**2. Biochemical functions of malaria parasite mitochondria**

#### **2.1. ATP production in canonical eukaryotes**

uninfected RBCs. These stages are called the erythrocytic stages, where the parasites multiply asexually. Following the establishment of infection, some parasites differentiate to gameto‐ cytes [4]. The gametocyte stage is essential for subsequent transmission because this is the only stage where the organism undergoes sexual development in the mosquito vector. Therefore, *Plasmodium* has a complex life cycle, which seems to be an adaptation to its host environment [5]. In addition to the complex life cycle, the malaria parasites have evolved sophisticated

pathways of energy transduction to adapt to their hosts.

18 An Overview of Tropical Diseases

**Figure 1.** Life cycle of the human malaria parasite *Plasmodium falciparum.*

Mitochondria, an organelle arising from alpha-proteobacterium engulfed by a eukaryotic progenitor [6], play a key role in energy transduction of eukaryotic cells. In vertebrates, that can become a host for malaria parasites, mitochondria have been reported to contribute to cellular responses such as autophagy, apoptosis, and ATP production [7]. The vertebrate mitochondrion comprises two separate and functionally distinct outer and inner membranes that form cristae, and it also contains its own circular genome, the mitochondrial genome (mtDNA). With few exceptions, vertebrate mtDNA is approximately 16 kb in size, encoding Conventionally, a mitochondrion is the cell's powerhouse, in which energy stored in chemical bonds is turned into ATP via oxidative phosphorylation. ATP production can be divided into three pathways: glycolysis, mitochondrial tricarboxylic acid (TCA) cycle, and mitochondrial electron transport chain (mtETC). Glycolysis breaks down one molecule of glucose into two molecules of pyruvate, generating two molecules of ATP. Pyruvate then moves into the mitochondrion where it is converted to acetyl-CoA and carbon dioxide by pyruvate dehydro‐ genase complex (PDH). Subsequently, acetyl-CoA enters the TCA cycle. The mtETC involves the passage of electrons from TCA-cycle NADH or from succinate via mtETC complexes to oxygen, with concomitant translocation of protons into the mitochondrial intermembrane space. Generally, the mtETC comprises four integral membrane enzyme complexes in the mitochondrial inner membrane: NADH-ubiquinone oxidoreductase (complex I), succinateubiquinone oxidoreductase (SQR, complex II), ubiquinol-cytochrome *c* oxidoreductase (complex III or cytochrome *bc*1), and cytochrome *c* oxidase (complex IV). Ubiquinone (Q) and cytochrome *c* (complex IV) function as electron carriers and the complexes I, III, and IV function as sites generating potential. The resultant potential across the mitochondrial inner membrane is used to drive ATP synthesis.

#### **2.2. ATP production in malaria parasites**

Similar to canonical eukaryotes, in the mosquito stages of malaria parasites, the organisms produce ATP in their mitochondria [13]. In the erythrocytic stages, however, the mitochondrial energy transduction system for oxidative phosphorylation is downregulated to adapt to host environments and produce ATP mainly via glycolysis using blood glucose [14, 15]. As a consequence, in malaria parasite-infected patients, plasma lactate levels tend to be high and highly variable, ranging from 2 to 26.7 mM [16, 17], compared with plasma lactate levels (0.3-1.3 mM) in normal individuals. Apart from the minor flux of carbon backbone derived from glucose, TCA metabolism of *Plasmodium* was believed to involve a branched architecture bifurcating from 2-oxoglutarate until recently [18]; however, this report was subsequently retracted [19]. More recently, the malaria parasites have been reported to use the canonical oxidative mitochondrial TCA cycle to catabolize host glucose and glutamate (Figure 2) [20], even during asexual multiplication. The TCA cycle begins with malate generated by anapler‐ otic reactions and 2-oxoglutarate produced from glutamine as well as conversion of acetyl-CoA to citrate by citrate synthase [20-22]. In general, pyruvate, the end product of glycolysis, is transported via the monocarboxylate transporter (MCT) family [23]. *Plasmodium* possesses two MCT genes (PF3D7\_0210300 and PF3D7\_0926400) identified in its genome (PlasmoDB version 11.0, website: http://plasmadb.org/plasma/). Although further evidence is required, these MCTs are considered to be associated with the transport of pyruvate across the mito‐ chondrial membrane [24]. To convert pyruvate into acetyl-CoA, *Plasmodium* retains branched chain ketoacid dehydrogenase (BCKDH), the only enzyme implicated in branched chain amino acid degradation [22]. PDH complex, linking cytoplasmic glycolysis to the TCA cycle in canonical eukaryotes, is not localized to the mitochondrion but to a plastid, apicoplast, in *Plasmodium* [25]. The function of the *Plasmodium* PDH complex seems to include the provision of acetyl-CoA for *de novo* fatty acid synthesis within the apicoplast.

