Identifying the molecular mechanisms for phagolysosome biogenesis. (360G-Wellcome-079705_Z_06_Z)

Phagocytosis is an essential process by which professional phagocytes engulf invading pathogens, apoptotic cells and other foreign particles. Phagocytosis triggers the activation of multiple transmembrane signaling pathways, leading to the reorganization of the actin cytoskeleton and the formation of a sealed intracellular compartment, the phagosome. The newly formed phagosome matures by undergoing multiple fusion events with endocytic organelles and finally fusion events with lysosomes to form the phagolysosome. It is within the phagolysosome that hydrolytic enzymes kill pathogens and allow phagocytosed material to be 'digested'. Pathogen survival mechanisms involve escaping from the phagocytic pathway (Shigella, Listeria), preventing phagolysosome biogenesis (Mycobacterium, Salmonella) or surviving the harsh phagolysosome environment itself (Leishmania). Unlike other endocytic events, the molecular mechanisms in phagosome maturation remain essentially descriptive and in particular, the molecular machinery involved in phagolysosome biogenesis is ill defined. The focus of this proposal is therefore to determine the molecular mechanisms for phagolysosome formation by developing an in vitro phagosome-lysosome fusion assay. The assay in conjunction with genetic and proteomic screens will be used to investigate the mechanism(s) by which pathogens survive intracellularly, in particular those pathogens that arrest phagolysosome formation.