Interferon and Interferon Inducers in Protozoal Infections

Several interferon inducers (Newcastle disease virus, statolon, and poly rI:poly rC) as well as exogenous mouse interferon protect mice from sporozoite-induced Plasmodium berghei malaria, as long as they are administered before the end of the preerythrocytic phase of development of the parasite. The protective effect of the interferon inducers was related to their interferon-inducing effect; the protective effect of the interferon preparations was related to the interferon titer of the preparations, and it exhibited other attributes of interferon such as species specificity. In contrast to sporozoite-induced infection, blood forms-induced P. berghei malaria was only weakly susceptible to the protective effect of interferon inducers. This difference may provide an approach to study the mechanism of protection. The growth in cell cultures of another intracellular protozoon, Toxoplasma gondii, is also inhibited by interferon (22). The fact that P. berghei and T. gondii (as well as another group of intracellular parasites susceptible to interferon, the Chlamydia) have their own ribosomes raises questions, concerning the role of host cell ribosomes in the host cell-parasite relationship of these intracellular parasites and in the mechanism of interferon action against them, that can be approached experimentally. The possibility of therapeutic or prophylactic application of interferon or of its inducers to certain protozoal diseases of man and of other animals is still remote, but it has to be considered for long range planning.     

The 2‐methylcitrate cycle is implicated in the detoxification of propionate in Toxoplasma gondii

Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa phylum. The Coccidia are obligate intracellular pathogens that establish infection in their mammalian host via the enteric route. These parasites lack a mitochondrial pyruvate dehydrogenase complex but have preserved the degradation of branched‐chain amino acids (BCAA) as a possible pathway to generate acetyl‐CoA. Importantly, degradation of leucine, isoleucine and valine could lead to concomitant accumulation of propionyl‐CoA, a toxic metabolite that inhibits cell growth. Like fungi and bacteria, the Coccidia possess the complete set of enzymes necessary to metabolize and detoxify propionate by oxidation to pyruvate via the 2‐methylcitrate cycle (2‐MCC). Phylogenetic analysis provides evidence that the 2‐MCC was acquired via horizontal gene transfer. In T. gondii tachyzoites, this pathway is split between the cytosol and the mitochondrion. Although the rate‐limiting enzyme 2‐methylisocitrate lyase is dispensable for parasite survival, its substrates accumulate in parasites deficient in the enzyme and its absence confers increased sensitivity to propionic acid. BCAA is also dispensable in tachyzoites, leaving unresolved the source of mitochondrial acetyl‐CoA.     

Plant hormone cytokinins control cell cycle progression and plastid replication in apicomplexan parasites

Cytokinins are plant hormones that are involved in regulation of cell proliferation, cell cycle progression, and cell and plastid development. Here, we show that the apicomplexan parasites Toxoplasma gondii and Plasmodium berghei, an opportunistic human pathogen and a rodent malaria agent, respectively, produce cytokinins via a biosynthetic pathway similar to that in plants. Cytokinins regulate the growth and cell cycle progression of T. gondii by mediating expression of the cyclin gene TgCYC4. A natural form of cytokinin, trans-zeatin (t-zeatin), upregulated expression of this cyclin, while a synthetic cytokinin, thidiazuron, downregulated its expression. Immunofluorescence microscopy and quantitative PCR analysis showed that t-zeatin increased the genome-copy number of apicoplast, which are non-photosynthetic plastid, in the parasite, while thidiazuron led to their disappearance. Thidiazuron inhibited growth of T. gondii and Plasmodium falciparum, a human malaria parasite, suggesting that thidiazuron has therapeutic potential as an inhibitor of apicomplexan parasites.     

The protective effect of CD40 ligand–CD40 signalling is limited during the early phase of Plasmodium infection

γδ T cells are essential for eliminating Plasmodium berghei XAT. Because administration of the agonistic anti-CD40 antibody can induce elimination of P. berghei XAT parasites in γδ T cell-deficient mice, we considered that γδ T cells might activate dendritic cells via CD40 signalling during infection. Here we report that administration of the anti-CD40 antibody to γδ T cell-deficient mice 3–10 days post-P. berghei XAT infection could eliminate the parasites. Our data suggest that dendritic cell activation via γδ T cells expressing CD40 ligand is critical during the early phase of infection.     

