Anthrax Toxins Impair Phagocyte Actin-based Motility

炭疽毒素损害吞噬细胞肌动蛋白的运动

• 批准号: 7148643
• 负责人: Frederick s Southwick
• 金额: $ 30.19万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7148643
• 关键词: actins; anthrax toxin; clinical research; neutrophil; phagocytes; proteins; toxin

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.

描述（由申请人提供）：中性粒细胞和巨噬细胞经常被全身性炭疽感染所淹没。炭疽毒素、保护性抗原（PA）与致死因子（LF）和水肿因子（EF）结合能够进入吞噬细胞的细胞质并损害其功能。先天免疫的丧失使得芽孢杆菌在宿主体内不受控制地生长，导致快速死亡。我们发现 PA + LF (50 ng/ml)，称为致死毒素 (LT)，会显着损害人类中性粒细胞趋化性和趋化剂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来削弱先天免疫力。我们建议： 1. 探索炭疽毒素对基于吞噬细胞肌动蛋白的运动过程的影响。 A. 使用 Alexa-鬼笔环肽染色和 FAC 分析比较 LT 对甲酰基-甲硫氨酸-亮氨酰-苯丙氨酸 (FMLP)、血小板活化因子 (PAF) 和 IgG-免疫复合物诱导中性粒细胞肌动蛋白组装的影响。 B. 检查 PA + EF 和 PA + LF + EF 对中性粒细胞趋化性、吞噬作用和肌动蛋白组装的影响。 C. 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。 D. 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 的蛋白质组特征并鉴定毒素处理细胞中上调和下调的蛋白质。 B. 使用 PH-GFP 构建体探索 LT 对 PI-3 激酶信号传导的影响，使用结合活性 Rac 的 GFP 探针检查 Rac 信号传导，并使用特定抗体和抑制剂研究 p38 MAPK 信号传导。 C. 使用罗丹明肌动蛋白和延时视频显微镜检查 LT 对受感染细胞以及细胞质和重构提取物中基于李斯特菌和志贺氏菌肌动蛋白的运动的影响。 D. 检查 LT 对肌动蛋白调节蛋白 Hsp27 体外功能的影响。 LT 阻断肌动蛋白调节蛋白 Hsp27 的磷酸化。因此，我们将通过测定纯化的重组野生型和突变体 Hsp27 对芘缀合肌动蛋白组装和解组装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麻痹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策略。