PASTEURELLA MULTOCIDA TOXIN--STRUCTURE AND ACTIVITY

多杀性巴氏杆菌是一种致病细菌，与 农业疾病巴氏杆菌病，出血性败血症， 皮肤病和进行性萎缩性鼻炎。多杀性巴氏杆菌可引起 动物咬伤或抓伤引起的人类感染的严重并发症， 呼吸道感染，以及在怀孕期间接触动物。P. 多杀性毒素是多杀性巴氏杆菌重要的毒力因子。 仅纯化的PMT就足以在实验中诱导进展 萎缩性鼻炎。PMT似乎通过受体介导进入细胞 内吞作用与信号转导事件和DNA的激活 综合。我们实验室最近的研究已经确定了Gα 蛋白质作为PMT作用的主要靶点，激活 磷脂酰肌醇特异性磷脂酶C-β1和肌醇 非洲爪哇卵母细胞中的三磷酸途径。我们实验室的研究表明 还表明PMT的N末端对这一活动很重要，以及 我们提出了PMT细胞内作用的模型。我们已经克隆了 多杀性巴氏杆菌的整个ToxA基因(1285个残基)已经产生了 一些缺失突变体，编码残基1-73、1-293、1-506、506- 1285和1059-1285。我们的长期目标是利用这些重组 了解PMT的结构和作用机制的蛋白质 分子和生化层面，都是为了促进未来 多杀性巴氏杆菌致病机制的治疗干预 ，以及提供对分子信号事件的洞察 参与控制细胞的生长和分化。在……里面 特别是，我们希望展示PMT作为一种新的 用于研究细胞内信号通路的生化工具 GQ调节蛋白家族。为了实现我们的目标，我们建议 以下内容： (1)确定蛋白质的功能结构域，以确定 毒素的哪个结构域负责(1)与 真核细胞受体和(2)刺激细胞内信号 转导通路。 (2)阐明PMT激活GQ的分子机制。 蛋白质，通过确定PMT对Gq蛋白的激活是否导致 通过共价修饰或通过非共价相互作用。 (3)检验PMT解偶联配体调控的假设 利用非洲爪哇卵母细胞研究受体与Gq蛋白的相互作用 系统过度表达外源性5-HT2受体和GqAlpha蛋白。

Pasteurella multocida is a pathogenic bacterium associated with the agricultural diseases pasteurellosis, hemorrhagic septicemia, dermonecrosis, and progressive atrophic rhinitis. P. multocida can cause severe complications in human infections from animal bites or scratches, respiratory infections, and exposure to animals during pregnancy. P. multocida toxin is an important virulence factor of P. multocida, and purified PMT alone is sufficient to experimentally induce progressive atrophic rhinitis. PMT appears to enter cells via receptor-mediated endocytosis and causes activation of signal transduction events and DNA synthesis. Recent studies from our laboratory have identified G alpha protein as the primary target of PMT action that activates the phosphatidylinositol-specific phospholipase C-beta 1 and the inositol triphosphate pathway in Xenopus oocytes. Studies from our laboratory have also shown that the N-terminus of PMT is important for this activity, and we have proposed a model for PMT's intracellular action. We have cloned the entire toxA gene (1285 residues) from P. multocida and have generated a number of deletion mutants, encoding residues 1-73, 1-293, 1-506, 506- 1285, and 1059-1285. Our long range goals are to use these recombinant proteins to understand the structure and mechanism of action of PMT at the molecular and biochemical level, both to facilitate future therapeutic intervention in the bacterial pathogenesis of P. multocida , as well as to provide insight into the molecular signalling events involved in the control of cell growth and differentiation. In particular, we hope to demonstrate the utility of PMT as a new biochemical tool for studying intracellular signalling pathways involving the Gq family of regulatory proteins. To achieve our goals, we propose the following: (1) To define the functional domains of the protein, so as to determine which of the toxin's domains are responsible for (1) binding to the eukaryotic cell receptors and (2) stimulating the intracellular signal transduction pathways. (2) To elucidate the molecular mechanism by which PMT activates Gq- protein, by determining whether PMT's activation of Gq-protein is caused by a covalent modification or by noncovalent interaction. (3) To test the hypothesis that PMT uncouples the ligand-regulated interaction between receptor and Gq-protein, using the Xenopus oocyte system overexpressing exogenous 5-HT2 receptor and Gqalpha-protein.