Functional analysis of kinases in the malaria parasite Plasmodium falciparum

Despite massive control efforts and extensive successes in reducing the malaria burden in the past decades, malaria still causes more than 400’000 deaths worldwide each year with the highest toll on African children under the age of five. The protozoan parasite Plasmodium falciparum is responsible for the majority of disease burden and deaths and its complex life cycle represents a challenge in disease control and treatment. During the intraerythrocytic asexual cycle, a subset of parasites undergoes sexual commitment and their progeny develop into gametocytes, the parasite form transmissible from humans to Anopheles vectors. Extensive research has identified that a drop in the level of the host lipid lysophosphatidylcholine is perceived by the parasite and induces sexual commitment by activating the expression of the transcription factor PfAP2-G, the master regulator of sexual conversion.
In this PhD project, I aimed at elucidating a potential upstream molecular pathway that induces expression of PfAP2-G. In more detail, using a PfAP2-G reporter cell line and other techniques to study sexual commitment, we investigated the involvement of nine P. falciparum kinases in this putative signalling pathway. We therefore generated a variety of transgenic parasite lines including kinase knockout as well as conditional knockdown, overexpression and inducible knockout mutants. However, investigating the effect of increased and decreased kinase expression levels did not conclusively identify any of these kinases as pathway component involved in sexual commitment signalling.
Nevertheless, having generated a variety of transgenic cell lines, I further studied the effect of changes in kinase expression levels on asexual and sexual intraerythrocytic parasite development. Interestingly, I identified that while the mitogen-activated protein kinase (MAPK) PfMAP-2 is dispensable for asexual parasite development, it is essential for male gametogenesis. Furthermore, we confirmed the essential role of the casein kinase 2 catalytic subunit PfCK2α in erythrocyte invasion by merozoites and likely also asexual parasite development. In addition, we discovered that PfCK2α is indispensable for sexual development and to my knowledge, this is the first kinase identified as being essential for gametocyte maturation. Finally, I studied the function of PfPKAc, the catalytic subunit of the cAMP-dependent protein kinase A. I confirmed that PfPKAc is required for asexual parasite growth due to its importance in erythrocyte invasion. I further showed that conditional overexpression of PfPKAc is lethal for intraerythrocytic asexual parasite development. Interestingly, however, selection of parasites tolerant to PfPKAc overexpression was possible. All six independently obtained survivor populations, but none of the unselected overexpression-sensitive mother clones, carried mutations in the putative serine/threonine kinase Pf3D7_1121900. We identified Pf3D7_1121900 as the P. falciparum orthologue of the 3-phosphoinositide-dependent protein kinase-1 (PfPDK1). In model eukaryotes, the PDK1 kinase is known to phosphorylate and activate various AGC kinases including PKA. Using targeted mutagenesis, I was able to show the essential role for PfPDK1-dependent phosphorylation of PfPKAc. This phosphorylation seems to be crucial for PfPKAc activity in P. falciparum.
The present PhD thesis describes new insights into the function of three essential kinases in asexual and sexual development of P. falciparum. Hence, this work broadens our knowledge on kinase function and regulation in malaria parasites in general and provides new potential antimalarial drug targets. Finally, the tools developed during this work will provide important resources for future research on sexual commitment und protein function in P. falciparum.