Kinetic Flux Profiling Elucidates Two Independent Acetyl-CoA Biosynthetic Pathways in Plasmodium falciparum

The malaria parasite Plasmodium falciparum depends on glucose to meet its energy requirements during blood-stage development. Although glycolysis is one of the best understood pathways in the parasite, it is unclear if glucose metabolism appreciably contributes to the acetyl-CoA pools required for tricarboxylic acid metabolism (TCA) cycle and fatty acid biosynthesis. P. falciparum possesses a pyruvate dehydrogenase (PDH) complex that is localized to the apicoplast, a specialized quadruple membrane organelle, suggesting that separate acetyl-CoA pools are likely. Herein, we analyze PDH-deficient parasites using rapid stable-isotope labeling and show that PDH does not appreciably contribute to acetyl-CoA synthesis, tricarboxylic acid metabolism, or fatty acid synthesis in blood stage parasites. Rather, we find that acetyl-CoA demands are supplied through a “PDH-like” enzyme and provide evidence that the branched-chain keto acid dehydrogenase (BCKDH) complex is performing this function. We also show that acetyl-CoA synthetase can be a significant contributor to acetyl-CoA biosynthesis. Interestingly, the PDH-like pathway contributes glucose-derived acetyl-CoA to the TCA cycle in a stage-independent process, whereas anapleurotic carbon enters the TCA cycle via a stage-dependent phosphoenolpyruvate carboxylase/phosphoenolpyruvate carboxykinase process that decreases as the parasite matures. Although PDH-deficient parasites have no blood-stage growth defect, they are unable to progress beyond the oocyst phase of the parasite mosquito stage.     

Diversity in mitochondrial metabolic pathways in parasitic protists Plasmodium and Cryptosporidium

Apicomplexans are obligate intracellular parasites and occupy diverse niches. They have remodeled mitochondrial carbon and energy metabolism through reductive evolution. Plasmodium lacks mitochondrial pyruvate dehydrogenase and H⁺-translocating NADH dehydrogenase (Complex I, NDH1). The mitochondorion contains a minimal mtDNA (～ 6 kb) and carries out oxidative phosphorylation in the insect vector stages, by using 2-oxoglutarate as an alternative means of entry into the TCA cycle and a single-subunit flavoprotein as an alternative NADH dehydrogenase (NDH2). In the blood stages of mammalian hosts, mitochondrial enzymes are down-regulated and parasite energy metabolism relies mainly on glycolysis. Mitosomes of Cryptosporidium parvum and Cryptosporidium hominis (human intestine parasites) lack mtDNA, pyruvate dehydrogenase, TCA cycle enzymes except malate-quinone oxidoreductase (MQO), and ATP synthase subunits except α and β. In contrast, mitosomes of Cryptosporidium muris (a rodent gastric parasite) retain all TCA cycle enzymes and functional ATP synthase and carry out oxidative phosphorylation with pyruvate-NADP⁺ oxidoreductase (PNO) and a simple and unique respiratory chain consisting of NDH2 and alternative oxidase (AOX). Cryptosporidium and Perkinsus are early branching groups of chromoalveolates (apicomplexa and dinoflagellates, respectively), and both Cryptosporidium mitosome and Perkinsus mitochondrion use PNO, MQO, and AOX. All apicomplexan parasites and dinoflagellates share MQO, which has been acquired from ε-proteobacteria via lateral gene transfer. By genome data mining on Plasmodium, Cryptosporidium and Perkinsus, here we summarized their mitochondrial metabolic pathways, which are varied largely from those of mammalian hosts. We hope that our findings will help in understanding the apicomplexan metabolism and development of new chemotherapeutics with novel targets.     

Transfection of Eimeria and Toxoplasma using heterologous regulatory sequences

Eimeriatenella and Toxoplasmagondii are Apicomplexan protozoa and share many similarities in biology and genomics. While the latter parasites are easily cultured in vitro and genetically manipulated, many Eimeria species are difficult to grow in vitro. We hypothesised that molecular tools for the genetic manipulation of T. gondii could be applied to the study of Eimeria parasites. Here we show that three different promoter sequences originating from E. tenella could function effectively not only in other species of the Eimeria genus (histone H4) but also in T. gondii (histone H4, actin and tubulin). Similarly, promoters of the “housekeeping” gene (tubulin) and differentially regulated gene (surface antigen gene, sag1) of T. gondii were effective in driving the expression of the yellow fluorescent protein (YFP) maker gene in E. tenella. The transfection efficiency with heterologous regulatory sequences was similar to that with homologous promoters; while the promoter strength of heterologous vectors is slightly weaker than the homologous vectors in both E. tenella and T. gondii. The results suggest that 5′ regulatory sequences are functionally conserved not only among the Eimeria species, but also between T. gondii and E. tenella, and that T. gondii could be used as a novel transfection check system for Eimeria-rooted vectors, accelerating the development of reverse genetics in Eimeria spp.     

