Abstract
BackgroundAspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is thus indispensable for the growth of Plasmodium parasites at the asexual blood stages. OAA can be produced in malaria parasites via two routes: (i) from phosphoenolpyruvate (PEP) by phosphoenolpyruvate carboxylase (PEPC) in the cytosol, or (ii) from fumarate by consecutive reactions catalyzed by fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO) in the mitochondria of malaria parasites. Although PEPC-deficient Plasmodium falciparum and Plasmodium berghei (rodent malaria) parasites show a growth defect, the mutant P. berghei can still cause experimental cerebral malaria (ECM) with similar dynamics to wild-type parasites. In contrast, the importance of FH and MQO for parasite viability, growth and virulence is not fully understood because no FH- and MQO-deficient P. falciparum has been established. In this study, the role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined.ResultsFirst, FH- and MQO-deficient parasites were generated by inserting a luciferase-expressing cassette into the fh and mqo loci in the genome of P. berghei ANKA strain. Second, the viability of FH-deficient and MQO-deficient parasites that express luciferase was determined by measuring luciferase activity, and the effect of FH or MQO deficiency on the development of ECM was examined. While the viability of FH-deficient P. berghei was comparable to that of control parasites, MQO-deficient parasites exhibited considerably reduced viability. FH activity derived from erythrocytes was also detected. This result and the absence of phenotype in FH-deficient P. berghei parasites suggest that fumarate can be metabolized to malate by host or parasite FH in P. berghei-infected erythrocytes. Furthermore, although the growth of FH- and MQO-deficient parasites was impaired, the development of ECM was suppressed only in mice infected with MQO-deficient parasites.ConclusionsThese findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.
Highlights
Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is indispensable for the growth of Plasmodium parasites at the asexual blood stages
Generation of fumarate hydratase (FH)‐disrupted and malate:quinone oxidoreductase (MQO)‐disrupted Plasmodium berghei To investigate the role of FH and MQO in malaria parasite growth in erythrocytes, fh- and mqo-disrupted P. berghei were generated
These findings demonstrate that the luciferase-expressing cassette was successfully introduced into fh and mqo locus
Summary
Aspartate, which is converted from oxaloacetate (OAA) by aspartate aminotransferase, is considered an important precursor for purine salvage and pyrimidine de novo biosynthesis, and is indispensable for the growth of Plasmodium parasites at the asexual blood stages. The role of FH and MQO in the pathogenicity of asexual-blood-stage Plasmodium parasites causing cerebral malaria was examined. Plasmodium spp. possesses all of the genes necessary for the tricarboxylic acid (TCA) cycle, [4] and most of the genes needed for electron transport chain (ETC) enzymes, asexual-bloodstage malaria parasites rely mainly on cytosolic glycolysis with limited contribution from mitochondrial oxidative phosphorylation for ATP synthesis [5, 6]. Two of the eight mitochondrial TCA cycle enzymes, fumarate hydratase (FH) and malate:quinone oxidoreductase (MQO), could not be genetically ablated in asexual-blood-stage Plasmodium falciparum, suggesting that these two enzymes are promising drug targets [9]
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