Correction: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

[This corrects the article DOI: 10.1371/journal.ppat.1011517.].

Analysis of Plasmodium falciparum Mitochondrial Electron Transport Chain Activity Using Seahorse XFe96 Extracellular Flux Assays.

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 (O2). 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 O2 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 O2 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.

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

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.

Real-Time Analysis of Mitochondrial Electron Transport Chain Function in Toxoplasma gondii Parasites Using a Seahorse XFe96 Extracellular Flux Analyzer.

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 TgQCR11 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.

Substrate-mediated regulation of the arginine transporter of Toxoplasma gondii.

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.

Coordinated action of multiple transporters in the acquisition of essential cationic amino acids by the intracellular parasite Toxoplasma gondii.

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.

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

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.

Measuring Solute Transport in Toxoplasma gondii Parasites.

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.

The tyrosine transporter of Toxoplasma gondii is a member of the newly defined apicomplexan amino acid transporter (ApiAT) family.

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.

Characterization of the ATP4 ion pump in Toxoplasma gondii.

The Plasmodium falciparum ATPase PfATP4 is the target of a diverse range of antimalarial compounds, including the clinical drug candidate cipargamin. PfATP4 was originally annotated as a Ca2+ 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, TgATP4. We show that TgATP4 is a plasma membrane protein. Knockdown of TgATP4 had no effect on resting pH or Ca2+ but rendered parasites unable to regulate their cytosolic Na+ concentration ([Na+]cyt). PfATP4 inhibitors caused an increase in [Na+]cyt and a cytosolic alkalinization in WT but not TgATP4 knockdown parasites. Parasites in which TgATP4 was knocked down or disrupted exhibited a growth defect, attributable to reduced viability of extracellular parasites. Parasites in which TgATP4 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.

Elucidating the mitochondrial proteome of Toxoplasma gondii reveals the presence of a divergent cytochrome c oxidase.

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 TgApiCox25, 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.

Identification of cryptic subunits from an apicomplexan ATP synthase.

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.

Cationic amino acid transporters play key roles in the survival and transmission of apicomplexan parasites.

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.

Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake.

Human erythrocytes have a low basal permeability to L-glutamate and are not known to have a functional glutamate transporter. Here, treatment of human erythrocytes with arsenite was shown to induce the uptake of L-glutamate and D-aspartate, but not that of D-glutamate or L-alanine. The majority of the arsenite-induced L-glutamate influx was via a high-affinity, Na(+)-dependent system showing characteristics of members of the "excitatory amino acid transporter" (EAAT) family. Western blots and immunofluorescence assays revealed the presence of a member of this family, EAAT3, on the erythrocyte membrane. Erythrocytes infected with the malaria parasite Plasmodium falciparum take up glutamate from the extracellular environment. Although the majority of uptake is via a low-affinity Na(+)-independent pathway there is, in addition, a high-affinity uptake component, raising the possibility that the parasite activates the host cell glutamate transporter.