**Figure 2.** TCA cycle and oxidative phosphorylation of malaria parasites. The TCA cycle of malaria parasites begins with malate, 2-oxoglutarate, and citrate [20-22].

In the *Plasmodium* TCA cycle, succinate and malate are oxidized by SQR and malate-ubiqui‐ none oxidoreductase (MQO), respectively, with transporting electrons to the matrix [26] (Figure 2). Similar to SQR of most eukaryotes, the *Plasmodium* SQR comprises four polypep‐ tides: a flavoprotein (Fp) subunit, iron-sulfur (Ip) subunit [27], and two cytochrome *b* (cyt*b*) subunits (CybL and CybS) [28]. Fp and Ip form the catalytic portion of the complex. This portion acts as a succinate dehydrogenase (SDH), catalyzing the oxidation of succinate by water-soluble electron acceptors such as phenazine methosulfate in SQR, and is bound to the matrix side of the mitochondrial inner membrane via the membrane-anchoring proteins CybL and CybS. Because the mitochondria of erythrocytic stage parasites show both SQR and SDH activities [27, 29, 30], complex II has been considered to have some role in parasite survival. These activities, however, are very low, compared with those of the other eukaryotes (Table 1) [31-35]. Furthermore, our previous studies have demonstrated that disruption of the Fp subunit genes *pfsdha* and *Pbsdha* does not affect growth in the erythrocytic stages *in vitro* [36] and *in vivo* [37], respectively. These findings reveal that complex II is not essential for survival of the erythrocytic stage parasites, and this appears to be associated with relatively low activities of SQR and SDH in these developmental stages.

bifurcating from 2-oxoglutarate until recently [18]; however, this report was subsequently retracted [19]. More recently, the malaria parasites have been reported to use the canonical oxidative mitochondrial TCA cycle to catabolize host glucose and glutamate (Figure 2) [20], even during asexual multiplication. The TCA cycle begins with malate generated by anapler‐ otic reactions and 2-oxoglutarate produced from glutamine as well as conversion of acetyl-CoA to citrate by citrate synthase [20-22]. In general, pyruvate, the end product of glycolysis, is transported via the monocarboxylate transporter (MCT) family [23]. *Plasmodium* possesses two MCT genes (PF3D7\_0210300 and PF3D7\_0926400) identified in its genome (PlasmoDB version 11.0, website: http://plasmadb.org/plasma/). Although further evidence is required, these MCTs are considered to be associated with the transport of pyruvate across the mito‐ chondrial membrane [24]. To convert pyruvate into acetyl-CoA, *Plasmodium* retains branched chain ketoacid dehydrogenase (BCKDH), the only enzyme implicated in branched chain amino acid degradation [22]. PDH complex, linking cytoplasmic glycolysis to the TCA cycle in canonical eukaryotes, is not localized to the mitochondrion but to a plastid, apicoplast, in *Plasmodium* [25]. The function of the *Plasmodium* PDH complex seems to include the provision

**Figure 2.** TCA cycle and oxidative phosphorylation of malaria parasites. The TCA cycle of malaria parasites begins

with malate, 2-oxoglutarate, and citrate [20-22].

of acetyl-CoA for *de novo* fatty acid synthesis within the apicoplast.

20 An Overview of Tropical Diseases


\*Activity values of *P. falciparum*, *T. cruzi*, *A. suum*, rat liver, bovine heart, and human cell are obtained from references [29, 31, 32, 33, 34], and [35], respectively.

