Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Takashima, Eizo; Takamiya, Shinzaburo; Takeo, Satoru; Mi-ichi, Fumika; Amino, Hisako; Kita, Kiyoshi

doi:10.1016/s1383-5769(01)00085-x

Cited by 42 publications

(39 citation statements)

References 30 publications

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“…In addition, because N2 cavitation is known to be among the mildest ways to maintain membrane integrity [e.g. in a study dealing with plasmodial mitochondria (Takashima et al, 2001)], these results suggest that the plasmodial DGAT enzyme and its substrate DAG are compartmentalized on parasite membranes.…”

Section: Composition and Distribution Of Neutral Lipid Species In Infmentioning

confidence: 92%

“…When indicated, isolated parasite cells prepared by saponin treatment were disrupted through N2 cavitation method with a 4639 cell disruption bomb from Parr Instrument (IL, USA) (Takashima et al, 2001) and stored at -80°C until use. The DGAT activity assay was performed as described (Coleman and Bell, 1976) with slight modifications.…”

Section: Dgat Activity Assaymentioning

confidence: 99%

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Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies inPlasmodium falciparum-infected erythrocytes

Palacpac

Hiramine

Mi-ichi

et al. 2004

Journal of Cell Science

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Triacylglycerol (TAG) serves as a major energy storage molecule in eukaryotes. In Plasmodium, however, this established function of TAG appears unlikely, despite detecting previously considerable amount of TAG associated with intraerythrocytic parasites, because plasmodial cells have very little capacity to oxidize fatty acids. Thus, it is plausible that TAG and its biosynthesis in Plasmodium have other functions. As a first step in understanding the biological significance of TAG and its biosynthesis to the intraerythrocytic proliferation of Plasmodium falciparum, we performed detailed characterization of TAG metabolism and trafficking in parasitized erythrocyte. Metabolic labeling using radiolabeled-oleic and palmitic acids in association with serum albumin, which have been shown to be among the serum essential factors for intraerythrocytic proliferation of P. falciparum, revealed that accumulation of TAG was strikingly pronounced from trophozoite to schizont, whereas TAG degradation became active from schizont to segmented schizont; the consequent products, free fatty acids, were released into the medium during schizont rupture and/or merozoite release. These results were further supported by visualization of lipid bodies through immunofluorescence and electron microscopy. At the schizont stages, there is some evidence that the lipid bodies are partly localized in the parasitophorous vacuole. Interestingly, the discrete formation and/or trafficking of lipid bodies are inhibited by brefeldin A and trifluoperazine. Inhibition by trifluoperazine hints at least that a de novo TAG biosynthetic pathway via phosphatidic acid contributes to lipid body formation. Indeed, biochemical analysis reveals a higher activity of acyl-CoA:diacylglycerol acyltransferase, the principal enzyme in the sn-glycerol-3-phosphate pathway for TAG synthesis, at trophozoite and schizont stages. Together, these results establish that TAG metabolism and trafficking in P. falciparum-infected erythrocyte occurs in a stage-specific manner during the intraerythrocytic cycle and we propose that these unique and dynamic cellular events participate during schizont rupture and/or merozoite release.

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Section: Composition and Distribution Of Neutral Lipid Species In Infmentioning

confidence: 92%

Section: Dgat Activity Assaymentioning

confidence: 99%

Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies inPlasmodium falciparum-infected erythrocytes

Palacpac

Hiramine

Mi-ichi

et al. 2004

Journal of Cell Science

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“…Following the establishment of the mitochondrial isolation method in the malaria parasite [14], we further tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant and sensitive clones in the presence of a wide concentration range of atovaquone for the sensitivity of the enzymes to the drug which might be altered by the cytochrome b mutations. We found that mutations in the quinone binding site of the cytochrome b gene resulted in a various sensitivity to atovaquone and provide a direct evidence for the atovaquone inhibitory mechanism in the parasite cytochrome bc 1 complex.…”

Section: Introductionmentioning

confidence: 99%

Direct evidence for the atovaquone action on the Plasmodium cytochrome bc 1 complex

