The Leishmania Phagosome: Isolation by Dual-Fluorescence Sorting

This project developed a novel, efficient technique for isolating Leishmania donovani-containing phagosomes to enable in-depth proteomic analysis, offering critical insight into host-pathogen interactions and the macrophage host response to infection. Building upon methods used for isolating other pathogen-containing vacuoles (like those of Salmonella and Legionella), and adapting prior work on other Leishmania species, our technique uses Fluorescence-Activated Cell Sorting (FACS). Mouse bone marrow-derived macrophages (BMDMs) were infected for 24 hours with a TdTomato fluorescent L. donovani line. The macrophages were then lysed via nitrogen cavitation, a gentle method that preserves intracellular amastigotes and the integrity of the phagosomes. The phagosomal membrane was stained using CellVue Burgundy, and L. donovani-containing phagosomes were subsequently isolated by FACS based on dual fluorescence (parasite and phagosome membrane stain). The established method successfully yields highly intact, isolated parasite-containing phagosomes, providing a strong foundation for future research. This successful isolation technique creates a reliable basis not only for the initial goal of proteomics but also for broader ‘omics’ studies, including lipidomics and interactomics, to fully characterize the Leishmania donovani-containing phagosome environment. While the results are promising, future optimization steps are outlined to enhance efficiency and validity for high-throughput applications.