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Charles University in Prague, Faculty of Science Department of Parasitology Prokaryotic proteins of antioxidant defense in Trichomonas vaginalis hydrogenosomes Ph.D. Thesis Mgr. Tamara Smutná Thesis supervisor: Doc. RNDr. Ivan Hrdý, Ph.D. Prague, 2016 Data presented in this thesis resulted from team collaboration at the Laboratory of Molecular and Biochemical Parasitology of Charles University in Prague and from collaboration with our partners. I declare that the involvement of Mgr. Tamara Smutná in this work was substantial and that she contributed significantly to the presented results. ................................................................... Doc. RNDr. Ivan Hrdý, Ph.D. Thesis supervisor Declaration of the author: I declare that I elaborated the PhD thesis independently. I also proclaim that the literary sources were cited properly and neither this work nor the substantial part of it has been used to reach the same or any other academic degree. Prehlásenie autora: Prehlasujem, že som záverečnú prácu zpracovala samostatne a že som uviedla všetky použité informačné zdroje a literatúru. Táto práca ani jej podstatná časť nebola predložená k získaniu iného alebo rovnakého akademického titulu. V Prahe, 10. 3. 2016 ........................................................................... Mgr. Tamara Smutná Many thanks to my supervisor Ivan Hrdý, for his helpful support and time he invested into me during that long time way. Special thanks to my friends and fellows Honza Mach and Eva Nývltová for their support and help, to laboratory manager Michaela Marcinčíková and the entire laboratory team for great and friendly atmosphere. I declare that I substantially contributed to the results presented in this thesis. Tamara Smutná Contents Contents............................................................................................................................1 Abstract ............................................................................................................................2 Abstrakt............................................................................................................................4 1. Introduction .................................................................................................................6 2. Reactive oxygen species................................................................................................7 3. Enzymes involved in ROS detoxification ....................................................................9 3.1 Superoxide dismutases (SOD) ..................................................................................9 3.2 Catalases ...............................................................................................................11 3.3 Glutathione and glutathione-related enzymes .........................................................11 3.4 Thioredoxin-linked detoxification system................................................................13 3.4.1 Peroxiredoxins ................................................................................................14 3.5 Flavodiiron proteins (FDPs) ..................................................................................15 3.6 Iron-sulfur flavoproteins ........................................................................................17 3.7 Osmotically inducible proteins (OsmC) and Organic hydroperoxide resistance protein (Ohr)................................................................................................................18 4. Antioxidant defense in anaerobic/ microaerophilic parasitic protists......................20 4.1 Entamoeba histolytica ............................................................................................21 4.2 Giardia intestinalis.................................................................................................25 4.3 Trichomonas vaginalis ...........................................................................................28 5. Aims ............................................................................................................................33 6. List of publications and author contribution ............................................................34 6.1 Publication 1: Smutná et al., 2009..........................................................................35 6.2 Publication 2: Smutná et al., 2014..........................................................................36 6.3 Publication 3: Nývltová et al., 2015........................................................................37 7. Conclusions.................................................................................................................38 7.1 TvFDP ...................................................................................................................38 7.2 TvIsf3 .....................................................................................................................39 7.3 TvOsmC .................................................................................................................40 8. References ..................................................................................................................42 1 Abstract Parasitic protists with modified mitochondria represent important and exciting group of organisms, not only from the view of eukaryotic cell evolution but also because these parasites are causative agents of serious and widespread diseases. The study and understanding of their biology is thus necessary for the development of new antiparasitic drugs. These organisms reside in host body cavities with low concentrations of oxygen and while they lack typical mitochondria, they possess mitochondrion-related organelles which still integrate many physiologically important processes. Trichomonas vaginalis is an anaerobic flagellate inhabiting mucosal surface of vagina. Instead of canonical mitochondria, T. vaginalis possesses organelles termed hydrogenosomes. These organelles harbor pathways of ATP-generating metabolism via substrate-level phosphorylation, dependent on enzymes prone to oxidative damage, such as pyruvate:ferredoxin oxidoreductase and Fe-Fe hydrogenase. Because the environment of trichomonads is not fully anaerobic, the parasite had to develop complex strategies to cope with both oxygen and reactive oxygen species (ROS) generated by host immune system cells. Recent data from T. vaginalis proteomic and genomic analyses revealed the presence of bacterial-type proteins potentially