Comparative ex vivo infection with Trypanosoma cruzi and Toxoplasma gondii of human,canine and ovine placenta: Analysis of tissue damage and infection efficiency

Trypanosoma cruzi, the causative agent of Chagas disease, and Toxoplasma gondii, which is responsible for Toxoplasmosis, are two parasites that cause significant protozoan zoonoses and consequently important economic losses in human, companion animals and livestock. For the congenital transmission to occur, both parasites must cross the barrier present in the mammalian placenta, which differs between species. Particularly, hemochorial, endotheliochorial and epitheliochorial placental barriers are present, respectively, in human, dog and sheep. The type of placental barrier has been associated with the probability of transmission of pathogens. In this study, we used experimental placental ex vivo infection models of T. cruzi and T. gondii in the above-mentioned mammals in order to study tissue alterations and to compare infection efficiency. Here, we infected placental term explants from human, dog and sheep and analyzed tissue damage by standard histological and histochemical methods. Comparative infection efficiency was determined by quantitative PCR. Both parasites are able to infect the different placental explants; however, more T. gondii parasites were detected, and T. gondii causes a more severe tissue damage in human and canine explants than T. cruzi. The histopathological changes observed in ovine placenta explants were similar in presence of both parasites. We conclude that the infection efficiency of T. gondii is higher, compared to T. cruzi, during the ex vivo infection of human, canine and ovine placental explants.In addition, the ex vivo infection of mammalian placental explants constitutes an interesting experimental approach to study part of the infection mechanisms as well as host responses during congenital infection of both parasites.     

Kinetics of IL-23 and IL-12 Secretion in Response to Toxoplasma gondii Antigens from THP-1 Monocytic Cells

IL-23 and IL-12 are structurally similar and critical for the generation of efficient cellular immune responses. Toxoplasma gondii induces a strong cell-mediated immune response. However, little is known about IL-23 secretion profiles in T. gondii-infected immune cells in connection with IL-12. We compared the patterns of IL-23 and IL-12 production by THP-1 human monocytic cells in response to stimulation with live or heat-killed T. gondii tachyzoites, or with equivalent quantities of either T. gondii excretory/secretory proteins (ESP) or soluble tachyzoite antigen (STAg). IL-23 and IL-12 were significantly increased from 6 hr after stimulation with T. gondii antigens, and their secretions were increased with parasite dose-dependent manner. IL-23 concentrations were significantly higher than those of IL-12 at the same multiplicity of infection. IL-23 secretion induced by live parasites was significantly higher than that by heat-killed parasites, ESP, or STAg, whereas IL-12 secretion by live parasite was similar to those of ESP or STAg. However, the lowest levels of both cytokines were at stimulation with heat-killed parasites. These data indicate that IL-23 secretion patterns by stimulation with various kinds of T. gondii antigens at THP-1 monocytic cells are similar to those of IL-12, even though the levels of IL-23 induction were significantly higher than those of IL-12. The detailed kinetics induced by each T. gondii antigen were different from each other.     

G-strand binding protein 2 is involved in asexual and sexual development of Plasmodium berghei

G-strand binding protein 2 (GBP2) is a Ser/Arg-rich (SR) protein involved in mRNA surveillance and nuclear mRNA quality control in yeast. However, the roles of GBP2 in virulence and sexual development in Plasmodium parasites are unclear, although GBP2 is involved in the asexual development of Plasmodium berghei, the rodent malaria parasite. In this study, we investigated the role of GBP2 in virulence and sexual development of P. berghei using gbp2-deleted P. berghei (Δgbp2 parasites). Then, to identify factors affected by gbp2 deletion, we performed a comparative proteomic analysis of the Δgbp2 parasites. We found that GBP2 was not associated with the development of experimental cerebral malaria during infection with P. berghei, but asexual development of the parasite was delayed with deletion of gbp2. However, the development of P. berghei gametocytes was significantly reduced with deletion of gbp2. Comparative proteomic analysis revealed that the levels of adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) in Δgbp2 parasites were significantly higher than those in wild-type (WT) parasites, suggesting that biosynthesis of purine nucleotides may be involved in function of GBP2. Therefore, we investigated the effect of purine starvation on the sexual development and proteome. In nt1-deleted P. berghei (Δnt1 parasites), the production of male and female gametocytes was significantly reduced compared to that in WT parasites. Moreover, we found that protein levels of GBP2 in Δnt1 parasites were markedly lower than in WT parasites. These findings suggest that GBP2 is primarily involved in the sexual development of malaria parasites, and its function may be suppressed by purine starvation.     

