Isolation, Purification And Proteomics Research Of The Acidocalcisomes In Plasmodium Falciparum

Background and Purpose:Plasmodium is a genus of parasitic protozoa. Infection with these parasites is known as malaria. Especially Plasmodium falciparum, over 75% of cases were due to P. falciparum. Malaria is prevalent in 102 countries and regions. High mortality of falciparum malaria killed about 100 million people every year, annual statistics show that 400 million people are infected. Although most patients can be cured, but there are still one million people dead, of which children accounted for the majority. In our country, there are still nearly 300 million people in 15 provinces (autonomous regions) 546 counties (cities) in the threat of malaria so far, the number of falciparum malaria cases is increasing in the south, therefore, the prevention and treatment of malaria still occupies an important position in infectious diseases. Artemisinin- based combination therapy (ACT) is the most powerful weapon for treating falciparum malaria currently, the efficacy of which can reach to more than 90%, but the resistance of artemisinin and chloroquine in P. falciparum has appeared in border area of Cambodia and Thailand recently. This is a wake-up call for malaria prevention and treatment. Therefore, The development of new drugs against malaria is not only essential but also urgent. In order to discover new antimalarial drug target, we need to further study the structure and properties of the parasite to find effective drug targets and set foundation for the development of antimalarial drugs.Many special organelles unique to the parasite do not exist within the host. Thus, through in-depth study of the structure and function of these organelles, it is possible to discover specific targets for these parasites. A new organelle -acidocalcisome (Ac) is one of them, which is discovered and named in recent studies. Acidocalcisome is a special kind of organelles,which is first discovered and named in Trypanosoma by Dr. Professor Docampo Roberto’s team from the University of Illinois in the United States. Because of its abundance of calcium and phosphate, acidic, so it was named for acidocalcisome. They are subsequently found present in Leishmania, Toxoplasma gondii, Plasmodium, Amy protozoa, green algae, fungi, bacteria and human platelets. Compared with the well known organelles, such newly discovered organelle provides more possibilities for our study to develop antimalarial drugs.Study found that there are many pumps in the membrane of acidocalcisomes, for example Ca2+ -ATPase, Vacuolar-H+ -ATPase, Vacuolar-H+-pryophosphatase, Na+/H+ exchanger, Ca2+/H+ exchanger, aquaporin and ion channels. Among them, Vacuolar-H+-pryophosphatase (V-PPase) is unique to acidocalcisome, which is considered as a sign of isolation and purification of acidocalcisome. These proteins lay structure foundation for the main physiological function of acidocalcisome. Acidocalcisomes function as storage sites for cations and phosphorus, and participate in PPi and poly P metabolism, calcium homeostasis, maintenance of intracellular pH, and osmoregulation. These play important roles in virulence, metabolism, and invasion of host. Among the studies of acidocalcisomes, which of Trypanosomes and Toxoplasma gondii are studied and investigated more thoroughly. The presence of acidocalcisomes in Plasmodium has also been confirmed, however, the study of them are much less than those of Toxoplasma gondii and Trypanosoma. The structure, biological function and metabolic characteristics are still not clear. To find out more protein can be used as antimalarial drug targets, we need to do proteomic analysis for Plasmodium falciparum acidocalcisome.Methods and Results:It is difficult to separate and purify acidocalcisome from Plasmodium falciparum with traditional methods, because the in vitro culture of Plasmodium falciparum is highly affected by the serum and difficult to standardize, it is difficult to obtain a sufficient amount of parasites. Our group established a method of serum-free culture medium with the use of AlbuMAX I to in vitro continuous Plasmodium falciparum culture in the long-term of research, with which the highest infection rate of erythrocytes in vitro continuous culture can reach 40%. This method has been proven to meet the large-scale isolation and purification of the parasite, can provide adequate parasites. When the infection rate of erythrocytes reached 20%, blood was collected and separated using 60% percoll isolation liquid to isolate erythrocytes infected by trophozoites and schizonts (iRBCs). iRBCs were then cracked by saponin and washed repeatedly to obtain pure Plasmodium falciparum. Parasites were cracked by mechanical grinding with silicon carbide along with cell lysis buffer and the supernatant was then collected. According to the previous research of Plasmodium berghei by our team, the optimal discontinuous density gradients of iodixanol for isolation and purification of acidocalcisomes from Plasmodium bergheias were 15%, 20%,25%,30%,34%,38%, using RCF 30000g. We used the same discontinuous density gradients of iodixanol and adjusted the RCF to 50000g, then the solution in centrifugal tube was divided into 13 layer, acidocalcisomes were enriched in sediments in the bottom. We fixed and directly observed the sediment sample with electron microscope, found abundant sizes of circular electron dense granules. We also embedded and sliced the separated iRBCs and observed, vacuoles resemble inclusion and a large number of round dense particles ranging in size were visible in the electron