LISTERIA PHOSPHOLIPASE ACTIVATION & CELL-TO-CELL SPREAD

李斯特菌磷脂酶激活

• 批准号: 6171025
• 负责人: HELENE MARQUIS
• 金额: $ 10.57万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-01 至 2003-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171025
• 关键词: Listeria; Listeria infections; bacteria infection mechanism; bacterial cytopathogenic effect; cysteine endopeptidases; enzyme activity; fluorescence microscopy; host organism interaction; immunofluorescence technique; metalloendopeptidases; phospholipase C; pore forming protein; protein localization; protein purification; site directed mutagenesis; vesicle /vacuole; virulence; western blottings

Listeria monocytogenes is a facultative intracellular bacterial pathogen that causes serious illness in pregnant women, neonates, elderly, and immunocompromised individuals. Listeriosis is among the leading causes of death from contaminated food products in US. In the last decade, L.monocytogenes has served as an excellent model system for exploring the interactions that take place between an intracellular parasite and its host. The overall goal of this proposal is to define the mechanisms by which L.monocytogenes is capable of spreading from cell to cell without exposure to the extracellular environment. In previous studies, a broad-range bacterial phospholipase C (PC-PLC) was shown to be necessary for efficient bacterial cell-to-cell spread. PC-PLC is secreted as an inactive precursor (proPC-PLC), and proteolytic cleavage at its N-terminus generates the active form of the enzyme. Recently, we obtained genetic and biochemical evidence that the intracellular activation of pro PC-PLC is mediated by a bacterial metalloprotease (Mpl), which is also active in broth culture, and a cysteine protease, whose activity can only be detected during intracellular infection. The activity of PC-PLC generated by either protease is essentially the same, although there is a small shift in substrate preference. Furthermore, proPC-PLC activation by either pathway is dependent on bacterial localization to a vacuole, and on vacuolar acidification. These observations support a model of bacterial escape from double membrane vacuoles formed during cell-to-cell spread that is dependent on host and bacterial determinants. In this proposal, a multidisciplinary approach will be used to test this model of bacterial cell-to-cell spread. Intravacuolar activation of proPC-PLC will serve as a probe to define the host and bacterial requirements for efficient and rapid lysis of double membrane vacuoles. More specifically, this proposal will define (I) the vacuolar compartment in which proPC-PLC activation occurs, (II) the influence of other bacterial virulence determinants on vacuolar maturation and proPC-PLC activation, (III) the origin and identity of the intracellular-specific proPC-PLC activating cysteine protease, and (IV) the relative importance of the two activating proteases. The long term objective of this research is to define the mechanism by which L.monocytogenes spreads from cell to cell. This may provide a novel target for development of drugs to treat or prevent intracellular microbial infections in humans.

单核细胞增生李斯特菌是一种细胞内细菌病原体 这会导致孕妇，新生儿，老年和 免疫功能低下的个体。 李斯特氏病是主要原因之一 在美国受污染的食品死亡。 在过去的十年中， L. mumocytogenes已成为探索的出色模型系统 在细胞内寄生虫和 它的主人。 该提议的总体目标是定义机制 L. munocytogenes能够从细胞传播到细胞 没有暴露于细胞外环境。 在先前的研究中， 广泛的细菌磷脂酶C（PC-PLC）被证明是 有效的细菌细胞对细胞扩散所必需的。 PC-PLC是 分泌为无活性前体（PROPC-PLC）和蛋白水解裂解 在其N末端产生酶的活性形式。 最近， 我们获得了细胞内的遗传和生化证据 ProC-PLC的激活是由细菌金属蛋白酶介导的 （MPL），在肉汤培养和半胱氨酸蛋白酶中也活跃， 只能在细胞内感染期间检测其活性。这 通过任何一种蛋白酶生成的PC-PLC的活性本质上都是相同的， 尽管底物偏好发生了很小的转变。 此外， 两种途径的ProPC-PLC激活取决于细菌 定位到液泡和液泡酸化。 这些 观察结果支持从双膜逃脱的细菌模型 在细胞到细胞扩散期间形成的液泡取决于宿主和 细菌决定因素。 在此提案中，一种多学科的方法 将用于测试这种细菌细胞间扩散模型。 ProPC-PLC的内腔激活将作为定义的探针 宿主和细菌要求有效，快速裂解 双膜液泡。 更具体地说，该建议将定义 （i）发生ProPC-PLC激活的液泡室，（ii） 这 其他细菌毒力决定因素对液泡的影响 成熟和ProPC-PLC激活，（iii）的起源和身份 细胞内特异性Propc-PLC激活半胱氨酸蛋白酶，和 （iv）两个激活蛋白酶的相对重要性。 长 这项研究的术语目的是定义该机制 L. mumocytogenes从细胞传播到细胞。 这可能会提供小说 用于开发药物以治疗或预防细胞内的靶标 人类中的微生物感染。