MITOCHONDRIAL HETEROGENEITY IN THE ASEXUAL AND SEXUAL BLOOD STAGES OF PLASMODIUM FALCIPARUM

The in vitro culture of the P. falciparum sexual blood stage in our laboratory (Petmitr & Krungkrai, 1995) was established using the methods of Ifediba & Vanderberg (1981) and Sinden (1982), which provides sexual-stage parasites for the isolation of mitochondria. In parallel, mitochondria were isolated from asexual blood stages, mostly trophozoite stages, in which there is active synthesis of DNA, RNA and protein.

A tubular-like cristate mitochondrion has been demonstrated in the asexual stage parasite (Fig. 1). The sexual stage parasite has a mitochondrion with more numbers of the cristate structures (Fig. 2). Based on the electron density of the matrices, and the numbers and appearances of tubular-cristate structure, 3 different types of mitochondria have been identifed in these 2 blood stages (Krungkrai et al. 1999a). Figure 1 shows a clearly defined double membrane mitochondrion in the trophozoite stage of the asexual stage parasite with tubular cristae formed by infolding of the inner membrane and similar to other protozoan mitochondrion. The elongated organelle shows a limited number of cristae. All mitochondria so far examined, in the asexual blood stages have none or a few tubular cristae. They are classified as ‘type I’ mitochondria (Fig. 1B).

Fig. 1. Transmission electron micrograph of the asexual stage of Plasmodium falciparum (trophozoite stage). A single mitochondrion is observed with a clearly visible double membrane organelle, and an elongated form which is preparing for binary fission (A). A single tubular cristate structure, marked by the arrowhead, is seen in the organelle at higher magnification (B). N, nucleus; M, mitochondrion; P, pigment.

Fig. 2. Transmission electron micrograph of the sexual blood stage of Plasmodium falciparum (gametocyte stage IV). Numerous mitochondria are observed in a single parasite (A). A higher magnification of two organelles containing several cristae (arrowhead) is shown (B). M, mitochondrion; P, pigment.

The mature gametocyte stage (Fig. 2) has ~4–8 mitochondria, containing marked proliferation and pronounced infolding of the inner membrane giving rise to large numbers of tubular cristae with clear intracristal spaces. They are classified as ‘type II’ mitochondria (Fig. 2B). This type II organelle is also seen in the asexual blood stage of P. berghei (Krungkrai et al. 1993). Some mature gametocytes possess higher numbers of cristae, and contain electron-dense tubular cristae. They are classified as ‘type III’ mitochondria.

Until recently, only the organelles in the asexual blood stage parasite had been isolated from 2 species of rodent parasites (P. yoelii, P. berghei) and the human parasite P. falciparum. Most organelles are type I structures, with a definite double-membrane and are found to be metabolically active (Fry & Beesley, 1991; Krungkrai et al. 1993; Krungkrai, 1995; Takashima et al. 2001). More recently, the mitochondria from the sexual stage parasite have been isolated (Krungkrai et al. 2000). Figure 3 shows the isolated organelles, typically type II mitochondria. The different variations in the mitochondria of asexual and sexual blood stages suggest that the gametocytes might have a higher demand for energy transduction and are also more metabolically active than the asexual stages. The more active mitochondria, producing more energy in the sexual stages, may be necessary for their survival during transmission into the mosquito vector. This remains to be studied.

Fig. 3. Transmission electron micrograph of the mitochondrial organelles isolated from sexual blood stages of Plasmodium falciparum. (A) The organelles contain 6–8 tubular cristate structures (arrowhead), as was found in the intact parasites. (B) A higher magnification of an isolated organelle indicate the mitochondrion is still intact with clearly defined double membranes, marked by the double arrowheads. M, mitochondrion.

Our ultrastructural analysis has confirmed the existence of stage-specific variations in mitochondria when comparing asexual and sexual stages in the blood (Krungkrai et al. 1999a; Learngaramkul et al. 1999; Krungkrai, 2000; Krungkrai et al. 2000). Similarly, in Haemogregarina myoxocephali, an apicomplexan blood parasite, there are different numbers of mitochondria in asexual and sexual stages, 1–2 and 4–6 organelles for the asexual and sexual stages, respectively (Siddall & Desser, 1992). The ultrastructural differences in mitochondria prompted us to carry out a molecular and biochemical investigation of mitochondria isolated from these 2 developmental stages in malaria parasites.

