Characterisation of Plasmodium falciparum proteins expressed on infected red blood cell surfaces as potential drug targets for severe malaria therapy

Abstract

The most severe form of malaria in humans is caused by the intracellular parasite Plasmodium falciparum. The African continent bears the greatest burden of malaria with 90% of all malaria deaths occurring in sub-Saharan Africa where the high risk populations include pregnant woman and children under the age of five. Fatal cases of malaria are often a result of the progression of the disease to a life threatening syndrome where intravenous quinine or artesunate are administered as an emergency treatment, however a 15-20% mortality rate is still observed among treated individuals. Pathogenesis of severe malaria is associated with the mature or late trophozoite stage of the parasite s intra-erythrocyte life cycle. At this stage the intracellular parasite expresses parasite derived proteins on the surface of the red blood cell (RBCs) that bind to host endothelial receptors. This cytoadhesion ultimately allows the parasite to multiply unhindered by the host resulting in high parasitaemia levels which is associated with the extent of the symptoms associated with severe malaria. There is currently no effective vaccine available for malaria and available antimalarial drugs are often compromised by rapidly developing drug resistance. Therefore there is an urgent need to identify novel drug targets for the treatment of severe malaria and to explore therapeutic agents that can be used as adjuncts to the currently available anti-malarial drugs. Statins, also known as 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, have been identified as a potential adjunctive therapy for severe malaria due to their pleiotropic effects which include anti-cytoadhesive activity. The aim of this study was to characterise cytoadhesive related parasite proteins that are expressed on the surface of parasitised RBCs and then to assess the effects of a classic statin, lovastatin, on these parasite proteins. This was done in order to provide improved insight and extend existing knowledge on the potential role statins may have in treating severe malaria. The P. falciparum 3D7 strain was efficiently synchronised and cultured according to standard culturing procedures and selective harvesting provided a 90% enrichment of narrowly synchronised cultures at the late trophozoite stage (35 -40 hours post invasion). Scanning electron microscopy (SEM) was used to successfully illustrate parasite induced morphological changes to the surface of parasitised RBCs which included parasitic knob structures, a characteristic feature of severe malaria. Isolation of membranes of equivalent RBCs and parasitised RBCs that were untreated and treated with 10 ?M of lovastatin was optimised and surface proteins were fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Bands of interest that were visually apparent on Stain Free imaged gels and Coomassie brilliant blue stained gels were excised and in-gel trypsinised followed by peptide sequencing by liquid chromatography tandem mass spectrometry (LC-MS/MS). Advanced proteomic software was then used to successfully confirm the identity of key RBC structural membrane proteins as well as several parasite surface proteins, such as P. falciparum erythrocyte membrane protein 1 (PfEMP1), P. falciparum erythrocyte membrane protein 3 (PfEMP3), P. falciparum knob associated histidine rich protein (PfKAHRP), P. falciparum mature infected erythrocyte surface antigen (PfMESA) and P. falciparum cytoadhesion linked asexual gene (PfCLAG) proteins. These cytoadhesive related proteins were identified on the membranes of parasitised RBCs that were treated with lovastatin thus suggesting that although lovastatin has the ability to retard cytoadhesion it is apparently not through the inhibition of expression of parasite derived adhesion proteins. The study also highlighted the need for stringent controls when using label-free proteomics to assess differential protein expression. The effects of lovastatin on protein abundance were inconclusive due to the low abundance of these proteins. This study demonstrated the potential of combining pharmacological studies with advanced proteomic techniques where the effects of physiological relevant concentrations of a drug were successfully assessed at the parasite protein expression level.