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Drug resistance against antimalarials is rendering them increasingly ineffective and so there is a need for the development of new antimalarials. To discover new antimalarial chemotypes a phenotypic screen of the Janssen Jumpstarter library against the P. falciparum asexual stage was undertaken, uncovering the cyclopropyl carboxamide structural hit class. Structure-activity analysis revealed that each structural moiety was largely resistant to change, although small changes led to the frontrunner compound, WJM280, which has potent asexual stage activity (EC 50 40 nM) and no human cell cytotoxicity. Forward genetics uncovered that cyclopropyl carboxamide resistant parasites have mutations and an amplification in the cytochrome b gene. Cytochrome b was then verified as the target with profiling against cytochrome b drug-resistant parasites and a mitochondrial oxygen consumption assay. Accordingly, the cyclopropyl carboxamide class was shown to have slow-acting asexual stage activity and activity against male gametes and exoerythrocytic forms. Enhancing metabolic stability to attain efficacy in malaria mouse models remains a challenge in the future development of this antimalarial chemotype., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Parasites, such as the malaria parasite P. falciparum, are critically dependent on host nutrients. Interference with nutrient uptake can lead to parasite death and, therefore, serve as a successful treatment strategy. P. falciparum parasites cannot synthesise cholesterol, and instead source this lipid from the host. Here, we tested whether cholesterol uptake pathways could be 'hijacked' for optimal drug delivery to the intracellular parasite. We found that fluorescent cholesterol analogues were delivered from the extracellular environment to the intracellular parasite. We investigated the uptake and inhibitory effects of conjugate compounds, where proven antimalarial drugs (primaquine and artesunate) were attached to steroids that mimic the structure of cholesterol. These conjugated antimalarial drugs improved the inhibitory effects against multiple parasite lifecycle stages, multiple parasite species, and drug-resistant parasites, whilst also lowering the toxicity to human host cells. Steroids with introduced peroxides also displayed antimalarial activity. These results provide a proof-of-concept that cholesterol mimics can be developed as a drug delivery system against apicomplexan parasites with the potential to improve drug efficacy, increase therapeutic index, and defeat drug resistance., (© 2024. The Author(s).)

Succinate dehydrogenase (SDH) is a protein complex that functions in the tricarboxylic acid cycle and the electron transport chain of mitochondria. In most eukaryotes, SDH is highly conserved and comprises the following four subunits: SdhA and SdhB form the catalytic core of the complex, while SdhC and SdhD anchor the complex in the membrane. Toxoplasma gondii is an apicomplexan parasite that infects one-third of humans worldwide. The genome of T. gondii encodes homologues of the catalytic subunits SdhA and SdhB, although the physiological role of the SDH complex in the parasite and the identity of the membrane-anchoring subunits are poorly understood. Here, we show that the SDH complex contributes to optimal proliferation and O 2 consumption in the disease-causing tachyzoite stage of the T. gondii life cycle. We characterize a small membrane-bound subunit of the SDH complex called mitochondrial protein ookinete developmental defect (MPODD), which is conserved among myzozoans, a phylogenetic grouping that incorporates apicomplexan parasites and their closest free-living relatives. We demonstrate that Tg MPODD is essential for SDH activity and plays a key role in attaching the Tg SdhA and Tg SdhB proteins to the membrane anchor of the complex. Our findings highlight a unique and important feature of mitochondrial energy metabolism in apicomplexan parasites and their relatives.

The mitochondrial electron transport chain (ETC) is a multi-component pathway that mediates the transfer of electrons from metabolic reactions that occur in the mitochondrion to molecular oxygen (O 2 ). The ETC contributes to numerous cellular processes, including the generation of cellular ATP through oxidative phosphorylation, serving as an electron sink for metabolic pathways such as de novo pyrimidine biosynthesis and for maintaining mitochondrial membrane potential. Proper functioning of the mitochondrial ETC is necessary for the growth and survival of apicomplexan parasites including Plasmodium falciparum , a causative agent of malaria. The mitochondrial ETC of P. falciparum is an attractive target for antimalarial drugs, due to its essentiality and its differences from the mammalian ETC. To identify novel P. falciparum ETC inhibitors, we have established a real-time assay to assess ETC function, which we describe here. This approach measures the O 2 consumption rate (OCR) of permeabilized P. falciparum parasites using a Seahorse XFe96 flux analyzer and can be used to screen compound libraries for the identification of ETC inhibitors and, in part, to determine the targets of those inhibitors. Key features • With this protocol, the effects of candidate inhibitors on mitochondrial O 2 consumption in permeabilized asexual P. falciparum parasites can be tested in real time. • Through the sequential injection of inhibitors and substrates into the assay, the molecular targets of candidate inhibitors in the ETC can, in part, be determined. • The assay is applicable for both drug discovery approaches and enquiries into a fundamental aspect of parasite mitochondrial biology., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (©Copyright : © 2023 The Authors; This is an open access article under the CC BY-NC license.)