participating in antioxidant defense. In this thesis, we characterized three hydrogenosomal proteins involved in oxygen and ROS detoxification. Flavodiiron protein (TvFDP), iron-sulfur flavoprotein (TvIsf3) and TvOsmC (a member of OsmC/Ohr protein family) represent proteins rarely encountered in eukaryotes, that were probably acquired by T. vaginalis predecessor trough lateral gene transfer from a prokaryotic donor. TvFDP, the terminal hydrogenosomal oxidase, catalyzes reduction of oxygen to water. TvIsf3, in addition to its oxygen-reducing activity, is able to detoxify the nitro-antibiotics metronidazole and chloramphenicol. TvOsmC is a thioldependent peroxidase homologues of which were believed to be restricted to prokaryotes. Unexpectedly, in addition to T. vaginalis we identified members of OsmC/Ohr proteins in a number of eukaryotic species. We also describe new and unexpected physiological function of H and L protein homologues of incomplete glycine decarboxylase complex (GDC) in hydrogenosomes. The proteins serve as electron donors for peroxidase activity of TvOsmC and together 2 constitute the defense system against both hydrogen peroxide and organic hydroperoxides in hydrogenosomes of T. vaginalis. 3 Abstrakt Parazitickí protisti s modifikovanou mitochondriou reprezentujú dôležitú a zaujímavú skupinu organizmov, a to nielen z pohľadu evolúcie eukaryotickej bunky, ale naviac predstavujú pôvodcov nebezpečných a rozšírených ochorení, a preto poznatky z oblasti ich biológie predstavujú doležitý nástroj k vývoju nových chemoterapeutík s antiparazitárnym účinkom. Vzhľadom na to, že tieto organizmy osídľujú telesné dutiny hostiteľského organizmu vyznačujúce sa nízkou koncentráciou kyslíka, nie je u nich prítomná typická mitochondria. Namiesto nej obsahujú redukované, mitochondriám príbuzné organely, v ktorých však stále prebiehajú mnohé fyziologicky dôležité procesy. Trichomonas vaginalis je anaerobný bičíkovec osídľujúci vaginálny epitel. Namiesto obvyklých mitochondriám. mitochondrií Hydrogenozómy obsahuje obsahujú hydrogenozómy, metabolické dráhy organely príbuzné generujúce ATP prostredníctvom substrátovej fosforylácie s využitím enzýmov citlivých k poškodeniu kyslíkom, ako sú pyruvát:feredoxín oxidoreduktáza a Fe-Fe hydrogenáza. Vzhľadom na to, že trichomonády nežijú v absolútne anaeróbnom prostredí, museli si vyvinúť komplexné stratégie ako čeliť kyslíku a reaktívnym formám kyslíka (ROS) produkovaným bunkami imunitného systému hostiteľa. Nedávne proteómové a genómové analýzy potvrdilli u T. vaginalis prítomnosť bakteriálnych proteínov potenciálne participujúcich na antioxidatívnej obrane. V tejto práci charakterizujeme tri hydrogenozomálne proteíny podieľajúce sa na detoxifikácii kyslíku a ROS. Dvojželezný flavoproteín (TvFDP), železo-sírny flavoproteín (TvIsf3) a TvOsmC (proteín zo skupiny peroxidáz indukovaných osmotickým stresom/ podieľajúcich sa na rezistencii k organickým peroxidom) predstavujú u eukaryot zriedkavo sa vyskytujúce proteíny. T. vaginalis pravdepodobne získala tieto gény cestou laterálneho génového prenosu z prokaryotického donora. TvFDP, terminálna hydrogenozomálna oxidáza, katalyzuje redukciu kyslíku na vodu. TvIsf3 je okrem redukcie kyslíka schopný redukovať antibiotiká s nitro skupinou, metronidazol a chloramfenikol. TvOsmC predstavuje peroxidázu homologickú k thiol-dependentným peroxidázam, ktoré boli považované za výhradne prokaryotické enzýmy. Neočakávane sa nám podarilo identifikovať členov OsmC/Ohr proteínovej rodiny medzi eukaryotickými druhmi. Súčasne popisujeme novú fyziologickú funkciu homológov H a L proteínov neúplného glycín-dekarboxylázového komplexu na úrovni hydrogenozómu. Tieto proteíny 4 slúžia ako donory elektrónov pre peroxidázovú aktivity TvOsmC a aktívne sa podieľajú na ochrane hydrogenozómov T. vaginalis pred účinkami organických i anorganických peroxidov. 5 1. Introduction About 2.4 billion years ago one of the most important events in history of our planet occurred, transition from reducing to oxidizing environment called the Great Oxygenation Event (GEO) (87,110). At that time the oxygenated atmosphere produced by cyanobacteria appeared and the evolution of aerobic metabolism and ultimately multicellular organisms with complex body plans, with the capacity for aerobic respiration, started. This powerful change of environs brought a big challenge to most then existing (anaerobic) organisms and is associated with their massive extinction. Although the first oxygen-producing cyanobacteria appeared 2.7 billion years ago or earlier, the evolution of oxygenated atmosphere was not straightforward. First oxygen disappeared almost as soon as it was formed - sopped up by Earth’s reducing atmosphere in reactions with volcanic gases or crustal minerals. Oxygen remained uncommon for the next few hundred million years (38,40,101). During this time, the reducing environment on the Earth was getting weaker and the oxygen progressively building up in the atmosphere. Simultaneously, the evolution of eukaryotes proceeded in the oceans, independent of increasing levels of oxygen in atmosphere, because all of the ocean’s waters except for the uppermost layers had remained anoxic for more than a billion years after atmospheric oxygen made its first appearance (36). Despite the first eukaryotes emerged from anoxic environment, progressive oxygenation of water column probably accelerated the evolution of more complex organisms since oxygen is thought to be a pre-requisite for the evolution of life as we know it today (39,202). Adaptation to new (oxidizing) conditions included not only the development of mechanisms to control pitfalls of oxygenated environment (the reactive oxygen species spawned from this molecule), but also the opportunity of more effective energy metabolism leading to expansion of multicellularity and boom of “higher organisms” (202). 