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Mechanism of Anthrax Lethal Factor Toxicity

炭疽致死因子毒性机制

基本信息

批准号：
8280315
负责人：
Usamah S Kayyali
金额：
$ 19.88万
依托单位：
TUFTS MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/8280315
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

项目摘要

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.

描述（由申请人提供）：炭疽因其可能被用作生物武器而构成相当大的公共卫生负担。尽管针对细菌的疫苗接种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数量的炭疽毒素，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，特别是致命因子（LF）的作用来损害组织，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通透性屏障的调节，并已经确定了削弱屏障和增强屏障的信号通路。我们已经证明，p38 MAP激酶激活导致HSP27被MK2激酶磷酸化，通过增强由肌动蛋白应激纤维和vimentin中间丝网络形成介导的粘附力，增强内皮通透性屏障。由于炭疽毒素LF成分的主要分子活性是阻断p38的激活，因此假定下游HSP27的磷酸化及其屏障增强作用在炭疽毒素反应中被阻断。本提案要验证的总体假设是，炭疽毒素通过阻断p38-MK2激活和HSP27磷酸化，破坏内皮通透性屏障，从而产生一些影响。因此，phospho-HSP27无法通过作用于肌动蛋白和波形蛋白来形成和介导通透性屏障增强，导致内皮通透性增加和水肿。炭疽致死毒素引起的内皮屏障通透性和血管渗漏也被认为可以通过激活HSP27磷酸化来阻断或逆转。由于炭疽热的大部分症状是由LF引起的，我们将描述致死毒素（LT）的作用，它是LF和保护性抗原（PA）的结合。PA是炭疽毒素的另一种成分，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，因为它们在培养中形成了一个紧密的渗透性屏障，而LF被认为直接作用于内皮细胞。此外，我们将在Fisher 244大鼠中进行体内实验，这些大鼠已被证明对炭疽LF敏感。在目的1中，我们将确定炭疽LT作用于p38-MK2-HSP27信号传导和与内皮单层通透性相关的细胞骨架重塑的动力学。在目的2中，我们将评估HSP27磷酸化的诱导作为一种在细胞培养和体内防止炭疽热lt诱导的通透性和水肿的机制。我们希望我们的实验能够证明，通过LT阻断HSP27磷酸化，通过抑制肌动蛋白和静脉蛋白介导的屏障增强，导致通透性和泄漏。此外，我们期望我们的实验表明，靶向HSP27磷酸化是一种机制上合理的治疗炭疽的方法，并证明该方法在炭疽动物模型中的有效性。