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

为了生存和复制作为一个寄生种群，L。主要的无鞭毛体必须经历感染、细胞内增殖、宿主细胞退出和进入新宿主细胞的循环。通过结合体外人类活细胞成像、退出过程的流式细胞术定量和小鼠模型中感染部位的活体双光子成像，我们旨在确定利什曼原虫持续存在、繁殖和发病机制的基本过程的机制。在SPP的第一个资助期，我们发现利什曼原虫在体内和体外的退出是由寄生虫的增殖状态触发的。高度增殖的无鞭毛体是参与退出的主要形式，并且促炎性巨噬细胞被发现是强烈增加增殖的优选生态位。证明了体外人巨噬细胞和体内小鼠组织中的细胞凋亡的参与，其中大多数在细胞材料从最初感染的细胞转移的背景下观察到退出。这表明直接的细胞间转移，仅最小程度地暴露于细胞外环境。此外，除了细胞凋亡外，细胞凋亡也可能有助于这一过程。此外，我们发现的证据表明，利什曼原虫增殖调节宿主细胞的代谢，如脂质受体CD 36的表达，这可能会影响宿主细胞死亡，以及对其他cells.We现在的目标是阐明参与细胞凋亡和pyroptosis在病原体退出的分子水平上使用BLaER 1细胞系和细胞死亡特异性caspase-3和Gasdermin D基因敲除。这些实验将在小鼠模型中使用胱天蛋白酶-3抑制和Gasdermin D缺陷宿主细胞进行补充。使用增殖报告基因和KBMA系统，我们希望剖析支持无鞭毛体有效退出的宿主细胞表型，这将使FACS分选富集和随后的质谱分析以及更详细的退出机制的功能和相关成像成为可能。最后，为了研究生产性细胞间转移所需的代谢变化，我们将表征宿主细胞的脂质代谢，并解决CD 36缺乏对病原体退出以及新宿主细胞对病原体摄取的影响。

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.