LISTERIA USES HOST CELL ACTIN TO SPREAD CELL TO CELL

李斯特菌利用宿主细胞肌动蛋白在细胞之间传播

• 批准号: 2069376
• 负责人: Frederick s Southwick
• 金额: $ 22.1万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2069376
• 关键词: Bacillus subtilis; Listeria; Listeria infections; Xenopus oocyte; actin binding protein; actins; bacterial proteins; gel filtration chromatography; gelsolin; gene mutation; genetic transduction; host organism interaction; immunofluorescence technique; ion exchange chromatography; laboratory rabbit; microinjections; nucleic acid probes; nucleic acid sequence; protein purification; protein structure function; tissue /cell culture; transfection; video recording system

Listeria monocytogenes is an intracellular parasite which causes bacteremia and meningitis in immunocompromised patients. The incidence of listeriosis in AIDS is 100-250 times higher than the normal population. Listeria invades the host cell cytoplasm and induces host cell actin to assemble into a rocket tail which propels the bacterium rapidly through the cytoplasm so it can spread from cell to cell. To address the question, "How do Listeria interact with the host cells to accomplish this remarkable task?", we plan to: I. Determine which actin-binding proteins are critical for Listeria intracellular movement and cell-to-cell spread. PtK2 cell (epithelial cells) and J774 cells (macrophage-like cells) will be infected with Listeria and immunofluorescence used to examine the localization of actin and actin-binding proteins at early and late stages of infection. In addition to normal wild type strains, the MACK mutant strain which constitutively overproduces surface proteins will be studied. To better synchronize the infection bacteria will be microinjected into cells. Listeria motility in Xenopus extracts will be studied by microscopy. A living demonstration of how the actin severing protein, gelsolin, affects Listeria motility will be provided by infecting 3T3 fibroblasts permanently transfected with human gelsolin cDNA. Bodipy phalloidin (an actin filament stain) will be used to measure bacteria rocket tail lengths (an indirect measure of bacterial speed). Cells will be microinjected with fluorescein labeled actin so that actin assembly and disassembly rates as well as migration speed can be measured using time lapse video image analysis. II. Purify ActA protein and investigate its mechanism of action. A bacterial surface protein, ActA, which is necessary for Listeria induction of actin assembly, will be purified from the Listeria MACK strain, using high salt extraction, ion exchange and gel filtration chromatography. Alternatively the pET12 protein expression vector can be used to produce recombinant protein. Purified ActA will be microinjected into living cells. The protein will be added to macrophage extracts and actin assembly monitored using the pyrenyl actin. ActA will be coated on polystyrene beads and motility examined in cell extracts. An ActA affinity column will be used to purify host cell ActA-binding proteins. III. Search for additional genes important for Listeria intracellular movement and cell-to-cell spread. The actA gene will be transduced into Bacillus subtilis (hly) to determine if the ActA gene alone can mediate actin assembly. Other genes important for actin assembly will be sought by screening Tn9l6 Listeria mutants using phase contrast microscopy of infected PtK2 cells. The genetic basis for the immotile Listeria mutant M117 will be determined using Tn9l6 probes and nucleic acid sequencing. Understanding how Listeria utilizes the host cell's contractile system to infect new host cells should be applicable to other intracellular pathogens such as Shigella, Trypanosoma Cruzi, and Rickettsia. These discoveries may lead to the development new methods for treating intracellular infections in AIDS patients.

单核细胞增生李斯特菌是一种细胞内寄生虫，可引起 免疫功能低下患者的菌血症和脑膜炎。 发病率 艾滋病患者中李斯特菌感染率比正常人高100-250倍 人口。李斯特菌侵入宿主细胞的细胞质并诱导宿主 细胞肌动蛋白组装成火箭尾，推动细菌 快速穿过细胞质，因此可以从一个细胞传播到另一个细胞。 到 解决这个问题，“李斯特菌如何与宿主细胞相互作用 完成这项非凡的任务吗？”，我们计划： I. 确定哪些肌动蛋白结合蛋白对李斯特菌至关重要 细胞内运动和细胞间传播。 PtK2细胞（上皮细胞 细胞）和 J774 细胞（巨噬细胞样细胞）将被感染 李斯特菌和免疫荧光用于检查肌动蛋白的定位 以及感染早期和晚期的肌动蛋白结合蛋白。 在 除正常野生型菌株外，MACK突变菌株 将研究组成性过量产生表面蛋白。 为了更好 同步感染细菌将被显微注射到细胞中。 将通过显微镜研究爪蟾提取物中的李斯特菌运动性。一个 生动地展示了肌动蛋白切割蛋白、凝溶胶蛋白如何影响 李斯特菌的运动能力将通过感染 3T3 成纤维细胞来提供 用人凝溶胶蛋白 cDNA 永久转染。 鬼笔环肽（Bodipy phalloidin） 肌动蛋白丝染色剂）将用于测量细菌火箭尾部 长度（细菌速度的间接测量）。 细胞将是 显微注射荧光素标记的肌动蛋白，以便肌动蛋白组装和 拆卸率和迁移速度可以用时间来衡量 延时视频图像分析。 二.纯化ActA蛋白并研究其作用机制。 一个 细菌表面蛋白 ActA，这是李斯特菌所必需的 诱导肌动蛋白组装，将从李斯特菌 MACK 中纯化 菌株，采用高盐提取、离子交换和凝胶过滤 色谱法。 或者，pET12蛋白表达载体可以是 用于生产重组蛋白。 纯化的 ActA 将进行显微注射 进入活细胞。 该蛋白质将被添加到巨噬细胞提取物中并 使用芘基肌动蛋白监测肌动蛋白组装。 ActA将被涂在 在细胞提取物中检查聚苯乙烯珠和运动性。法案A 亲和柱将用于纯化宿主细胞 ActA 结合蛋白。 三． 寻找对胞内李斯特菌重要的其他基因 运动和细胞间传播。 actA基因将被转导至 枯草芽孢杆菌 (hly) 确定 ActA 基因是否可以单独介导 肌动蛋白组装。 将寻找对肌动蛋白组装重要的其他基因 通过使用相差显微镜筛选 Tn9l6 李斯特菌突变体 感染PtK2细胞。 不动的李斯特菌突变体的遗传基础 M117将使用Tn916探针和核酸测序来确定。 了解李斯特菌如何利用宿主细胞的收缩系统 感染新的宿主细胞应该也适用于其他细胞内 病原体，例如志贺氏菌、克氏锥虫和立克次体。 这些 发现可能会导致开发新的治疗方法 艾滋病患者的细胞内感染。