PASTEURELLA MULTOCIDA TOXIN--STRUCTURE AND ACTIVITY

多杀性巴氏杆菌是一种致病性细菌， 农业疾病巴氏杆菌病，出血性败血症， 皮肤坏死和进行性萎缩性鼻炎 多杀性巴氏杆菌可导致 动物咬伤或抓伤引起的人类感染的严重并发症， 呼吸道感染和怀孕期间接触动物。 p. 多杀性巴氏杆菌毒素是巴氏杆菌的重要毒力因子， 单独纯化的PMT足以在实验上诱导进行性 萎缩性鼻炎PMT似乎通过受体介导的细胞内信号传导进入细胞。 内吞作用并引起信号转导事件和DNA的激活 合成.我们实验室最近的研究发现G α 蛋白作为PMT作用的主要靶点， 磷脂酰肌醇特异性磷脂酶C-β 1和肌醇 非洲爪蟾卵母细胞的三磷酸途径。我们实验室的研究 还表明PMT的N-末端对于该活性是重要的，并且 我们提出了PMT细胞内作用的模型。我们已经克隆 多杀性巴氏杆菌的完整toxA基因（1285个残基），并产生了 许多缺失突变体，编码残基1 - 73、1 - 293、1 - 506、506 - 508、508 - 509、509 - 509、5 1285，1059 - 1285。我们的长期目标是利用这些重组 蛋白质，以了解PMT的结构和作用机制， 分子和生物化学水平，这两者都有助于未来 多杀性巴氏杆菌细菌发病机制的治疗干预 ，以及提供对分子信号事件的深入了解， 参与控制细胞生长和分化。在 特别是，我们希望证明PMT作为一种新的 用于研究细胞内信号传导途径的生化工具， Gq家族的调节蛋白。为了实现我们的目标，我们建议 如下： (1)确定蛋白质的功能结构域，从而确定 毒素的哪些结构域负责（1）与 真核细胞受体和（2）刺激细胞内信号 转导途径。 (2)为了阐明PMT激活Gq的分子机制， 蛋白质，通过确定是否引起PMT的Gq-蛋白质的活化， 通过共价修饰或通过非共价相互作用。 (3)为了检验PMT解偶联配体调节的 受体和Gq蛋白之间的相互作用，使用非洲爪蟾卵母细胞 过量表达外源性5-HT 2受体和Gq α蛋白的系统。

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.