The genomes of malaria parasites contain many genes of unknown function. To assist drug development through the identification of essential genes and pathways, we have measured competitive growth ...rates in mice of 2,578 barcoded Plasmodium berghei knockout mutants, representing >50% of the genome, and created a phenotype database. At a single stage of its complex life cycle, P. berghei requires two-thirds of genes for optimal growth, the highest proportion reported from any organism and a probable consequence of functional optimization necessitated by genomic reductions during the evolution of parasitism. In contrast, extreme functional redundancy has evolved among expanded gene families operating at the parasite-host interface. The level of genetic redundancy in a single-celled organism may thus reflect the degree of environmental variation it experiences. In the case of Plasmodium parasites, this helps rationalize both the relative successes of drugs and the greater difficulty of making an effective vaccine.
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•Two-thirds of Plasmodium berghei genes contribute to normal blood stage growth•The core genome of malaria parasites is highly optimized for rapid host colonization•Essential parasite genes and pathways are identified for drug target prioritization•Low functional redundancy reflects the constant environment encountered by a parasite
An in vivo genetic screen in a mouse model of malaria reveals the essential genes and pathways required by Plasmodium parasite, with a surprising two-thirds of the genome being required for normal parasite growth in the blood.
Mitochondria in malaria parasites have some unusual evolutionary and functional features. The drastic reduction in the size of their mitochondrial genome, encoding just three proteins, appears to ...have originated at the point of divergence of dinoflagellates and apicomplexan parasites from ciliates and may have accompanied the acquisition of plastids by the former. Unusual translational machinery as revealed by the highly fragmented mitochondrial ribosomal RNA genes also appears to have originated at this deflection point. Some of the biochemical properties of malarial mitochondria also appear to be unconventional. Although tricarboxylic acid cycle enzymes are encoded by the genome, they do not appear to be involved in the full oxidation of glucose to fuel mitochondrial ATP synthesis in the blood stages of malaria parasites. A critical role of the mitochondrial electron transport chain appears to be to serve pyrimidine biosynthesis. In spite of their minimal nature, Plasmodium mitochondria are attractive targets for antimalarial drugs.
New antimalarial drugs are urgently needed to control drug-resistant forms of the malaria parasite Plasmodium falciparum. Mitochondrial electron transport is the target of both existing and new ...antimalarials. Herein, we describe 11 genetic knockout (KO) lines that delete six of the eight mitochondrial tricarboxylic acid (TCA) cycle enzymes. Although all TCA KOs grew normally in asexual blood stages, these metabolic deficiencies halted life-cycle progression in later stages. Specifically, aconitase KO parasites arrested as late gametocytes, whereas α-ketoglutarate-dehydrogenase-deficient parasites failed to develop oocysts in the mosquitoes. Mass spectrometry analysis of 13C-isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development.
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•Six of the eight TCA cycle enzymes were knocked out without affecting asexual growth•Metabolic labeling was analyzed in nine TCA KOs via 13C-labeling and mass spectrometry•The TCA cycle is adaptable, and the effect of a disrupted TCA cycle is stage specific
Mitochondria of malaria parasites have features that are divergent from their host’s mitochondria. Ke et al. show that six of the TCA cycle enzymes can be disrupted without affecting asexual stages of Plasmodium falciparum. The TCA cycle is adaptable and is essential in insect stages of the parasite.
Highlights • Toxoplasma and Plasmodium possess two organelles of endosymbiotic origin: the apicoplast, and the mitochondrion. • The mitochondrion hosts a complete TCA cycle and an electron transport ...chain lacking complex I. Glutamine catabolism contributes to the TCA cycle. • Haem biosynthesis is an extremely unusual chimerical pathway shared between the two organelles and the cytoplasm. • Isoprenoid precursor synthesis and fatty acid synthesis are essential roles of the apicoplast. Different life stages show differential dependencies on these pathways.
The mitochondrion of malaria parasites is an attractive antimalarial drug target, which require mitoribosomes to translate genes encoded in the mitochondrial (mt) DNA. Plasmodium mitoribosomes are ...composed of highly fragmented ribosomal RNA (rRNA) encoded in the mtDNA. All mitoribosomal proteins (MRPs) and other assembly factors are encoded in the nuclear genome. Here, we have studied one putative assembly factor, RSM22 (Pf3D7_1027200) and one large subunit (LSU) MRP, L23 (Pf3D7_1239100) in Plasmodium falciparum. We show that both proteins localize to the mitochondrion. Conditional knock down (KD) of PfRSM22 or PfMRPL23 leads to reduced cytochrome bc1 complex activity and increased sensitivity to bc1 inhibitors such as atovaquone and ELQ-300. Using RNA sequencing as a tool, we reveal the transcriptomic changes of nuclear and mitochondrial genomes upon KD of these two proteins. In the early phase of KD, while most mt rRNAs and transcripts of putative MRPs were downregulated in the absence of PfRSM22, many mt rRNAs and several MRPs were upregulated after KD of PfMRPL23. The contrast effects in the early phase of KD likely suggests non-redundant roles of PfRSM22 and PfMRPL23 in the assembly of P. falciparum mitoribosomes. At the late time points of KD, loss of PfRSM22 and PfMRPL23 caused defects in many essential metabolic pathways and transcripts related to essential mitochondrial functions, leading to parasite death. In addition, we enlist mitochondrial proteins of unknown function that are likely novel Plasmodium MRPs based on their structural similarity to known MRPs as well as their expression profiles in KD parasites.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Spiroindolone and pyrazoleamide antimalarial compounds target Plasmodium falciparum P-type ATPase (PfATP4) and induce disruption of intracellular Na
homeostasis. Recently, a PfATP4 mutation was ...discovered that confers resistance to a pyrazoleamide while increasing sensitivity to a spiroindolone. Transcriptomic and metabolic adaptations that underlie this seemingly contradictory response of P. falciparum to sublethal concentrations of each compound were examined to understand the different cellular accommodation to PfATP4 disruptions.
