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）：炭疽病由于其可能用作生物武器，造成相当大的公共卫生负担。尽管接种疫苗可能提供最好的保护，但仍然迫切需要成功治疗炭疽并发症。抗生素可以阻止感染的进展，但一旦细菌产生了破坏性或致命量的炭疽毒素，抗生素的作用就有限了。炭疽毒素被认为是通过其蛋白质成分的作用，特别是致死因子（LF），导致严重的血管渗漏和组织水肿，从而损害组织。我们的实验室一直在研究肺血管内皮细胞通透性屏障的调节，并已确定信号通路，削弱了屏障和那些增强it. We已经表明，p38 MAP激酶激活导致HSP 27磷酸化的激酶MK2增强内皮细胞通透性屏障，通过加强粘附力介导的肌动蛋白应力纤维和波形蛋白中间丝网络的形成。由于炭疽毒素的LF组分的主要分子活性是阻断p38活化，因此假定下游HSP 27磷酸化及其屏障增强作用响应于炭疽毒素而被阻断。在该提议中待测试的总体假设是炭疽毒素通过阻断p38-MK2活化和HSP 27磷酸化破坏内皮渗透屏障来产生其一些作用。因此，磷酸-HSP 27不能通过其对肌动蛋白和波形蛋白的作用形成和介导渗透性屏障增强，导致内皮渗透性增加和水肿。炭疽致死毒素引起的内皮屏障通透性和血管渗漏也被假定通过激活HSP 27磷酸化而被阻断或逆转。由于LF引起炭疽的大部分症状，我们将描述致命毒素（LT）的作用，它是LF和保护性抗原（PA）的组合。PA是炭疽毒素的另一种成分，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，因为它们在培养中形成紧密的渗透屏障，并且LF被认为直接作用于内皮细胞。此外，我们将在对炭疽LF敏感的Fisher 244大鼠中进行体内实验。在目的1中，我们将确定炭疽LT对p38-MK2-HSP 27信号传导和细胞骨架重塑的动力学作用，这些作用与内皮单层通透性有关。在目标2中，我们将评估诱导HSP 27磷酸化作为一种机制，以防止炭疽LT诱导的渗透性和水肿在细胞培养和体内。我们希望我们的实验表明，通过LT阻断HSP 27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通透性和渗漏。此外，我们希望我们的实验表明，靶向HSP 27磷酸化是治疗炭疽的一种机械上合理的方法，并证明该方法在炭疽动物模型中的有效性。 公共卫生关系：由于炭疽暴露的致命威胁，迫切需要成功治疗其并发症，虽然抗生素可以阻止感染的进展，但一旦细菌产生了破坏性或致命量的炭疽毒素，抗生素的使用就有限了。本申请中提出的研究集中于炭疽毒素可引起渗透性和血管渗漏的潜在机制，这与其致死性有关。通过评估阻断炭疽毒素对内皮通透性屏障调节途径的分子作用的方法，这些研究可能导致成功治疗炭疽和引起通透性和血管渗漏的类似药物。