Anaerobic peroxisomes in Entamoeba histolytica metabolize myo-inositol.

Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.

Anaerobic peroxisomes in Mastigamoeba balamuthi.

The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either ß-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.

Investigation of the Secretory Pathway in Trichomonas vaginalis Argues against a Moonlighting Function of Hydrogenosomal Enzymes.

The enzymes pyruvate ferredoxin oxidoreductase (PFO), malic enzyme (ME), and the α- and ß-subunits of succinyl-CoA synthetase (SCS) catalyze key steps of energy metabolism in Trichomonas vaginalis hydrogenosomes. These proteins have also been characterized as the adhesins AP120 (PFO), AP65 (ME), AP33, and AP51 (α- and ß-SCS), which are localized on the cell surface and mediate the T. vaginalis cytoadherence. However, the mechanisms that facilitate the targeting of these proteins to the cell surface via the secretory pathway and/or to hydrogenosomes are not known. Here we adapted an in vivo biotinylation system to perform highly sensitive tracing of protein trafficking in T. vaginalis. We showed that α- and ß-SCS are biotinylated in the cytosol and imported exclusively into the hydrogenosomes. Neither α- nor ß-SCS is biotinylated in the endoplasmic reticulum and delivered to the cell surface via the secretory pathway. In contrast, two surface proteins, tetratricopeptide domain-containing membrane-associated protein and tetraspanin family surface protein, as well as soluble-secreted ß-amylase-1 are biotinylated in the endoplasmic reticulum and delivered through the secretory pathway to their final destinations. Taken together, these results demonstrate that the α- and ß-SCS subunits are targeted only to the hydrogenosomes, which argues against their putative moonlighting function.

Mitochondrial pyruvate carrier in Trypanosoma brucei.

Pyruvate is a key product of glycolysis that regulates the energy metabolism of cells. In Trypanosoma brucei, the causative agent of sleeping sickness, the fate of pyruvate varies dramatically during the parasite life cycle. In bloodstream forms, pyruvate is mainly excreted, whereas in tsetse fly forms, pyruvate is metabolized in mitochondria yielding additional ATP molecules. The character of the molecular machinery that mediates pyruvate transport across mitochondrial membrane was elusive until the recent discovery of mitochondrial pyruvate carrier (MPC) in yeast and mammals. Here, we characterized pyruvate import into mitochondrion of T. brucei. We identified mpc1 and mpc2 homologs in the T. brucei genome with attributes of MPC protein family and we demonstrated that both proteins are present in the mitochondrial membrane of the parasite. Investigations of mpc1 or mpc2 gene knock-out cells proved that T. brucei MPC1/2 proteins facilitate mitochondrial pyruvate transport. Interestingly, MPC is expressed not only in procyclic trypanosomes with fully activated mitochondria but also in bloodstream trypanosomes in which most of pyruvate is excreted. Moreover, MPC appears to be essential for bloodstream forms, supporting the recently emerging picture that the functions of mitochondria in bloodstream forms are more diverse than it was originally thought.

Malleable mitochondrion of Trypanosoma brucei.

The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms.

Lineage-specific activities of a multipotent mitochondrion of trypanosomatid flagellates.

Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each representing a novel yet globally distributed and/or species-rich clade, and compare them with well-studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarentolae. Differences in structure and activities of respiratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phylogenetic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for purposes other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypanosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.

Comparative analysis of respiratory chain and oxidative phosphorylation in Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and procyclic stage of Trypanosoma brucei.

Trypanosomatids are unicellular parasites living in a wide range of host environments, which to large extent shaped their mitochondrial energy metabolism, resulting in quite large differences even among closely related flagellates. In a comparative manner, we analyzed the activities and composition of mitochondrial respiratory complexes in four species (Leishmania tarentolae, Crithidia fasciculata, Phytomonas serpens and Trypanosoma brucei), which represent the main model trypanosomatids. Moreover, we measured the activity of mitochondrial glycerol-3-phosphate dehydrogenase, the overall oxygen consumption and the mitochondrial membrane potential in each species. The comparative analysis suggests an inverse relationship between the activities of respiratory complexes I and II, as well as the overall activity of the canonical complexes and glycerol-3-phosphate dehydrogenase. Our comparative analysis shows that mitochondrial functions are highly variable in these versatile parasites.

Characterization of two mitochondrial flavin adenine dinucleotide-dependent glycerol-3-phosphate dehydrogenases in Trypanosoma brucei.

Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD(+) reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion.