Siregar

Kurisu

Tamaki

et al. 2015

Parasitology International

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Atovaquone, a coenzyme Q analogue has been indicated to specifically target the cytochrome bc1 complex of the mitochondrial respiratory chain in the malarial parasite and other protozoan. Various mutations in the quinone binding site of the cytochrome b gene of Plasmodium spp. such as M133I, L144S, L271V, K272R, Y268C, Y268S, Y268N, and V284F are suggesting to associate with resistance to atovaquone. There is no direct evidence of relation between the mutations and resistance to atovaquone in Plasmodium parasite that has been available. Technical difficulties in isolating active assayable mitochondria in the malarial parasite hinder us to obtain direct biochemical evidence to support the relation between the mutations and drug resistance. The establishment of a mitochondrial isolation method for the malaria parasite has allowed us to test the degree of resistance of Plasmodium berghei isolates to atovaquone directly. We have tested the activity of dihydroorotate (DHO)-cytochrome c reductase in various P. berghei atovaquone resistant clones in the presence of a wide concentration range of atovaquone. Our results show the IC(50) of P. berghei atovaquone resistant clones is much higher (1.5 up to 40 nM) in comparison to the atovaquone sensitive clones (0.132-0.465 nM). The highest IC(50) was revealed in clones carrying Y268C and Y268N mutations (which play an important role in atovaquone resistance in Plasmodium falciparum), with an approximately 100-fold increase. The findings indicate the importance of the mutation in the quinone binding site of the cytochrome b gene and that provide a direct evidence for the atovaquone inhibitory mechanism in the cytochrome bc1 complex of the parasite.

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“…The latter would require either an efficient alternate source of quinol oxidation in place of the cyt bc 1 complex, or that DHODH utilize an alternate readily-available electron acceptor other than ubiquinone. While succinate dehydrogenase [Complex II] can function in "reverse" to oxidize ubiquinol [27], its maximal capacity to do so is unknown; the capability of the Type II DHODH of Plasmodium to employ other electron acceptors has not yet been demonstrated in intact parasites.…”

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confidence: 99%

A drug-selected Plasmodium falciparum lacking the need for conventional electron transport

Smilkstein

Forquer

Kanazawa

et al. 2008

Molecular and Biochemical Parasitology

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Mitochondrial electron transport is essential for survival in Plasmodium falciparum, making the cytochrome (cyt) bc 1 complex an attractive target for antimalarial drug development. Here we report that P. falciparum cultivated in the presence of a novel cyt bc 1 inhibitor underwent a fundamental transformation in biochemistry to a phenotype lacking a requirement for electron transport through the cyt bc 1 complex. Growth of the drug-selected parasite clone (SB1-A6) is robust in the presence of diverse cyt bc 1 inhibitors, although electron transport is fully inhibited by these same agents. This transformation defies expected molecular-based concepts of drug resistance, has important implications for the study of cyt bc 1 as an antimalarial drug target, and may offer a glimpse into the evolutionary future of Plasmodium.Unlike most other eukaryotic cells, Plasmodia do not rely on mitochondrial electron transport for energy production. Nonetheless, inhibition of cyt bc 1 complex electron transport is normally lethal to the parasite, presumably by interruption of essential links to de novo pyrimidine biosynthesis, via dihydroorotate dehydrogenase (DHODH), and to maintenance of the mitochondrial membrane potential. Although these interrelationships determine the viability of the parasite and our ability to reduce that viability, they are only generally understood. Review of Plasmodial electron transport and cyt bc 1 complex function are beyond the scope of this report, but in-depth discussions are available [1][2][3].The anti-malarial drug atovaquone occupies the quinol oxidase (Q o ) site of mitochondrial cyt b, inhibiting electron flux through the cyt bc 1 complex (ubiquinol:cytochrome c oxidoreductase or complex III) and collapsing mitochondrial membrane potential with a potency 1000-fold greater in Plasmodium than mammalian cells [4,5]. Unfortunately, high rates of recrudescent infection and treatment failure were seen after anti-malarial use of atovaquone alone [6], and treatment failures after atovaquone-proguanil combination therapy (Malarone®) were soon evident [7,8] despite only limited worldwide anti-malarial use. The rapid development and the diversity of atovaquone-resistant phenotypes and genotypes (perhaps a consequence of its mechanism of action [9]), the parallel evolution of resistance-encoding mutations [10] and their appearance with or without atovaquone exposure [11] in dispersed geographic locations, all indicate the strong propensity for atovaquone resistance. The hope that this risk can be overcome by appropriate combination therapy or by novel inhibitors continues to drive efforts to discover and develop cyt bc 1 inhibitor anti-malarials [12].Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that ...

show abstract

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration

Cited by 42 publications

References 30 publications

Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies inPlasmodium falciparum-infected erythrocytes

Developmental-stage-specific triacylglycerol biosynthesis, degradation and trafficking as lipid bodies inPlasmodium falciparum-infected erythrocytes

Direct evidence for the atovaquone action on the Plasmodium cytochrome bc 1 complex

A drug-selected Plasmodium falciparum lacking the need for conventional electron transport

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