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

铜绿假单胞菌感染机制尚不完全清楚。我们发现，铜绿假单胞菌激活了Toll样受体(TLR)信号通路(MyD88-IRAK通路和TRIF-IRF3/7通路)。随后，激活信号被允许宿主细胞返回到基础休眠状态的负信号所抑制。因此，TLR介导的激活和随后的抑制是高度有序的、连续的分子事件。然而，关于激活的TLR信号在铜绿假单胞菌感染中是如何下调的，人们知之甚少。 不可逆的蛋白质降解和可逆的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成分都受蛋白质降解的调节。因此，铜绿假单胞菌触发的自噬可能作为下调TLR信号的负调控因子。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PTP1B缺乏导致了铜绿假单胞菌感染中TLR信号的增强。在这里，我们建议研究TLR信号下调的两种可能机制，自噬介导的蛋白质降解和蛋白酪氨酸磷酸酶1B(PTP1B)介导的蛋白质去磷酸化。 1.检测自噬是否调节MyD88-IRAK4和TRIF-IRF3/7通路对铜绿假单胞菌感染的反应。尽管TLRs和TLR信号分子MyD88和TRIF在触发自噬中起到了一定的作用，但自噬是否在控制TLR信号中起到反馈调节机制的作用还知之甚少。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。TLR信号通路的激活以及MyD88和TRIF通路的各种成分的降解将被确定。TLR信号分子与自噬标记物的关联将被检测。TLR通路的激活或降解以及与自噬标志物的关联将在自噬促进剂或抑制剂处理的细胞和未处理的细胞之间进行比较。 2.研究PTP1B在铜绿假单胞菌诱导的MyD88-IRAK4和TRIF-IRF3/7通路激活中的作用。PTP1B缺陷的巨噬细胞和肥大细胞以及野生型对照会受到铜绿假单胞菌感染的刺激。MyD88-IRAK4和TRIF-IRF3/7通路的激活将通过各种生化检测来检测，并在PTP1B缺陷细胞和野生型细胞之间进行比较。免疫沉淀法将检测PTP1B与TLR信号通路组件之间的直接物理相互作用。 这些研究将为TLR信号在铜绿假单胞菌感染中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.