Molecular and cellular characterization of cell death pathways involved in Leishmania host cell exit and cell-to-cell transfer

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In order to survive and replicate as a parasite population, L. major amastigotes have to undergo cycles of infection, intracellular proliferation, host cell exit and entry into new host cells. By the combination of both in vitro human live cell imaging, flow cytometry quantification of the exit process and intravital 2-photon imaging of the infection site in the mouse model, we aim to identify mechanisms underlying this fundamental process crucial for Leishmania persistence, propagation and pathogenesis.Using a genetically encoded proliferation reporter system and growth-inhibited KBMA pathogens, we found in the first funding period of the SPP that Leishmania exit, both in vivo and in vitro, is triggered by the parasite’s proliferation state. Highly proliferating amastigotes are the predominating form involved in exit and pro-inflammatory macrophages were found as the preferred niche for strongly increased proliferation. Involvement of apoptosis in human macrophages in vitro and in the mouse tissue in vivo was demonstrated, whereby exit was mostly observed in the context of cell material transfer from the initially infected cell. This suggests a direct cell-to-cell transfer with only minimal exposure to the extracellular milieu. Also, pyroptosis, in addition to apoptosis, might contribute to the process. Furthermore, we found evidence that Leishmania proliferation modulates the host cell metabolism such as expression of the lipid receptor CD36, which might impact both on host cell death, as well as on efferocytosis by other cells.We now aim to elucidate the involvement of both apoptosis and pyroptosis in pathogen exit on a molecular level by using the BLaER1 cell line and cell death-specific caspase-3 and Gasdermin D knockouts. These experiments will be complemented using caspase-3 inhibition and Gasdermin D deficient host cells in the mouse model. Using both the proliferation reporter and KBMA systems, we want to dissect the host cell phenotypes supporting efficient exit of amastigotes, which will enable FACS sorting enrichment and consequent mass spectrometry analyses as well as functional and correlative imaging of the exit mechanism in more detail. Finally, to investigate the metabolic changes required for a productive cell-to-cell transfer, we will characterize host cell lipid metabolism and address the impact of CD36 deficiency both for pathogen exit as well as for pathogen uptake by new host cells.