DIFFERENTIAL EXPRESSION OF THE MITOCHONDRIAL GENE IN THE ASEXUAL AND SEXUAL BLOOD STAGES OF PLASMODIUM FALCIPARUM

The 6·0 kb mitochondrial DNA of P. falciparum encodes only 3 subunits of the electron transport chain. These are 2 subunits (cox I and cox III) of cytochrome c oxidase (complex IV) and a cytochrome b subunit (cyt b) of ubiquinol-cytochrome c reductase (cytochrome bc₁ or complex III) (Feagin, 1992; Joseph et al. 1989) (Fig. 4). Most mitochondrial proteins are encoded by nuclear genes (Gardner et al. 2002; Bender et al. 2003). Interestingly, the cox II protein of complex IV is encoded by 2 independent nuclear genes, cox2a (chromosome 13) and cox2b (chromosome 14) in P. falciparum, and this is different from most eukaryotic cox II subunit which is encoded by a single gene on the mitochondrial genome (Funes et al. 2002). The cox2a and cox2b genes encode N-terminal domain and C-terminal domain of the cox II protein, respectively (Funes et al. 2002; Gardner et al. 2002). A lateral transfer of these genes from the green alga nucleus to P. falciparum nuclear genome has been suggested (Funes et al. 2002). A similar division of the cox2 gene is also seen in the unicellular alga, Chlamydomonas reihardtii (Perez-Martinex et al. 2001; Funes et al. 2002). Coordinated expression of the cox2a and cox2b genes, mitochondrial targeting and assembling of complex IV have not been studied in P. falciparum. The cyt b gene which encodes the mitochondrial cytochrome b protein of complex III does not derive from the green alga genome. Mitochondrial DNA synthesis occurs coincidentally with that of chromosomal DNA throughout the growth and multiplication cycle of the asexual parasite in its host cell (Feagin & Drew, 1995). It has a phage-like replication mechanism and undergoes extensive recombination like the mitochondrial DNA element of some other lower eukaryotes (Preiser et al. 1996). The transcripts of protein-coding genes of the mitochondrial DNA are least abundant in the ring and early trophozoite stages and most abundant in the late trophozoite and schizont stages (Feagin & Drew, 1995).

Fig. 4. A map of the organization of the 6 kb mitochondrial DNA of Plasmodium falciparum. Arrows indicate the direction of transcription. The gene regions cox I, cox III and cyt b encoding cytochrome oxidase subunits I, III and cytochrome b subunit of ubiquinol-cytochrome c reductase, respectively, are shown in shaded boxes. Fragmented large and small subunits rRNA genes are indicated as filled and open boxes respectively.

The expression profiles of both chromosomal and mitochondrial DNAs are largely unknown during gametocytogenesis, exflagellation and gametogenesis of the sexual-stage parasites. Abundant stage-specific proteins with molecular masses of 27 and 25 kDa have been shown in P. falciparum during gametocytogenesis (Carter et al. 1989). We have recently determined the copy number, the nucleotide sequence and detailed molecular characterization of the 1·3 kb cyt b gene in the mitochondrial DNA. We also determined these for the chromosomal dihydrofolate reductase (dhfr) gene, a single copy gene, in both asexual and sexual blood stages of P. falciparum, using established methods (Cowman et al. 1988; Wiesner, 1992). Increased amounts of both mitochondrial DNA templates (using quantitative polymerase chain reaction, PCR) and transcripts (using quantitative reverse transcription PCR) are found in the sexual blood stages and not in the asexual blood stages (Petmitr & Krungkrai, 1995; Learngaramkul et al. 1999). These findings imply that both gene dosage and the transcription play an important role in the expression of the mitochondrial DNA during development of the parasites in the asexual and sexual blood stages. Gene regulation by gene dosage and transcription level have also been advocated in mammalian mitochondrial biogenesis (Nagley, 1991).

