Analysis On The Function And Intracellular Transportation Mechanism Of The Plasmodium Falciparum Membrane Protein Pf332

Malaria is one of the most important infectious diseases caused by Plasmodiumparasites. In recent years, due to the emerging and quick spreading of parasites withresistance to classical drugs, the prevention and treatment of malaria has becomemore difficult. Therefore, a safe and effective vaccine is one of the most desiredmeans to prevent the disease. According to the life cycle of the malaria parasite,current studies on malaria vaccine have been mainly focused on the erythrocyticstage of the parasite. The erythrocytic stage vaccine can directly reduce the morbidityand mortality of the host, thus dissection of the properties of the parasite proteinsinside the red blood cell and their interaction with erythrocyte membrane as well asthe skeleton would be the essential step for identification of vaccine candidates.Plasmodium falciparum can synthesise and export a large number of proteinsto the cytoplasm of red blood cell, among these, Pf332is the largest protein whichcontains DBL, TM and WR domains. For further analysis the function andintracellular transportation of P. falciparum Pf332molecule, we constructed plasmidscontaining genes encoding the DBL, TM, WR, TM-WR and DBL-TM domains of thePf332fused with the green fluorescent protein (GFP), which were transfected into the3D7strain. The expression and distribution of different domains were monitoredusing a fluorescence microscope. The potential bending of the domains witherythrocyte actomyosin β-actin were detected by IFA colocalization and ELISA.Further, vesicles serected from the infected erythrocytes were purified and analysedby both Western-blot and electromicroscopy.The results showed that the recombinant plasmids encoding the DBL, TM, WR,TM-WR and DBL-TM domains could be expressed in the parasite-infected RBCs, butthe expressed proteins were all trapped in the parasitophorous vacuole, failing to transfer to the cytoplasm of the infected RBCs. Thus colocalization of the fusionproteins with β-actin in the infected erythrocyte was not observed, indicating thatmultiple functional domains are needed for the translocation of the molecule fromPVM to the cytoplasma. The binding assay in vitro showed that the WR domain ofPf332molecules can combine to β-actin of erythrocytes. Thus, through anchoring onred blood cell skeleton, Pf332molecule may change red blood cell rigidity.Further, we, at the first time, demonstrated that P. falciparum can secretemicro-vesicles which carry parasite-derived functional molecules. The micro-vesiclesmediated translocation of these molecules between different parasite cells. The datais importance in dissection of the mechanism of parasite biology. In conclusion, thisstudy resolved the expression and transportation of DBL, TM and WR domains of P.falciparum Pf332molecule inside infected erythrocyte. The binding domain of thismolecule with erythrocyte cytoskeleton was confirmed. The identification ofmicro-vesicles that mediated molecular transportation between different cells willpromote the deep dissection of the parasite biology and vaccine development.