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

参与利什曼原虫宿主细胞退出和细胞间转移的细胞死亡途径的分子和细胞特征

• 批准号: 446496937
• 负责人: Professor Dr. Andreas Müller
• 金额: --
• 依托单位: Institut für Molekulare und Klinische Immunologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/446496937?language=en
• 关键词: Molecular; cellular; characterization; cell; death

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.

为了作为寄生虫种群生存和复制，主要无鞭毛虫必须经历感染、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、流式细胞术定量退出过程和小鼠模型中感染部位的活体双光子成像，我们旨在确定这一基本过程的潜在机制，该过程对利什曼原虫的持续、繁殖和致病至关重要。使用基因编码的增殖报告系统和生长抑制KBMA病原体，我们在SPP的第一个资助期发现，无论是体内还是体外，利什曼原虫的退出都是由寄生虫的增殖状态触发的。高度增殖的无鞭毛体是退出的主要形式，促炎症的巨噬细胞被发现是强烈促进增殖的首选利基细胞。在体外的人巨噬细胞和体内的小鼠组织中，证实了细胞凋亡的参与，因此，大多数情况下，EXIT是在最初感染的细胞的细胞物质转移的背景下观察到的。这表明细胞间的直接转移只需极少的细胞外环境暴露。此外，除细胞凋亡外，上睑下垂可能也参与了这一过程。此外，我们还发现了利什曼原虫增殖调节宿主细胞代谢的证据，如脂类受体CD36的表达，这可能影响宿主细胞的死亡，也可能影响其他细胞的胞吐作用。我们现在的目标是通过BLaER1细胞株和细胞死亡特异性caspase-3和Gasdermin D基因敲除，在分子水平上阐明细胞凋亡和嗜热症在病原体退出中的作用。这些实验将通过抑制caspase-3和在小鼠模型中缺乏Gasdermin D的宿主细胞来补充。使用增殖报告系统和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分选浓缩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，为了研究有效的细胞间转移所需的代谢变化，我们将表征宿主细胞的脂代谢，并解决CD36缺乏对病原体退出以及新宿主细胞摄取病原体的影响。