| Background and Purpose:Malaria remains a serious hazard to human health. This epidemic killed approximately1million people each year, resulting in400million people infected. In tropical and subtropical regions, malaria is not only a common disease, but also a serious public health problem [1]. The statistics show that each year there are billions of people worldwide infected with malaria. Although the majority of patients can be cured, there are still about one million people died, including children in the majority. World Health Organization has paid close attention to malaria [2]Curbing malaria has become a global aim. China also proposed to eliminate malaria in most of regions by2015and malaria elimination will be achieved by2020nationwide [3]. Effective drugs are the main method in curing malaria. However, the parasite resistance to antimalarial medicines and mosquito resistance to insecticides for malaria control have become a global threat for malaria control. Artemisinin-based combination therapy (ACT) is currently the most powerful weapon for treating malignant malaria which is the most deadly form of the disease. Its efficacy can reach more than90%, but artemisinin resistance has appeared in border area of Cambodia and Thailand recently [4,5]. In view of drug resistance has arised in all current malaria drugs. Therefore, developing new malaria drugs is not only crucial, but also urgent.Looking for effective drug target is the basis of developing anti-malarial drugs. Parasite retained many special organelles to adapt different external environment and the host environment in the long period of evolutionary process. These organelles which are 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 of specific targets for these parasites. A new organelle-acidic calcium body (Acidocalcisome, Ac) which is discovered and named in recent studies belongs to one of them. Acidocalcisome is an acidic electron dense particles, rich in calcium and polyphosphate. It is also an ions, amino acids, inorganic phosphate and energy storage. It plays a important role in phosphorus metabolism, calcium balance, acid-base balance and osmotic pressure regulating[6-101Acidocalcisomes are first discovered and named in Trypanosoma by team Dr. Professor Docampo Roberto from the University of Illinois in the United States [11,12]. They are subsequently found present in Leishmania [13], Apicomplexa:Toxoplasma gondii [14-17], Plasmodium [18-20], Amy protozoa [21], green algae [22],fungi[23], bacteria [24,25] and human platelets [26].This organelle contains large amounts of calcium and polyphosphates which is similar to "particles"(granules) reported before. Although these "particles" had reported as early as1904[27], until recent twenty years it was named acidocalcisomes and its structure and function are systematically studied [28-30]. Many parasitic protozoa or bacteria possessing acidocalcisomes are the causative agent. Acidocalcisomes are characterized in its pyrophosphatase activity. Drugs which are able to remove the activity pyrophosphatase could possibly killed pathogens containing such organelles [31,32]. Therefore, it is of great value to study acidocalcisomes.In the studies of acidocalcisomes, trypanosomes and Toxoplasma gondii acidocalcisomes are studied and investigated more thoroughly. 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 [30]. These parasitic components play an important role in toxicity, metabolism, and invasion of host. Gene knockout of Ca2+-ATPase (TgAl) in Toxoplasma gondii by homologous recombination can cause disorder in the calcium balance, the toxicity of Toxoplasma gondii significantly reduced[17]. RNA interference (RNAi) confirmed that Ca2+-ATPase in Trypanosoma brucei causative agent of African sleeping sickness is of great importance in maintaining their normal metabolism [33]. The same experiments confirmed that H+-ATPase in Trypanosoma cruzi, the Chagas disease pathogens, and Trypanosoma brucei (TcHA1/2; TbHA1/2) is necessary for the maintenance of pH balance and parasite growth. More important finding is that, TcHAl/2and TbHA1/2are different from mammalian P-type H+-ATPase. Thus both of them are consideredas new therapeutic drug targets[34,35].The presence of acidocalcisomes in Plasmodium has also been confirmed [18-20], however, their studies are much less than Toxoplasma gondii and Trypanosoma. The specific structure, biological function and metabolic characteristics are still unclear. Due to the limitation of sources of fresh blood and unstable growth of parasite in vitro and so on, it is difficult to enlarge parasite cultivation. However, the rodent malaria parasite Plasmodium berghei is similar with human malaria parasite in morphology, genetic composition, physical characteristics and cell cycle, so we use Plasmodium berghei as the biological model to study acidocalcisomes.Methods and ResultsFirstly, we cultivate Plasmodium berghei in Kunming mice. When the mice got sick, fresh blood was collected from the eyeballs.60%percollseparation liquid are used to isolate erythrocytes infected by trophozoites and schizonts. Erythrocytes were cracked by saponin and washed repeatedly to obtain pure Plasmodium. Parasites were cracked by silicon carbide mechanical grinding and washed repeatedly with buffer A to