Suppression of experimental cerebral malaria by disruption of malate:quinone oxidoreductase

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1898-5) contains supplementary material, which is available to authorized users.

Biochemical characterization and essentiality of fumarate hydratase

“…A study that appeared recently reports on the knockout of fh gene in P. berghei, with the knockout parasites exhibiting slow growth phenotype (39). Though the authors show the absence of fh gene expression in the knockout strain, the genotyping for confirmation of knockout that was carried out with oligonucleotide primers corresponding to the homology arm used for recombination cannot confirm the site of integration.…”

“…Apart from the length of the homology arm used for recombination, the key difference is with regard to the strains of mice used. While our study has used BALB/c, Niikura et al, (39) have used the C57BL/6 strain of mice. P. berghei is known to exhibit differences in growth and infectivity across different strains of mice (40)(41)(42).…”

Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella

“…In other apicomplexan parasites, such as Plasmodium falciparum or Toxoplasma gondii , which are closely related parasite to Eimeria spp., the parasite’s mitochondria are also key organelles involved in cellular metabolic pathways essential to parasite’s adaptation and survival within different hosts’ environment. Amongst the mitochondrial metabolic pathways, the ETC is believed to be the key pathway for energy generation, pyrimidine de novo biosynthesis, and purine salvage pathways (through fumarate cycle), at least in P. falciparum [12,13,14]. In addition to ubiquinone, a low potential quinone species involved in anaerobic respiration, menaquinone, has been detected in P. falciparum [15].…”

Biochemical characterization and essentiality ofPlasmodiumfumarate hydratase

“…The PCRs with primers P9 and P10 (Fig 6 e) encompassing the full-length gene yielded the expected size band with genomic DNA from all 17 clones, indicating that all clones contain full-length fh gene. A study that appeared recently reports on the knockout of fh gene in P. berghei, with the knockout parasites exhibiting slow growth phenotype (38). Though the authors show the absence of fh gene expression in the knockout strain, the genotyping for confirmation of knockout that was carried out with oligonucleotide primers corresponding to the homology arm used for recombination cannot confirm the site of integration.…”

“…Apart from the length of the homology arm used for recombination, the key difference is with regard to the strains of mice used. While our study has used BALB/c, Niikura et al, (38) have used the C57BL/6 strain of mice. P. berghei is known to exhibit differences in growth and infectivity across different strains of mice (39)(40)(41).…”