National Institute of General Medical Sciences (U.S.) (Center for Integrative Synthetic Biology Grant P50GM098792)

A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes

Our current understanding of mitochondrial functioning is largely restricted to traditional model organisms, which only represent a fraction of eukaryotic diversity. The unusual mitochondrion of malaria parasites is a validated drug target but remains poorly understood. Here, we apply complexome profiling to map the inventory of protein complexes across the pathogenic asexual blood stages and the transmissible gametocyte stages of Plasmodium falciparum. We identify remarkably divergent composition and clade-specific additions of all respiratory chain complexes. Furthermore, we show that respiratory chain complex components and linked metabolic pathways are up to 40-fold more prevalent in gametocytes, while glycolytic enzymes are substantially reduced. Underlining this functional switch, we find that cristae are exclusively present in gametocytes. Leveraging these divergent properties and stage dynamics for drug development presents an attractive opportunity to discover novel classes of antimalarials and increase our repertoire of gametocytocidal drugs.

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Composition and stage dynamics of mitochondrial complexes in Plasmodium falciparum.

Respiration is a core biological energy–converting process whose last steps are carried out by a chain of multisubunit complexes in the inner mitochondrial membrane. To probe the functional and structural diversity of eukaryotic respiration, we examined the respiratory chain of the ciliate Tetrahymena thermophila (Tt). Using cryo–electron microscopy on a mixed sample, we solved structures of a supercomplex between Tt complex I (Tt-CI) and Tt-CIII2 (Tt-SC I+III2) and a structure of Tt-CIV2. Tt-SC I+III2 (~2.3 megadaltons) is a curved assembly with structural and functional symmetry breaking. Tt-CIV2 is a ~2.7-megadalton dimer with more than 50 subunits per protomer, including mitochondrial carriers and a TIM83-TIM133–like domain. Our structural and functional study of the T. thermophila respiratory chain reveals divergence in key components of eukaryotic respiration, thereby expanding our understanding of core metabolism. Description Big complexes from single-celled eukaryote Mitochondria use a series of membrane-embedded enzymes to transform chemical energy first into an electrochemical gradient and then into ATP. The series of chemical steps that take place are conserved in aerobic organisms, but there is considerable structural divergence in different lineages. Working with extracted mitochondrial membranes, Zhou et al. determined structures of mitochondrial electron transfer chain complexes from a model ciliate (see the Perspective by Huynen and Elurbe). Complex I and a dimer of complex III form a supercomplex as in many other eukaryotes, but both have important fundamental differences in architecture and functional apparatus. Complex IV is massive relative to previously known structures, with more than half of the mass composed of components not seen in homologous structures, including mitochondrial carrier proteins and a translocase that may be involved in assembly or may be a relic of such components. —MAF Structural analysis of Tetrahymena mitochondrial respiratory complexes illuminates the diversity of eukaryotic cellular respiration.

Structures of Tetrahymena’s respiratory chain reveal the diversity of eukaryotic core metabolism

There is an urgent need for new antimalarial drugs with novel mechanisms of action to deliver effective control and eradication programs. Parasite resistance to all existing antimalarial classes, including the artemisinins, has been reported during their clinical use. A failure to generate new antimalarials with novel mechanisms of action that circumvent the current resistance challenges will contribute to a resurgence in the disease which would represent a global health emergency. Here we present a unique generation of quinolone lead antimalarials with a dual mechanism of action against two respiratory enzymes, NADH:ubiquinone oxidoreductase (Plasmodium falciparum NDH2) and cytochrome bc1. Inhibitor specificity for the two enzymes can be controlled subtly by manipulation of the privileged quinolone core at the 2 or 3 position. Inhibitors display potent (nanomolar) activity against both parasite enzymes and against multidrug-resistant P. falciparum parasites as evidenced by rapid and selective depolarization of the parasite mitochondrial membrane potential, leading to a disruption of pyrimidine metabolism and parasite death. Several analogs also display activity against liver-stage parasites (Plasmodium cynomolgi) as well as transmission-blocking properties. Lead optimized molecules also display potent oral antimalarial activity in the Plasmodium berghei mouse malaria model associated with favorable pharmacokinetic features that are aligned with a single-dose treatment. The ease and low cost of synthesis of these inhibitors fulfill the target product profile for the generation of a potent, safe, and inexpensive drug with the potential for eventual clinical deployment in the control and eradication of falciparum malaria.