Pyruvate kinase type-II isozyme in Plasmodium falciparum localizes to the apicoplast

Bioinformatics research on Plasmodium falciparum revealed two isoforms of pyruvate kinase: type-I and type-II enzymes. The type-I enzyme shows typical glycolytic properties, while type-II enzyme is involved in fatty acid type-II biosynthesis and has been predicted to localize to the apicoplast with the targeting signal in its N-terminus. The type-I and type-II isoforms have the same evolutionary origin as Toxoplasma gondii isozymes, TgPyKI and TgPyKII, respectively; however, TgPyKII localizes to both the mitochondrion and the apicoplast. Accordingly, we made a recombinant full length of P. falciparum pyruvate kinase type-II protein using a wheat germ cell-free expression system and obtained a specific antibody against the type-II protein. Fluorescent microscopic analysis revealed that P. falciparum type-II enzyme was localized only to the apicoplast, not to the mitochondrion. The data suggest differences in localization and metabolic pathways between P. falciparum and T. gondii pyruvate kinase isoforms.     

Analysis of Toxoplasma gondii surface antigen 2 gene (SAG2). Relevance of genotype I in clinical toxoplasmosis

Toxoplasma gondii is one of the most successful protozoan parasites given its ability to manipulate the immune system and establish a chronic infection. It is a parasite with a significant impact on human health, mainly in immunocompromised patients. In Europe and North America, only a few clonal genotypes (I, II and III) seem to be responsible for the vast majority of Toxoplasma infections. Surface antigen 2 gene (SAG2) has been extensively used for genotyping T. gondii isolates. The analysis of this locus reveals that in Northern hemisphere, human disease causing isolates are mainly type II, whereas T. gondii isolated from different animals are both type II and III. Since the immune response depends on parasite genotype, it seems relevant to characterize parasites producing human toxoplasmosis in different geographical areas. The growing information about the prevalent T. gondii genotypes in South America mostly refers to domestic animals. This is the first report of genetic characterization of T. gondii isolates from clinical samples in Chile, South America. All the samples analyzed corresponded to SAG2 type I isolates, and they differ from classic SAG2 type I by genetic polymorphisms. This study contributes to the scarce available information on T. gondii at South America, and reinforces an emerging concept suggesting that SAG2 type I, rather than II, parasites are a frequent cause of clinical toxoplasmosis in this continent.     

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD⁺ or NADH at about the same operating redox potential as the NADH/NAD⁺ pool and comprise the NADH/NAD⁺ isopotential enzyme group. Complex I (specifically the flavin, site I_F) is often regarded as the major source of matrix superoxide/H₂O₂ production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H₂O₂ production. To differentiate the superoxide/H₂O₂-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H₂O₂ production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H₂O₂ production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)⁺ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H₂O₂ at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H₂O₂ was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site I_F of complex I. Depending on the substrates present, the dominant sites of superoxide/H₂O₂ production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.     

Secondary mutations correct fitness defects in Toxoplasma gondii with dinitroaniline resistance mutations

Dinitroanilines (oryzalin, trifluralin, ethafluralin) disrupt microtubules in protozoa but not in vertebrate cells, causing selective death of intracellular Toxoplasma gondii parasites without affecting host cells. Parasites containing α1-tubulin point mutations are dinitroaniline resistant but show increased rates of aberrant replication relative to wild-type parasites. T. gondii parasites bearing the F52Y mutation were previously demonstrated to spontaneously acquire two intragenic mutations that decrease both resistance levels and replication defects. Parasites bearing the G142S mutation are largely dependent on oryzalin for viable growth in culture. We isolated 46 T. gondii lines that have suppressed microtubule defects associated with the G142S or the F52Y mutations by acquiring secondary mutations. These compensatory mutations were α1-tubulin pseudorevertants or extragenic suppressors (the majority alter the β1-tubulin gene). Many secondary mutations were located in tubulin domains that suggest that they function by destabilizing microtubules. Most strikingly, we identified seven novel mutations that localize to an eight-amino-acid insert that stabilizes the α1-tubulin M loop, including one (P364R) that acts as a compensatory mutation in both F52Y and G142S lines. These lines have reduced dinitroaniline resistance but most perform better than parental lines in competition assays, indicating that there is a trade-off between resistance and replication fitness.     

