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

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.