eliminate a large number of malaria pigment interfering the experiment. By reviewing literature and varing experimental conditions, we determined the optimal discontinuous density gradient for separation and purification of acidocalcisomes from Plasmodium bergheias as15%,20%,25%,30%,34%,38%and got some sedimentation. We fixed, embedded and sliced the separated red blood cells infected with trophozoites and merozoites and directly observed the slices and sedimentation under electron microscope (FEI Tecnai TM G2Spirit transmission electron microscope,80kv). We also analyzed the X-ray element of the sample grid by Norvar thin window detector. Vacuoles resemble inclusion and a large number of round dense particles ranging in size were visible in the electron microscope. X-ray spectroscopy analysis showed that the particles are rich in chemical elements calcium, phosphorus and magnesium.Digging a new protein of acidocalcisomes through proteomics approach is an important aspect of this research, which is of great value in in-depth study of the features of acidocalcisomes. Therefore, the separated samples were lysed, protein were concentrated and subjected to SDS electrophoresis and LC-MS/MS detection. Then we searched the matched protein in the database created by Mascot2.3.02, and obtained a series of protein identification information. By statistically analyzing, we obtained125,804spectra,1183identified spectra,472identified peptides and369identified proteins. The identified proteins were subjected to bioinformatics analysis, including GO annotation, COG analysis and passway analysis. In the GO annotation analysis results, the identified proteins were revealed to have a catalytic activity (41.05%) and binding activity (42.47%)(molecule-function analysis), be more concentrated in organelles (23.83%) and membrane (23.83%)(subcellular positioning) and be involved in the metabolic pathway (27.85%), cellular processes (29.84%), signal transduction (2.69%) and other channels (biological process analysis).We compared the identified proteins in COG database, predicted their functions and analysed statistically, results from which showed that they function mainly in energy generation and conversion, lipid transport and metabolism, inorganic ion transport and metabolism. Referring to a large number of reported results, a variety of subcellular localization prediction software results and GO annotation results, we statistically analysed the subcellular localization of the identified proteins to make a more accurate subcellular localization map of the identified proteins. Finally, by consolidating results of GO analysis, COG analysis and subcellular localization, we initially predicted the18proteins locate in acidocalcisomes in Plasmodium berghei. To verify whether the18proteins do locate in acidocalcisomes, we prepared monoclonal antibody against vacuolar proton pump pyrophosphatase, an iconic protein of acidocalcisomes to do immunofluorescence. Pyrophosphataseis a membrane protein containing lots of transmembrane domain, which make us choose to produce antibody antigen by synthesizing epitope peptide immunogens rather than by prokaryotic expression system. First, we predicted the transmembrane domains of pyrophosphatase in Plasmodium berghei by TMHMM and15transmembrane domains were found, and the latter should be avoid in the choosing of antigen peptides. Meanwhile, B-cell epitope analysis was done with IEDB software.6antigen epitopes were predicted for their scores higher than the red line, which indicates the critical value0.350. Next, V-PPase amino acid sequences of Plasmodium berghei, Plasmodium falciparum were compared for homology, and a conserved sequence was ultimately selected as the target antigen peptide(YTKAADCGADLSGKNEYGIPEDDDIM).In consideration of its weak antigenic due to its small molecular weight, the synthetic peptide was conjugated to BSA andthen used to immunize BALB/c mice to prepare monoclonal antibodies. We used the classic method to prepare monoclonal antibody against pyrophosphatase, hybridoma cell lines were positively screened by direct ELISA. At last,9monoclonal antibodies were construct, among which3D3,4E7,1H5hybridoma lines were strong positive mice, and then they were intraperitoneally injected into mice to produce ascites. Identifying ascites by western blot, we found the line4E7has an interaction with all of the proteins (weighted about80kd) in Toxoplasma, Plasmodium falciparum and Plasmodium berghei.CONCLUSIONWe have established an approach for separation and purification of acidocalcisomes in plasmodium berghei by utilizing discontinuous density gradient of iodixanol and obtained acidocalcisomes successfully. By proteomic analysis on the acidocalcisomes obtained, we got369proteins identified,18among which may locate in acidocalcisomes in Plasmodium berghei. We have also successfully produced a monoclonal antibody against a targeting protein of acidocalcisomes(vacuolar proton pump pyrophosphatase), which provides an effective tool for subcellular locating and further functional identification for other proteins in acidocalcisomes. Finally, our results pave the way for related basic research on plasmodium and discovery of new antimalarial drugs targeting them. |
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