Functional characterization of both MAP kinases of the human malaria parasite Plasmodium falciparum by reverse genetics

The kinome of the human malaria parasite Plasmodium falciparum includes two genes encoding mitogen-activated protein kinase (MAPK) homologues, pfmap-1 and pfmap-2, but no clear orthologue of the MAPK kinase (MAPKK) family, raising the question of the mode of activation and function of the plasmodial MAPKs. Functional studies in the rodent malaria model Plasmodium berghei recently showed the map-2 gene to be dispensable for asexual growth and gametocytogenesis, but essential for male gametogenesis in the mosquito vector. Here, we demonstrate by using a reverse genetics approach that the map-2 gene is essential for completion of the asexual cycle of P. falciparum, an unexpected result in view of the non-essentiality of the orthologous gene for P. berghei erythrocytic schizogony. This validates Pfmap-2 as a potential target for chemotherapeutic intervention. In contrast, the other P. falciparum MAPK, Pfmap-1, is required neither for in vitro schizogony and gametocytogenesis in erythrocytes, nor for gametogenesis and sporogony in the mosquito vector. However, Pfmap-2 protein levels are elevated in pfmap-1(-) parasites, suggesting that Pfmap-1 fulfils an important function in asexual parasites that necessitates compensatory adaptation in parasites lacking this enzyme.

SAM domain-dependent activity of PfTKL3, an essential tyrosine kinase-like kinase of the human malaria parasite Plasmodium falciparum

Over the last decade, several protein kinases inhibitors have reached the market for cancer chemotherapy. The kinomes of pathogens represent potentially attractive targets in infectious diseases. The functions of the majority of protein kinases of Plasmodium falciparum, the parasitic protist responsible for the most virulent form of human malaria, remain unknown. Here we present a thorough characterisation of PfTKL3 (PF13_0258), an enzyme that belongs to the tyrosine kinase-like kinase (TKL) group. We demonstrate by reverse genetics that PfTKL3 is essential for asexual parasite proliferation in human erythrocytes. PfTKL3 is expressed in both asexual and gametocytes stages, and in the latter the protein co-localises with cytoskeleton microtubules. Recombinant PfTKL3 displays in vitro autophosphorylation activity and is able to phosphorylate exogenous substrates, and both activities are dramatically dependent on the presence of an N-terminal "sterile alpha-motif" domain. This study identifies PfTKL3 as a validated drug target amenable to high-throughput screening.

Birkhäuser-Verlag, Springer2010

Malaria: targeting parasite and host cell kinomes

Malaria still remains one of the deadliest infectious diseases, and has a tremendous morbidity and mortality impact in the developing world. The propensity of the parasites to develop drug resistance, and the relative reluctance of the pharmaceutical industry to invest massively in the developments of drugs that would offer only limited marketing prospects, are major issues in antimalarial drug discovery. Protein kinases (PKs) have become a major family of targets for drug discovery research in a number of disease contexts, which has generated considerable resources such as kinase-directed libraries and high throughput kinase inhibition assays. The phylogenetic distance between malaria parasites and their human host translates into important divergences in their respective kinomes, and most Plasmodium kinases display atypical properties (as compared to mammalian PKs) that can be exploited towards selective inhibition. Here, we discuss the taxon-specific kinases possessed by malaria parasites, and give an overview of target PKs that have been validated by reverse genetics, either in the human malaria parasite Plasmodium falciparum or in the rodent model Plasmodium berghei. We also briefly allude to the possibility of attacking Plasmodium through the inhibition of human PKs that are required for survival of this obligatory intracellular parasite, and which are targets for other human diseases.

2010

Interactome and reverse genetics studies of regulatory subunits of Plasmodium falciparum casein kinase

Guillem Dayer

Malaria is the most devastating parasitic infection killing each year between 1 and 3 million of people. Furthermore this decease has a huge socio-economic impact in affected countries. The emergence of resistance against currently used drug makes the development of a new treatment an urgent task. Considering the success of kinase inhibitors in treatment of various cancers and considering the divergence between the parasites and human kinome, proteins kinases have become an attractive potential target. In this project we have used mass spectrometry analysis to study the Interactome of three P.flaciparum kinases: PfCK2beta1; PfCK2beta2 and PfGSK3. The genetically modified lines that we have used possess haemaglutinin tag. We have used this haemaglutinin tag to immunoprecipitate our proteins and their potentials interactors. Samples were then separated on acrylamide gel and analysed at the proteomics core facility at EPFL. Finally we have analysed the potential interactors and study the pathway in which they are involved. Using these methods we have found that the three PfCK2 subunits were immunoprecipitate together. These were already deemed known results since it has already been shown that these subunits interact together. But since there is, to the best of our knowledge, no example of this kind of study in P.falciparum, this project tends to demonstrate that mass spectrometry can be used to study Interactome of the parasites proteins. For our three proteins of interest we have identified some potential interactors but our results are too preliminary to conclude that those proteins are real interactors. A better understanding of the parasite life cycle and its metabolic pathway is critical in the development of a treatment against Malaria

2010