Functional characterization of phospholipases in the malaria parasite Plasmodium falciparum

疟原虫，疟疾的病原体，感染肝细胞和红细胞（红细胞）。在这些细胞内，寄生虫被寄生液泡膜（PVM）包围。因此，为了从宿主细胞中释放出来并传播感染，寄生虫必须破坏PVM和宿主细胞膜。这种膜破裂是如何在分子水平上发生的，以及它是如何被控制的，目前我们只部分了解，而且迄今为止，只有非常有限数量的寄生虫蛋白参与了这一过程。我们之前的研究表明，在寄生虫从肝细胞排出时，寄生虫磷脂酶介导PVM破裂。寄生虫的磷脂酶是否同样参与了寄生虫从红细胞中的释放尚不清楚，而且大多数疟原虫的磷脂酶没有被表征。因此，本项目拟从功能上分析人类疟疾寄生虫恶性疟原虫在红细胞内发育过程中磷脂酶的作用。首先，我们将通过靶向基因破坏探讨在无性血期寄生虫中显示表达的那些酶的冗余性。常规敲除技术无法删除的关键基因将进一步使用条件系统进行分析，分析其功能缺失表型并阐明其细胞定位。为了揭示不同磷脂酶之间潜在的相互作用和协同功能，将产生并分析针对酶子集的双敲除寄生虫。最后，我们的目的是研究寄生虫磷脂酶是否通过与出口相关的枯草杆菌样丝氨酸蛋白酶SUB1的蛋白水解裂解而被激活。为此，将测试在几种假定的磷脂酶中确定的预测的SUB1切割位点的功能重要性。总之，我们的工作将增加我们对导致寄生虫从宿主细胞释放的分子机制的了解，并可能导致寄生虫特异性干预策略的新靶点的鉴定。

Plasmodium parasites, the causative agents of malaria, infect hepatocytes and red blood cells (RBCs). Within these cells, parasites are surrounded by a parasitophorous vacuole membrane (PVM). Consequently, for their release from host cells and to propagate their infection, parasites have to disrupt the PVM and the host cell membrane. How this membrane rupture occurs on a molecular level and how it is controlled is only partially understood and only a very limited number of parasite proteins involved in this process have been identified so far. We previously showed that a parasite phospholipase mediates PVM rupture during parasite egress from hepatocytes. Whether parasite phospholipases are similarly involved in parasite release from RBCs is not known and most of the Plasmodium phospholipases are not characterized. In this project, it is thus proposed to functionally analyze the role of phospholipases of the human malaria parasite Plasmodium falciparum during its development within red blood cells. First, we will probe into the redundancy of those enzymes that show expression in asexual blood stage parasites by targeted gene disruption. Essential genes, which cannot be deleted using conventional knockout techniques, will further be analyzed using conditional systems, their loss-of-function phenotypes analyzed and their cellular localization elucidated. In order to reveal a potential interplay and synergistic functions between different phospholipases, double knockout parasites targeting a subset of enzymes will be generated and analyzed. Finally, we aim to investigate, whether parasite phospholipases are activated by proteolytic cleavage of the egress-associated subtilisin-like serine protease SUB1. For this, the functional importance of predicted SUB1 cleavage sites identified in several putative phospholipases will be tested. In conclusion, our work will increase our knowledge of the molecular machinery leading to parasite release from host cells and might lead to the identification of novel targets for parasite-specific intervention strategies.