Characterization and functional analysis of the Artemisinin resistance protein Kelch13 in Plasmodium falciparum

Abstract

The infectious disease malaria kills more than 400,000 people every year, mainly children younger than 5 years. The human malaria parasite Plasmodium falciparum is a unicellular parasite that belongs to the genus Apicomplexa. The parasite is transmitted to humans through the bite of an Anopheles mosquito. The following asexual reproduction of the parasite in erythrocytes of the human host cause the symptoms of the disease. Artemisinin and its derivatives (ART) are currently the most important agents for the control and treatment of malaria and are typically administered with a partner drug as an Artemisinin based combination therapy. ART are activated in the parasite by hemoglobin degradation products that result from host cell cytosol that the intracellular parasite endocytoses during blood stage growth. Alarmingly, since 2008, parasites with lower susceptibility to the drug (resistance) have emerged in Southeast Asia which has led to treatment failures and threatens recent success of malaria control. Artemisinin resistant parasites have single point mutations in a protein named Kelch13. The potential molecular mechanism of resistance was recently cleared up: less endocytic uptake of hemoglobin in resistant parasites leads to less hemoglobin that is available for digestion in the parasite and hence less artemisinin activation. To gain insight into the function and cellular location of Kelch13, former studies performed dimerization induced quantitative DIQ-BioID experiments with Kelch13 wild type parasites and proteins in close proximity to Kelch13 were identified. These proteins co-located with Kelch13 in an unknown compartment. Beside proteins such as UBP1 and Eps15, most of the other Kelch13 compartment proteins were Plasmodium specific (designated as Kelch13 interaction candidates (KICs)). To better understand the involvement of the Kelch13-defined compartment in ART resistance, it was assessed in this thesis whether other Kelch13 interacting candidates (KICs) were also involved in resistance which would substantiate their involvement in the Kelch13 pathway. These experiments showed that the disruption of three of the non-essential KICs (MCA2-TGD, KIC4-TGD and KIC5-TGD) led to reduced susceptibility to ART. It was shown recently that inactivation of UBP1, Eps15 and KIC7 resulted in decreased susceptibility of the parasites to ART and together with the results of the KIC-TGD cell lines it can be concluded that the Kelch13 compartment is involved in ART resistance. Furthermore, it is demonstrated in this thesis that the MCA2-TGD and KIC5-TGD parasites showed impaired fitness levels and that KIC7 and UBP1 are essential for parasite survival in ring as well as in trophozoite stages. Inactivation of KIC7 via knock sideways resulted in a similar phenotype to that of UBP1 inactivation when stage-specific parasite development was monitored using Giemsa smears, in support of a similar function of these proteins in parasite development. In contrast to KIC7 and UBP1, the data presented here also confirm previous findings that Kelch13 is essential for parasite survival in ring stages only. Moreover, the data presented in this thesis shows that inactivation of KIC7 leads to increased vesicle formation or accumulation in the parasite, supporting the hypothesis that endolysosomal transport of vesicles to the digestive vacuole might be impaired after KIC7 inactivation. Additionally, the data indicate that less hemozoin is present upon KIC7 inactivation, giving further evidence that KIC7 is involved in endocytosis. To further investigate the resistance causing mechanism and to assess how mutations in the Kelch13 protein influence ART resistance, it was determined whether a changed interaction profile of parasites with a resistance-conferring Kelch13 mutation could lead to resistance. Using DIQ-BioID experiments with the Kelch13 mutant parasites the interaction profile of mutated and wild type Kelch13 was compared. However, no notable differences were observed in the two cell lines, indicating that mutating Kelch13 does not change a specific interaction of the protein. Rather it was shown using quantitative immunoblot analysis and fluorescence intensity quantification that mutated Kelch13 is less abundant in the cell compared to wild type Kelch13. Moreover, using episomally expressed complementation constructs, it was shown in this thesis that increased Kelch13 abundance in resistant parasites leads to the loss of resistance. These findings indicated that Kelch13 protein levels influence resistance and gave evidence that mutated Kelch13 reduces its abundance and hence the overall Kelch13 activity per cell. In order to better understand the cellular function of Kelch13 and to investigate the functions of the individual domains of Kelch13, constructs consisting of different Kelch13 domain combinations were episomally expressed on the resistant Kelch13 mutant background. The importance of the domains for the cellular localization of Kelch13 was determined by fluorescence microscopy and the ability of constructs to maintain the function of Kelch13 was assessed by determining the resistance level, assuming that a functional construct reverts the parasites to sensitivity. These experiments indicated that the Plasmodium specific N-terminus of Kelch13 promotes function and that the conserved C-terminus containing the Kelch propeller domain does not display a role in protein function but rather might act as a regulator of Kelch13 stability. Semi-quantitative Western Blot analysis indicated that episomal constructs containing the Kelch propeller domain have significantly higher abundance in the cell than constructs lacking the Kelch propeller domain. These findings suggest a function of the Kelch propeller domain as a regulator of protein abundance of Kelch13. The data presented in this this thesis show that proteins of the Kelch13 defined compartment are involved in artemisinin resistance and that resistance is influenced by Kelch13 abundance. Furthermore, the data indicate that the N-terminal part of Kelch13 fulfills the function of the protein and the C-terminal part is a regulatory domain and influences Kelch13 abundance.