Anthrax Toxins Impair Phagocyte Actin-based Motility

描述(申请人提供)：中性粒细胞和巨噬细胞经常被系统性炭疽感染淹没。炭疽毒素、保护性抗原(PA)、致死因子(LF)和水肿因子(EF)能够进入吞噬细胞的细胞质，损害吞噬细胞的功能。先天免疫力的丧失会使芽孢杆菌在宿主体内肆无忌惮地生长，导致迅速死亡。我们发现，PA LF(50 ng/ml)，称为致死毒素(LT)，显著损害人中性粒细胞的趋化和趋化诱导的肌动蛋白组装。通过这种方式，炭疽毒素可以通过阻断吞噬细胞肌动蛋白的运动性来削弱先天免疫力。我们建议：1.探索炭疽毒素对吞噬细胞肌动蛋白运动过程的影响。通过Alexa-Palloidin染色和FACS分析比较LT对甲酰-甲基-亮氨酰-苯丙氨酸(FMLP)、血小板激活因子(PAF)和免疫复合物诱导中性粒细胞肌动蛋白组装的影响。检查PA EF和PA LF EF对中性粒细胞趋化、吞噬和肌动蛋白组装的影响。研究炭疽毒素阻断单核细胞、巨噬细胞和树突状肌动蛋白运动的能力。探索炭疽毒素损害血小板肌动蛋白组装和扩散的能力。2.确定炭疽毒素如何直接或间接改变一种或多种特定收缩蛋白的功能。A.比较LT的蛋白质组特征，并识别毒素处理细胞中上调和下调的蛋白质。B.使用PH-GFP构建探索LT对PI-3激酶信号的影响，使用结合活性RAC的GFP探针检测RAC信号，并使用特定抗体和抑制剂研究p38MAPK信号。C.使用罗丹明肌动蛋白和延时视频显微镜，检测LT对感染细胞中李斯特氏菌和志贺氏菌肌动蛋白以及细胞质和重组提取物的运动性的影响。检测LT对肌动蛋白调节蛋白Hsp27体外功能的影响。它阻止肌动蛋白调节蛋白Hsp27的磷酸化。因此，我们将通过分析纯化的重组野生型和突变体Hsp27对芘结合肌动蛋白的组装和拆解的影响来比较磷酸化和去磷酸化Hsp27的功能。这些研究有望为炭疽毒素介导免疫系统瘫痪的机制提供新的见解，并可能为系统性炭疽感染的诊断和治疗提供新的策略。

DESCRIPTION (provided by applicant): Neutrophils and macrophages are often overwhelmed by systemic anthrax infection. The anthrax toxins, protective antigen (PA) combined with lethal factor (LF) and edema factor (EF) are able to enter the cytoplasm of phagocytic cells and impair their function. Loss of innate immunity allows the bacillus to grow unchecked within the host, leading to rapid death. We have found that PA + LF (50 ng/ml), called lethal toxin (LT), markedly impairs human neutrophil chemotaxis and chemoattractant-induced actin assembly. In this way anthrax toxins can weaken innate immunity by blocking phagocyte actin-based motility. We propose to: 1. Explore anthrax toxins effects on phagocyte actin-based motile processes. A. Compare the effects of LT on formyl-methionly-leucyl-phenylalanine (FMLP), platelet activating factor (PAF) and IgG-immune complex induction of neutrophil actin assembly using Alexa-phalloidin staining and FACs analysis. B. Examine the effects of PA + EF, and PA + LF + EF on neutrophil chemotaxis, phagocytosis, and actin assembly. C. Study the ability of anthrax toxins to block monocyte, macrophage and dendritic actin-based motility. D. Explore anthrax toxins ability to impair platelet actin assembly and spreading. 2. Determine how anthrax toxins directly or indirectly alter the function of one or more specific contractile proteins. A. Compare the proteomic signatures of LT and identify the up-regulated and down-regulated proteins in toxin-treated cells. B. Explore the effects of LT on PI-3 kinase signaling using PH-GFP constructs, examine Rac signaling using a GFP- probe that binds active Rac, and investigate p38 MAPK signaling using specific antibodies and inhibitors. C. Examine the effects of LT on Listeria and Shigella actin-based motility in infected cells, as well as cytoplasmic and reconstituted extracts, using rhodamine actin and time lapse video microscopy. D. Examine LT's effects on the in vitro function of the actin-regulatory protein Hsp27. LT blocks phosphorylation of the actin-regulatory protein Hsp27. Therefore, we will compare the function of phosphorylated to dephosphorylated Hsp27 by assaying the effects of purified recombinant wild-type and mutant, Hsp27 on the assembly and disassembly of pyrene-conjugated actin. These investigations promise to provide new insights into the mechanisms by which anthrax toxins mediate paralysis of the immune system, and are likely to provide new strategies for the diagnosis and treatment of systemic anthrax infections.