Alternative NADH dehydrogenase (NDH2): intermembrane-space-facing counterpart of mitochondrial complex I in the procyclic Trypanosoma brucei.

The respiratory chain of the procyclic stage of Trypanosoma brucei contains the standard complexes I through IV, as well as several alternative enzymes contributing to electron flow. In this work, we studied the function of an alternative NADH : ubiquinone oxidoreductase (NDH2). Depletion of target mRNA was achieved using RNA interference (RNAi). In the non-induced and RNAi-induced cell growth, membrane potential change, alteration in production of reactive oxygen species, overall respiration, enzymatic activities of complexes I, III and/or IV and distribution of NADH : ubiquinone oxidoreductase activities in glycerol gradient fractions were measured. Finally, respiration using different substrates was tested on digitonin-permeabilized cells. The induced RNAi cell line exhibited slower growth, decreased mitochondrial membrane potential and lower sensitivity of respiration to inhibitors. Mitochondrial glycerol-3-phosphate dehydrogenase was the only enzymatic activity that has significantly changed in the interfered cells. This elevation as well as a decrease of respiration using NADH was confirmed on digitonin-permeabilized cells. The data presented here together with previously published findings on complex I led us to propose that NDH2 is the major NADH : ubiquinone oxidoreductase responsible for cytosolic and not for mitochondrial NAD+ regeneration in the mitochondrion of procyclic T. brucei.

Disparate phenotypic effects from the knockdown of various Trypanosoma brucei cytochrome c oxidase subunits.

The Trypanosoma brucei cytochrome c oxidase (respiratory complex IV) is a very divergent complex containing a surprisingly high number of trypanosomatid-specific subunits with unknown function. To gain insight into the functional organization of this large protein complex, the expression of three novel subunits (TbCOX VII, TbCOX X and TbCOX 6080) were down-regulated by RNA interference. We demonstrate that all three subunits are important for the proper function of complex IV and the growth of the procyclic stage of T. brucei. These phenotypes were manifested by the structural instability of the complex when these indispensible subunits were repressed. Furthermore, the impairment of cytochrome c oxidase resulted in other severe mitochondrial phenotypes, such as a decreased mitochondrial membrane potential, reduced ATP production via oxidative phoshorylation and redirection of oxygen consumption to the trypanosome-specific alternative oxidase, TAO. Interestingly, the inspected subunits revealed some disparate phenotypes, particularly regarding the activity of cytochrome c reductase (respiratory complex III). While the activity of complex III was down-regulated in RNAi induced cells for TbCOX X and TbCOX 6080, the TbCOX VII silenced cell line actually exhibited higher levels of complex III activity and elevated levels of ROS formation. This result suggests that the examined subunits may have different functional roles within complex IV of T. brucei, perhaps involving the ability to communicate between sequential enzymes in the respiratory chain. In summary, by characterizing the function of three hypothetical components of complex IV, we are able to assign these proteins as genuine and indispensable subunits of the procyclic T. brucei cytochrome c oxidase, an essential component of the respiratory chain in these evolutionary ancestral and medically important parasites.

Stage-specific requirement for Isa1 and Isa2 proteins in the mitochondrion of Trypanosoma brucei and heterologous rescue by human and Blastocystis orthologues.

IscA/Isa proteins function as alternative scaffolds for the assembly of Fe-S clusters and/or provide iron for their assembly in prokaryotes and eukaryotes. Isa are usually non-essential and in most organisms are confined to the mitochondrion. We have studied the function of TbIsa1 and TbIsa2 in Trypanosoma brucei, where the requirement for both of them to sustain cell growth depends on the life cycle stage. The TbIsa proteins are abundant in the procyclic form, which contains an active organelle. Both proteins are indispensable for growth, as they are required for the assembly of Fe-S clusters in mitochondrial aconitase, fumarase and succinate dehydrogenase. Reactive oxygen species but not iron accumulate in the procyclic mitochondrion upon ablation of the TbIsa proteins, but their depletion does not influence the assembly of Fe-S clusters in cytosolic proteins. In the bloodstream form, which has a downregulated mitochondrion, the TbIsa proteins are non-essential. The Isa2 orthologue of the anaerobic protist Blastocystis partially rescued the growth and enzymatic activities of TbIsa1/2 knock-down. Rescues of single knock-downs as well as heterologous rescues with human Isa orthologues partially recovered the activities of aconitase and fumarase. These results show that the Isa1 and Isa2 proteins of diverse eukaryotes have overlapping functions.

Experimental examination of EFL and MATX eukaryotic horizontal gene transfers: coexistence of mutually exclusive transcripts predates functional rescue.