6 2. Reactive oxygen species In 1954, the publication by Gerschman referred to deleterious properties of oxygen in consequence to its partial reduction to free radicals (75). Subsequently, in 1969 the discovery of the enzyme superoxide dismutase (SOD) by McCord and Fridovich (142) and in 1977 the work of Mittal and Murrad (148) describing the role of hydroxyl radical OH in activation of guanylate cyclase producing the “second messenger“, cyclic guanosine monophosphate (cGMP), pointed to the importance and dual role of free radicals in biologic systems. Reactive molecules derived from molecular oxygen (including the singlet oxygen, the non-reduced molecule in excited state), called reactive oxygen species (ROS), along with reactive nitrogen species (RNS) derived from nitric oxide, are molecules of „two faces“, since they can be either beneficial or harmful to organisms, all in the dependence of their equilibrium (19,177,209). In “higher” organisms low/moderate concentrations of ROS play cardinal functions in an array of biological processes, such as defense against infectious agents, cell signaling pathways and in the induction of a mitogenic response. Appropriate ROS concentration in physiological signaling enables modification of redox-sensitive amino acids in a variety of proteins including phosphatases, ion channels and transcription factors (217), thus fulfilling indispensable role. Even in ancient (in evolutionary terms) organisms ROS participate in important biochemical processes such as cross-linking of cellular matrix (55) or hardening of the fertilization envelope after egg-sperm fusion (213). (For example, the cross-linking mechanism of the sea urchin fertilization envelope requires synthesis of hydrogen peroxide by the egg and likely a scavenging system for residual hydrogen peroxide and its freeradical byproducts (215).) At high levels ROS cause oxidative stress usually leading to cellular damage. If uncontrolled, high reactivity of ROS with DNAs, proteins and lipids leads to the collapse of cellular homeostasis and cell death. The fragile balance between deleterious and beneficial effects of ROS is maintained by an array of mechanisms called „redox regulation“, which protects living organisms from various oxidative stresses and keeps „redox homeostasis“ in living system (52). 7 The most common ROS include singlet oxygen (1O2), superoxide anion radical (O2-), hydroxyl radical (HO) and hydrogen peroxide (H2O2), all of which are normal byproducts of oxygen metabolism. Oxygen has a unique molecular structure with unique electron configuration resulting from two unpaired electrons in ground state of O2 molecule (triplet oxygen). It readily accepts electrons generated by normal oxidative metabolism of the cell. Processes causing uncoupling of electron transport can enhance the production of ROS, with mitochondria being a major source (23,80). Flow of electrons and protons through mitochondrial electron transport system generates an electrochemical proton gradient, the pivotal driving force for ATP synthesis within most aerobic eukaryotic cells. In the process of oxidative phosphorylation, some electrons “leak” to oxygen prematurely, forming the anion and free radical, superoxide O2-. Superoxide causes the release of iron from iron-containing clusters of enzymes (FeS clusters) (208) or reduces Fe3+ via first step of Haber-Weiss reaction. In both cases reduced Fe2+ can participate in the Fenton reaction (Fe2+ + H2O2→Fe3+ + OH + OH-), generating highly reactive hydroxyl radical (125), the short-lived molecule able to attack biomolecules in a diffusion-limited reactions (84). Hydroxyl radical damages proteins (201), polysaccharides (84), nucleic acids (28) and membrane lipids (206). Superoxide does not readily cross the membranes and its effect is spatially rather limited, but is spontaneously or by superoxide dismutase converted to hydrogen peroxide, longer-lasting and membrane-diffusible molecule (82). H2O2 participates in chain reactions generating deleterious compounds such as hypochlorous acid (HOCl), singlet oxygen (1O2) and other species. Singlet molecular oxygen can be generated in biological systems by photoexcitation upon exposure of endogenous photosensitizers (porphyrins, flavins, quinones, etc.) to UVA (21) or by reactions involving peroxides such as lipid peroxidation, phagocytosis and the catalytic mechanisms of peroxidases (149). The decomposition of lipid hydroperoxides (LOOH) into peroxyl radicals is another potential source of singlet molecular oxygen (1O2) (149). Besides mitochondrial electron transporting chain, other significant sources of endogenous ROS are peroxisomes, endoplasmic reticulum and NAD(P)H oxidases (NOX) complexes localized in cell membranes. NAD(P)H oxidases localized in membranes of neutrophils are an important tool of immune system activated during respiratory burst and rapidly generating ROS in order to defend against pathogenic microorganisms (26,108). 8 3. Enzymes involved in ROS detoxification 3.1 Superoxide dismutases (SOD) Superoxide anion (O2-) and free radical is a commonly present product of oneelectron reduction of dioxygen O2. It is a reactive molecule with short half-life and principal element of a cascade generating deleterious ROS such as hydrogen peroxide, hypochlorite anion, peroxynitrate and hydroxyl radical (147). Biological systems massively produce O2- by NAD(P)H oxidases in phagocyting cells of immune system, during oxygen-dependent intracellular killing of invading pathogens. The enzymes specifically aimed at detoxification of superoxide are SODs. The SODs are widely distributed enzymes, present in all kingdoms of life. The family of enzymes with common name SOD consists of three unrelated enzyme types with the ability to catalyze dismutation of superoxide anion into either molecular oxygen O2 or less detrimental hydrogen peroxide H2O2. Three classes of SODs based on amino acid sequence homology are recognized to this date (Fig. 1) (22,73,104,117): Figure 1. Comparison of ribbon structures that characterize three classes of SODs. Source: (147). 9 NiSOD was first described in Streptomyces (221). Genes coding for homologous proteins were identified also in additional actinobacteria (191) and cyanobacteria species (57). No evidence for NiSOD in gram-positive bacteria, archaea or eukaryotes was found, except for eukaryotic green alga Ostreococcus tauri, the smallest free-living eukaryote known (191). Cu,ZnSOD is a cytoplasmic enzyme containing one Cu2+ and one Zn2+ ion in each of two identical subunits. Protists do not possess Cu,ZnSOD (216), while plants, animals and fungi all present this enzyme mostly in the cytosplasm. Plants contain two homologous groups of Cu,ZnSOD, cytoplasmic and chloroplast ones (70). Among animals, two different Cu,ZnSODs are present, each coded by a separate gene: dimeric cytoplasmic Cu,ZnSOD (143) and extracellular tetrameric glycosylated Cu,ZnSOD (11), which e.g. in Schistosoma provides protection of this parasite against oxidants produced by host phagocytes (88). FeSOD is present in diverse evolutionarily ancient organisms, absent from fungi and animals and present in chloroplasts of plants. FeSOD is probably an ancient version of enzyme which was subsequently modified to use manganese, probably due to diminished bioavailability of iron and participation of this metal ion in deleterious hydroxyl radicalproducing Fenton reaction under conditions of oxidative stress (147). FeSOD is present in protists, including the parasites such as E. histolytica (128), trichomonads (59,171), trypanosomes (216), plasmodia (25) and Perkinsus marinus (16), providing these organisms the protection against oxidative stress produced by host immune system. MnSOD is present in eukaryotic mitochondria, but it was also described in photosynthetic protist Euglena gracilis, tightly associated with thylakoid membranes, reminiscent of membrane-bound MnSOD of filamentous cyanobacteria (18,100). The human malaria parasite Plasmodium falciparum represents an inventive arrangement, where both cyanide-sensitive Cu,ZnSOD acquired from the host cell cytoplasm and an endogenous cyanide-resistant FeSOD are present (64,173). 