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

铜绿假单胞菌感染的机制尚未完全确定。我们发现toll样受体（TLR）信号通路（MyD88-IRAK通路和TRIF-IRF3/7通路）被P. aeruginosa激活。激活信号随后被负面信号抑制，从而使宿主细胞恢复到基本的静息状态。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，对于铜绿假单胞菌感染中激活的TLR信号是如何下调的，我们知之甚少。******不可逆蛋白降解和可逆磷酸化是控制信号转导的两个基本过程。自噬是一种细胞内降解过程。TLR信号通路中的许多信号成分受蛋白质降解调控。因此，P. aeruginosa引发的自噬可能作为负调节因子下调TLR信号。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缺失导致铜绿假单胞菌感染中TLR信号的增强。在此，我们提出研究TLR信号下调的两种可能机制，自噬介导的蛋白降解和蛋白酪氨酸磷酸酶1B （PTP1B）介导的蛋白去磷酸化。* * * * * * 1。研究自噬是否在铜绿假单胞菌感染时调节MyD88-IRAK4和TRIF-IRF3/7通路。虽然TLR和TLR信号分子MyD88和TRIF的作用与触发自噬有关，但自噬是否作为控制TLR信号的反馈调节机制尚不清楚。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信号通路的激活和MyD88和TRIF通路的各种成分的降解。我们将研究TLR信号分子与自噬标志物的关联。TLR通路的激活或降解以及与自噬标志物的关联将在接受自噬促进剂或抑制剂治疗的细胞和未接受治疗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通路激活调控中的作用。ptp1b缺陷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分析进行检测，并在ptp1b缺陷细胞和野生型细胞之间进行比较。PTP1B与TLR信号通路组分之间的直接物理相互作用将通过免疫沉淀法检测。***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. ***********