Functional Analysis Of The Plasmodium Falciparum Dynamin Like Protein 1

Malaria is one of the most infectious diseases to human health in the world.Thereare an estimated 500 million cases and up to 3 million deaths from malaria each year.The mortality levels are greatest in sub-Saharan Africa,where children under 5 years ofage and pregnant women account for ninety percent of all deaths due to malaria.Despitemore than a century of efforts have been taken to eradicate or control malaria,the diseaseremains a major and growing threat to the public health and economic development ofcountries in the tropical and subtropical regions of the world.The emergence and spreadof drug-resistant parasites coupled with the absence of an effective vaccine makesmalaria treatment more complicated,and thus the development of new antimalarial drugsand new vaccine targets is one of the urgent tasks in malaria research.P.falciparum dynamin like protein 1 was a new plasmodium gene identified by ourgroup.Our previous work suggested that P.falciparum dynamin like protein 1 mayparticipate in some important trafficking process in the Plasmodiumfalciparum:theendocytosis and secretion process;It also suggested that P.fDYN 1 may be essential for thesurvival of the parasites during the erythrocytic stage based on RNAi experiment.Based on the previous work,this study focus on the functional analysis of theP.fDYN1.The results are as follows:1) Two targeting plasmids were constructed and tansfected into P.falciparum 3D7strain and the two stable transfected parasites were firmly established.PCRbased genetic analysis showed that the whole P.fdynamin like proteinl geneknockout was not success.The truncated knockout experiment was notcompleted but it was still worth to continue.2) The expression of the whole protein of P.fdynlin Escherichia.coli system and Picha pastoris system was tested but failed.Finally,the whole protein of P.f dynlwas expressed in E.coli based on codon optimization by using the DNAworkssoftware.The whole protein and the truncated protein showed the samecharacters of their GTPase activity in vitro assay.It seems that the C terminal ofthe P.fDYN1 protein was not important for its GTPase activity.3) A dynamin GTPase inhibitor named dynasore can also inhibit the GTPaseactivity of P.fDYN1 with the concentration of 80μM.4) Treated the P.falciparum 3D7 strain culture with 80μM dynasore resulted incell growth delay.We supposed that dynasore inhibited the GTPase activity ofthe P.fDYN1,thus the endocytosis of the parasite was inhibited and the nutrientacquisition such as the hemoglobin uptake was arrested.5) Biochemical and morphological study supported our hypothesis.Theaccumulated hemoglobin and haemozoin in the parasite was remarkablydecreased when the parasite was treated with 80μM dynasore.FITC-dextranendocytosis assay also showed that 80μM dynasore can inhibit the endocytosisof the plasmodium falciparum.The ultrastructure showed that the vesicle likestructure was increased in the parasite when treated with 80μM dynasorecompared with the negative control.Taken together,we concluded that P.falciparum dynamin like protein 1 participated inthe endocytic trafficking pathway in the malaria parasite Plasmodium falciparum andplayed an important role.These work demonstrated that the P.fDYN 1 had the potential tobe a drug target for malaria chemotherapy.