Generation of quinolone antimalarials targeting the Plasmodium falciparum mitochondrial respiratory chain

The mitochondrion is critical for the survival of apicomplexan parasites. Several major anti-parasitic drugs, such as atovaquone and endochin-like quinolones, act through inhibition of the mitochondrial electron transport chain at the coenzyme Q:cytochrome c oxidoreductase complex (Complex III). Despite being an important drug target, the protein composition of Complex III of apicomplexan parasites has not been elucidated. Here, we undertake a mass spectrometry-based proteomic analysis of Complex III in the apicomplexan Toxoplasma gondii. Along with canonical subunits that are conserved across eukaryotic evolution, we identify several novel or highly divergent Complex III components that are conserved within the apicomplexan lineage. We demonstrate that one such subunit, which we term TgQCR11, is critical for parasite proliferation, mitochondrial oxygen consumption and Complex III activity, and establish that loss of this protein leads to defects in Complex III integrity. We conclude that the protein composition of Complex III in apicomplexans differs from that of the mammalian hosts that these parasites infect.

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Divergent features of the coenzyme Q:cytochrome c oxidoreductase complex in Toxoplasma gondii parasites.

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their assignment as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC and F1Fo-ATP synthase complexes and sets the stage for future structural and drug discovery studies.

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Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex.

The mitochondrial electron transport chain (mETC) and F1Fo-ATP synthase are of central importance for energy and metabolism in eukaryotic cells. The Apicomplexa, important pathogens of humans causing diseases such as toxoplasmosis and malaria, depend on their mETC in every known stage of their complicated life cycles. Here, using a complexome profiling proteomic approach, we have characterised the Toxoplasma mETC complexes and F1Fo-ATP synthase. We identified and assigned 60 proteins to complexes II, IV and F1Fo-ATP synthase of Toxoplasma, of which 16 have not been identified previously. Notably, our complexome profile elucidates the composition of the Toxoplasma complex III, the target of clinically used drugs such as atovaquone. We identified two new homologous subunits and two new parasite-specific subunits, one of which is broadly conserved in myzozoans. We demonstrate all four proteins are essential for complex III stability and parasite growth, and show their depletion leads to decreased mitochondrial potential, supporting their role as complex III subunits. Our study highlights the divergent subunit composition of the apicomplexan mETC complexes and sets the stage for future structural and drug discovery studies.

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Complexome profile of Toxoplasma gondii mitochondria identifies a divergent cytochrome bc1 complex

Mitochondrial ribosomes are fundamental to mitochondrial function, and thus survival, of nearly all eukaryotes. Despite their common ancestry, mitoribosomes have evolved divergent features in different eukaryotic lineages. In apicomplexans, the mitochondrial rRNA is extremely fragmented raising questions about its evolution, protein composition and structure. Apicomplexan mitochondrial translation and the mitoribosomes are essential in all parasites and life stages studied, highlighting mitoribosomes as a promising target for drugs. Still, the apicomplexan mitoribosome is understudied, with one of the obstacles being that its composition is unknown. Here, to facilitate the study of apicomplexan mitoribosomes, we identified and validated components of the mitoribosomal large subunit in the model apicomplexan Toxoplasma gondii.

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Mariana F. Silva

Papers

Complexome profile of Toxoplasma gondii mitochondria identifies divergent subunits of respiratory chain complexes including new subunits of cytochrome bc1 complex.

Complexome profile of Toxoplasma gondii mitochondria identifies a divergent cytochrome bc1 complex

Identification and Validation of Toxoplasma gondii Mitoribosomal Large Subunit Components