Anthrax Toxins Impair Phagocyte Actin-based Motility

炭疽毒素损害吞噬细胞基于肌动蛋白的运动

基本信息

批准号：
7890545
负责人：
Frederick s Southwick
金额：
$ 23.55万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-08-01 至 2012-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7890545
关键词：
1-Phosphatidylinositol 3-Kinase Actins Admission activity Affect Affinity Agonist Anthrax disease Antigen-Antibody Complex Antigens Apoptosis Bacillus (bacterium)Bacillus anthracis Binding Biological Assay Blood Blood Platelets Breathing Bypass Cause of Death Cell division Cells Cessation of life Chemotactic Factors Chemotaxis Contractile Proteins Cytoplasm Dendritic Cells Diagnosis Edema Endocytosis Filament Growth Hela Cells Hospitals Host Defense Human Immune Immune system Immunoglobulin G Impairment In Vitro Infection Inflammation Investigation Lead Lesion Listeria Listeria monocytogenes MAP Kinase Gene MAPK14 gene MEKKs Mediating Metalloproteases Microfilaments Natural Immunity Paralysed Pathogenesis Pathway interactions Phagocytes Phagocytosis Phase Phosphatidylinositols Phosphorylation Phosphotransferases Platelet Activating Factor Play Process Protein Dephosphorylation Proteins Proteomics Pyrenes Receptor Mediated Signal Transduction Recombinants Research Personnel Rhodamine Rhodamines Role Sepsis Shigella Shigella flexneri Signal Transduction Signal Transduction Pathway Speed Staining method Stains Stream Tail Therapeutic Time Toxin Video Microscopy anthrax lethal factor anthrax toxin antibody inhibitor antigen processing base cell motility cell type edema factor genetic regulatory protein human MAP3K1 protein in vivo insight leucyl-phenylalanine macrophage methionyl-leucyl-phenylalanine migration monocyte monomer mutant neutrophil novel diagnostics pathogen programs protective effect receptor reconstitution research study response

项目摘要

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）：全身性炭疽感染通常会淹没中性粒细胞和巨噬细胞。炭疽毒素，保护性抗原（PA）与致命因子（LF）和水肿因子（EF）结合使用，能够进入吞噬细胞的细胞质并损害其功能。先天免疫的丧失使杆菌在宿主中不受限制地生长，从而迅速死亡。我们发现PA + LF（50 ng/ml），称为致命毒素（LT），显着损害了人类嗜中性粒细胞趋化性和化学吸引剂诱导的肌动蛋白组装。这样，炭疽毒素可以通过阻断基于吞噬肌动蛋白的运动性来削弱先天免疫。我们建议：1。探索炭疽毒素对基于吞噬肌动蛋白的运动过程的影响。 A.比较LT对使用Alexa-phalloidin染色和FACS分析的LT对嗜中性粒细胞肌动蛋白组装的甲酰金属二硫代 - 苯基丙氨酸（FMLP），血小板激活因子（PAF）和IgG免疫复合物诱导的影响。 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，巨噬细胞和基于树突状肌动蛋白的运动的能力。 D.探索炭疽毒素能够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的收缩蛋白的功能。答：比较LT的蛋白质组学特征，并确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，并使用特定的抗体和抑制剂研究p38 MAPK信号传导。 C.使用Rhodamine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建提取物中基于李斯特氏蛋白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，我们将通过分析纯化的重组野生型和突变体的影响，Hsp27对吡啶偶联肌动蛋白的组装和拆卸，将磷酸化与去磷酸化的Hsp27的功能进行比较。这些研究有望提供有关炭疽毒素介导免疫系统瘫痪的机制的新见解，并可能为全身性炭疽感染的诊断和治疗提供新的策略。