**3. The mitochondrial genome of malaria parasites**

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 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

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

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

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.

the potential of targeting NDH2 as an antimalarial drug remains controversial.

pyrimidine biosynthetic pathway rather than for contributing to the ATP pool [11].

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

gradients on the mitochondrial inner membrane.

generated.

22 An Overview of Tropical Diseases

Malaria parasites possess a mitochondrial genome in the form of circular and/or tandemly repeated linear elements of 6 kb, the smallest in size among eukaryotic cells [58]. Copy numbers for this element are approximately 20-fold and 150-fold of the nuclear genomes in the human malaria parasite *Plasmodium falciparum* [58] and the rodent malaria parasite *P. yoelii* [59], respectively. These differences in the copy number may reflect differences in oxidative phosphorylation activities as noted previously (see Section 2.3). The 6-kb element contains only three mitochondrial protein-coding genes in addition to the large subunit (LSU) and small subunit (SSU) rRNA genes [60, 61, 62] (Figure 3). The three protein-coding genes are cyt*c* oxidase subunit 1 (*cox1*) and subunit 3 (*cox3*), members of the cyt*c* oxidase complex (complex IV), and cyt*b* (*cob*), a member of cytochrome *bc1* complex. In all eukaryotic cells possessing mitochondria, *cox1* and *cob* are encoded by the mitochondrial genome. Because the organisms possessing mitochondrion-like organelles without its own DNA (e.g., hydrogenosome and mitosome) do not have *cox1* and *cob*, these two genes appear to be essential for maintenance of the mtETC.

**Figure 3.** Mitochondrial (mt) genome structure of malaria parasites. Mt-genome organization is perfectly conserved among 23 *Plasmodium* species [63]. Elements within the mt genome of *Plasmodium* are tandemly repeated, so the desig‐ nation of both termini is arbitrary. Light green and light magenta boxes indicate fragments of LSU and SSU rRNA genes, respectively.

The two rRNA genes of the *Plasmodium* mitochondrial genome are highly fragmented [63], and the fragmentation is the most extreme example of any described rRNA fragmentation. Recently, transcription of almost all intergenic regions of the *Plasmodium* mitochondrial genome has been demonstrated [63]. The results show that 27 small rRNA fragments (12 SSU rRNAs and 15 LSU rRNAs), ranging from 23 to 190 nt, are present in its mitochondrial genome (Figure 4). All the rRNAs are predicted to pair with at least one of the other rRNA, creating interactions that would help maintain the appropriate location and orientation of each rRNA. Notably, among the *Plasmodium* genera, the nucleotide sequences of noncoding regions, as well as fragmented rRNA gene regions, are more conserved when compared with those of the protein-coding gene regions [10]. It thus appears that these highly conserved sequence regions code for functional RNAs, including additional fragmented rRNAs.

In addition to the highly fragmented rRNAs, the mitochondria of malaria parasites have a unique property—transfer RNA (tRNA) is absent; therefore, protein translation in the

**Figure 4.** Fragmentation of the mitochondrial LSU and SSU rRNA genes of *Plasmodium falciparum*. Red line indicates rRNA regions. Purple lines indicate recently identified additional fragmented rRNA candidates[63].

mitochondrion was to date considered as being impossible. However, recently, extramito‐ chondrial phenylalanyl-tRNA synthesis has been found in mitochondria of the erythrocytic stages, suggesting that the parasite mitochondrion can import tRNAs from the cytoplasmic tRNA pool [64]. These findings referring to the parasite rRNAs and tRNAs would make the parasite mitochondrial protein translation a desirable organelle to target as an antimalarial drug.

In malaria parasites, mtDNA is replicated via rolling circle replication to generate the linear concatemers, similar to the replication mechanism used by some bacteriophages and plasmids [58]. This replication manner is remarkably different from that of the vertebrate mtDNA, which is replicated by a theta mechanism. Furthermore, mitochondrial DNA polymerase, which has been characterized as a γ-like DNA polymerase, is strongly resistant to 2,3-dideoxythymi‐ dine-5-triphosphate and, in this aspect, differs from its vertebrate homolog [65], suggesting structural differences between the *Plasmodium* and vertebrate DNA polymerase. Further research on translation and replication mechanisms of the parasite mtDNA may help identify potential targets for drug candidates.