10 3.2 Catalases Catalases are widely distributed enzymes capable of H2O2 decomposition by heterolytic cleavage of the peroxidic bond in hydrogen peroxide (H-O-O-H) as well as in some small organic peroxides (R-O-O-H). Moreover, they are able to evolve molecular oxygen (O2) by oxidation of hydrogen peroxide. This is how the catalases decompose hydrogen peroxide by dismutation. Catalases are divided into three protein families. Two of the families consist of heme-containing enzymes, typical “monofunctional” catalases (hydrogen peroxide:hydrogen peroxide oxidoreductases) and “bifunctional” catalase-peroxidases (donor:hydrogen peroxide oxidoreductases). Members of these protein families have no sequence similarity, very different active site and both tertiary and quaternary structure, although both families possess high catalase activity. Typical catalases comprise the most abundant group found in Eubacteria, Archaeabacteria, Protista, Fungi, Plantae and Animalia and possess catalatic activity that includes degradation of two molecules of hydrogen peroxide to water and molecular oxygen (H2O2 → 2H2O + O2) (222). Bifunctional catalase-peroxidases possess, besides their catalatic activity, also the activity similar to that of conventional peroxidases (electron donor + H2O2 → oxidized donor + 2H2O). Nonheme manganese-containing enzymes (Mn-catalases) represent the third, minor group of enzymes with catalatic activity present only in bacteria (222). 3.3 Glutathione and glutathione-related enzymes Glutathione (GSH) is one of the most important antioxidants within the cell. GSH is a tripeptide consisting of amino acids γ-L-glutamate, L-cysteine and glycine that donates two electrons (one electron per one molecule GSH) into reactions and is reversibly converted into oxidized form (GSSG) consisting from two GSH molecules linked with disulfide bond. Human cells contain GSH in high concentrations, facilitating involvement of GSH in detoxification reactions (94). GSH can react directly with O2- and some other ROS, RNS and xenobiotics but its indispensable function lies in regeneration of other antioxidants. For example, it is involved in reduction of dehydroascorbic acid, formed during 11 reconversion of α-tocopheroxyl radical to α-tocopherol, an important lipophilic antioxidant scavenging various ROS and lipid oxy-radicals (167,174). The level of GSH in the cell changes depending on its utilization, recycling and cellular export. This circulation is called GSH cycle and connects some other important enzymes as glutathione peroxidase (GPx) and glutathione S-transferase (GST). GPx is an important selenium-dependent enzyme (protein containing selenocysteine amino acid residue), able to scavenge H2O2 and lipid hydroperoxides by using two molecules of GSH and producing oxidized GSSG (15). This molecule is subsequently regenerated/reduced by glutathione reductase (GR) at the expense of NADPH (Fig. 2). Figure 2. Reduction of peroxides by glutathione peroxidase. GPx, glutathion peroxidase; GSH, reduced glutathione; GSSG, oxidized glutathione; GR, glutathione reductase. Source: Figure based on (15). Schistosoma mansoni GPx is the only human pathogen possessing real seleniumincorporating GPx, but the enzyme is more likely involved in egg maturation than in antioxidant defense (180). The major function of glutathione S-transferase (GST) is detoxification of harmful electrophilic compounds, xenobiotics and products of oxidative stress, by conjugation with GSH molecule (Fig. 3). Such conjugates are subsequently exported from the cell by appropriate transporters. 12 Figure 3. Schematic depiction of GST enzymatic activity. GSH, reduced glutathione; GST, glutathione S-transferase; R, reactant; GS-R, conjugate with glutathione. Source: figure based on (15). Glutathione is also a cofactor and substrate for multiple glutaredoxins (Fig. 4), reducing disulfide bond in the presence of NADPH and GR (69). Figure 4. Schematic depiction of GRx enzymatic activity. GRx, glutaredoxin; GSH, reduced glutathione; GSSG, oxidized glutathione; Pr, protein with disulfide bond. Source: figure based on (69). 3.4 Thioredoxin-linked detoxification system In parallel with the glutathione reducing system, the thioredoxin/thioredoxin reductase (Trx/TrxR) system represents another important detoxifying mechanism. Thioredoxin reductases (TrxRs) are thiol-dependent flavoreductases belonging to the flavoprotein family of pyridine nucleotide-disulphide oxidoreductases (156). These proteins represent active homodimers where each monomer contains an FAD-binding domain, NADPH-binding domain and an active site containing a redox-active disulfide (156). Electrons are transferred from NADPH via FAD to the active-site disulphide of TrxR, which then reduces the substrate (thioredoxin). 13 All parasitic protists possess antioxidant system such as GSH/GR or Trx/TrxR due to their constant exposition to ROS originating from their own metabolism or from the defense reactions of the host (85). Among anaerobic parasitic protists the GSH/GR system is absent and Trx/TrxR are the main components of their antioxidant defense systems. E. histolytica, T. vaginalis and G. intestinalis all possess a complete thioredoxin system, consisting of thioredoxin (Trx), Trx peroxidase and Trx reductase (14,45,92,119), although the physiological functionality of this system in G. intestinalis was not experimentally confirmed (see 4.2 Giardia intestinalis) (140). Plasmodium, the agent of malaria, possesses both glutathione- and thioredoxin-linked redox systems (102,103,157). In contrast, the internal thiol homeostasis of trypanosomatids depends exclusively on trypanothione [N1,N8-bis(glutathionyl)spermidine] (65), the polyamine spermidineGSH conjugate specific for kinetoplastida, and a tryparedoxin/trypanothione reductase system (66). 