Mechanism of Anthrax Lethal Factor Toxicity

炭疽致死因子毒性机制

• 批准号: 8177455
• 负责人: Usamah S Kayyali
• 金额: $ 23.85万
• 依托单位: TUFTS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-15 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8177455
• 关键词: Actins; Adherent Culture; Adhesives; Aftercare; Animal Model; Animals; Anthrax disease; Anthrax exposure; Antibiotics; Antigens; Bacteria; Biological; Biological Models; Biological Warfare; Blood; Blood Vessels; Cell Culture Techniques; Cells; Development; Disease; Edema; Effectiveness; Endothelial Cells; Exposure to; HSPB1 gene; Histamine; Hypoxia; Infection; Inflammation Mediators; Intermediate Filaments; Kinetics; Laboratories; Lead; Life; Link; Lung; MAPK14 gene; Mediating; Molecular; Pathway interactions; Permeability; Phosphorylation; Phosphotransferases; Proteins; Public Health; Pulmonary Edema; Pulmonary artery structure; Rattus; Regulation; Signal Pathway; Signal Transduction; Site; Stress Fibers; Symptoms; Testing; Therapeutic; Tissues; Toxic effect; Vaccination; Vascular Permeabilities; Vimentin; acetopyrrothine; anthrax lethal factor; anthrax toxin; cytokine; edema factor; in vivo; mitogen-activated protein kinase p38; monolayer; overexpression; research study; response; sound; treatment effect; weapons

DESCRIPTION (provided by applicant): Anthrax poses a considerable public health burden because of its potential use as a biological weapon. Although vaccination against the bacteria might offer the best protection, there remains a pressing need to successfully treat anthrax complications. Antibiotics can stop the progression of the infection, but are of limited use once a damaging or lethal amount of anthrax toxin has been produced by the bacteria. Anthrax toxin is believed to damage tissues through the action of its protein components particularly the Lethal Factor (LF), which causes severe vascular leak and tissue edema. Our laboratory has been studying the regulation of the pulmonary vascular endothelial permeability barrier and has identified signaling pathways that weaken the barrier and those that augment it. We have shown that p38 MAP kinase activation leading to HSP27 phosphorylation by the kinase MK2 augments the endothelial permeability barrier through strengthening adhesive forces mediated by actin stress fiber and vimentin intermediate filament network formation. Since a major molecular activity of the LF component of anthrax toxin is blocking p38 activation, downstream HSP27 phosphorylation and its barrier augmenting effect are postulated to be blocked in response to anthrax toxin. The overall hypothesis to be tested in this proposal is that anthrax toxin produces some of its effects through disrupting the endothelial permeability barrier via blocking p38-MK2 activation and HSP27 phosphorylation. As a result phospho-HSP27 is unable to form and mediate permeability barrier augmentation through its action on actin and vimentin, resulting in increased endothelial permeability and edema. Endothelial barrier permeability and vascular leak caused by anthrax lethal toxin are also postulated to be blocked or reversed by activating HSP27 phosphorylation. Since LF causes most of the symptoms of anthrax we will characterize the action of Lethal Toxin (LT) which is a combination of LF and Protective Antigen (PA). PA is another component of the anthrax toxin that mediates the entry of LF into cells. We will carry out experiments in rat pulmonary microvascular endothelial cells because they form a tight permeability barrier in culture and LF is believed to act directly on endothelial cells. In addition we will carry out in vivo experiments in Fisher 244 rats which have been shown to be sensitive to anthrax LF. In Aim 1 we will determine the kinetics of anthrax LT action on p38-MK2-HSP27 signaling and cytoskeletal remodeling as related to endothelial monolayer permeability. In Aim 2 we will evaluate induction of HSP27 phosphorylation as a mechanism to protect against anthrax LT-induced permeability and edema in cell culture and in vivo. We expect our experiments to demonstrate that blocking HSP27 phosphorylation by LT causes permeability and leak through inhibiting actin- and vimentin-mediated barrier augmentation. Furthermore, we expect our experiments to show that targeting HSP27 phosphorylation is a mechanistically sound approach to treat anthrax and to demonstrate the effectiveness of that approach in animal models of anthrax. PUBLIC HEALTH RELEVANCE: Because of the lethal threat of anthrax exposure there is a pressing need to successfully treat its complications, and while antibiotics can stop the progression of the infection, they are of limited use once a damaging or lethal amount of anthrax toxin has been produced by the bacteria. The studies proposed in this application focus on a potential mechanism by which the anthrax toxin can cause permeability and vascular leak, which has been associated with its lethality. By evaluating approaches that block the molecular action of anthrax toxin on endothelial permeability barrier-regulating pathways these studies might lead to successful treatment of anthrax and similar agents that cause permeability and vascular leak.

