LISTERIA MONOCYTOGENES AND PHAGOSOME MEMBRANE TRAFFIC

包括单核细胞增生李斯特菌（Listeria monocytogenes，LM）在内的多种人类病原体通过干扰膜运输事件而在宿主细胞内驻留和繁殖。 LM被内化到吞噬体中，在吞噬体中它积极地抑制吞噬体的成熟。 毒力LM逃逸到细胞质中。缺乏大肠杆菌溶血素的李斯特菌突变体（LMhly-）不能进入细胞质，但保留了阻断吞噬体成熟的能力。 新形成的吞噬体通过一系列连续的膜融合事件，随后是吞噬体-溶酶体融合，通过动态重塑而成熟。 每个融合事件似乎都受到RabGT 3的调控。 Rab 5a是吞噬体-内体融合所必需的。 活LMhly通过干扰Rab 5a功能来阻断吞噬体成熟。 因此，Rab 5a的分析提供了一个有吸引力的机会来研究吞噬体成熟的调节和LM干扰该过程的机制。 我们的中心假设是Rab 5a的GTP/GDP循环与吞噬体成熟和有效吞噬体-溶酶体融合所需的下游GTP酶的激活紧密耦合。 干扰素γ通过选择性诱导Rab 5a合成增强LM的细胞内杀伤。 我们的目标是确定LM和Rab 5a在吞噬体成熟中的作用，确定干扰素γ在促进该过程中的作用，并描述下游包括Rab 7和Rab 11的GTP酶的作用。 具体目的包括确定LM吞噬过程中控制鸟嘌呤核苷酸状态Rab 5a的信号转导机制。 我们还将描绘蛋白激酶B/akt的作用，Rab 5的一个已知的调节器。 第二个具体目标集中于IFN γ刺激吞噬体成熟和杀伤的机制。 干扰素γ选择性诱导Rab 5a合成和加工。 我们将研究IFN γ提高Rab 5a的异戊二烯化的机制。 我们将使用吞噬体-溶酶体融合测定来确定IFN γ处理是否增强Rab 5a与下游Rab GTP酶Rab 7和Rab 11的偶联。 我们将使用缺乏IFN γ受体的基因敲除小鼠来证实该受体所起的作用。 由于IFN γ处理选择性地诱导Rab 5a而不是Rab 5 b或Rab 5c，并且由于活LM导致Rab 5a在吞噬体上积累，因此我们将探索内吞装置由不同Rab 5同种型标记的亚室组成的可能性。Rab 5a可以特异性地将内吞装置连接到发育中的吞噬体，而其他Rab 5同种型可能具有不同的功能。 使用表位标记的Rabs结合光学和电子显微镜，并使用活细胞中的GFP-Rab 5同种型，我们将鉴定在吞噬体-内体融合中起作用的内体的子集。 我们将使用GFP-Rab 5在真实的时间观察GFP-Rab 5同种型标记的内体与新形成的携带活的或死的单核细胞增生李斯特菌的吞噬体的对接和融合。 我们将使用GFP-Rab 5、GFP-Rab 7和GFP-Rab 11确定IFN γ处理对囊泡运输进出LM吞噬体的影响。

A wide variety of human pathogens including Listeria monocytogenes (LM) take up residence and thrive within host cells by interfering with membrane trafficking events. LM is internalized into phagosomes where it actively inhibits maturation of the phagosome. Virulent LM escapes to the cytoplasm. Listeria mutants (LMhly-) that lack listeriolysin fail to access the cytoplasm but retain the ability block phagosome maturation. Newly formed phagosomes mature by dynamic remodeling via a series of sequential membrane fusion events followed by phagosome-lysosome fusion. Each fusion event appears to be regulated by a RabGTPase. Rab5a is required for phagosome- endosome fusion. Live LMhly- blocks phagosome maturation by interfering with Rab5a function. Thus, analysis of Rab5a provides an attractive opportunity to examine the regulation of phagosome maturation and the mechanism by which LM interferes with the process. Our central hypothesis is that the GTP/GDP cycle of Rab5a is tightly coupled to phagosome maturation and the activation of downstream GTPases required for efficient phagosome-lysosome fusion. Interferon gamma enhances intracellular killing of LM by selectively inducing Rab5a synthesis. Our goal is to determine how LM and Rab5a function in phagosome maturation, to define the role of interferon gamma in facilitating the process and to delineate the role of GTPases operating down-stream including Rab7 and Rab11. The Specific Aims include identifying the signal transduction mechanisms that control the guanine nucleotide status Rab5a during phagocytosis of LM. We will also delineate the role of protein kinase B/akt, a known regulator of Rab5. The second specific aim focuses on the mechanism by which IFNgamma stimulates phagosome maturation and killing. Interferon gamma selectively induces Rab5a synthesis and processing. We will investigate the mechanism by which IFNgamma elevates the prenylation of Rab5a. We will use phagosome-lysosome fusion assays to determine whether IFNgamma treatment enhances coupling of Rab5a to downstream Rab GTPases, Rab7 and Rab11. We will use knock out mice lacking the IFNgamma receptor to confirm the role played by this receptor. Since IFNgamma treatment selectively induces Rab5a but not Rab5b or Rab5c and since live LM causes Rab5a to accumulate on phagosomes, we will explore the possib ility that the endocytic apparatus is composed of sub-compartments marked by different rab5 isoforms. Rab5a may specifically connect the endocytic apparatus to the developing phagosomes whereas other Rab5 isoforms may have different functions. Using epitope tagged Rabs coupled with both light and electron microscopy and using GFP-Rab5 isoforms in living cells, we will identify a subset of endosomes that function in phagosome-endosome fusion. We will use GFP-Rab5 to observe in real time the docking and fusion of GFP-Rab5 isoform-marked endosomes to newly formed phagosomes harboring live or dead Listeria monocytogenes. We will determine the effects of IFNgamma treatment on vesicular traffic into and out of LM phagosomes using GFP-Rab5, GFP-Rab7 and GFP-Rab11.