3.4.1 Peroxiredoxins 2-Cys peroxiredoxin (Prx) family of enzymes are thiol-specific proteins able to reduce H2O2 at a very high rate (219). They are widespread all over the taxa of all kingdoms and in pathogens are more common than, e.g., glutathione peroxidase and catalase (72). In addition to their H2O2 reducing activity, Prx are able to decompose alkylhydroperoxides and peroxinitrites (32). The 2-Cys Prxs are dimers, where the peroxidatic activity of the enzyme is dependent on two redox-active cysteines (a peroxidatic and resolving cysteine) in its active site. In reaction with H2O2, the peroxidatic cysteine (Cp-SH) is oxidized to a sulfenic acid (Cp-SOH), which is subsequently condensed with resolving cysteine from the other subunit (Fig. 5) (5,86,193,218,219). In bacteria, the group of flavoproteins (AhpF) fulfils the task of disulfide reductase to recycle the bacterial peroxiredoxin (AhpC) during catalysis (158). In eukaryotes, oxidized cysteine residues of Prx are specifically reduced by thioredoxins (Trx) (41) (in the context of thioredoxin-linked reduction system peroxiredoxins are also termed thioredoxin peroxidases -TrxP). Peroxiredoxin-linked detoxification system was confirmed in protist parasites including trypanosomatids, Plasmodium falciparum and E. histolytica. T. vaginalis possesses thiol-dependent peroxidase system both in cytoplasm and hydrogenosomes. This duplicity underscores the relevance of thioredoxin system for redox homeostasis in Trichomonas, which lacks many of conventional detoxification enzymes (catalase, GSH 14 system) (45,146). Intestinal parasite G. intestinalis encodes 2 genes for 2-Cys peroxiredoxins; however, as mentioned above, the physiological reducing partner is unknown (140). Figure 5. Schematic representation of peroxiredoxin enzymatic activity. (1) Formation of cysteine sulfenic acid intermediate – peroxidatic cysteine in peroxiredoxin reacts with peroxide, (2) condensation of sulfenic acid with the resolving cysteine, (3) peroxiredoxin reduction by thioredoxin; ROOH, peroxide; TrxR, thioredoxin reductase; Trx, thioredoxin. Source: figure adapted from (219). 3.5 Flavodiiron proteins (FDPs) Flavodiiron proteins, first named A-type flavoproteins, are a large family of enzymes widespread among anaerobic and some aerobic prokaryotes (Bacteria including cyanobacteria, and Archaea) and anaerobic protists with modified mitochondria. The data from genome sequencing projects also revealed the genes coding for FDPs in some photosynthetic eukaryotes (4). All known FDPs consist of conserved core, built by two structural domains: the Nterminal metallo-β-lactamase-like domain, harboring a non-heme diiron catalytic center, and the C-terminal flavodoxin-like domain, containing a flavin mononucleotide (FMN) moiety (210). In addition to common core, some FDPs harbor C-terminal extensions. Based on overall structure, four classes of FDPs are recognized to date (189) (Fig. 6). 15 Figure 6. Classificition of FDPs and their modular organisation. Source: (4). The most numerous group of FDPs is Class A, which represents the minimal core structure. Members of this class are found in Bacteria, Archaea and protists. Class B contains C-terminal extension similar to type I rubredoxins, such as in flavorubredoxin enzyme from E. coli (74,76). Genes of Class B flavodiiron proteins are present in genomes of Enterobacteria. Class C consists of proteins exclusively found in genomes of photosynthetic cyanobacteria, with flavin-binding NAD(P)H:flavin oxidoreductase domain fused to the C-terminus. Bacteria of the Clostridium genus and T. vaginalis are the main representatives possessing Class D proteins. Proteins of this class consist of two domains condensed in C-terminal extension of core module into a single polypeptide chain. This functional extension incorporates NADH:rubredoxin oxidoreductase module and a rubredoxin module (4). The native FDPs are arranged as homodimers or homotetramers in a “head to tail” configuration, with flavin moiety and diiron center in close proximity, which enables fast electron transfer between the cofactors. The electron flow and number of redox partners depends on the modular arrangement of FDPs. The more complex protein is, the fewer redox partners are needed for electron transfer. E.g. rubredoxin, necessary for reduction of 16 Class A proteins, does not participate as a soluble protein in reduction of Class B proteins as it is an integral part of enzyme’s polypeptide chain. The described role of FDPs in anaerobic organisms is the reduction of deleterious O2 to water or NO to N2O (210). While in some species FDPs preferably reduce NO (74,76), in others the enzyme prefers O2 (51,195,198), whereas some FDPs can catalyze both reactions (197). All protist FDPs display up-shifted reduction potential of diiron centre, which can play a role in their substrate selectivity. This hypothesis is supported by recent study of two amino acid residues in close spatial proximity to the diiron site. These amino acids differ between NO-selective FDPs from bacteria (a serine and an aspartate) and O2-selective FDPs from protists (a tyrosine and a lysine, respectively). Study of site-directed mutants affirmed that the residues in these positions contribute to substrate selectivity of FDPs (77). 3.6 Iron-sulfur flavoproteins Iron-sulfur flavoprotein (MtIsf) from Methanosarcina thermophila, a strictly anaerobic methane-producing thermophilic archaeon, is the prototype of a recently described family of flavoproteins (116,223). Sequences coding for homologous genes have been identified in genomes of numerous anaerobic prokaryotes belonging to the domains Bacteria and Archaea (223). The proteins are characterized as α2-homodimers with one FMN moiety and 4Fe-4S or 3Fe-4S cluster per monomer. The iron sulfur cluster is bound to the protein by highly conserved motif of four cysteines (CX2CX2CX5C) (223). Ferredoxin was identified as an electron donor, reducing the low-potential FeS cluster and subsequently flavin (24). The function of Isf as a component of acetate fermentation pathway has been postulated primarily (116) until the evidence of the role of the protein from M. thermophila in the oxidative stress management was published. The protein is believed to reduce O2 and H2O2 to water and thus probably plays an important role in ROS detoxification in anaerobic prokaryotes (46). However, the presence of multiple homologues in metabolically diverse anaerobic organisms may suggest broader physiological functions (9). Recent genomic data revealed the presence of these proteins in anaerobic eukaryotes as well. (37,128) Upregulation of three genes encoding Isfs was described in E. histolytica under oxidative stress generated by H2O2 (211). Genome of 17 T. vaginalis codes for 7 Isf homologues (37), one of which was functionally characterized and localized in hydrogenosome ((199) and publication 2 in this thesis). 