LISTERIA MONOCYTOGENES AND PHAGOSOME MEMBRANE TRAFFIC

包括单核细胞增多性李斯特氏菌在内的多种人类病原体通过干扰膜转运活动在宿主细胞内定居和繁衍。LM内化到吞噬小体中，在那里它积极地抑制吞噬小体的成熟。毒力强的Lm逃逸到细胞质中。缺乏李斯特溶素的李斯特菌突变体(LMhly-)不能进入细胞质，但保留了阻止吞噬小体成熟的能力。新形成的吞噬小体通过一系列连续的膜融合事件动态重塑成熟，随后是吞噬小体-溶酶体融合。每个融合事件似乎都受到RabGTP酶的调控。吞噬小体-内小体融合需要Rab5a。活性LMhly-通过干扰Rab5a功能来阻止吞噬小体成熟。因此，对Rab5a的分析为研究吞噬小体成熟的调节以及LM干扰这一过程的机制提供了一个有吸引力的机会。我们的中心假设是，Rab5a的GTP/GDP循环与吞噬小体成熟和下游GTP酶的激活密切相关，这是有效的吞噬小体-溶酶体融合所必需的。干扰素通过选择性地诱导Rab5a的合成来增强对LM的细胞内杀伤作用。我们的目标是确定Lm和Rab5a在吞噬小体成熟过程中的作用，确定干扰素在促进这一过程中的作用，并描述包括Rab7和Rab11在内的下游GTP酶的作用。具体目的包括确定在LM吞噬过程中控制鸟嘌呤核苷酸状态Rab5a的信号转导机制。我们还将描述蛋白激酶B/AKT的作用，蛋白激酶B/AKT是Rab5的已知调节因子。第二个具体目标集中在干扰素刺激吞噬小体成熟和杀伤的机制上。干扰素-γ选择性地诱导Rab5a的合成和加工。我们将研究IFNGamma上调Rab5a预烯基化的机制。我们将使用吞噬小体-溶酶体融合分析来确定IFNGamma处理是否增强了Rab5a与下游Rab GTP酶Rab7和Rab11的偶联。我们将使用缺乏IFNGamma受体的基因敲除小鼠来证实该受体所起的作用。由于IFNGamma处理选择性地诱导Rab5a，而不诱导Rab5b或Rab5c，并且由于live LM导致Rab5a积聚在吞噬小体上，我们将探索内吞机构由不同Rab5亚型标记的亚室组成的可能性。Rab5a可以特异性地将吞噬小体与内吞细胞器连接，而其他Rab5亚型可能具有不同的功能。利用表位标记的RAB结合光镜和电子显微镜，并在活细胞中使用GFP-Rab5亚型，我们将鉴定在吞噬小体-内小体融合中起作用的内小体的一个子集。我们将使用GFP-Rab5来实时观察GFP-Rab5亚型标记的内小体与新形成的含有活的或死亡的单核细胞增多性李斯特菌的吞噬小体的对接和融合。我们将使用GFP-Rab5、GFP-Rab7和GFP-Rab11来确定IFNGamma处理对进出LM噬菌体的囊泡运输的影响。

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.