microscope. These results were consistent with previous literature, which indicated that the isolation and purification of acidocalcisome from Plasmodium falciparum were successful.The purified Plasmodium falciparum acidocalcisome was then detected using proteomic analysis. The samples were lysed, concentrated and subjected to electrophoresis and LC-MS/MS detection. The matched protein were then searched in the database created by Mascot2.3.02, and 283 identified proteins were obtained. Among which, there were 77 hypothetical proteins,62 conserved proteins and 144 detected proteins. GO annotation analysis results revealed that, most identified proteins were of a catalytic activity (33.33%) or a binding activity (55.56%) in Molecular Function analysis, about 16.67% of the identified proteins were located in organelles in Cellular Component analysis, and about 16.47% of the identified proteins were involved in metabolic process,16.47% involved in cellular process and 12.50% involved in single-organism process in Biological Process analysis. The identified proteins were then compared in COG database to predict their functions, results from which showed that they functioned mainly in energy generation and conversion, intracellular trafficking, secretion, and vesicular transport, General function prediction only. Through literature retrieval and bioinformatic analysis, we initially predicted the 17 proteins, including V-type H(+)-translocating pyrophosphatase (V-PPase), Phosphoribosyl pyrophosphate synthetase, P-type calcium transporting ATPase、ABC transporter (MDR family), small GTP-binding protein sari, calcium-dependent protein kinase 4 (CDPK4),14-3-3 protein family, aquaglyceroporin (AQP), vacuolar proton translocating ATPase, vesicle-associated membrane protein, phospholipid-transporting ATPase, vacuolar ATP synthetase, located in acidocalcisomes of Plasmodium falciparum. Our team had predicted 18 proteins located in acidocalcisome including aquaglyceroporin, CCR4-associated factor 16, calcium dependent protein kinase 4, multidrug resistance protein, vacuolar ATP synthase subunits, phospholipid-transporting ATPase, phosphatidylinositol 3-kinase, PLA2s and V-type H(+)-translocating pyrophosphatase. As Plasmodium berghei is similar with Plasmodium falciparum in morphology, genetic composition, physical characteristics and cell cycle, we use Plasmodium berghei as a guide to study acidocalcisomes. By comparison of the 17 predicted proteins of P. falciparum and the 18 predicted proteins of P. berghei, we found 8 homologous proteins were predicted to locate in acidocalcisomes of both P. falciparum and P. berghei. Among which,7 proteins were found to be of a homology higher than 60% by homology comparison with ClustalW2.By proteomics analysis of P. falciparum acidocalcisome, we chose aquaglycerporins of Plasmodium falciparum (PfAQP) for further study. At the same time antibody against Plasmodium falciparum vacuolar proton pyrophosphatase (PfVP1) was needed for localization research. Both of them were difficult to express in E. coli for the too much transmembrane domains. Therfore, we chose an approach of synthetic peptide conjugated with a carrier protein for antigen preparation to monoclonal antibodies generation. For follow-up research, we compared the amino acid sequences of T. gondii vacuole proton phosphatase (TgVP1) and PfVP1 and selected the homologous sequence for synthetic peptides to produce monoclonal antibodies. For PfAQP monoclonal antibody generation, a homologous peptide of PfAQP and Plasmodium berghei aquaglycerporin (PbAQP) was selected to synthesized. The full-length of PfAQP is 258 amino acids, the molecular mass is about 27kDa, while TgVPl is 816 amino acids long and 85kDa. For PfAQP, after IEBD, BCPREDS Server 1.0, TMHMM Server v 2.0 and homology analysis, we selected a peptide with high homology between P. falciparum and P. berghei, outside of the 6 transmembrane domains of PfAQP, CPLVDLANNEKDGVDL,243 to 258 amino acids located at the C-terminal of PfAQP peptide chain. Finally we got mAb 2C3 and mAb 6H10, which can react with both synthetic peptides and the native P. falciparum protein of 27kDa, consistent with the predicted molecular mass and literatures of PfAQP. And for TgVPl, we use IEBD, TMHMM Server v 2.0 and homology analysis to selected a peptide with high homology between T. gondii and P. falciparum, outside the 17 transmembrane domains. Finally we got mAb 1D7-H11, mAb 3A6-E4 and mAb 3G6-F9, which can react with synthetic peptides, native T. gondii protein of 85kDa and the native P. falciparum protein of 76kDa. When immunofluorescence, the above 5 mAbs can produce specific fluorescence, but the co-localization of PfAQP and TgVP1 fluorescence remained to be verified.CONCLUSION:A method of serum-free culture medium with the use of AlbuMAX I to in vitro continuous Plasmodium falciparum culture was established in this study, with which adequate parasites can be collected. After successful isolation and purification of Plasmodium falciparum acidocalcisome, proteomic analysis was performed and identified 283 proteins. Among which 17 P. falciparum proteins were predicted to be located in acidocalcisome by primary bioinformatic analysis. Then 2 mAbs against P. falciparum aquaglycerporin (PfAQP) and 3 mAbs against T. gondii vacuolar proton pyrophosphatase (TgVP1). By identification, this 5 antibodies can specifically bind to the target protein, and the anti-TgVP1 antibody can also specifically bind to PfVPl. These results will lay foundation for the further study of P. faldparum acidocalcisomes and the development of antimalarial drugs targets.