Characterization of Anthrax Lethal Toxin

描述(申请人提供)：本申请的总体目标是阐明炭疽致死毒素在疾病过程中的作用机制。致死毒素是炭疽芽孢杆菌定植并杀死宿主的一种基本毒力因子。此外，致命性毒素造成的损害可能是存活患者表现出长期病理的一个促成因素。因此，更好地了解毒素与宿主的相互作用是至关重要的。致死因子是毒素的酶组分，由第二个毒素组分保护性抗原传递到哺乳动物细胞胞浆后，裂解有丝分裂原激活的蛋白激酶激酶。致死因子的酶活性如何导致与毒素相关的细胞效应尚不清楚。这项提议的第一个目的是确定致命性毒素如何干扰血管生成因子的表达。我们发现致命性毒素会破坏原代人内皮细胞中IL-8mRNA的稳定性。已有研究表明，许多转录本在转录后受其3‘非翻译区的特定元件和结合这些区域的蛋白质的调控。我们将确定致命毒素作用的调控蛋白和信使核糖核酸元件；并确定其他被毒素破坏稳定的转录本。我们的第二个目标是确定致命性毒素如何以及为什么在人巨噬细胞中诱导caspase非依赖性程序性细胞死亡，而不是在它们衍生出的未成熟的单核细胞中。我们将结合使用细胞分离和免疫荧光技术来识别介导细胞死亡的蛋白质；并比较毒素敏感和耐药细胞，以确定赋予致命毒素敏感性的细胞属性。我们的最终目标是分离出能够切割某些底物而不能切割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。为了做到这一点，我们将筛选随机致命因子突变体，寻找能够杀死某些类型的细胞，但不能杀死其他类型的细胞。这项工作可能会确定新的底物结合决定因素，可以作为治疗学的靶点。 相关性：我们研究的目标是了解炭疽致命毒素是如何损害人类细胞的。更好地了解这种毒素的作用方式将有助于医生治疗炭疽病患者，并帮助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.