Mitochondria and apicoplast of Plasmodium falciparum: Behaviour on subcellular fractionation and the implication
Introduction
Malaria, by far the most important tropical parasitic disease, is caused by a group of parasites Plasmodium spp. belonging to the phylum Apicomplexa. Currently, various anti-malarial drug resistant parasite strains are reported and there is a long way for the development of vaccine. Emergence of insecticide resistant mosquito vector limits the current control schemes as well (Greenwood et al., 2005). In order to control this world problem, studies seeking for unique properties of the parasite are indispensable.
Previous study reported malaria parasites obtain most of their energy from glycolysis, if not all (Roth et al., 1988) and malaria parasite possesses one mitochondrion with various shapes at different stages of the intra-erythrocytic development and it is acristae (Slomianny and Prensier, 1986). Mitochondria of Plasmodium species carries 6-kb genome, which is the smallest mitochondrial genome ever been reported and encoding only 3 open reading frames with homology to classical mitochondrial protein, cytochrome c oxidase subunit I, cytochrome c oxidase subunit III and cytochrome b, as well as abbreviated rRNA genes (Vaidya et al., 1989, Feagin, 1992). Thus this organelle heavily depends on most of the proteins and all tRNAs supplied from the outside.
Biochemical analysis suggested that Plasmodium falciparum might lack TCA cycle in the erythrocytic stage (see the review by Sherman, 1979). Recent completion of malaria genome project has revealed that the genes necessary for a complete TCA cycle were present in P. falciparum (Gardner et al., 2002). However, it still remains unclear whether the TCA cycle is responsible for the further oxidation of glycolysis product. Nevertheless, the activity of the electron transport chain and the membrane potential of this organelle are indispensable for the survival of the parasite. For example, dihydroorotate dehydrogenase (DHOD) involved in the parasite’s de novo biosynthesis of pyrimidine requires the functional electron transport chain on the mitochondrial membrane as the electron disposal sink (Gutteridge and Trigg, 1970, Gero et al., 1984, Prapunwattana et al., 1988). More recent study showed that the membrane potential of mitochondria formed by respiration is essential for parasite growth (Srivastava et al., 1999) and complex III (ubiquinol-cytochrome c reductase) inhibitor, atovaqone, an anti-malarial that is currently in use is reported to disrupt mitochondrial membrane potential resulting in parasite growth reduction (Srivastava et al., 1997).
Aikawa (1966) carried out an extensive morphological study by electron microscope and found a distinctive organelle in the cell of the malaria parasite. This organelle is multi-membrane bound, always observed adjacent to the mitochondrion (see the review by Bannister et al., 2000). Later, a non-mitochondrial extra-chromosomal DNA encoding a set of genes characteristic of the plastid genome was found in apicomplexan parasites including Plasmodium spp. Toxoplasma gondii and Theileria spp. (Wilson et al., 1996, Kohler et al., 1997) localized the plastid genome-like DNA to the multi-membrane organelle in T. gondii by in situ hybridization, revealing that the distinctive multi-membrane organelle is the plastid of the apicomplexan parasite. The apicomplexan plastid, which is non-photosynthetic, is often called “the apicoplast” for abbreviation. The genome of the apicoplast is one of the smallest known plastid genomes (Wilson et al., 1996, Gardner et al., 2005). The apicoplast depends heavily on proteins imported post-translationally from the cytosol (see the review by Ralph et al., 2004), as does the mitochondrion.
For biochemical studies of each organelle of Plasmodium spp., it is necessary to obtain the pure sample. Fry and Beesley (1991) reported a method to prepare the mitochondria from Plasmodium spp. by Percoll density gradient centrifugation. Takashima et al. (2001) reported another preparation method using nitrogen cavitation. The mitochondrial preparation by the latter method exhibited a significantly higher succinate dehydrogenase activity than that by the former method (Takashima et al., 2001). By contrast, no method for preparing the plasmodial apicoplast with a significant purity has been reported.
In this study, we combined nitrogen cavitation method with two different fractionation methods, Percoll density gradient centrifugation or fluorescence-activated organelle sorting (FOS), to prepare the mitochondrion of higher purity from P. falciparum. Surprisingly, we found that the mitochondrion and the apicoplast were recovered in the same fraction by each fractionation methods, most likely because the two organelles are bound each other. To our knowledge, this is the first report that suggests the presence of a physical connection between the mitochondrion and the apicoplast of P. falciparum.
Section snippets
Parasite cultivation and handling
Plasmodium falciparum (Honduras-1 strain and 3D7 strain) was cultured following the method reported by Trager and Jensen (1976) with modifications. The culture was maintained with 3% hematocrit type A human red blood cell (RBC) in RPMI 1640 medium (Invitrogen) supplemented with 10% (v/v) type A human serum. Prior to the preparation of crude mitochondrial fraction, parasites were synchronised by 5% (w/v) sorbitol as it was described previously (Lambros and Vanderberg, 1979).
Preparation of the crude P. falciparum mitochondria fraction
Plasmodium falciparum
The mitochondria and apicoplast co-fractionated by the Percoll density gradient centrifugation
Previously, Fry and Beesley (1991) reported a method to prepare plasmodial mitochondria using a density gradient in 22% (v/v) Percoll formed by centrifugation at 10,000g for 5 min. As this method has been successfully used in other laboratories (Wilson et al., 1992, Krungkrai, 1995, Krungkrai et al., 1997), we preliminarily tested if this method is directly applicable to improve the purity of mitochondria prepared by nitrogen cavitation method, which showed higher enzyme activities of
Discussion
The biochemical study of plasmodial mitochondria has been limited due to the difficulty to prepare sample with high quality and quantity. Previously we have reported the improved mitochondrial preparation from P. falciparum using nitrogen cavitation method (Takashima et al., 2001). In this study, we investigated the method to obtain a mitochondrial preparation of better quality by combining nitrogen cavitation with another fractionation method.
Percoll density gradient centrifugation is a well
Acknowledgements
The authors thank late Dr. Masamichi Aikawa for comments on morphological observations by electron microscopy and wish to dedicate this paper to him. The blood and plasma used in this study is a kind donation from Tokyo Metropolitan Red Cross Blood Centre. WR99210, the drug used for the screening of transfected parasites was a kind gift from Jacobus Pharmaceutical Co., Inc. This study was supported by a Grant-in-Aid for scientific research on priority areas and for Creative Scientific Research
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