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B 5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Hayward et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The mitochondrial electron transport chain (ETC) of apicomplexan parasites differs considerably from the ETC of the animals that these parasites infect, and is the target of numerous anti-parasitic drugs. The cytochrome c oxidase complex (Complex IV) of the apicomplexan Toxoplasma gondii ETC is more than twice the mass and contains subunits not found in human Complex IV, including a 13 kDa protein termed TgApiCox13. TgApiCox13 is homologous to a human iron-sulfur (Fe-S) cluster-containing protein called the mitochondrial inner NEET protein (HsMiNT) which is not a component of Complex IV in humans. Here, we establish that TgApiCox13 is a critical component of Complex IV in T. gondii, required for complex activity and stability. Furthermore, we demonstrate that TgApiCox13, like its human homolog, binds two Fe-S clusters. We show that the Fe-S clusters of TgApiCox13 are critical for ETC function, having an essential role in mediating Complex IV integrity. Our study provides the first functional characterisation of an Fe-S protein in Complex IV., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2023 Leonard et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The development of new antimalarials is required because of the threat of resistance to current antimalarial therapies. To discover new antimalarial chemotypes, we screened the Janssen Jumpstarter library against the P. falciparum asexual parasite and identified the 7- N -substituted-3-oxadiazole quinolone hit class. We established the structure-activity relationship and optimized the antimalarial potency. The optimized analog WJM228 ( 17 ) showed robust metabolic stability in vitro , although the aqueous solubility was limited. Forward genetic resistance studies uncovered that WJM228 targets the Q o site of cytochrome b (cyt b ), an important component of the mitochondrial electron transport chain (ETC) that is essential for pyrimidine biosynthesis and an established antimalarial target. Profiling against drug-resistant parasites confirmed that WJM228 confers resistance to the Q o site but not Q i site mutations, and in a biosensor assay, it was shown to impact the ETC via inhibition of cyt b . Consistent with other cyt b targeted antimalarials, WJM228 prevented pre-erythrocytic parasite and male gamete development and reduced asexual parasitemia in a P. berghei mouse model of malaria. Correcting the limited aqueous solubility and the high susceptibility to cyt b Q o site resistant parasites found in the clinic will be major obstacles in the future development of the 3-oxadiazole quinolone antimalarial class.

The mitochondrial respiratory chain is an essential pathway in most studied eukaryotes due to its roles in respiration and other pathways that depend on mitochondrial membrane potential. Apicomplexans are unicellular eukaryotes whose members have an impact on global health. The respiratory chain is a drug target for some members of this group, notably the malaria-causing Plasmodium spp. This has motivated studies of the respiratory chain in apicomplexan parasites, primarily Toxoplasma gondii and Plasmodium spp. for which experimental tools are most advanced. Studies of the respiratory complexes in these organisms revealed numerous novel features, including expansion of complex size. The divergence of apicomplexan mitochondria from commonly studied models highlights the diversity of mitochondrial form and function across eukaryotic life., Competing Interests: Declaration of interests No interests are declared., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Diverse compounds target the Plasmodium falciparum Na + pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4 G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na + regulation. The G358S mutation reduces the affinity of PfATP4 for Na + and is associated with an increase in the parasite's resting cytosolic [Na + ]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4 G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development., (© 2022. The Author(s).)

