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 i nclude 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.
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