Expression of the dhfr gene in the sexual blood stage is greatly reduced, compared to the relatively high expression in the asexual blood stage. In contrast to the dhfr gene, the chromosomal tubulin gene encoding the microtubule protein does not appear to be developmentally regulated as indicated when RNA from the asexual stages and from the mosquito midgut stages (i.e., gametes, zygotes) are analysed by Northern hybridization (Wesseling et al. 1989). However, there is no report of the tubulin gene expression in the sexual blood stage, which contains prominent microtubules near the parasite membrane during their development, as demonstrated by transmission electron microscopy techniques (Krungkrai et al. unpublished results). More recently, the signal-transducing protein kinases belonging to families known to regulate differentiation have been found in the sexual-stage parasite (Doerig et al. 1995; Bracchi et al. 1996; Li & Baker, 1998; Dorin et al. 1999). However, whether or not these enzymes directly influence gametocytogenesis and exflagellation remains to be determined.

It is also known that the 2 extrachromosomal genomes, the mitochondrial DNA and the apicoplast 35·0 kb circular DNA, are probably inherited only through the female gamete (Creasey et al. 1993). The molecular mechanism that regulates gene expression during the switching of the asexual stage to the gametocytic stage in the host erythrocyte remains to be elucidated (Horrocks et al. 1998; Fang et al. 2004).

ELECTRON TRANSPORT SYSTEM AND BIOCHEMICAL FUNCTIONS OF MITOCHONDRIA OF THE ASEXUAL AND SEXUAL BLOOD STAGES OF PLASMODIUM FALCIPARUM

The asexual blood stage of P. falciparum is a microaerophillic organism and requires low oxygen tension (0·5–3% oxygen) for optimal growth and development in in vitro culture (Scheibel, Ashton & Trager, 1979; Scheibel, 1988). The gametocytes also have low oxygen requirement for their development in human erythrocytes (Sinden & Smalley, 1979; Petmitr & Krungkrai, 1995; Krungkrai et al. 1999a). We investigated whether the mitochondria in the gametocytes of P. falciparum are biochemically active. The coupling of mitochondrial electron transport system (mtETS) and oxygen consumption through complex IV are well characterized in the asexual blood stages of both P. berghei and P. falciparum (Fry & Beesley, 1991; Krungkrai, 1993; Krungkrai et al. 1997, 1998, 1999a,b, 2002; Murphy et al. 1997; Krungkrai et al. 2000; Suraveratum et al. 2000). Experimental evidence indicates the biochemical functions of the mitochondria of the malarial parasite. The mitochondrial oxygen consumption of asexual and sexual blood stages of P. falciparum is similar. They have relatively low activities (~20–24 nmol/min/10⁷ parasites), compared to human leukocytes and mouse neuroblastoma cells which show oxygen consumption of ~110 and ~130 nmol/min/10⁷ cells, respectively. Naive human erythrocytes show no detectable mitochondrial oxygen consumption. Murphy et al. (1997) measured the rate of mitochondrial oxygen consumption in the asexual stage of P. falciparum and found it to be relatively low, which is similar to our results. However, a branched pathway with relatively low activity of alternative oxidase, transferring electrons directly from ubiquinol to oxygen, presumably located in mitochondria, has also been demonstrated in P. falciparum (Murphy et al. 1997). A gene encoding the alternative oxidase has not been detected in the P. falciparum genome, indicating the following possibilities: the gene might be lost during evolution, the sequence diversity of the gene might not be detected by homology searching, and the oxidase activity might be catalysed by another and unknown protein. This controversial finding remains to be studied. Such a gene has been found in Trypanosoma brucei and Cryptosporidium parvum (Suzuki et al. 2004).

Based on the above findings, it is suggested that the abundant mitochondria in gametocytes are biochemically active, at least in oxygen utilization. Comparison of the morphological and biochemical characteristics of the mitochondria in the asexual and sexual blood stages suggests that the metabolic activities of the developing sexual blood stages may be different from those of the asexual stages. The specific activities of the mitochondrial DHODase and the respiratory mtETS complexes III and IV were measured in mitochondria isolated from both asexual and sexual stages. The DHODase and the activities of complexes III and IV of the sexual-stage mitochondria are ~8 to 10-fold higher than those of the asexual-stage organelles, indicating that the mitochondria in the asexual stage are less metabolically active than those of the sexual blood parasite (Learngaramkul et al. 1999). Our results suggest that the P. falciparum mitochondria from both stages are still under developed forms, which exist in the uncoupling state between mitochondrial oxidation and ATP synthesis.