The relationship between the presence of antibodies and direct detection of Toxoplasma gondii in slaughtered calves and cattle in four European countries

In cattle, antibodies to Toxoplasma gondii infection are frequently detected, but evidence for the presence of T. gondii tissue cysts in cattle is limited. To study the concordance between the presence of anti-T. gondii IgG and viable tissue cysts of T. gondii in cattle, serum, liver and diaphragm samples of 167 veal calves and 235 adult cattle were collected in Italy, the Netherlands, Romania and the United Kingdom. Serum samples were tested for anti-T. gondii IgG by the modified agglutination test and p30 immunoblot. Samples from liver were analyzed by mouse bioassay and PCR after trypsin digestion. In addition, all diaphragms of cattle that had tested T. gondii-positive (either in bioassay, by PCR on trypsin-digested liver or serologically by MAT) and a selection of diaphragms from cattle that had tested negative were analyzed by magnetic capture quantitative PCR (MC-PCR). Overall, 13 animals were considered positive by a direct detection method: seven out of 151 (4.6%) by MC-PCR and six out of 385 (1.6%) by bioassay, indicating the presence of viable parasites. As cattle that tested positive in the bioassay tested negative by MC-PCR and vice-versa, these results demonstrate a lack of concordance between the presence of viable parasites in liver and the detection of T. gondii DNA in diaphragm. In addition, the probability to detect T. gondii parasites or DNA in seropositive and seronegative cattle was comparable, demonstrating that serological testing by MAT or p30 immunoblot does not provide information about the presence of T. gondii parasites or DNA in cattle and therefore is not a reliable indicator of the risk for consumers.     

Toxoplasma gondii: Proteomic analysis of antigenicity of soluble tachyzoite antigen

The obligate intracellular parasite Toxoplasma gondii is an important pathogen of humans and animals. The tachyzoite of T. gondii is the main life-cycle stage that is responsible for toxoplasmosis. Study of the antigenicity of soluble tachyzoite antigen (STAg) is important for discovery of protective antigens which will aid in the detection and prevention of toxoplasmosis. At present, no complete proteome map of T. gondii STAg is established, although a large-scale whole proteomic analysis of tachyzoites is underway. In this study, 1227 protein spots of T. gondii soluble tachyzoite antigen (STAg) were fractionated by 2-dimensional electrophoresis (2-DE) at pH range 3-10. By mass spectrometry (MS) analysis, among the separated 1227 protein spots, 426 were identified by searching the Swissport and NCBI nr databases. Two hundred and thirty of these identified spots (230/426, 54%) were demonstrated to be T. gondii protein by MS. Of the 21 Toxoplasma protein spots identified by Western blot with rabbit anti-T. gondii serum, 16 had immunoregulatory functions and five had immune defense functions. Due to multiple spots for a single protein, these 16 spots represented 11 proteins: a putative protein disulfide isomerase (PDI), heat shock protein 60 (Hsp60), a pyruvate kinase (PK), a putative glutamate dehydrogenase (GDH), a coronin, a heat shock protein 70 (Hsp70), a protein kinase C receptor 1 (RACK1), a malate dehydrogenase (MDH), a major surface antigen 1 (SAG1), an uridine phosphorylase (UPase) and a peroxiredoxin (Prx). Among the identified 11 proteins, except that the antigenicity and immunogenicity of the SAG1 has been reported and antigenicity of Hsp70 has been disputed, the remaining antigenic proteins were first identified in this study. In conclusion, we obtained nine novel types of immunogenic proteins that might be potential candidates of vaccine development for toxoplasmosis, which we will confirm in later studies.     

Type II NADH Dehydrogenase Inhibitor 1-Hydroxy-2-Dodecyl- 4(1H)Quinolone Leads to Collapse of Mitochondrial Inner- Membrane Potential and ATP Depletion in Toxoplasma gondii

