Preliminary Analysis Of Glucose Permeation Pathway In Plasmodium Falciparum Blood Stage

Malaria is still the major epidemic influencing millions of people in tropical and subtropical regions.However,there are no effective antimalarial vaccines available for widespread clinical uses,and antimalarial drugs,including artemisinin,are facing serious resistance situations.To solve the problems,we urgently need in-depth study to better understand the etiology of malaria parasite and pathogenesis of malaria.Plasmodium falciparum(P.falciparum)is the highest fatality rate of plasmodium that infects humans.The infection in human bodies includes two stages: hepatocyte stage and erythrocyte stage.Erythrocyte stage is the main phase of the development and proliferation of P.falciparum and the appearance of clinical symptoms,and its energy metabolism is mainly dependent on the absorption of glucose in the blood.Firstly,glucose goes through Glucose transporter 1(GLUT1)located on the host erythrocyte plasma membrane(EPM)into the red blood cell cytoplasm,then goes into the parasite plasma membrane(PPM)through P.falciparum hexose transporter(Pf HT)to complete absorption of glucose for malaria parasite.Previous researches on carcinoma reveal that Vitamin C(Ascorbic acid)could inhibit the development of many cancer cells including colorectal cancer.The mechanism is related to the increased uptake of vitamin C(dehydroascorbic acid,DHA)in tumor cells.Cells can absorb DHA through GLUT1.And some tumor cells have increased GLUT1 expression due to increased energy demand,so that they can absorb more DHA than normal cells.A large amount of absorption of reductive DHA in cells increases the consumption of GSH,resulting in oxidative stress,thus inhibiting cell growth.Similarly,the expression of GLUT1 is abundant in the erythrocyte membrane,and the infected erythrocytes may need to adjust intake and metabolism of glucose for the growth of the parasite.Meanwhile,the infected erythrocytes may intake more DHA.Therefore,we decided to give vitamin C treatment to the cultured erythrocytes infected with P.falciparum in vitro,which were treated with three conditions:(1)different doses;(2)different administration time;(3)different delivery time.We found that(1)Vitamin C above 3mM had inhibitory effect on the growth of P.falciparum.(2)20mM Vitamin C treating 3 hours can inhibits the parasites growth or even kill them;(3)Vitamin C could inhibit the parasites growth at different times,but in different degrees.Moreover,after the treatment,P.falciparum showed morphological changes of nucleus shrink and cytoplasm reduction.Those results confirmed that high dose of Vitamin C in the physiological function of time can effectively restrain the growth of the malaria parasite.GLUT1 is the red cell membrane channel protein that could transport both glucose and Vitamin C.Previous studies have shown that there is no significant difference in the expression of GLUT1 between uninfected and infected erythrocytes,and their levels of phosphorylation may be different.The glucose transporter on human red cell membrane has important influence on the growth and proliferation of P.falciparum.To further understand the function of GLUT1 and the possible mechanism of such growth inhibition,we use Mass Spectrometry detection to compare the levels of GLUT1 expression and phosphorylation between infected and uninfected erythrocyte.We discovered 2 phosphorylation sites on GLUT1 of from the infected samples,however none from the uninfected ones,and such results coincide with the previous study.When we tried to measure the quantity of GLUT1 expression by immunoblot experiments,we found the existing commercial antibodies have poor sensitivity in the detection GLUT1,and cannot be used for immunoprecipitation experiments.In addition,there is still no commercially available antibody to identify the protein,PfHT,which is the glucose transporter locating on the malaria parasite.To further study the crucial proteins for glucose transportation during P.falciparum blood stage,we carried out on the protein sequence analysis,synthetic peptides corresponding to the specific identification of GLUT1 and PfHT protein antibody.By the technologies of culture and synchronization of P.falciparum,we acquired the infected erythrocytes with more than 90% parasitemia.Molecular clone and Western blot analysis revealed that endogenous GLUT1 and PfHT can be recognized by such antibodies specifically.GLUT1 antibodies obtained here are more efficient than commercial GLUT1 antibodies.Our study confirmed that high dose of vitamin C can effectively inhibit the growth of plasmodium.The antibodies we generated against GLUT1 and PfHT can be effectively used in the immunoblot experiment,and may provide more choices for immunoprecipitation and immunofluorescence experiments.These results help to further investigate the role of glucose transportation pathway in the process of P.falciparum blood stage infection and provide the necessary conditions for the exploration of new targets for antimalarial drugs.