PASTEURELLA MULTOCIDA TOXIN--STRUCTURE AND ACTIVITY

多杀性巴氏杆菌毒素——结构和活性

• 批准号: 2457834
• 负责人: Brenda A. Wilson
• 金额: $ 9.49万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2457834
• 关键词: 3T3 cells; G protein; Pasteurella multocida; SDS polyacrylamide gel electrophoresis; Xenopus oocyte; bacterial proteins; bacterial toxicology; bacterial toxins; biological signal transduction; covalent bond; gene deletion mutation; microinjections; polymerase chain reaction; protein sequence; protein structure function; receptor binding; receptor coupling; recombinant proteins; transfection; western blottings

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.

多杀性巴氏杆菌是一种致病细菌