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

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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.