描述（由申请人提供）：由于其潜在用作生物武器，炭疽病带来了相当大的公共卫生负担。尽管针对细菌的疫苗接种可能会提供最佳的保护，但仍然需要成功治疗炭疽并发症。抗生素可以阻止感染的进展，但是一旦细菌产生了造成损害或致死量的炭疽毒素的使用有限。据信，炭疽毒素通过其蛋白质成分的作用，尤其是致命因子（LF）来损害组织，这会导致严重的血管渗漏和组织水肿。我们的实验室一直在研究肺血管内皮渗透性屏障的调节，并确定了削弱障碍物和增强屏障的信号通路。我们已经表明，通过增强肌动蛋白胁迫纤维和Vimentin Intermendiper丝网网络形成，激酶MK2通过增强内皮渗透性屏障的p38 MAP激酶激活导致HSP27磷酸化。由于炭疽毒素的LF成分的主要分子活性正在阻止p38激活，因此假设下游HSP27磷酸化及其屏障增强效果被认为会响应炭疽毒素而阻塞。该提案中要检验的总体假设是，炭疽毒素通过阻断p38-MK2激活和HSP27磷酸化而破坏内皮渗透性屏障来产生其某些作用。结果，通过对肌动蛋白和波形蛋白的作用，磷酸-HSP27无法形成并介导渗透性屏障增强，从而导致内皮通透性和水肿增加。炭疽致死毒素引起的内皮屏障渗透性和血管泄漏也被假定通过激活HSP27磷酸化来阻断或逆转。由于LF导致大多数炭疽病症状，因此我们将表征致命毒素（LT）的作用，该作用是LF和保护性抗原（PA）的组合。 PA是炭疽毒素的另一个成分，它介导LF进入细胞。我们将在大鼠肺微血管内皮细胞中进行实验，因为它们在培养物中形成紧密的渗透性屏障，而LF被认为直接作用于内皮细胞。此外，我们将在Fisher 244只大鼠中进行体内实验，这些实验已证明对炭疽LF敏感。在AIM 1中，我们将确定与内皮单层渗透性相关的P38-MK2-HSP27信号传导和细胞骨架重塑的动力学。在AIM 2中，我们将评估HSP27磷酸化的诱导，以防止细胞培养和体内的炭疽LT诱导的渗透性和水肿。我们预计我们的实验可以证明，通过LT阻断HSP27磷酸化，通过抑制肌动蛋白和波形蛋白介导的屏障增强引起渗透性和泄漏。此外，我们预计我们的实验表明，靶向Hsp27磷酸化是一种机械性的方法，可用于治疗炭疽并证明该方法在炭疽动物模型中的有效性。 公共卫生相关性：由于炭疽暴露的致命威胁，迫切需要成功治疗其并发症，尽管抗生素可以阻止感染的进展，但一旦细菌产生了受损或致命量的炭疽病毒素，它们的使用有限。该应用中提出的研究集中于一种潜在的机制，通过该机制，炭疽毒素会导致渗透性和血管泄漏，这与其致死性有关。通过评估阻止炭疽毒素对内皮渗透性屏障调节途径的分子作用的方法，这些研究可能会导致对炭疽和类似药物的成功治疗，从而导致渗透率和血管泄漏。