Niikura Mamoru, Komatsuya Keisuke, Inoue Shin-Ichi, Matsuda Risa, Asahi Hiroko, Inaoka Daniel Ken, Kita Kiyoshi, Kobayashi Fumie
Department of Infectious Diseases, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo, 181-8611, Japan.
School of Tropical Medicine and Global Health, Nagasaki University, Nagasaki, 852-8523, Japan.
Malar J. 2017 Jun 12;16(1):247. doi: 10.1186/s12936-017-1898-5.
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 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.
First, 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.
These findings suggest that MQO-mediated mitochondrial functions are required for development of ECM of asexual-blood-stage Plasmodium parasites.
天冬氨酸由天冬氨酸转氨酶从草酰乙酸（OAA）转化而来，被认为是嘌呤补救合成和嘧啶从头生物合成的重要前体，因此对于疟原虫在无性血液阶段的生长不可或缺。OAA可通过两条途径在疟原虫中产生：（i）由细胞质中的磷酸烯醇丙酮酸羧化酶（PEPC）从磷酸烯醇丙酮酸（PEP）生成，或（ii）由疟原虫线粒体中的延胡索酸水合酶（FH）和苹果酸：醌氧化还原酶（MQO）催化的连续反应从延胡索酸生成。尽管缺乏PEPC的恶性疟原虫和伯氏疟原虫（啮齿动物疟疾）寄生虫表现出生长缺陷，但突变的伯氏疟原虫仍可引起实验性脑疟疾（ECM），其动态与野生型寄生虫相似。相比之下，由于尚未建立缺乏FH和MQO的恶性疟原虫，FH和MQO对寄生虫生存能力、生长和毒力的重要性尚未完全了解。在本研究中，研究了FH和MQO在导致脑疟疾的无性血液阶段疟原虫致病性中的作用。
首先，通过将表达荧光素酶的盒插入伯氏疟原虫ANKA株基因组中的fh和mqo基因座，产生了缺乏FH和MQO的寄生虫。其次，通过测量荧光素酶活性来确定表达荧光素酶的缺乏FH和缺乏MQO的寄生虫的生存能力，并研究FH或MQO缺乏对ECM发展的影响。虽然缺乏FH的伯氏疟原虫的生存能力与对照寄生虫相当，但缺乏MQO的寄生虫的生存能力显著降低。还检测到了源自红细胞的FH活性。这一结果以及缺乏FH的伯氏疟原虫寄生虫中无表型表明，在感染伯氏疟原虫的红细胞中，延胡索酸可被宿主或寄生虫的FH代谢为苹果酸。此外，尽管缺乏FH和MQO的寄生虫的生长受到损害，但仅在感染缺乏MQO的寄生虫的小鼠中，ECM的发展受到抑制。
这些发现表明，MQO介导的线粒体功能是无性血液阶段疟原虫ECM发展所必需的。
