"Plasmodium falciparum" transfection technology for the analysis of "var" gene regulation and knockout investigation

Abstract

Malaria is an infectious disease caused by protozoans of the genus Plasmodium, which are injected by the bite of an infected female Anopheles mosquito during a blood meal. Out of the four species that infect humans, P. falciparum is the most important. About 40% of the world’s population is at risk and 500 million cases of malaria occur every year, mainly in sub- Saharan Africa. Due arising resistance of mosquitoes against insecticides, the lack of a malaria vaccine, and emerging resistance of parasites against established drugs, research into new drugs and vaccine targets is most important. Morbidity is associated with adherence of infected red blood cells (iRBC) to endothelial tissue thereby obstructing the blood flow. The major protein conferring this cytoadherence is the P. falciparum erythrocyte membrane protein 1 (PfEMP1) anchored in the erythrocyte membrane of infected red blood cells (iRBCs). PfEMP1 is encoded by the var gene family that consists of approximately 60 members in the haploid genome of the 3D7 strain. var genes are expressed mutually exclusive, i.e. only one var gene is expressed in a parasite at a time and the rest is silenced. In this thesis we were interested in the regulation of expression and silencing of var genes. For this purpose we generated transgenic parasite lines that harbored plasmids expressing luciferase under the control of various fragments of the var gene upstream region. By comparing luciferase activities in the different lines we identified the core promoter, two activator-binding sites and a repressorbinding site. Additionally, we identified a regulatory sequence on the var upstream region that interacts with the var intron during silencing. Using quantitative RT-PCR with specific primers for every var gene we were unable to confirm that the var upstream regions on the transfected plasmids were recognized by the machinery that ensures mutually exclusive transcription. In the second part of this thesis, we evaluated phosphodiesterase 1 (PDE1) as a possible drug target in P. falciparum by creating a knockout parasite line. PDEs are known drug targets in humans where selective PDE inhibitors are being used to treat a wide range of diseases. In trypanosomiasis research PDE inhibitors are promising drug candidates against sleeping sickness, Nagana or Chagas’ disease. Out of the four PDEs described for P. falciparum we focused on PfPDE1, which is expressed in blood stage parasites and in gametocytes and sporozoites. We observed a slightly faster growth of the knockout parasite line compared to the wildtype indicating that the knockout parasite had a shorter erythrocytic lifecycle. We found that PfPDE1 is responsible for 20% of the total cGMP activity observed in late blood stage parasites and that there is no rescue mechanism of the remaining PDEs to compensate for the loss of activity. We were not able to localize PfPDE1 in the parasite. The fact that we could delete PfPDE1 clearly shows that it is not an essential gene in blood stage forms of P. falciparum and hence not a good drug target. Nevertheless we created a useful tool to investigate the role of PfPDE1 in the development of sexual parasite forms.