In the asexual stage, the major pathways of carbohydrate metabolism are essentially anaerobic (Sherman, 1979; Scheibel, 1988). It is possible that carbohydrate metabolism in the sexual-stage parasites is similar to the asexual-stage parasites. In addition, our results also demonstrate a clear difference between the parasite and the mammalian mitochondria, the latter having a tight coupling oxidative phosphorylation for ATP production with a P[ratio ]O ratio of ~2 in the presence of succinate and ADP (Krungkrai et al. 2000). In contrast, the asexual blood stage of the rodent parasite P. berghei has the ability to produce ATP through oxidative phosphorylation with a P[ratio ]O ratio of ~2, in the presence of either succinate or dihydroorotate and ADP (Uyemura et al. 2000, 2004), suggesting the coupling oxidative phosphorylation for ATP production may operate in other malaria parasites. This is consistent with our results indicating increased numbers of subunits in each mtETS complex in the rodent parasite, e.g., 2 subunits (P. falciparum) versus 4 subunits (P. berghei) of complex II (Suraveratum et al. 2000; Takeo et al. 2000; Krungkrai et al. unpublished results), or 6 subunits (P. falciparum) versus 8 subunits (P. berghei) of complex IV (Krungkrai et al. 1993, 1997, 1998).

Measurement of mitochondrial oxygen consumption of asexual (mostly trophozoites, Fig. 1) and sexual (mostly gametocytic stages IV, Fig. 2) blood stages was performed in the presence of specific inhibitors of the 4 mtETS complexes. These inhibitors were well characterized as previously described (Hatefi, 1985). By using a cell density of 1×10⁷ parasites in each oxygen consumption reaction, all mitochondrial inhibitors affecting the respiratory complexes I–IV were found to inhibit the oxygen consumption to a different degree when calculating 50% effective concentrations (EC₅₀ values) in both asexual and sexual blood stages (Table 1). The sexual blood-stage parasite is more sensitive to rotenone and cyanide, affecting the respiratory complexes I and IV respectively, than the asexual blood-stage parasite. In contrast, both stages have a similar response to thenoyltrifluoroacetone and antimycin A, which affect the respiratory complexes II and III respectively.

In the sexual-stage parasite, the mtETS may be similar to the asexual-stage parasite in which electrons from donating enzymes, including DHODase, are transferred via coenzyme Q or ubiquinone and a series of respiratory electron transporting complexes and oxygen is the final electron acceptor (Gutteridge et al. 1979; Krungkrai et al. 1991, 1993, 1999a,b, 2000; Krungkrai, 1993a, 1995; Kita et al. 2002). It is proposed that, in P. falciparum, these asexual and sexual developmental stages have functional mitochondria with major biochemical activities in mtETS complexes I, II, III and IV, and which provide other metabolic activities operating in the organelle (Fig. 5).

Fig. 5. A diagram of the proposed arrangement of the mitochondrial electron transport components and its association with pyrimidine biosynthesis via dihydroorotate dehydrogenase in the asexual and sexual blood stages of Plasmodium falciparum. The mitochondrial electron transporting complexes are NADH-ubiquinone oxidoreductase (NADH dehydrogenase, a component of complex I), succinate-ubiquinone oxidoreductase (succunate dehydrogenase, complex II), ubiquinol-cytochrome c reductase (cytochrome bc1 or complex III) and cytochrome c oxidase (complex IV). Alternative oxidase as a cyanide (CN−) insensitive-branched pathway (shown in a broken line) and glycerol-3-phosphate (glycerol-3-P) dehydrogenase are also included in the respiratory chain. Ubiquinone and cytochrome c (Cyt c, a component of complex III), are shown serving as electron and proton transferring components. Glycolysis, links to the tricarboxylic acid (TCA) cycle, is operating in the cytosol of P. falciparum. The tree-bars indicate known active sites of inhibitors and the antimalarial drug atvaquone. The diagram is adapted from web site: http://sites.huji.ac.il/malaria.

