1B缺陷型和野生型细胞之间进行比较。将通过免疫沉淀试验检查PTP 1B与TLR信号传导途径组分之间的直接物理相互作用。 这些研究将为铜绿假单胞菌感染中TLR信号的调控提供新的信息。

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.