Amy Sorensen and Dr. James B. Jensen, Microbiology

The protozoa, Plasmodium falciparum, causes malaria, a disease that rivals tuberculosis in being the world’s largest killer. In order to combat malaria, new methods of chemotherapy, as well as better control of its causative agent are necessary. Knowing the main source from which P. falciparum gains its electrons may provide a new method for its control.

The mitochondrion, commonly known as the “powerhouse” of the cell, is the organelle where high energy electrons from glucose and other sources are passed down the electron transport chain to the final electron acceptor, oxygen, forming water. The energy of the “falling” electrons is captured as chemical energy in the form of ATP.

Observations with the transmission electron microscope show that the malaria parasite, P. falciparum, has an abnormal mitochondrial structure. Electron transport occurs along the cristate membrane. Plasmodium falciparum, however, lacks cristae. This suggests that the organelle’s function is altered with respect to this process. It has also been shown that P. falciparum’s mitochondrion lacks site one of the electron transport pathway. This, and the fact that no Kreb’s cycle enzymes have been identified, lends further evidence that this organelle might be significantly different from classical mitochondria.

Because the principal role played by the Kreb’s cycle is to provide high energy electrons for the electron transport, and since the parasite seems to lack this critical pathway in its entirety, the question naturally arises as to the source of electrons for the electron transport. There are two possibilities. Recent studies have shown that, unlike most eukaryotic mitochondria, P. falciparum’s mitochondria apparently have no site one where most electrons enter the electron transport chain. 1 However, site two is active and allows the entrance of electrons into the electron transport pathway from the electron-rich compound succinate, normally a Kreb’s cycle intermediate. At this site succinate is oxidized to fumarate. Plasmodium falciparum probably uses the enzyme fumarate reductase, a rare enzyme not found in vertebrate cells, to reduce fumarate to succinate and gleans the electron for its own use. However, since the parasite lacks a Kreb’s cycle, it is necessary to determine how the parasite obtained such Kreb’s cycle intermediates. It is possible that P. falciparum possesses the enzyme fumarate reductase, a common occurrence in invertebrates and bacteria. Tapeworm Hymenolepis diminuta and protozoa Leishmania major have fumarate reductase. The function of fumarate reductase is unique. Electrons are brought into the parasite’s mitochondria via the malate shuttle. In the cytosol, electrons are transferred from NADH to oxaloacetate, forming malate. Malate crosses the inner mitochondrial membrane and is dehydrated to fumarate by fumarate hydratase. Fumarate is then reduced to succinate by fumarate reductase. Electrons from succinate can then enter the electron transport chain at complex II.

The other possible source of electrons for the parasite is from the pyrimidine precursor, orotic acid. In the trophozoite stage, the parasite synthesizes new DNA in order to replicate itself. Orotic acid is a major pyrimidine precursor, and during pyrimidine synthesis electrons are released for the parasite to use or disperse. Excess electrons from the conversion of orotic acid to pyrimidines might be released down the electron transport chain, where it joins with oxygen, forming water.

This experiment consisted of four distinct phases: culturing parasites, synchronizing the cultures, collecting media for assaying, and performing glucose assays. A commercially prepared glucose assay was used to analyze the concentration of glucose in the sampled medium. The concentration of glucose present was compared against data from previous research which showed when electron transport was highest through the use of fluorescent dye DiOC6 and flow cytometry. 1

Our aim in this experiment was to show that glucose may be a main source for electrons in P. falciparum. Our results show that metabolic activity mimics electron transport. Previous research indicates that the mitochondria in the ring stage and the trophozoit/schizont stages take up the cationic dye DiOC6.1 This means that the parasite clearly has a source of electrons other than the pyrimidine biosynthetic pathway. Pyrimidines are only synthesized in the late trophozoite/ early schizont phase of the parasite’s life- cycle. Therefore, if the electrons for electrons transport in P. falciparum were to come solely from the pyrimidine biosynthetic pathway, electron transport would not occur during the ring phase. However, figure one clearly illustrates that electron transport occurs during every phase of the parasite’s life-cycle. Also, the data suggest that the rate of electron transport doubles between the ring and trophozoite phases. This clearly indicates that electron transport occurs during the ring phase. Therefore, we can conclude, as hypothesized, that glucose is reasonable as a main source of electrons for electron transport, while the pyrimidine biosynthetic pathway is not.