Many eukaryotic genes do not follow simple vertical inheritance. Elongation factor 1α (EF-1α) and methionine adenosyl transferase (MAT) are enzymes with complicated evolutionary histories and, interestingly, the two cases have several features in common. These essential enzymes occur as two relatively divergent paralogs (EF-1α/EFL, MAT/MATX) that have patchy distributions in eukaryotic lineages that are nearly mutually exclusive. To explain such distributions, we must invoke either multiple eukaryote-to-eukaryote horizontal gene transfers (HGTs) followed by functional replacement or presence of both paralogs in the common ancestor followed by long-term coexistence and differential losses in various eukaryotic lineages. To understand the evolution of these paralogs, we have performed in vivo experiments in Trypanosoma brucei addressing the consequences of long-term coexpression and functional replacement. In the first experiment of its kind, we have demonstrated that EF-1α and MAT can be simultaneously expressed with EFL and MATX, respectively, without affecting the growth of the flagellates. After the endogenous MAT or EF-1α was downregulated by RNA interference, MATX immediately substituted for its paralog, whereas EFL was not able to substitute for EF-1α, leading to mortality. We conclude that MATX is naturally capable of evolving patchy paralog distribution via HGTs and/or long- term coexpression and differential losses. The capability of EFL to spread by HGT is lower and so the patchy distribution of EF-1α/EFL paralogs was probably shaped mainly by deep paralogy followed by long-term coexistence and differential losses.

Complex I (NADH:ubiquinone oxidoreductase) is active in but non-essential for procyclic Trypanosoma brucei.

The requirement of complex I (NADH:ubiquionone oxidoreductase) for respiration in Trypanosoma brucei is controversial. Recent identification of homologues of its subunits in mitochondrial proteome resolved a question of its presence or absence. However, with one exception, no data have been available concerning the function(s) of complex I or its subunits. Here we present a functional RNAi study of three (NUBM, NUKM, NUEM) putative subunits of this complex. Although no changes were detected in growth, mitochondrial membrane potential or reactive oxygen species production in cell lines depleted for target transcript, the NUBM and NUKM RNAi knock-downs showed decreased specific NADH:ubiquinone oxidoreductase activity. Moreover, glycerol gradients of all cell lines revealed the presence of two distinct peaks of NADH dehydrogenase activity, with shifted sensitivity to inhibitors of complex I upon RNAi induction. Thus complex I is not only present in the procyclic stage of T. brucei 29-13 strain, but it does participate in electron transport chain.

Mitochondrial membrane potential-based genome-wide RNAi screen of Trypanosoma brucei.

We have screened the Trypanosoma brucei genome-wide RNAi library by staining the procyclics with the dye JC-1 followed by sorting the differentially stained cells by flow cytometry. This allowed us to highly enrich for cells in which mitochondrial membrane potential was decreased. We have further validated a subset of selected clones by a reverse approach in which we showed that cloning the selected genomic regions into another RNAi vector also results in a drop in mitochondrial membrane potential.

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida).

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.

Downregulation of the nuclear-encoded subunits of the complexes III and IV disrupts their respective complexes but not complex I in procyclic Trypanosoma brucei.

The function, stability and mutual interactions of selected nuclear-encoded subunits of respiratory complexes III and IV were studied in the Trypanosoma brucei procyclics using RNA interference (RNAi). The growth rates and oxygen consumption of clonal cell lines of knock-downs for apocytochrome c1 (apoc1) and the Rieske Fe-S protein (Rieske) of complex III, and cytochrome c oxidase subunit 6 (cox6) of complex IV were markedly decreased after RNAi induction. Western analysis of mitochondrial lysates using specific antibodies confirmed complete elimination of the targeted proteins 4-6 days after induction. The Rieske protein was reduced in the apoc1 knock-down and vice versa, indicating a mutual interdependence of these components of complex III. However, another subunit of complex IV remained at the wild-type level in the cox6 knock-down. As revealed by two-dimensional blue native/SDS-PAGE electrophoresis, silencing of a single subunit resulted in the disruption of the respective complex, while the other complex remained unaffected. Membrane potential was reproducibly decreased in the knock-downs and the activities of complex III and/or IV, but not complex I, were drastically reduced, as measured by activity assays and histochemical staining. Using specific inhibitors, we have shown that in procyclics with depleted subunits of the respiratory complexes the flow of electrons was partially re-directed to the alternative oxidase. The apparent absence in T. brucei procyclics of a supercomplex composed of complexes I and III may represent an ancestral state of the respiratory chain.