LISTERIA USES HOST CELL ACTIN TO SPREAD CELL TO CELL

单核细胞增生李斯特菌是一种细胞内寄生虫， 免疫功能低下患者的菌血症和脑膜炎。 发生率 艾滋病患者的艾滋病毒感染率是正常人的100-250倍， 人口李斯特菌侵入宿主细胞质并诱导宿主细胞凋亡， 细胞肌动蛋白组装成火箭尾， 快速通过细胞质，这样它就可以在细胞间传播。 到 解决这个问题，“李斯特菌如何与宿主细胞相互作用， 完成这项艰巨的任务？“，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扩散。 PtK 2细胞（上皮 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 除了正常的野生型菌株外， 将研究组成型过量产生表面蛋白的方法。 更好地 同步将感染细菌显微注射到细胞中。 将通过显微镜研究非洲爪蟾提取物中的李斯特菌运动性。一 一个活生生的例子，证明了肌动蛋白切割蛋白，凝溶胶蛋白， 通过感染3 T3成纤维细胞提供李斯特菌运动性 用人凝溶胶蛋白cDNA永久转染。 Bodipy鬼笔环肽（一种 肌动蛋白丝染色）将用于测量细菌火箭尾 长度（细菌速度的间接测量）。 细胞将被 显微注射荧光素标记的肌动蛋白， 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 李斯特菌所必需的细菌表面蛋白ActA 诱导肌动蛋白组装，将从李斯特菌MACK中纯化 菌株，使用高盐提取、离子交换和凝胶过滤 层析 或者，pET 12蛋白表达载体可以是 用于生产重组蛋白。 将微量注射纯化的ActA 转化成活细胞 将蛋白质加入巨噬细胞提取物中， 使用芘基肌动蛋白监测肌动蛋白组装。 ActA将被涂覆在 在细胞提取物中检测聚苯乙烯珠和运动性。的ActA 亲和柱将用于纯化宿主细胞ActA结合蛋白。 三. 寻找对李斯特菌细胞内重要的其他基因 运动和细胞间传播。actA基因将被导入 枯草芽孢杆菌（hly），以确定ActA基因单独是否可以介导 肌动蛋白组装 其他对肌动蛋白组装重要的基因也将被寻找 通过使用相差显微镜筛选Tn 916李斯特菌突变体， 感染PtK 2细胞。 不动李斯特菌突变体的遗传基础 将使用Tn 916探针和核酸测序测定M117。 了解李斯特菌如何利用宿主细胞的收缩系统， 感染新宿主细胞应该适用于其它细胞内 病原体如志贺氏菌、克氏锥虫和立克次体。 这些 这些发现可能会导致新的治疗方法的发展。 艾滋病患者的细胞内感染。

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.