The mitochondrial electron transport chain (ETC) performs several critical biological functions, including maintaining mitochondrial membrane potential, serving as an electron sink for important metabolic pathways, and contributing to the generation of ATP via oxidative phosphorylation. The ETC is important for the survival of many eukaryotic organisms, including intracellular parasites such as the apicomplexan Toxoplasma gondii . The ETC of T. gondii and related parasites differs in several ways from the ETC of the mammalian host cells they infect, and can be targeted by anti-parasitic drugs, including the clinically used compound atovaquone. To characterize the function of novel ETC proteins found in the parasite and to identify new ETC inhibitors, a scalable assay that assesses both ETC function and non-mitochondrial parasite metabolism ( e.g. , glycolysis) is desirable. Here, we describe methods to measure the oxygen consumption rate (OCR) of intact T. gondii parasites and thereby assess ETC function, while simultaneously measuring the extracellular acidification rate (ECAR) as a measure of general parasite metabolism, using a Seahorse XFe96 extracellular flux analyzer. We also describe a method to pinpoint the location of ETC defects and/or the targets of inhibitors, using permeabilized T. gondii parasites. We have successfully used these methods to investigate the function of T. gondii proteins, including the apicomplexan parasite-specific protein subunit Tg QCR11 of the coenzyme Q:cytochrome c oxidoreductase complex (ETC Complex III). We note that these methods are also amenable to screening compound libraries to identify candidate ETC inhibitors., Competing Interests: Competing interestsThe authors declare that no competing interests exist., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Since Nicolle, Manceaux and Splendore first described Toxoplasma gondii as a parasite of rodents and rabbits in the early 20th century, a diverse and vigorous research community has been built around studying this fascinating intracellular parasite. In addition to its importance as a pathogen of humans, livestock and wildlife, modern researchers are attracted to T. gondii as a facile experimental system to study many aspects of evolutionary biology, cellular biology, host-microbe interactions, and host immunity. For new researchers entering the field, the extensive literature describing the biology of the parasite, and the interactions with its host, can be daunting. In this review, we examine four foundational studies that describe various aspects of T. gondii biology, presenting a 'journal club'-style analysis of each. We have chosen a paper that established the beguiling life cycle of the parasite (Hutchison et al., 1971), a paper that described key features of its cellular biology that the parasite shares with related organisms (Gustafson et al., 1954), a paper that characterised the origin of the unique compartment in which the parasite resides within host cells (Jones and Hirsch, 1972), and a paper that established a key mechanism in the host immune response to parasite infection (Pfefferkorn, 1984). These interesting and far-reaching studies set the stage for subsequent research into numerous facets of parasite biology. As well as providing new researchers with an entry point into the literature surrounding the parasite, revisiting these studies can remind us of the roots of our discipline, how far we have come, and the new directions in which we might head., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Intracellular parasites of the phylum Apicomplexa are dependent on the scavenging of essential amino acids from their hosts. We previously identified a large family of apicomplexan-specific plasma membrane-localized amino acid transporters, the ApiATs, and showed that the Toxoplasma gondii transporter TgApiAT1 functions in the selective uptake of arginine. TgApiAT1 is essential for parasite virulence, but dispensable for parasite growth in medium containing high concentrations of arginine, indicating the presence of at least one other arginine transporter. Here we identify TgApiAT6-1 as the second arginine transporter. Using a combination of parasite assays and heterologous characterisation of TgApiAT6-1 in Xenopus laevis oocytes, we demonstrate that TgApiAT6-1 is a general cationic amino acid transporter that mediates both the high-affinity uptake of lysine and the low-affinity uptake of arginine. TgApiAT6-1 is the primary lysine transporter in the disease-causing tachyzoite stage of T. gondii and is essential for parasite proliferation. We demonstrate that the uptake of cationic amino acids by TgApiAT6-1 is 'trans-stimulated' by cationic and neutral amino acids and is likely promoted by an inwardly negative membrane potential. These findings demonstrate that T. gondii has evolved overlapping transport mechanisms for the uptake of essential cationic amino acids, and we draw together our findings into a comprehensive model that highlights the finely-tuned, regulated processes that mediate cationic amino acid scavenging by these intracellular parasites., Competing Interests: The authors have declared that no competing interests exist.

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite's external environment, increasing in response to decreased [Arg]. Using a luciferase-based 'biosensor' strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient., Competing Interests: The authors have declared that no competing interests exist.

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms., Competing Interests: The authors have declared that no competing interests exist.