The apicomplexan parasite Toxoplasma gondii expresses type II NADH dehydrogenases (NDH2s) instead of canonical complex I at the inner mitochondrial membrane. These non-proton-pumping enzymes are considered to be promising drug targets due to their absence in mammalian cells. We recently showed by inhibition kinetics that T. gondii NDH2-I is a target of the quinolone-like compound 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ), which inhibits T. gondii replication in the nanomolar range. In this study, the cationic fluorescent probes Mitotracker and DiOC₆(3) (3,3′-dihexyloxacarbocyanine iodine) were used to monitor the influence of HDQ on the mitochondrial inner membrane potential (ΔΨm) in T. gondii. Real-time imaging revealed that nanomolar HDQ concentrations led to a ΔΨm collapse within minutes, which is followed by severe ATP depletions of 30% after 1 h and 70% after 24 h. ΔΨm depolarization was attenuated when substrates for other dehydrogenases that can donate electrons to ubiquinone were added to digitonin-permeabilized cells or when infected cultures were treated with the F_o-ATPase inhibitor oligomycin. A prolonged treatment with sublethal concentrations of HDQ induced differentiation into bradyzoites. This dormant stage is likely to be less dependent on the ΔΨm, since ΔΨm-positive parasites were found at a significantly lower frequency in alkaline-pH-induced bradyzoites than in tachyzoites. Together, our studies reveal that oxidative phosphorylation is essential for maintaining the ATP level in the fast-growing tachyzoite stage and that HDQ interferes with this pathway by inhibiting the electron transport chain at the level of ubiquinone reduction.The apicomplexan parasite Toxoplasma gondii contains a single mitochondrion of an elongated tubular structure (28, 32), which shows several significant metabolic differences from the mammalian counterpart (see references 24 and 33 for review). Although the T. gondii mitochondrion harbors the complete set of enzymes for the tricarboxylic acid cycle (15), it lacks the pyruvate dehydrogenase complex (7, 14, 18), which is typically a central enzyme in carbohydrate metabolism that catalyzes the decarboxylation from pyruvate to acetyl coenzyme A. Other mitochondrial pathways for mitochondrial acetyl coenzyme A generation, such as the 2-methylcitrate cycle, are currently under investigation (33). The T. gondii genome predicts the presence of all components necessary for a respiratory chain. Biochemical evidence for oxidative phosphorylation was provided by extracellular T. gondii tachyzoites that were permeabilized with digitonin (39). However, the overall contribution of oxidative phosphorylation to energy production in relation to other ATP-generating pathways has not been satisfactorily clarified for intracellular T. gondii so far.A fundamental difference of the T. gondii and also the Plasmodium falciparum electron transport chains (ETCs) as opposed to the mammalian ETC is the lack of multisubunit complex I, which couples the transfer of electrons from NADH to ubiquinone with the translocation of protons (6). Instead, P. falciparum expresses one isoform (2) and T. gondii expresses two isoforms (22) of so-called “alternative” or type II NADH dehydrogenases (NDH2s). These single-subunit enzymes do not transport protons across the membrane, and they are, in contrast to the NADH-oxidizing activity of complex I, not rotenone sensitive (21, 27). NDH2s can occur in two topological orientations with respect to the inner mitochondrial membrane. Internal enzymes are facing with their active site toward the mitochondrial matrix and use mitochondrial NAD(P)H as the electron donor, while external enzymes use cytosolic NAD(P)H. Up to now, the orientation of the apicomplexan isoforms is unknown.Due to their absence in the mammalian host, NDH2s were proposed to be promising drug targets against Mycobacterium tuberculosis (40). Their suitability as a drug target in Plasmodium is controversial and has been the subject of discussion (16, 17, 38). Previously, it was demonstrated that low-affinity NDH2 inhibitors in micromolar concentrations were able to inhibit the activity of the P. falciparum NDH2 and led to a collapse of the mitochondrial membrane potential (ΔΨm) (2). The only high-affinity NDH2 inhibitors described so far are 1-hydroxy-2-alkyl-4(1H)quinolones with long alkyl-site chains, for example, 1-hydroxy-2-dodecyl-4(1H)quinolone (HDQ) (C₁₂), which possesses structural similarity to ubiquinone. These compounds were shown to inhibit the activities of Yarrowia lipolytica NDH2 (13) and T. gondii NDH2-I (22) with 50% inhibitory concentrations of between 200 and 300 nM. HDQ was also shown to effectively inhibit T. gondii and P. falciparum replication in nanomolar concentrations in tissue cultures (31).We demonstrate in this study that HDQ treatment in nanomolar concentrations leads to a depolarization of the T. gondii ΔΨm within minutes. The subsequent lack of oxidative phosphorylation leads to a ∼70% reduction of the intracellular ATP level within 24 h. This suggests an indispensable role of NDH2 activity in the maintenance of the ΔΨm and in energy metabolism in the tachyzoite stage.     

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F₁-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.