Identification and characterization of novel factors mediating Plasmodium falciparum egress from the red blood cell

红细胞内的原生动物寄生虫恶性疟原虫的复制以及这些细胞的相关转化和破坏是疟疾临床症状的原因。在这些细胞中，寄生虫在寄生虫空泡（PV）内繁殖，直到它在外出过程中破坏空泡和宿主细胞膜，这对于感染的传播是必不可少的。虽然这一生物过程提供了有前途的干预策略，只有少数寄生虫蛋白参与出口已被确定到目前为止，并没有很好地了解潜在的分子机制。出口与被称为外丝体的顶端分泌细胞器的释放有关，外丝体含有两种已知的出口调节剂，枯草杆菌蛋白酶样丝氨酸蛋白酶SUB1和天冬氨酸蛋白酶浆蛋白酶X（PMX）。SUB1被PMX蛋白水解切割，并从外排体释放到PV，在PV中它激活参与外出的其他蛋白质。考虑到这两种蛋白质对寄生虫释放的中心功能以及它们是迄今为止唯一已知的外丝体成分的事实，在该项目中提出将邻近依赖性生物素化技术应用于恶性疟原虫寄生虫以鉴定另外的外丝体蛋白。为了实现这一点，将工程化寄生虫，使其在外排体中条件性表达生物素连接酶miniTurbo或抗坏血酸过氧化物酶APEX 2，从而允许纯化和随后基于质谱法鉴定外排体蛋白。这种分析将补充类似的蛋白质组学研究的另一组顶端分泌细胞器称为“微线”，通常涉及的入侵，但也被证明有助于出口相关的顶复门寄生虫弓形虫。随后，将使用条件性敲低和敲除技术定位和功能表征选定数量的推定的新型外消旋体和微消旋体蛋白。这将允许识别和验证代表寄生虫增殖基石的出口级联中的新参与者。

The replication of the protozoan parasite Plasmodium falciparum within red blood cells and the associated transformation and destruction of these cells is responsible for the clinical symptoms of malaria. In these cells, the parasite multiplies within a parasitophorous vacuole (PV) until it ruptures the vacuole and the host cell membrane in the process of egress, which is essential for propagation of the infection. Although this biological process offers promising intervention strategies, only a few parasite proteins involved in egress have been identified so far and the underlying molecular mechanisms are not well understood. Egress is linked to the release of apical secretory organelles termed exonemes, which contain two known regulators of egress, the subtilisin-like serine protease SUB1 and the aspartic protease plasmepsin X (PMX). SUB1 is proteolytically cleaved by PMX and released from exonemes to the PV, where it activates other proteins involved in egress. Given the central function of both of these proteins for parasite release and the fact that they are the only known constituents of exonemes so far, it is proposed in this project to apply proximity-dependent biotinylation techniques to P. falciparum parasites in order to identify additional exoneme proteins. To achieve this, parasites will be engineered that conditionally express the biotin ligase miniTurbo or the ascorbate peroxidase APEX2 in the exonemes that allows purification and subsequent mass spectrometry based identification of exoneme proteins. This analysis will be complemented with a similar proteomic study of another set of apical secretory organelles termed “micronemes” that are generally implicated in invasion but which were also shown to contribute to egress in the related Apicomplexan parasite Toxoplasma gondii. Subsequently, a selected number of putative novel exoneme and microneme proteins will be localized and functionally characterized using conditional knockdown and knockout techniques. This will allow to identify and validate novel players in the egress cascade that is representing a corner stone for parasite proliferation.