Activity of Pseudomonas Type III Toxins

简介(申请人提供)：铜绿假单胞菌是一种 一种表达多种毒力决定因素的机会性病原体。 我们的工作集中在一种胞外细菌产物的贡献上 从发病机制上讲，胞外酶S是链球菌胞外酶家族中的一员。 ADP-核糖基转移酶。胞外酶S的产生与 铜绿假单胞菌传播或经上皮传播的能力 移居地点转移到感染者的血液中，导致 发展为致命的败血症。我们最初的醉酒机制模型 因为EXOS是简单化的，并且基于EXOS将显示A：B的概念 结构。然而，我们随后表明，EXOS被传递到 真核细胞的胞浆通过III型中毒机制。这些 观察开辟了新的调查领域，导致了克隆和 假单胞菌III型系统的序列分析 类型III器分泌的胞外蛋白，并发现两个 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的表达与急性 培养细胞的细胞毒性反应与体内肺损伤。ExoU没有 已知的基序、酶活性或与数据中其他蛋白质的同源性 碱基，可能代表一种新的毒素。结构功能分析表明 ExoU的细胞毒性反应至少编码在两个结构域中，这两个结构域可以 在哺乳动物细胞内的反式作用。新数据，在此提供 应用，证明ExoU对酵母有毒性。我们将使用酵母作为一种 建立遗传生化系统模型，确定ExoU毒性靶标。 ExoY具有腺苷环化酶活性，并与腺苷有关 炭疽杆菌和百日咳杆菌的环化酶毒素。尽管 ExoY的活性是已知的，ExoY表达与ExoY的关系 活性尚未与铜绿假单胞菌的致病机制相关。 了解ExoU和ExoY的作用机制将有助于设计 可供选择的疗法。

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.