Listeria phospholipase C: sorting, activation, release

李斯特菌磷脂酶 C：分选、激活、释放

• 批准号: 6610230
• 负责人: HELENE MARQUIS
• 金额: $ 18.05万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6610230
• 关键词: Listeria infections; SDS polyacrylamide gel electrophoresis; autoradiography; bacteria infection mechanism; bacterial proteins; electrospray ionization mass spectrometry; enzyme activity; enzyme induction /repression; fluorescence microscopy; gram positive bacteria; host organism interaction; immunoprecipitation; intracellular transport; matrix assisted laser desorption ionization; metalloendopeptidases; microorganism growth; phospholipase C; polymerase chain reaction; protein structure function; two dimensional gel electrophoresis; virulence; western blottings; zymogens

Listeria monocytogenes is a facultative intracellular Gram-positive bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has become a significant public health problem and has caused several epidemics in the United States and Europe. The virulence of L. monocytogenes is directly related to its ability to grow in the cytosol of host cells and its efficacy in spreading from cell to cell without leaving the intracellular milieu. We are interested in the mechanism regulating the activity of a bacterial phosphotipase C (PC-PLC), whose function increases the efficacy of bacterial cell-to-celt spread. PC-PLC is made as a proenzyme whose activation requires cleavage of a Nterminal prodomain. During intracellular growth, bacteria cumulate a pool of PC-PLC that is rapidly secreted in its active form upon a decrease in intracellular pH. A bacterial metalloprotease (Mpl) is involved in the regulation of PC-PLC activation and secretion. However, factors other than pH and Mpl control PC-PLC activation and secretion since a decrease in pH has little effect on the status of bacteria-associated PC-PLC in vitro. Our results reveal the existence of a previously undescribed mechanism regulating the activity of a virulence factor in a Gram-positive bacterial pathogen. This mechanism can be divided into three major steps: sorting, activation, and release of the virulence factor. Premature activation and secretion of PC-PLC in the cytosol of infected cells is cytotoxic, emphasizing the importance of this control mechanism for the intraceltular survival of L. monocytogenes. Our ultimate goal is to define at the cellular, molecular, and biochemical levels the mechanism regulating the activity of L. monocytogenes PC-PLC during infection. The specific aims of this proposal are to (I) Define functional regions of L. monocytogenes PC-PLC and Mpl that promote their association with the bacterial celt, (II) Identify and analyze bacterial factors accessory to the regulation of PC-PLC activity, and (III) Define environmental signals influencing the regulalation of PC-PLC activity. On a broader scope, these studies will contribute to our understanding of protein secretion in Grampositive bacteria.

单核细胞增生李斯特菌是一种细胞内革兰氏阳性细菌，在易感宿主中引起侵入性，通常是致命的疾病。作为食物出生的病原体，细菌已成为一个重大的公共卫生问题，并在美国和欧洲引起了几种流行病。单核细胞增生李斯特氏菌的毒力与其在宿主细胞的细胞质中生长的能力直接相关，及其在不离开细胞内环境的情况下扩散到细胞的功效。我们对调节细菌磷酸蛋白酶C（PC-PLC）活性的机制感兴趣，后者的功能会提高细菌细胞对循环扩散的功效。 PC-PLC作为蛋白酶，其激活需要裂解N端 Prodomain。在细胞内生长期间，细菌累积了一池PC-PLC，其在细胞内pH值下降后以其活性形式迅速分泌。细菌金属蛋白酶（MPL）参与了PC-PLC激活和分泌的调节。然而，除了pH和MPL控制PC-PLC激活和分泌以外的其他因素，由于pH值的降低对细菌相关的PC-PLC体外的状态几乎没有影响。我们的结果揭示了先前未描述的机制，该机制调节了革兰氏阳性细菌病原体中毒力因子的活性。该机制可以分为三个主要步骤：毒力因子的分类，激活和释放。 PC-PLC在受感染细胞的胞质溶胶中的过早激活和分泌是细胞毒性的，强调了这种控制机制对于该控制机制的重要性 单核细胞增生乳杆菌的胸内存活。我们的最终目标是在细胞，分子和生化水平上定义调节在感染过程中单核细胞增生李斯特菌的活性的机制。该提案的具体目的是（i）定义单核细胞增生李斯特氏菌PC-PLC和MPL的功能区域，这些区域促进了它们与细菌凯尔特的关联，（ii）识别和分析细菌因子附件，以调节PC-PLC活性的调节，（III）定义了影响PC-PLC活性调节的环境信号。在更广泛的范围上，这些研究将有助于我们对息肉呈蛋白质分泌的理解 细菌。