Phagocytosis by macrophages is essential for immune surveillance and response. Although postphagocytic delivery of microbes into macrophage lysosomes typically leads to their degradation, some pathogenic microorganisms survive phagocytosis by altering the course to lysosomes. These pathogens could be counteracted by therapies that redirect phagosomes toward lysosomes or that deliver drugs specifically into those phagosomes. The goal of the work described here is to design such therapies through in-proved understanding of organelle dynamics inside macrophages. We will first describe the normal course of phagosomes toward lysosomes. In our previous studies of macropinosomes, we found that their intracellular path to the lysosome includes intermediate stages. if phagosomes proceeded toward lysosomes similarly, by progressively acquiring and losing features of endosomes, then phagosome- lysosome fusion would be only the last of a series of vesicle fusion and fission events, any of which might be inhibited by a pathogen in a phagosome. We will examine phagocytosis of IgG-opsonized particles to determine if phagosomes have properties of early or late encosomes before they fuse with lysosomes. Phagosomes of different ages will be identified by pulse-labeling with fluorescein dextran during phagocytosis, then chasing without fluorophore for defined intervals before fixing and preparing cells for immunofluorescent localization of markers for early and late endosomes and for lysosomes. The rate of phagosome acidification will be measured by quantitative fluroescence microscopy and correlated with the morphology. We will then measure the effects of various factors on the course or rate of phagosomes progression. Next, we will describe the course of phagosomes containing Salmonella typhimurium, which enter mouse macrophages via macropinocytosis. We will determine where along the path phagosomes containing Salmonella slow their progression toward lysosomes. We will then examine progression of phagosomes containing mutants of S. typhimurium that fail to induce macropinocytosis. We will also ask if Salmonella -phagosomes become removed from the traffic of other endocytic organelles, and if their permeability or transport properties differ from those of other phagosomes. Finally, we will study phagocytosis and the intracellular progression of various liposomes, modifying them to optimize uptake, to delay their delivery to lysosomes, and to target them to phagosomes containing Salmonella. We will also devise strategies to deliver molecules from liposomes into macrophage cytoplasm. Although these studies aim to kill Salmonella typhimurium in its phagosome, the information gained in the process should be applicable to other organisms that evade macrophage defense machanisms. This research should therefore provide both basic cell biology and clinically relevant therapeutic strategies.
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