Mechanism of Anthrax Lethal Factor Toxicity

描述(由申请人提供)：炭疽病可能被用作生物武器，因此对公众健康造成相当大的负担。尽管接种针对这种细菌的疫苗可能提供最好的保护，但仍迫切需要成功地治疗炭疽并发症。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死量的炭疽毒素，抗生素的使用就有限了。炭疽毒素被认为是通过其蛋白质成分，特别是致死因子(LF)的作用来损害组织，从而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细胞通透性屏障的调节，并发现了减弱和增强该屏障的信号通路。我们已经证明，p38 MAP激活导致HSP27被MK2磷酸化，通过加强肌动蛋白应激纤维介导的粘附力和波形蛋白中间细丝网络的形成来增强内皮通透性屏障。由于炭疽毒素的LF组分的一个主要分子活性是阻止p38的激活，因此推测炭疽毒素可以阻断HSP27下游的磷酸化及其屏障增强作用。这项建议中要检验的总体假设是，炭疽毒素通过阻断p38-MK2激活和HSP27磷酸化来破坏内皮通透性屏障，从而产生一些影响。因此，磷酸化的HSP27不能通过作用于肌动蛋白和波形蛋白而形成和介导通透性屏障的增加，从而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可能通过激活HSP27的磷酸化而被阻断或逆转。由于Lf引起炭疽的大部分症状，我们将描述致死毒素(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，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紧密的通透性屏障，而LF被认为直接作用于内皮细胞。此外，我们还将在Fisher 244大鼠身上进行体内实验，这些大鼠已被证明对炭疽LF敏感。在目标1中，我们将确定炭疽热休克蛋白对p38-MK2-HSP27信号和与内皮单层通透性相关的细胞骨架重塑的作用动力学。在目标2中，我们将在细胞培养和体内评估HSP27磷酸化的诱导作为一种机制来保护炭疽杆菌LT诱导的通透性和水肿。我们希望我们的实验能够证明，LT通过抑制肌动蛋白和波形蛋白介导的屏障增强来阻断HSP27的磷酸化，从而导致通透性和泄漏。此外，我们希望我们的实验表明，靶向HSP27磷酸化是治疗炭疽病的一种机械上合理的方法，并在炭疽病的动物模型中证明该方法的有效性。

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.