Activity of Pseudomonas Type III Toxins

假单胞菌 III 型毒素的活性

• 批准号: 6733550
• 负责人: Dara W. Frank
• 金额: $ 33.64万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6733550
• 关键词: Pseudomonas aeruginosa; adenylate cyclase; bacterial toxins; enzyme activity; exoenzyme; gene expression; yeast two hybrid system; yeasts

DESCRIPTION (provided by the applicant): Pseudomonas aeruginosa is an opportunistic pathogen that expresses a wide variety of virulence determinants. Our work has focused on the contribution of one extracellular bacterial product to pathogenesis, exoenzyme S. Exoenzyme S is a member of the family of ADP-ribosyltransferase enzymes. Production of exoenzyme S is correlated with the ability of P. aeruginosa to spread or disseminate from epithelial colonization sites to the bloodstream of infected individuals, resulting in the development of a fatal sepsis. Our initial models of the intoxication mechanism for ExoS were simplistic and based on the notion that ExoS would exhibit an A:B structure. We subsequently showed, however, that ExoS was delivered into the cytosol of eukaryotic cells by a type III mechanism of intoxication. These observations opened new areas of investigation resulting in the cloning and sequence analysis of the Pseudomonas type III system, characterizing the major extracellular proteins secreted by the type III apparatus, and discovering two new toxins, ExoU and ExoY. The long-term goals of this new proposal are to determine the mechanism of action of ExoU and to begin expression and biochemical studies on ExoY. ExoU expression is responsible for the acute cytotoxic response in cultured cells and lung injury in vivo. ExoU possesses no known motifs, enzymatic activity, or homology to other proteins in the data base and likely represents a novel toxin. Structure-function analysis indicates that the cytotoxic response of ExoU is encoded in at least two domains that can function in trans within mammalian cells. New data, presented in this application, demonstrate that ExoU is toxic to yeast. We will use yeast as a model genetic and biochemical system to identify the target of ExoU toxicity. ExoY possesses adenylate cyclase activity and is related to the adenylate cyclase toxins of Bacillus anthracis and Bordetella pertussis. Although the activity of ExoY is known, the association of ExoY expression and the ExoY activity have not been examined relative to the pathogenesis of P. aeruginosa. Understanding the mechanisms of action of ExoU and ExoY will aid in the design of alternative treatments.

描述（由申请人提供）： 铜绿假单胞菌是一种 表达多种毒力决定子的机会致病菌。 我们的工作重点是一种细胞外细菌产物的贡献 外切酶 S 是发病机制中的一员。 ADP-核糖基转移酶。外切酶 S 的产生与 铜绿假单胞菌从上皮细胞传播或播散的能力 定植位点进入受感染个体的血液，从而导致 发展为致命的败血症。我们最初的中毒机制模型 对于 ExoS 来说是简单化的，并且基于 ExoS 将表现出 A:B 的概念 结构。然而，我们随后表明，ExoS 被传递到 通过 III 型中毒机制进入真核细胞的细胞质。这些 观察开辟了新的研究领域，导致克隆和 假单胞菌 III 型系统的序列分析，表征了主要 III 型装置分泌的细胞外蛋白，并发现了两种 新毒素 ExoU 和 ExoY。这项新提案的长期目标是 确定 ExoU 的作用机制并开始表达 ExoY 的生化研究。 ExoU 表达负责急性 培养细胞的细胞毒性反应和体内肺损伤。 ExoU 不具备 已知的基序、酶活性或与数据中其他蛋白质的同源性 碱基，可能代表一种新型毒素。结构功能分析表明 ExoU 的细胞毒性反应是在至少两个结构域中编码的 在哺乳动物细胞内以反式发挥作用。本报告中提供的新数据 应用，证明 ExoU 对酵母有毒。我们将使用酵母作为 模型遗传和生化系统以确定 ExoU 毒性的目标。 ExoY具有腺苷酸环化酶活性并且与腺苷酸有关 炭疽杆菌和百日咳博德特氏菌的环化酶毒素。虽然 ExoY 的活性已知，ExoY 表达与 ExoY 的关联 尚未检查与铜绿假单胞菌发病机制相关的活性。 了解 ExoU 和 ExoY 的作用机制将有助于设计 的替代疗法。