3.7 Osmotically inducible proteins (OsmC) and Organic hydroperoxide resistance protein (Ohr) This superfamily of proteins encompasses a group of mainly bacterial proteins (both Gram-positive and Gram-negative) involved in detoxification of organic hydroperoxides (17) generated as by-products of bacterial aerobic metabolism, and as reaction intermediates during host defense responses following the release of lysosomal contents within inflammatory cells and the neutrophil oxidative burst (17,120). OsmC protein was first identified in E. coli as a protein responding to osmotic stress (81,121). Ohr is an organic peroxide-specific defense enzyme that is under the control of the organic peroxide sensing repressor OhrR (17). Protein sequences of each subfamily present high mutual identity (40% - 70%), while they share no significant sequence similarity to other prokaryotic and eukaryotic proteins. The amino acid sequence consists of more conserved C-terminal region encoding two crucial cysteine residues involved in hydroperoxide reduction (120). The catalytic mechanism is similar to that of peroxiredoxins. Peroxide reacts with cysteine and is reduced to alcohol, while the cysteine is oxidized to a cysteine sulfenic acid intermediate. Subsequently, cysteine sulfenic acid condenses with the second cysteine, leading to the formation of an intramolecular disulfide bond and releasing water. Finally, oxidized protein is regenerated by intracellular reductant/dithiol (121) (Fig. 7). 18 Figure 7. Schematic depiction of OsmC/Ohr enzymatic activity. (1) Formation of cysteine sulfenic acid intermediate – cysteine in protein reacts with peroxide; (2) condensation of sulfenic acid with the second cysteine; (3) protein regeneration; ROOH, peroxide; R(SH)2, intracellular reductant/dithiol. Source: Figure based on (121). Until the recent work of Cussiol, the natural electron donor for OsmC/Ohr proteins was unknown; Dithiols such as DTT were found to support the peroxidase activity in vitro (120). Subsequently, lipoate (as a component of lipoylated enzymes), an important dithiol/disulfide redox agent, was established as a source of electrons, reducing Ohr of Xylella fastidiosa (47). Most species encode only one member of these two enzyme subfamilies, however, occurrence of both enzymes in one organism is also documented (Pseudomonas aeruginosa, Deinococcus radiodurans and data in publication 3 in this thesis), despite the fact that both protein groups appear structurally and functionally similar. The simultaneous presence of both enzymes could be possibly due to distinct subcellular localization (such as in protein clusters and microcompartments, cytoskeletal elements and organelle-like bodies (184)) of each protein, which may be beneficial if, for example, one enzyme is primarily responsible for detoxification of exogenous peroxides produced by host immune system, while the other scavenges the peroxide byproducts of bacterial metabolism. It is also possible that OsmC and Ohr have evolved to metabolize different substrates. Recent structural studies refer to different distribution of hydrophobic residues and threedimensional shape of active site cavities between Ohr and OsmC proteins, thus supporting the hypothesis that these proteins metabolize structurally different substrates (121). 19 4. Antioxidant defense in anaerobic/ microaerophilic parasitic protists Current available evidence suggests that all extant eukaryotes possess some form of mitochondrial organelle. The last eukaryotic common ancestor (LECA) gained mitochondrion by endosymbiosis of an ancestral α-proteobacterium with a methaneproducing archaeon or possibly a more advanced cell already with higher level of subcellular organization (105,107,136,138,170). Such organisms could derive benefits of increased ATP production via oxidative phosphorylation coupled to aerobic respiration, using oxygen as the final electron acceptor. Despite of these advantages, many species of protists subsequently adapted to (or never left) anaerobic habitats and now possess modified, reduced mitochondria collectively called mitochondrion-related organelles (MROs) and represented by hydrogenosomes and mitosomes. These organelles are found in a wide range of free-living as well as parasitic/endosymbiotic protists (mitosomes seem to be restricted to endobionts) residing in anaerobic/microaerophilic niches such as anoxic sediments or body cavities of their hosts (134). MROs lack most of enzymes involved in energy conservation via the tricarboxylic acid cycle, oxidative phosphorylation and β-oxidation of fatty acids that are typical for canonical mitochondria (134), and are no longer able to utilize oxygen as a terminal electron acceptor in pathways of energy metabolism. However, some MROs retained components of respiratory chain and can still produce ATP via substrate-level phosphorylation (50,53,115,154). The most conserved function of mitochondrial organelles appears to be the Fe-S cluster biosynthesis (134). The group of anaerobic/ microaerophilic protists unites the eukaryotic organisms of diverse evolutionary affinities living in the environment with low levels of oxygen, typically up to 10% of atmospheric O2 saturation (67). Although some of these organisms tolerate oxygen or even flourish in low oxygen tensions, their energy metabolism relies on enzymes that are susceptible to oxygen and ROS, including, e.g., PFOR and hydrogenase (134,154). Living in the habitat they are permanently confronted with the threat of oxidative damage, these organisms had to develop effective strategies enabling them to survive. 