Iron-sulfur (Fe-S) clusters are prosthetic groups on proteins that function in a range of enzymatic and electron transfer reactions. Fe-S cluster synthesis is essential for the survival of all eukaryotes. Independent Fe-S cluster biosynthesis pathways occur in the mitochondrion, plastid, and cytosolic compartments of eukaryotic cells. Little is known about the cytosolic Fe-S cluster biosynthesis in apicomplexan parasites, the causative agents of diseases such as malaria and toxoplasmosis. NBP35 serves as a key scaffold protein on which cytosolic Fe-S clusters assemble, and has a cytosolic localization in most eukaryotes studied thus far. Unexpectedly, we found that the NBP35 homolog of the apicomplexan Toxoplasma gondii (TgNBP35) localizes to the outer mitochondrial membrane, with mitochondrial targeting mediated by an N-terminal transmembrane domain. We demonstrate that TgNBP35 is critical for parasite proliferation, but that, despite its mitochondrial localization, it is not required for Fe-S cluster synthesis in the mitochondrion. Instead, we establish that TgNBP35 is important for the biogenesis of cytosolic Fe-S proteins. Our data are consistent with TgNBP35 playing a central and specific role in cytosolic Fe-S cluster biosynthesis, and imply that the assembly of cytosolic Fe-S clusters occurs on the cytosolic face of the outer mitochondrial membrane in these parasites., (© 2020 John Wiley & Sons Ltd.)

Toxoplasma gondii and Plasmodium falciparum parasites both extrude L-lactate, a byproduct of glycolysis. The P. falciparum Formate Nitrite Transporter, PfFNT, mediates L-lactate transport across the plasma membrane of P. falciparum parasites and has been validated as a drug target. The T. gondii genome encodes three FNTs that have been shown to transport L-lactate, and which are proposed to be the targets of several inhibitors of T. gondii proliferation. Here, we show that each of the TgFNTs localize to the T. gondii plasma membrane and are capable of transporting L-lactate across it, with TgFNT1 making the primary contribution to L-lactate transport during the disease-causing lytic cycle of the parasite. We use the Xenopus oocyte expression system to provide direct measurements of L-lactate transport via TgFNT1. We undertake a genetic analysis of the importance of the tgfnt genes for parasite proliferation, and demonstrate that all three tgfnt genes can be disrupted individually and together without affecting the lytic cycle under in vitro culture conditions. Together, our experiments identify the major lactate transporter in the disease causing stage of T. gondii, and reveal that this transporter is not required for parasite proliferation, indicating that TgFNTs are unlikely to be targets for anti-Toxoplasma drugs.

Toxoplasmosis is caused by Toxoplasma gondii, an apicomplexan parasite that is able to infect any nucleated cell in any warm-blooded animal. Toxoplasma gondii infects around 2 billion people and, whilst only a small percentage of infected people will suffer serious disease, the prevalence of the parasite makes it one of the most damaging zoonotic diseases in the world. Toxoplasmosis is a disease with multiple manifestations: it can cause a fatal encephalitis in immunosuppressed people; if first contracted during pregnancy, it can cause miscarriage or congenital defects in the neonate; and it can cause serious ocular disease, even in immunocompetent people. The disease has a complex epidemiology, being transmitted by ingestion of oocysts that are shed in the faeces of definitive feline hosts and contaminate water, soil and crops, or by consumption of intracellular cysts in undercooked meat from intermediate hosts. In this review we examine current and future approaches to control toxoplasmosis, which encompass a variety of measures that target different components of the life cycle of T. gondii. These include: education programs about the parasite and avoidance of contact with infectious stages; biosecurity and sanitation to ensure food and water safety; chemo- and immunotherapeutics to control active infections and disease; prophylactic options to prevent acquisition of infection by livestock and cyst formation in meat; and vaccines to prevent shedding of oocysts by definitive feline hosts., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: The authors have declared that no competing interests exist.

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase ( Tg UroD). We found that Tg UroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c -type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c / c 1 heme lyase 1 ( Tg CCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c -type cytochromes. Tg CCHL1 depletion reduced parasite proliferation and decreased the abundance of c -type cytochromes. We further sought to characterize the overall importance of Tg UroD and Tg CCHL1 for both mitochondrial and general parasite metabolism. Tg UroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of Tg CCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites., (© 2020 Tjhin et al.)