Mitochondrial oxygen consumption in asexual and sexual stages has been measured in the presence of 5-fluoroorotate, a potent inhibitor of DHODase of the pyrimidine biosynthetic pathway of P. falciparum (Krungkrai et al. 1999a), and in the presence of the antimalarial drugs, atovaquone (Fry & Pudney, 1992; Krungkrai et al. 1997) and primaquine (Vaidya et al. 1993) that possibly act on complex III, and artemisinin that might target specifically to complex IV (Zhao et al. 1986; Kawai et al. 1993). Table 2 shows the EC₅₀ values of 5-fluoroorotate, atovaquone, primaquine and artemisinin on the parasites' oxygen consumption in both developing stages. The 5-fluoroorotate shows strong inhibitory effects on the oxygen consumption in both the asexual- and the sexual-stage parasites, suggesting that the parasite in the sexual stage has a link to the 2 metabolic pathways, pyrimidine biosynthesis and mtETS, through the enzyme DHODase of the pyrimidine pathway, similar to the asexual blood stage (Krungkrai, 1995). The association between the pyrimidine pathway and mitochondrial ETS in the asexual-stage parasite has been recently confirmed (Krungkrai et al. 1999a,b; Takashima et al. 2001). It is concluded that in P. falciparum in asexual and sexual stages there are functioning mitochondria that contribute significantly to the de novo synthesis of pyrimidine and less to the membrane potential maintenance and the energy metabolism of the parasite as proposed in Fig. 5. The role of the mitochondria in the sexual blood stages, e.g., for gametocytogenesis in the human host, for exflagellation and gametogenesis in the mosquito vector, remains to be determined.

The findings of the effects of antimalarial drugs on oxygen consumption are directly related to the lines of evidence that these drugs act specifically on the mitochondria of the malarial parasite. Atovaquone inhibits electron transport and depolarizes the mitochondria of the asexual stages of the rodent parasite P. yoelii (Srivastava et al. 1997). It has moderate gametocytocidal activity (Fleck, Pudney & Sinden, 1996), compared to its potent blood schizontocidal activity (Krungkrai et al. 1999a). Primaquine (Vaidya et al. 1993) and artemether (Kawai et al. 1993), a derivative of artemisinin, act specifically on the mitochondria of P. falciparum by causing morphological changes. Artesunate, a derivative of artemisinin, inhibits complex IV activity in P. berghei (Zhao et al. 1986). More recently, Eckstein-Ludwig et al. (2003) have shown that artemisinin targets sarco/endoplasmic reticulum Ca²⁺-ATPase of P. falciparum. These multiple sites of action by artemisinin derivatives remain to be elucidated. Nevertheless, our results would suggest that the mitochondrion in both the asexual and sexual blood stages is a potential drug target.

OTHER METABOLIC ROLES OF THE PLASMODIUM FALCIPARUM MITOCHONDRION

Studies of mitochondrial functions in malaria parasites have been limited (Divo et al. 1985; Fry & Beesley, 1992; Krungkrai, 1995; Srivastava et al. 1997; Sato, Rangachari & Wilson, 2003; Sato & Wilson, 2004). Using a flow cytometric assay with a lipophilic cation-fluorescent probe, the mitochondrial membrane potential in live P. yoelii-infected erythrocytes can be measured. The membrane potential, generated by the mtETS, is constantly maintained to allow transport of metabolites and proteins across the inner mitochondrial membrane (Srivastava et al. 1997).

P. falciparum can synthesize haem de novo (Varadharajan et al. 2002). Proteins encoded by 2 nuclear genes, δ-aminolevulinate synthase and ferrochelatase, the first and the last enzymes of the de novo pathway, are localized in the mitochondrion (Wilson, Smith & Baylon, 1996; Varadharajan et al. 2002; Dhanasekaran et al. 2004). The second enzyme δ-aminolevulinate dehydratase has been found to be localized in the apicoplast organelle (Dhanasekaran et al. 2004). Compartmentalization of the de novo haem synthetic pathway in the parasite remains to be studied.

