M AVIUM INHIBITION OF PHAGOSOME MEMBRANE TRAFFIC

鸟分枝杆菌已成为艾滋病的主要机会致病菌 患者鸟分枝杆菌通常位于细胞的液泡区室中， 巨噬细胞在那里它限制进入消化和加工 宿主细胞的能力。访问似乎受到限制， 膜蛋白运输进出鸟分枝杆菌， 结核分枝杆菌吞噬体。由于这一点以及 细胞内的鸟分枝杆菌与结核分枝杆菌之间，鸟分枝杆菌可能提供一种理想的 研究蛋白质运输的基本机制， 单核吞噬细胞新形成的吞噬体融合并交换膜 具有多个细胞内区室，包括溶酶体， 这种蛋白质的交换对于细胞内杀伤是至关重要的。我们有 开发了体外重建吞噬体融合事件的方法， 已经开始确定吞噬体所需的因子 膜交通在这些蛋白质中， 在吞噬体膜运输中的调节作用是GTP结合 蛋白（异三聚体G蛋白，Rab GTP结合蛋白和ADP- 核糖基化因子-ARF）和新描述的SNARE对接蛋白 (i.e., NEM敏感性融合蛋白的受体）。 我们最初 实验已经成功地重建了早期的体外融合， 吞噬体和内体。我们建议利用体外试验， 确定核内体融合的生化要求， 从正常巨噬细胞和感染的巨噬细胞分离的吞噬体 关于M. avium我们最初的努力是确定关键的GTP结合蛋白 和介导蛋白质运输进出的SNARE对接蛋白 正常和鸟型支原体吞噬体。利用鸟分枝杆菌的细胞膜和细胞质 感染的细胞，我们将研究可能的抑制剂产生的 有机体在我们最初的实验中，我们已经开始对 在吞噬过程中发生在吞噬体膜上的生化变化 成熟我们建议开发体外试验来重建融合 吞噬体的囊泡来源于trans-Golgi网络， 并使用这些测定来确定鸟分枝杆菌的产物是否 胞内感染损害膜转运。最后，我们将利用 使用鸟分枝杆菌的体外试验，以确定 活生物体对吞噬体与内体、囊泡融合的影响 来源于高尔基体网络和溶酶体。

Mycobacterium avium has emerged as a major opportunistic pathogen in AIDS patients. M.avium normally resides within the vacuolar compartment of the macrophage where it restricts access to the digestive and processing capabilities of the host cell. Access appears to be restricted by impaired membrane protein trafficking into and out of the M.avium and M.tuberculosis phagosome. Because of this and the similarity of intracellular M.avium to M.tuberculosis, M.avium may provide an ideal organism to investigate fundamental mechanism of protein traffic in mononuclear phagocytes. Newly formed phagosomes fuse and exchange membrane with multiple intracellular compartments including lysosomes and that the exchange of such proteins is critical to intracellular killing. We have developed methods to reconstitute phagosome fusion events in vitro and we have begun to identify the factors that are required for phagosome membrane traffic. Among the proteins that are likely to play structural and regulatory roles in phagosome membrane trafficking are the GTP binding proteins (Heterotrimeric G proteins, Rab GTP binding proteins and ADP- ribosylation factors-ARFs ) and the newly described SNARE docking proteins (i.e., receptors for the NEM-sensitive fusion protein ). Our initial experiments have successfully reconstituted the in vitro fusion of early phagosomes with endosomes. We propose to exploit the in vitro assay to determine the biochemical requirements for fusion of endosomes with phagosomes isolated from normal macrophages and from macrophages infected with M.avium. Our initial effort is to identify key GTP-binding proteins and SNARE docking proteins that mediate protein trafficking into and out of normal and M.avium phagosomes. Using membranes and cytosol from M.avium infected cells, we will investigate possible inhibitors produced by the organism. In our initial experiments we have begun to catalogue the biochemical changes occurring in phagosomal membranes during phagosome maturation. We propose to develop in vitro assays to reconstitute fusion of phagosomes with vesicles derived from the trans-Golgi Network and with lysosomes and to use these assays to determine whether products of M.avium intracellular infection impair membrane transport. Lastly, we will utilize in vitro assays using Mycobacterium avium to determine the influence of the live organism on the fusion of phagosomes with endosomes, vesicles derived from the trans Golgi Network and lysosomes.