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命毒素导致疾病过程的机制。致死毒素是炭疽芽孢杆菌定植并杀死宿主的重要毒力因子。此外，致命毒素造成的损害很可能是造成存活患者长期病理的一个因素。因此，更好地了解毒素如何与宿主相互作用是至关重要的。致死因子是毒素的酶成分，它通过第二毒素成分保护性抗原传递到哺乳动物细胞细胞质后，裂解有丝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尚不清楚。本提案的第一个目的是确定致命毒素如何干扰cvtokine的表达。我们已经发现，致命毒素破坏了原代人内皮细胞中白细胞介素-8 mRNA的稳定性。研究表明，许多转录本受其3'非翻译区的特定元件和结合这些区域的蛋白质的转录后调控。我们将确定致命毒素作用的调控蛋白和mRNA元件；找出其他被毒素破坏的转录本。我们的第二个目标是确定致命毒素如何以及为什么在人巨噬细胞中诱导caspase不依赖的程序性细胞死亡，而不是在巨噬细胞衍生的未成熟单核细胞中。我们将结合细胞分离和免疫荧光技术来鉴定介导细胞死亡的蛋白质；比较对毒素敏感的细胞和对毒素有抵抗力的细胞，以确定对致命毒素敏感的细胞特性。我们的最终目标是分离出能够切割某些底物而不是其他底物的致死因子突变体，以便评估单个底物对致病过程的贡献。为了实现这一目标，我们将随机筛选能够杀死某些细胞类型而不能杀死其他细胞类型的致命因子突变体。这项工作将有可能确定新的底物结合决定因素，这些决定因素可能是治疗方法的目标。

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.