M AVIUM INHIBITION OF PHAGOSOME MEMBRANE TRAFFIC

禽分枝杆菌已成为艾滋病的主要条件致病菌。 病人。禽类分枝杆菌通常驻留在 巨噬细胞限制其消化和加工的途径 宿主细胞的能力。访问似乎受到了损害的限制 膜蛋白进出禽分枝杆菌和 结核分枝杆菌噬菌体。因为这一点和 从细胞内的M.avium到结核分枝杆菌，M.avium可能提供一种理想的 生物将研究蛋白质运输的基本机制 单核巨噬细胞。新形成的噬菌体融合交换膜 有包括溶酶体在内的多个细胞内隔室， 这种蛋白质的交换对细胞内的杀伤至关重要。我们有 开发了在体外重建吞噬小体融合事件的方法 已经开始确定吞噬小体所需的因子 膜流量。在可能发挥结构性作用的蛋白质中 而吞噬体膜转运的调节作用是GTP结合 蛋白质(异三聚体G蛋白、Rab GTP结合蛋白和ADP- 核糖化因子与新发现的SNARE对接蛋白 (即NEM敏感融合蛋白的受体)。我们最初的 实验成功地重建了早期的体外融合 吞噬小体和内小体。我们建议利用体外试验来 确定与内体融合的生化要求 从正常巨噬细胞和感染巨噬细胞分离的吞噬小体 与M.avium。我们最初的努力是确定关键的GTP结合蛋白 和介导蛋白质进出的陷阱对接蛋白 正常和禽类分枝杆菌吞噬小体。利用M.avium的膜和胞浆 受感染的细胞，我们将调查可能的抑制物由 有机体。在我们最初的实验中，我们已经开始对 吞噬体膜在吞噬过程中的生化变化 成熟。我们建议发展体外试验来重建融合。 具有来自跨高尔基网络的囊泡的吞噬小体 并使用这些检测方法来确定禽类分枝杆菌的产物 细胞内感染损害了膜的运输。最后，我们将利用 用禽分枝杆菌进行体外测定，以确定不同浓度的金黄色葡萄球菌对 吞噬小体与内小体、囊泡融合的活生物体 来源于跨高尔基网络和溶酶体。

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.