20 4.1 Entamoeba histolytica Entamoeba histolytica, the causative agent of amoebiasis, is a unicellular parasite of humans widespread trough the world (187). The amoebiasis is the third leading cause of death by parasitic infection in almost all developing countries with insufficient quality of water, with 50 million clinical episodes of dysentery or amoebic liver abscess and ca. 100,000 deaths annually (WHO, http://who.int). The majority of infections is asymptomatic, with trophozoites colonizing the surface of mucus layer in the human colon, feeding on bacteria and food particles. This form of E. histolytica, called minuta, produces inert infectious cysts that serve to oro-fecal transmission. However, in an estimated 10-20% of cases, the trophozoites transform (in response to stimuli such as disturbed gut flora, diet, host immune status etc.) to form magna – tissue-invasive trophozoites that are unable to produce cysts. In extreme conditions the trophozoites of magna form can penetrate intestinal submucosa and cause symptoms ranging from severe bloody diarrhea to extraintestinal tissue invasion (187,203). During tissue invasion E. histolytica must cope with increased oxidative and nitrosative stress. The main source of reactive nitrogen species comprises host immune system responding to the presence of pathogen. Virulent E. histolytica strains display higher resistance to oxidative and nitrosative stress because of their elevated transcription of genes involved in response to ROS/RNS (133,172,188,211). E. histolytica is able to metabolize oxygen without accumulation of toxic hydrogen peroxide (155,176), however, it lacks most of typical eukaryotic ROS scavenging enzymes such as catalase, peroxidase, glutathione and glutathione-related enzymes (63,145). E. histolytica apparently lacks glutathione metabolism and the main reducing component is believed to be L-cysteine. This low-molecular weight thiol (63) is required for the survival, growth, attachment, motility, gene expression regulation, and oxidative stress adaptation (63,97). In view of the fact that trans-sulfuration pathways are absent in Entamoeba metabolism, L-cysteine is potentially synthetized de novo, which is a typical feature of bacteria and plants (34,186). This biosynthesis requires two enzymes: serine acetyltransferase (SAT) and cysteine synthase (CS; O-acetylserine(thiol)lyase). Although three paralogues of each SAT and CS are present in E. histolytica genome (2), the cultivation of trophozoites under cysteine deprivation resulted in undetectable intracellular levels of L-cysteine and L-cystine (93). It has been suggested that the endogenous L21 cysteine biosynthetic pathway in E. histolytica is inoperative or insufficient and probably serves to generate S-methylcysteine (product of O-acetylserine conversion) (93), which fulfills the task of sulfur storage (175). It thus appears that E. histolytica trophozoites depend on cysteine uptake from the environment (97). Although the cysteine is the major thiol in Entamoeba (when the trophozoites are cultured in cysteine-depleted medium, the intracellular ROS level increases 3-4fold), the reduced thiols in cysteine pose potential risk as they are strongly nucleophilic and reactive (166). Therefore intracellular concentration of cysteine must be maintained at relatively low level, balancing between its toxicity and requirement for protein synthesis and redox equilibrium maintenance. External L-cysteine is metabolized by E. histolytica trophozoites into thiazolidine derivatives, such as thiazolidine-4carboxylic acid (T4C), 2-methylthiazolidine-4-carboxylic acid (MT4C) and 2- ethylthiazolidine-4-carboxylic acid (ET4C) (condensation products of L-cysteine), or oxidized to L-cystine. T4C, also named thioproline, is a structural analogue of L-proline and it is considered an intracellular sulfhydryl antioxidant and scavenger of free radicals (60,214). The above mechanisms of L-cysteine uptake and conversion allow for regulation of its intracellular level: in response to metabolic demand, L-cysteine can be liberated back from its condensation products. Besides the elimination of ROS (214), thiazolidine derivatives also participate in mechanisms of detoxification of reactive metabolic byproducts, such as aldehydes (97). Major role in oxidative stress defense plays thioredoxin/ thioredoxin reductase system (129) localized in cytoplasm and 2-Cys peroxiredoxin associated with cell surface. E. histolytica genome contains a single TrxR gene; however, there are 22 Trx genes, with two of them fully characterized (13,14). E. histolytica TrxR also presents H2O2-generating NAD(P)H oxidoreductase activity in the presence of O2 with the catalytic mechanism different from the disulfide reduction (13). Unlike TrxRs from other organisms, which are strictly NADPH-specific, Entamoeba TrxR can utilize both NADPH and NADH as reductants, however, the affinity for NAPDH is 10-fold higher than that for NADH (3,13,43). Entamoebic peroxiredoxin is a thiol- specific 29 kDa membrane-associated antigen, which reduces and detoxifies peroxides and peroxynitrites under conditions of oxidative stress. The association with the cellular membrane is due to interaction with the cytoplasmic domain of the N-acetylgalactosamine-inhibitable (GalNAc) lectin, the major surface lectin mediating adherence to host cells. During host-parasite interaction the lectin 22 probably recruits peroxiredoxin to the parasite surface, a mechanism by which the parasite protects itself during tissue adherence and invasion from oxidative attacks of activated host phagocytic and epithelial cells. Therefore E. histolytica, which is, unlike nonpathogenic E. dispar, capable of tissue invasion, contains 50 times more peroxiredoxin (44). ROS detoxification system of Entamoeba comprises also an FeSOD (31), three NADPH oxidoreductases (95), iron-sulfur flavoproteins (211), flavodiiron proteins (212) and a rubrerythrin (a peroxidase) which is localized in the mitosome (6,135) (Fig.8). Mitosomal rubrerythrin represents bacterial-type peroxidase, harboring Fe (S-Cys)4 reactive centre and a non-sulfur oxo-bridged diiron centre that are essential for its function. Analogous to bacterial rubrerythrin, the in vitro activity of E. histolytica rubrerythrin (and also flavodiiron protein) is supported by electron transfer from reduced bacterial rubredoxin (135,212). However, the evidence for the presence of genes coding for rubredoxin in E. histolytica genome is lacking to date (128). The question of physiological reductant of rubrerythrin was proposed recently, by discovering an E. histolytica protein with rubrerythrin reductase activity. This protein is a NAD(P)H-dependent rubredoxin reductase (NROR) homologue which possesses a rubredoxin-like redox active domain and exhibits enzymatic activity with heterologous bacterial rubredoxins. The enzyme is able to reduce entamoebic rubrerythrin at the expense of either NADPH or NADH (35). It was established that both FDP and rubrerythrin could be reduced by E. histolytica [4Fe-4S] ferredoxins (Fdx1 and 2) (35). E. histolytica possesses four genes coding for FDP proteins with Fdp1 being abundantly expressed in cytoplasm under the conditions of increased exposition to oxygen. FDP1 represents the only amoebic enzyme directly reducing O2 to H2O (212). Recently, the presence of at least 7 independent genes for Isf proteins was described in Entamoeba genome. Three Isf paralogues are significantly upregulated upon oxidative stress conditions, suggesting their involvement in oxidative stress management (211). E. histolytica and T. vaginalis are the only known eukaryotes possessing Isf genes (109). Interestingly, the presence of trypanothione (a spermidine-glutathione conjugate, typically present in trypanosomatids) (160,161) and NADPH-dependent trypanothione reductase activity has been observed in E. histolytica (205). However, although the trypanothione reductase activity in trophozoite lysates was detected and partially purified by Tamayo et al. (205), these data are rather controversial because the genes homologous to trypanothione reductase are apparently absent from E. histolytica genome as well as from other Entamoeba species (96). 