**Table 1.** Specific activities of succinate-ubiquinone oxidoreductase of various organisms

MQO is an FAD-dependent membrane-associated protein that catalyzes the oxidation of malate to oxaloacetate [38]. The electrons are donated to quinones of the mtETC, and NAD is accepted as an electron donor. The MQO has been not observed in mammals but has been found in *Plasmodium* [39] and some bacteria [40]. This implies that the *Plasmodium* MQO could be a target for drug design. In addition to SQR and MQO in the TCA cycle, *Plasmodium* possesses three oxidoreductases in its mitochondrial inner membrane: type II NADH:ubiqui‐ none oxidoreductase (NDH2) [41], dihydroorotate dehydrogenase (DHODH) [42, 43], and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) [44, 45], all of which can reduce ubiquinone (Figure 2). Unlike the large multisubunit complex I in most mitochondria, the *Plasmodium* NDH2 is a single subunit enzyme, not involved in the direct pumping of protons across the membrane [46]. The absence of NDH2 in mammalian mitochondria shows that this enzyme would be a promising target of a novel antimalarial drug. Some antimalarial activities of NDH2 inhibitors, such as HQNO [47] and 1-hydroxy-2-dodecyl-4(1H) quinolone [48], have been reported. However, a recent *in vivo* study on *Plasmodium berghei* revealed that the *Plasmodium* NDH2 could be deleted by targeted gene disruption, indicating that it is dispen‐ sable in the erythrocytic stages [49]. This disproves that NDH2 is a candidate drug target. Thus, the potential of targeting NDH2 as an antimalarial drug remains controversial.

The other dehydrogenases (DHODH and G3PDH) transfer electrons from reduced com‐ pounds in the cytosol (Figure 2). In the erythrocytic stages of the parasite, DHODH plays two roles—a generator of reduced ubiquinone and the fourth enzyme in the pyrimidine biosyn‐ thetic pathway. Since *Plasmodium* cannot salvage pyrimidine [50], DHODH is essential for its survival [42]. Therefore, in the erythrocytic stages, the mtETC appears to be essential for the pyrimidine biosynthetic pathway rather than for contributing to the ATP pool [11].

As presented above, in *Plasmodium* mitochondria, five mitochondrial dehydrogenases (SQR, MQO, NDH2, DHODH, and G3PDH) can generate reduced ubiquinone, which in turn is reoxidized by cytochrome *bc*<sup>1</sup> complex (complex III). Complex III is inhibited by atovaquone [51], which collapses the mitochondrial membrane potential [52]. As an antimalarial, atova‐ quone is very effective; however, atovaquone-resistant parasites develop easily. Mechanisms of atovaquone resistance are described in Section 4. Similar to canonical eukaryotes, *Plasmo‐ dium* utilizes cytochrome *c* (cyt*c*) as electron carriers and complexes III and IV as sites gener‐ ating potential. The resultant potential across the mitochondrial inner membrane is used to drive ATP synthesis. *Plasmodium* ATP synthase is markedly different from that of its host [53] —it is assembled as a large dimeric complex in the erythrocytic stages. In the ciliates *Tetrahy‐ mena thermophila* and *Paramecium*, the structure and arrangement of dimeric ATP synthase have been suggested to determine the tubular morphology of the mitochondrial cristae [54, 55]. This could explain how the tubular cristae found in the mitochondria of erythrocytic stages are generated.

#### **2.3. Mitochondrial energy metabolism: a target of antimalarial drugs**

Recently, in addition to the genetic disruptions of SDH and NDH2 described above, it has been reported that six TCA cycle enzymes can be genetically disrupted in the erythrocytic stage or sexual development stage [45]. These reports suggest that the TCA cycle would not be essential for survival in these developmental stages. Hence, to develop an antimalarial drug, promising mitochondrial targets would be DHODH, which is associated with the pyrimidine biosynthe‐ sis pathway and mtETC, and the mitochondrial complexes III, IV, and V that generate electron gradients on the mitochondrial inner membrane.

On the other hand, it has been recently demonstrated that parasites derived directly from infected patients show three distinct gene expression states. One of these states demonstrates that the expression levels of the TCA cycle- or mtETC-related genes are increased [56]. Furthermore, mice infected with *P. berghei* or *Plasmodium yoelii* perform active oxidative phosphorylation [57], suggesting that, in some physiological conditions, malaria parasites may produce ATP via the mitochondrial TCA cycle and mtETC. Thus, we cannot exclude the possibility that all the mitochondrial enzymes are potential targets for antimalarial drugs.