The asexual blood stages of P. falciparum rely principally on anaerobic glycolysis for ATP production. Genes encoding all of the enzymes necessary for a functional glycolytic pathway have been identified (Gardner et al. 2002). Furthermore, the genes necessary for a complete tricarboxylic acid (TCA) cycle, including a complete pyruvate dehydrogenase complex, have been identified (Fig. 6). The mitochondrial TCA cycle in these asexual stages may not be involved ATP production, but may serve mainly to synthesize metabolites for other metabolic pathways (Gardner et al. 2002; Chan & Sim, 2003; Wrenger & Muller, 2003). The succinate dehydrogenase component of the mitochondrial TCA cycle and the complex II of P. falciparum is also involved in this anaerobic metabolism by acting as quinol-fumarate reductase (Kita et al. 2002). NADH dehydrogenase acts as NADH-ubiquinol reductase, a component of complex I (Krungkrai et al. 2002). The malarial enzyme may have similar properties to other types of single polypeptide NADH dehydrogenase as found in Saccharomyces cerevisae (Luttik et al. 1998), and in T. cruzi (Fang & Beattie, 2002). The role of the mitochondrial NADH dehydrogenase in P. falciparum remains to be studied.

Fig. 6. Mitochondrial tricarboxylic acid (TCA) cycle of Plasmodium falciparum. The TCA cycle is a part of the mitochondrial metabolism shown in Fig. 5, but its function remains to be studied. It is shown here that metabolites produced by the TCA cycle are important for other metabolic pathways. The diagram is taken from web site: http://sites.huji.ac.il/malaria.

Genes encoding F₀ and F₁ subunits of ATP synthase (complex V) have also been identified in the P. falciparum genome. A possible functioning TCA cycle, oxidative phosphorylation and ATP synthase in the gametocytes remain to be explored.

HOW MANY MITOCHONDRIAL PROTEINS ARE THERE IN PLASMODIUM FALCIPARUM?

Gardner et al. (2002) have identified 246 (4·6% of total parasite proteins) proteins targeted to the mitochondrion after translation by the TargetP and MitoProtII methods. Using the recently developed PlasMit program for prediction of mitochondrial transit peptides, Bender et al. (2003) have predicted 381 (7·1%) mitochondrial proteins, based on 5334 annotated genes in the P. falciparum genome, close to the numbers of the mitochondrial proteins in other organisms. Most of these protein sequences are identified as hypothetical proteins (see detail in web site: http://gecco.org.chemie.uni-frankfurt.de/plasmit/). Nevertheless, homology searching has identified some components of metabolic pathways, transporter and import proteins (Table 3).

PLASMODIUM FALCIPARUM MITOCHONDRIA AS POTENTIAL CHEMOTHERAPEUTIC TARGETS

Some currently used antimalarial drugs act specifically on the mitochondria, e.g., tetracycline (Prapunwattana et al. 1988), primaquine (Lanners, 1991; Vaidya et al. 1993), atovaquone (Fry & Pudney, 1992; Krungkrai et al. 1997, 1998, 2003; Srivastava et al. 1997; Srivastava et al. 1999; Peters et al. 2002; Kessl et al. 2003) and artemisinin derivatives (Zhao et al. 1986; Kawai et al. 1993). Evidence from the action of antimalarial drugs further supports the view that the mitochondrion is a potential chemotherapeutic target. As mentioned before, in both the asexual and the sexual blood stages of P. falciparum, the DHODase of pyrimidine biosynthesis catalysing dihydroorotate oxidation and generating 2 electrons is thought to link to mtETS, containing ubiquinone (coenzyme Q), and complexes I, II, III and IV (Fig. 5). Most of these enzymes, as shown in Fig. 5, have been examined in detail by our laboratory. The predominant forms of ubiquinones in the asexual stage parasite are Q₈ and Q₉, whereas the major form in the mammalian host tissues is Q₁₀ (Porter & Folkers, 1974; Ellis, 1994; de Macedo et al. 2002). The Q_8–9 coenzyme performs a pivotal role in the transfer of electrons between the mtETS complexes and acts as a proton carrier that supports ATP synthesis (Hatefi, 1985). Thus the inhibition of the electron transfer at this site represents an important target for chemotherapeutic attack (Wan, Porter & Folkers, 1974).