The uptake of host-derived nutrients is key to the growth and survival of Toxoplasma gondii parasites. Nutrients are acquired via solute transporters that localize to the plasma membrane of the parasites. In this chapter, we describe methodology by which the uptake of solutes via plasma membrane transporters may be monitored and characterized. These assays, used here to investigate the uptake of amino acids into parasites, have broad applicability in measuring the uptake of a diverse range of solutes.

Mitochondrial respiration is a critical process for the survival of many eukaryotes, including parasites in the phylum Apicomplexa. These intracellular parasites include the causative agents of numerous serious diseases in humans and animals, including toxoplasmosis (Toxoplasma gondii) and malaria (Plasmodium species). Emerging evidence indicates that the mitochondrial respiratory chain of apicomplexans has notable differences to that of the host cells they infect. These differences make the respiratory chain a prominent drug target in apicomplexans, with numerous inhibitors of this pathway in current use or development. This review highlights unique aspects of the respiratory chain of apicomplexans and provides perspective on emerging points of inquiry into this essential and therapeutically exploitable pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Apicomplexan parasites including Toxoplasma gondii and Plasmodium spp. manufacture a complex arsenal of secreted proteins used to interact with and manipulate their host environment. These proteins are organised into three principle exocytotic compartment types according to their functions: micronemes for extracellular attachment and motility, rhoptries for host cell penetration, and dense granules for subsequent manipulation of the host intracellular environment. The order and timing of these events during the parasite's invasion cycle dictates when exocytosis from each compartment occurs. Tight control of compartment secretion is, therefore, an integral part of apicomplexan biology. Control of microneme exocytosis is best understood, where cytosolic intermediate molecular messengers cGMP and Ca 2+ act as positive signals. The mechanisms for controlling secretion from rhoptries and dense granules, however, are virtually unknown. Here, we present evidence that dense granule exocytosis is negatively regulated by cytosolic Ca 2+ , and we show that this Ca 2+ -mediated response is contingent on the function of calcium-dependent protein kinases TgCDPK1 and TgCDPK3. Reciprocal control of micronemes and dense granules provides an elegant solution to the mutually exclusive functions of these exocytotic compartments in parasite invasion cycles and further demonstrates the central role that Ca 2+ signalling plays in the invasion biology of apicomplexan parasites., (© 2019 The Authors Cellular Microbiology Published by John Wiley & Sons Ltd.)

The Plasmodium falciparum ATPase Pf ATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. Pf ATP4 was originally annotated as a Ca 2+ transporter, but recent evidence suggests that it is a Na + efflux pump, extruding Na + in exchange for H + Here we demonstrate that ATP4 proteins belong to a clade of P-type ATPases that are restricted to apicomplexans and their closest relatives. We employed a variety of genetic and physiological approaches to investigate the ATP4 protein of the apicomplexan Toxoplasma gondii , Tg ATP4. We show that Tg ATP4 is a plasma membrane protein. Knockdown of Tg ATP4 had no effect on resting pH or Ca 2+ but rendered parasites unable to regulate their cytosolic Na + concentration ([Na + ] cyt ). Pf ATP4 inhibitors caused an increase in [Na + ] cyt and a cytosolic alkalinization in WT but not Tg ATP4 knockdown parasites. Parasites in which Tg ATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which Tg ATP4 had been disrupted showed reduced virulence in mice. These results provide evidence for ATP4 proteins playing a key conserved role in Na + regulation in apicomplexan parasites., (© 2019 Lehane et al.)

Apicomplexan parasites are auxotrophic for a range of amino acids which must be salvaged from their host cells, either through direct uptake or degradation of host proteins. Here, we describe a family of plasma membrane-localized amino acid transporters, termed the Apicomplexan Amino acid Transporters (ApiATs), that are ubiquitous in apicomplexan parasites. Functional characterization of the ApiATs of Toxoplasma gondii indicate that several of these transporters are important for intracellular growth of the tachyzoite stage of the parasite, which is responsible for acute infections. We demonstrate that the ApiAT protein TgApiAT5-3 is an exchanger for aromatic and large neutral amino acids, with particular importance for L-tyrosine scavenging and amino acid homeostasis, and that TgApiAT5-3 is critical for parasite virulence. Our data indicate that T. gondii expresses additional proteins involved in the uptake of aromatic amino acids, and we present a model for the uptake and homeostasis of these amino acids. Our findings identify a family of amino acid transporters in apicomplexans, and highlight the importance of amino acid scavenging for the biology of this important phylum of intracellular parasites., Competing Interests: The authors have declared that no competing interests exist.

