Characterization of Anthrax Lethal Toxin

炭疽致命毒素的表征

基本信息

批准号：
7796879
负责人：
JEREMY S MOGRIDGE
金额：
$ 25.46万
依托单位：
UNIVERSITY OF TORONTO
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-01 至 2012-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7796879
关键词：
3&apos Untranslated Regions Animals Anthrax disease Antigens Apoptosis Bacillus anthracis Bacteria Caspase Cell Death Cell physiology Cells Cessation of life Cleaved cell Cytosol Disease Elements Endothelial Cells Epitopes Fluorescent Antibody Technique Fractionation Goals Human Immune Immune system Impairment Indium Individual Injection of therapeutic agent Interleukin-8 MAP2K1 gene Mammalian Cell Mediating Messenger RNA Mitogen-Activated Protein Kinase Kinases Molecular Pathogenesis Pathology Patients Peptide Hydrolases Pharmaceutical Preparations Predisposition Process Property Protein Binding Proteins Regulation Research Research Personnel Resistance Symptoms Toxin Transcript Virulence Factors Work Zinc anthrax lethal factor antigen binding cell type cytokine genetic regulatory protein human MAP2K1 protein killings mRNA Stability macrophage mutant therapeutic target

项目摘要

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.

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导致疾病过程的机制。致命毒素是炭疽杆菌定殖并杀死宿主的重要毒力因子。此外，致命毒素造成的损害可能是导致幸存患者出现长期病变的一个因素。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促成分，在被第二种毒素成分（保护性抗原）递送到哺乳动物细胞胞浆后，裂解丝裂原激活的蛋白激酶激酶。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。该提案的首要目的是确定致命毒素如何干扰细胞因子的表达。我们发现致命毒素会破坏原代人内皮细胞中白细胞介素 8 mRNA 的稳定性。已经表明，许多转录物受到其3'非翻译区中的特定元件以及结合这些区域的蛋白质的转录后调节。我们将鉴定致命毒素作用所针对的调节蛋白和 mRNA 元件；并识别其他因毒素而不稳定的转录本。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不依赖半胱天冬酶的程序性细胞死亡，但在其来源的未成熟单核细胞中则不然。我们将结合使用细胞分级分离和免疫荧光技术来鉴定介导细胞死亡的蛋白质；并比较毒素敏感细胞和耐药细胞，以确定对致命毒素易感性的细胞特性。我们的最终目标是分离出可以裂解其某些底物但不能裂解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，我们将随机筛选致死因子突变体，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因素，这些决定因素可以作为治疗的目标。相关性：我们研究的目标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的作用原理将有助于医生治疗炭疽患者，并帮助研究人员发现新的药物治疗方法。