One class of compounds extensively investigated over 50 years for antimalarial activity is the quinone analog, hydroxynaphthoquinone (Hudson, 1984; Hudson et al. 1985). One of the compounds, atovaquone which is 2-[trans-4-(4′-chlorophenyl)cyclohexyl]-3-hydroxy-1,4-naphthoquinone, is currently used as a potent antimalarial drug (TDR/WHO, 1997). Atovaquone affects multiple sites in which Q_8–9 plays a significant catalytic role in the target enzyme, e.g., complex III (Krungkrai et al. 1997, 1998; Srivastava et al. 1997; Kessl et al. 2003), and DHODase (Krungkrai, 1995; Krungkrai et al. 2003). The malaria parasites have their own synthesis of ubiquinones and cannot salvage this from the host (see detail in Fig. 7). Atovaquone at micromolar levels also strongly inhibits ubiquinone biosynthesis in P. falciparum (Krungkrai et al. unpublished results), similar to its effect on Pneumocystis carinii (Kaneshiro, Sul & Hazra, 2000).

Fig. 7. Mitochondrial ubiquinone (coenzyme Q) biosynthetic pathway of Plasmodium falciparum. The de novo coenzyme Q synthetic pathway is a part of the mitochondrial metabolism shown in Fig. 5. Major forms occurring in the parasite are coenzyme Q8–9. The parasite cannot salvage coenzyme Q10, an abundant source from the host. It is also shown that metabolites derived by the de novo pathway are used for other metabolic pathways. The diagram is taken from web site: http://sites.huji.ac.il/malaria.

Many derivatives of 5-hydroxy-2-methyl-1,4-naphthoquinone have been synthesized and tested for their antimalarial activities on the in vitro growth of P. falciparum (Likhitwitayawuid et al. 1998). These include 15 quinoline quinones (Krungkrai, 2000). The lead compound 5-hydroxy-2-methyl-1,4-naphthoquinone exhibits a strong effect on the malarial mtETS complex I, II and mitochondrial oxygen consumption (Suraveratum et al. 2000; Krungkrai et al. 2002). These lines of evidence also show that the quinoline quinones will represent a new class of compounds with potent antimalarial activity.

CONCLUSION, FUTURE DIRECTIONS AND IMPLICATIONS FOR THE MITOCHONDRION OF PLASMODIUM FALCIPARUM

In conclusion, whether or not the mitochondrial heterogeneity, i.e. in ultrastructure, biochemistry of respiratory system, amplification and differential expression of the mitochondrial gene, which are observed in the asexual and sexual blood stages, have functional significance in P. falciparum growth and development remains unclear. It is necessary to study the biochemistry and physiology of the mitochondrion in more detail, for instance, membrane potential, differences in the mechanism of energy metabolism, the mitochondrial tricarboxylic acid cycle, functioning oxidative phosphorylation and ATP synthase, functional properties of the mitochondrial electron transport complexes, the absence of genes involving multi-subunits of complex I, ubiquinone biosynthesis, haem biosynthesis and the role of low oxygen tension on the survival of the parasites circulating in the human blood system (Divo et al. 1985; Ginsburg et al. 1986; Waters, Syin & McCutchan, 1989; Basco & Le Dras, 1994; Escalante et al. 1998; Lobo & Kumar, 1998; Uyemura et al. 2000, 2004; Kita et al. 2002). Basic understanding of the organelle biogenesis is still lacking. There is also evidence that the malaria parasites produce oxidative stress via the mitochondrial superoxide radical generation and is finely balanced against the parasite's antioxidant enzyme activities (Krungkrai, 1991). Also, this phenomenon exists in the mammalian mitochondria (Dileepan & Kennedy, 1985).

Based on the malarial genome databases, about 380 proteins are predicted to target to the mitochondrion post-translationally. These include the pyruvate dehydrogenase complex, the complete tricarboxylic acid cycle enzymes, many electron transport complexes and ATP synthase (Gardner et al. 2002; Bender et al. 2003). Functional analyses remain to be clarified e.g., gene deletion, RNA interference, transcriptomics and proteomics. The mitochondrion is a potential chemotherapeutic target for antimalarial drug development, for instance, the enzyme dihydroorotate dehydrogenase (Krungkrai, 1993a; Baldwin et al. 2002; Ridley, 2002). In this post-genomics era, proteomics should be performed with mitochondria from all stages of the human parasite, in the presence or absence of possible compounds affecting biogenesis and functions of the organelles.