A genetically engineered P. falciparum Dd2 strain (Dd2
) carrying an Ala211Val (A211V) mutation in PfATP4 was used to identify metabolic adaptations associated with the mutation that results in decreased sensitivity to PA21A092 (a pyrazoleamide) and increased sensitivity to KAE609 (a spiroindolone). First, sublethal doses of PA21A092 and KAE609 causing substantial reduction (30-70%) in Dd2
parasite replication were identified. Then, at this sublethal dose of PA21A092 (or KAE609), metabolomic and transcriptomic data were collected during the first intraerythrocytic developmental cycle. Finally, the time-resolved data were integrated with a whole-genome metabolic network model of P. falciparum to characterize antimalarial-induced physiological adaptations.
Sublethal treatment with PA21A092 caused significant (p < 0.001) alterations in the abundances of 91 Plasmodium gene transcripts, whereas only 21 transcripts were significantly altered due to sublethal treatment with KAE609. In the metabolomic data, a substantial alteration (≥ fourfold) in the abundances of carbohydrate metabolites in the presence of either compound was found. The estimated rates of macromolecule syntheses between the two antimalarial-treated conditions were also comparable, except for the rate of lipid synthesis. A closer examination of parasite metabolism in the presence of either compound indicated statistically significant differences in enzymatic activities associated with synthesis of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol.
The results of this study suggest that malaria parasites activate protein kinases via phospholipid-dependent signalling in response to the ionic perturbation induced by the Na
homeostasis disruptor PA21A092. Therefore, targeted disruption of phospholipid signalling in PA21A092-resistant parasites could be a means to block the emergence of resistance to PA21A092.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The origin of all mitochondria can be traced to the symbiotic arrangement that resulted in the emergence of eukaryotes in a world that was exclusively populated by prokaryotes. This arrangement, ...however, has been in continuous genetic flux: the varying degrees of gene loss and transfer from the mitochondrial genome in different eukaryotic lineages seem to signify an ongoing 'conflict' between the host and the symbiont. Eukaryotic parasites belonging to the phylum Apicomplexa provide an excellent example to support this view. These organisms contain the smallest mitochondrial genomes known, with an organization that differs among various genera; one genus, Cryptosporidium, seems to have lost the entire mitochondrial genome. Here we show that erythrocytic stages of the human malaria parasite Plasmodium falciparum seem to maintain an active mitochondrial electron transport chain to serve just one metabolic function: regeneration of ubiquinone required as the electron acceptor for dihydroorotate dehydrogenase, an essential enzyme for pyrimidine biosynthesis. Transgenic P. falciparum parasites expressing Saccharomyces cerevisiae dihydroorotate dehydrogenase, which does not require ubiquinone as an electron acceptor, were completely resistant to inhibitors of mitochondrial electron transport. Maintenance of mitochondrial membrane potential, however, was essential in these parasites, as indicated by their hypersensitivity to proguanil, a drug that collapsed the membrane potential in the presence of electron transport inhibitors. Thus, acquisition of just one enzyme can render mitochondrial electron transport nonessential in erythrocytic stages of P. falciparum.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The battle against malaria has been substantially impeded by the recurrence of drug resistance in Plasmodium falciparum, the deadliest human malaria parasite. To counter the problem, novel ...antimalarial drugs are urgently needed, especially those that target unique pathways of the parasite, since they are less likely to have side effects. The mitochondrial type II NADH dehydrogenase (NDH2) of P. falciparum, PfNDH2 (PF3D7_0915000), has been considered a good prospective antimalarial drug target for over a decade, since malaria parasites lack the conventional multi-subunit NADH dehydrogenase, or Complex I, present in the mammalian mitochondrial electron transport chain (mtETC). Instead, Plasmodium parasites contain a single subunit NDH2, which lacks proton pumping activity and is absent in humans. A significant amount of effort has been expended to develop PfNDH2 specific inhibitors, yet the essentiality of PfNDH2 has not been convincingly verified. Herein, we knocked out PfNDH2 in P. falciparum via a CRISPR/Cas9 mediated approach. Deletion of PfNDH2 does not alter the parasite's susceptibility to multiple mtETC inhibitors, including atovaquone and ELQ-300. We also show that the antimalarial activity of the fungal NDH2 inhibitor HDQ and its new derivative CK-2-68 is due to inhibition of the parasite cytochrome bc1 complex rather than PfNDH2. These compounds directly inhibit the ubiquinol-cytochrome c reductase activity of the malarial bc1 complex. Our results suggest that PfNDH2 is not likely a good antimalarial drug target.
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Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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