The mitochondrion of apicomplexan parasites is critical for parasite survival, although the full complement of proteins that localize to this organelle has not been defined. Here we undertake two independent approaches to elucidate the mitochondrial proteome of the apicomplexan Toxoplasma gondii . We identify approximately 400 mitochondrial proteins, many of which lack homologs in the animals that these parasites infect, and most of which are important for parasite growth. We demonstrate that one such protein, termed Tg ApiCox25, is an important component of the parasite cytochrome c oxidase (COX) complex. We identify numerous other apicomplexan-specific components of COX, and conclude that apicomplexan COX, and apicomplexan mitochondria more generally, differ substantially in their protein composition from the hosts they infect. Our study highlights the diversity that exists in mitochondrial proteomes across the eukaryotic domain of life, and provides a foundation for defining unique aspects of mitochondrial biology in an important phylum of parasites., Competing Interests: AS, LM, ER, ET, LD, VA, PF, AW, CT, Gv No competing interests declared, (© 2018, Seidi et al.)

The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex. However, sequence-based searches fail to identify genes encoding stator subunits in apicomplexan parasites like Toxoplasma gondii or the related organisms that cause malaria. Here, we identify 11 previously unknown subunits from the Toxoplasma ATP synthase, which lack homologs outside the phylum. Modeling suggests that two of them, ICAP2 and ICAP18, are distantly related to mammalian stator subunits. Our analysis shows that both proteins form part of the ATP synthase complex. Depletion of ICAP2 leads to aberrant mitochondrial morphology, decreased oxygen consumption, and disassembly of the complex, consistent with its role as an essential component of the Toxoplasma ATP synthase. Our findings highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities., Competing Interests: DH, ER, Gv, SL No competing interests declared, (© 2018, Huet et al.)

Apicomplexans are obligate intracellular parasites that scavenge essential nutrients from their hosts via transporter proteins on their plasma membrane. The identities of the transporters that mediate amino acid uptake into apicomplexans are unknown. Here we demonstrate that members of an apicomplexan-specific protein family-the Novel Putative Transporters (NPTs)-play key roles in the uptake of cationic amino acids. We show that an NPT from Toxoplasma gondii (TgNPT1) is a selective arginine transporter that is essential for parasite survival and virulence. We also demonstrate that a homologue of TgNPT1 from the malaria parasite Plasmodium berghei (PbNPT1), shown previously to be essential for the sexual gametocyte stage of the parasite, is a cationic amino acid transporter. This reveals a role for cationic amino acid scavenging in gametocyte biology. Our study demonstrates a critical role for amino acid transporters in the survival, virulence and life cycle progression of these parasites.

Outside of well characterized model eukaryotes, relatively little is known about the translocons that transport proteins across the two membranes that surround the mitochondrion. Apicomplexans are a phylum of intracellular parasites that cause major diseases in humans and animals and are evolutionarily distant from model eukaryotes such as yeast. Apicomplexans harbor a mitochondrion that is essential for parasite survival and is a validated drug target. Here, we demonstrate that the apicomplexan Toxoplasma gondii harbors homologues of proteins from all the major mitochondrial protein translocons present in yeast, suggesting these arose early in eukaryotic evolution. We demonstrate that a T. gondii homologue of Tom22 (TgTom22), a central component of the translocon of the outer mitochondrial membrane (TOM) complex, is essential for parasite survival, mitochondrial protein import, and assembly of the TOM complex. We also identify and characterize a T. gondii homologue of Tom7 (TgTom7) that is important for parasite survival and mitochondrial protein import. Contrary to the role of Tom7 in yeast, TgTom7 is important for TOM complex stability, suggesting the role of this protein has diverged during eukaryotic evolution. Together, our study identifies conserved and modified features of mitochondrial protein import in apicomplexan parasites., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)