Characterization of Anthrax Lethal Toxin

描述（由申请人提供）：本申请的总体目标是阐明炭疽致死毒素促进疾病过程的机制。致死毒素是炭疽杆菌在宿主体内定植和致死的重要毒力因子。此外，致命毒素造成的损伤可能是存活患者表现出长期病理的一个促成因素。因此，更好地了解毒素如何与宿主相互作用至关重要。致死因子是毒素的酶组分，其在通过第二毒素组分（保护性抗原）递送至哺乳动物细胞胞质溶胶后切割促分裂原活化的蛋白激酶激酶。致死因子的酶活性如何引起与毒素相关的细胞效应还知之甚少。该提议的第一个目的是确定致死毒素如何干扰cvtokine表达。我们已经发现，致命毒素不稳定的白细胞介素-8 mRNA在原代人内皮细胞。已经表明，许多转录物在转录后由其3'非翻译区中的特定元件和结合这些区域的蛋白质调节。我们将确定致命毒素作用靶向的调节蛋白和mRNA元件，并确定毒素不稳定的其他转录本。我们的第二个目标是确定致命毒素如何以及为何在人类巨噬细胞中诱导非半胱氨酸蛋白酶依赖性程序性细胞死亡，但在它们所来源的未成熟单胞细胞中却不诱导。我们将使用细胞分级分离和免疫荧光技术相结合，以确定介导细胞死亡的蛋白质，并比较毒素敏感和耐药细胞，以确定细胞特性，赋予致命毒素的敏感性。我们的最终目标是分离出可以切割其某些底物而不能切割其他底物的致死因子突变体，以便可以评估单个底物对致病过程的贡献。为了实现这一目标，我们将筛选随机致死因子突变体，以寻找能够杀死某些细胞类型但不能杀死其他细胞类型的突变体。这项工作将有可能确定新的底物结合决定簇，可以通过治疗靶向。 相关性：我们研究的目的是了解炭疽致命毒素如何损害人体细胞。更好地了解这种毒素是如何起作用的，将有助于医生治疗炭疽病患者，并有助于研究人员发现新的药物治疗方法。

DESCRIPTION (provided by applicant): The overall objective of this application is to elucidate mechanisms by which anthrax lethal toxin contributes to the disease process. Lethal toxin is an essential virulence factor that allows Bacillus anthracis to colonize and kill the host. Furthermore, damage caused by lethal toxin is likely a contributing factor to the long-term pathologies displayed by surviving patients. It is critical, therefore, to gain a better understanding of how the toxin interacts with the host. Lethal factor is the enzymatic component of the toxin that cleaves mitogen activated protein kinase kinases after being delivered to the mammalian cell cytosol by the second toxin component, protective antigen. How lethal factor's enzymatic activity causes the cellular effects associated with the toxin is poorly understood. The first aim of this proposal is to determine how lethal toxin interferes with cvtokine expression. We have discovered that lethal toxin destabilizes interleukin-8 mRNA in primary human endothelial cells. It has been shown that many transcripts are post-transcriptionally regulated by specific elements in their 3' untranslated regions and by the proteins that bind these regions. We will identify regulatory proteins and mRNA elements that are targeted by lethal toxin action; and identify other transcripts that are destabilized by the toxin. Our second aim is to determine how and why lethal toxin induces caspase-independent programmed cell death in human macrophages, but not in the immature monocvtes from which they were derived. We will use a combination of cellular fractionation and immunofluorescence techniques to identify proteins that mediate cell death; and compare toxin-sensitive and resistant cells to determine cellular properties that confer susceptibility to lethal toxin. Our final aim is to isolate lethal factor mutants that can cleave some of its substrates, but not others, so that the contributions of individual substrates to pathogenic processes can be assessed. To accomplish this, we will screen random lethal factor mutants for ones that are able to kill some cell types, but not others. This work will potentially identify new substrate binding determinants that could be targeted by therapeutics. Relevance: The goal of our research is to understand how anthrax lethal toxin damages human cells. A better understanding of how the toxin works will help doctors treat patients with anthrax and help researchers discover new drug treatments.