Pathophysiological Actions of the Anthrax Toxins

炭疽毒素的病理生理作用

• 批准号: 7732682
• 负责人: Stephen Leppla
• 金额: $ 89.94万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732682
• 关键词: Adenylate Cyclase; Animal Model; Animals; Anthrax Vaccines; Anthrax disease; Antigens; Bacillus anthracis; Bacteria; Binding; Calcium; Calmodulin; Cardiovascular system; Caspase-1; Cell surface; Cells; Cessation of life; Cleaved cell; Complex; Cultured Cells; Cyclic AMP; Cytolysis; Cytosol; Disease; Dose; Edema; Endocytic Vesicle; Endocytosis; Endopeptidases; Event; Heat-Shock Response; Hemorrhage; Imaging Techniques; Inbred F344 Rats; Infection; Injection of therapeutic agent; Kinetics; Lead; Mammalian Cell; Membrane Microdomains; Metalloproteases; Mitogen-Activated Protein Kinase Kinases; Modeling; Molecular Mechanisms of Action; Monitor; Monoclonal Antibodies; Mus; Pathway interactions; Peptide Hydrolases; Play; Population; Proteins; Pulmonary Edema; Rattus; Role; Signal Transduction; Surface; System; Testing; Therapeutic; Toxin; Virulence; Work; anthrax lethal factor; anthrax toxin; edema factor; experience; macrophage; multicatalytic endopeptidase complex; novel vaccines; receptor; response; uptake

Anthrax toxin protective antigen protein (PA, 83 kDa) binds to receptors on the surface of mammalian cells, is cleaved by the cell surface protease furin, and then captures either of the two other toxin proteins, lethal factor (LF, 90 kDa) or edema factor (EF, 89 kDa). The PA-LF and PA-EF complexes enter cells by endocytosis via lipid rafts and pass through endocytic vesicle populations, and then translocate LF and EF to the cytosol. EF is a calcium and calmodulin-dependent adenylyl cyclase that causes large and unregulated increases in intracellular cAMP concentrations. LF is a metalloprotease that cleaves several mitogen-activated protein kinase kinases (MEKs). The toxin proteins play a central role in the virulence of Bacillus anthracis and the PA protein is the key ingredient in anthrax vaccines. The toxins cause profound effects on cultured cells and in experimental animals. The mechanism underlying the rapid lysis of mouse macrophages by anthrax lethal toxin was shown by others to require the newly described inflammasome and activation of caspase-1. By detailed kinetic and pharmacological studies, we showed that these two factors are late events in the cascade of signaling events that lead to lysis. The proteasome, which we previously showed is also required for macrophage lysis, was shown in new studies to be required upstream of the inflammasome. In other studies, we showed that the heat shock response of cells protects cells from anthrax lethal toxin, and its does so by blocking the inflammasome-dependent caspase-1 activation. Finally, we also showed that the requirement for proteasome activity in macrophage lysis works through the N-end rule, suggesting that an unknown substrate having a destabilizing residue at the N-terminus must be a component of the pathway leading to lysis. Additional studies have been directed to understanding the response of rats and mice to lethal toxin injection. The death of Fischer rats in as little as 38 minutes following lethal toxin injection points to a unique pathological mechanism. With several collaborating groups, we showed that rats exposed to lethal toxin experience pulmonary edema and hemorrhage. Specific effects on the cardiovascular system were observed by continuous monitoring and imaging techniques. This rat model was also used to evaluate and document the efficacy of a monoclonal antibody developed by a commercial entity. The long-standing question of the interaction of the anthrax edema and lethal toxins was explored in DBA/2J mice. It was shown that pretreatment with small doses of edema toxin sensitizes the mice to a dose of lethal toxin given immediately or after some delay. A cooperative action of the two toxins may help to explain why the bacterium has these two toxins which share a common mechanisms of uptake.

炭疽毒素保护性抗原蛋白（PA，83 kDa）与哺乳动物细胞表面的受体结合，被细胞表面蛋白酶弗林蛋白酶切割，然后捕获其他两种毒素蛋白，致死因子（LF，90 kDa）或水肿因子（EF，89 kDa）。 PA-LF 和 PA-EF 复合物通过脂筏通过内吞作用进入细胞，并穿过内吞囊泡群，然后将 LF 和 EF 易位至胞质溶胶。 EF 是一种钙和钙调蛋白依赖性腺苷酸环化酶，可导致细胞内 cAMP 浓度大幅且不受调节地增加。 LF 是一种金属蛋白酶，可裂解多种丝裂原激活蛋白激酶激酶 (MEK)。毒素蛋白在炭疽杆菌的毒力中起着核心作用，PA蛋白是炭疽疫苗的关键成分。 这些毒素对培养细胞和实验动物造成深远影响。 其他人表明，炭疽致死毒素快速裂解小鼠巨噬细胞的机制需要新描述的炎症小体和 caspase-1 的激活。 通过详细的动力学和药理学研究，我们表明这两个因素是导致裂解的信号传导事件级联中的晚期事件。 我们之前表明蛋白酶体也是巨噬细胞裂解所必需的，新的研究表明蛋白酶体是炎症小体上游所必需的。 在其他研究中，我们表明细胞的热休克反应可以保护细胞免受炭疽致命毒素的侵害，这是通过阻断炎症小体依赖性 caspase-1 激活来实现的。 最后，我们还表明，巨噬细胞裂解中对蛋白酶体活性的要求通过 N 端规则起作用，这表明在 N 端具有不稳定残基的未知底物必定是导致裂解的途径的组成部分。 其他研究旨在了解大鼠和小鼠对致命毒素注射的反应。 Fischer 大鼠在注射致命毒素后短短 38 分钟内就会死亡，这表明了一种独特的病理机制。 通过与几个合作小组的合作，我们发现暴露于致命毒素的老鼠会出现肺水肿和出血。 通过连续监测和成像技术观察对心血管系统的具体影响。 该大鼠模型还用于评估和记录商业实体开发的单克隆抗体的功效。 在 DBA/2J 小鼠中探索了炭疽水肿和致命毒素之间相互作用的长期问题。 研究表明，用小剂量的水肿毒素进行预处理会使小鼠对立即或延迟一段时间后给予的致命毒素敏感。 这两种毒素的协同作用可能有助于解释为什么细菌具有这两种具有共同吸收机制的毒素。