23 These results are also in contradiction with common opinion that Entamoeba lacks glutathione-related metabolism, because GSH is an integral part of trypanothione molecule. Figure 8. Schematic representation of the antioxidant system in E. histolytica. NADPH ox., NADPH oxidoreductase; SOD, superoxide dismutase; Isf, iron-sulfur flavoprotein; 29 kDa Prx, membrane-associated peroxiredoxin; GalNAc, N- acetylgalactosamine-inhibitable lectin; Rbr, rubrerythrin; TrxR, thioredoxin reductase; FDP, flavodiiron protein. Source: figure adapted from (96). 24 4.2 Giardia intestinalis Giardia spp., the aetiological agent of giardiasis, is a unicellular flagellated parasite that inhabits upper small intestine of mammals, including humans. With 280 million symptomatic infections per year (1,10,114,162), giardiasis belongs to one of the most common intestinal infectious diseases worldwide; it is one of major contributors to diarrheal diseases and the second leading cause of death in children under five years of age worldwide (106,190). The clinical symptoms of infection may vary from watery diarrhea, nausea, abdominal discomfort and vomiting accompanied by dehydration to malabsorption and weight loss (33). Giardia intestinalis species is grouped into eight morphologically identical genetic assemblages (A-H, assemblages are currently distinguished by polymerase chain reaction (PCR) and DNA sequencing of genes such as the small-subunit ribosomal RNA (ssu-rRNA), β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) genes) (68,185), with assemblage A and B being infectious to humans. Simple life cycle consists of two forms: microaerophilic trophozoite inhabiting the mucosa of duodenum and jejunum (where trophozoites attach by ventral adhesive disk and reproduce via binary fission) and a resistant cyst serving as a source of oro-fecal transmission (1). Despite much higher tensions of oxygen (up to 50μM, (49,83,196)) as compared with distal tract of the gut, Giardia proliferates in proximal small intestine, the bile- and nutrients-rich niche (48). This part of the tract is also less colonized with microbial flora than the large intestine and thus represents less competitive environment. In addition to fluctuating levels of oxygen (peaking at every meal), the trophozoites must resist microbicidal nitric oxide (NO) produced by NO synthases (NOS) in intestinal epithelial cells and/or derived from reduction of dietary nitrate/nitrite (62,130). Besides that, the trophozoites possess enzymes such as NAD(P)H menadione oxidoreductase producing ROS in reactions with oxygen (122). Giardia is an “amitochondrial” parasite, which possesses double membrane bounded organelles, mitosomes, involved in iron-sulfur cluster assembly (204). Anaerobic, fermentative metabolism of Giardia trophozoites depends on enzymes susceptible to inactivation by O2, such as pyruvate:ferredoxin oxidoreductase (PFOR) (207) and hydrogenase (although the role of hydrogenase in Giardia metabolism is less clear) (127). Therefore, Giardia trophozoites must be endowed with efficient detoxifying 25 system enabling survival of cells in (semi)aerobic conditions of proximal small intestine mucosa. Giardia lacks the most common ROS scavenging enzymes such as catalase, superoxide dismutase and glutathione peroxidase. The trophozoites are known to display a notable cyanide-insensitive O2 consumption activity (58,126,163). This activity was initially attributed to a H2O-producing NADH oxidase with FAD cofactor and a redox active cysteine (29,30). The enzyme can utilize both NADH and NADPH as electron donor and it is inhibited by thiol inhibitors, flavoantagonists and metal chelators (Cu 2+ and Zn2+). NADH oxidase was supposed to be the only terminal oxidase, responsible for trophozoites` O2 consumption and providing for safe oxygen reduction without production of deleterious partially reduced oxygen species (30). However, later the gene coding for a FDP protein was identified in the genome of G. intestinalis. Giardia fdp, most likely acquired from a prokaryote by lateral gene transfer (7,8,128,150), codes for a protein with tetrameric structure consisting of two homodimers arranged in ‘head-to-tail’ configuration (for structure see paragraph 3.5. Flavodiiron proteins). The enzymatic activity of the protein expressed and purified from E. coli displayed significant O2-reductase activity (reducing molecular oxygen to water) and only marginal NO-reductase activity (51). These findings are in accordance with the data reported for the FDPs described from protist parasites Trichomonas vaginalis (198) and Entamoeba histolytica (212). It is as yet unknown how both FDP and NADH oxidase participate on overall cellular O2 consumption by Giardia and in what ratio. The fdp gene is expressed in Giardia cells under basal conditions (132,141) and no significant upregulation was noticed upon exposing the cells to higher tensions of oxygen (132). Interestingly, the nadhox and fdp genes were recently found to be upregulated in some albendazole-resistant clones of parasite (12), pointing to a possible linkage between drug resistance and O2 metabolism in Giardia (139). Although Giardia lacks SOD, it appears to possess a gene for superoxide reductase (SOR) in assemblage A. SOR is a non-heme iron containing enzyme, catalyzing conversion of superoxide into H2O2 without releasing O2 (98,124). This group of enzymes (1Fe- and 2-FeSORs (42,151)) was initially thought to be restricted solely to Bacteria and Archaea (169). In some species SOR gene is coded in the same operon as rubredoxin, which is considered to be a physiological redox partner of SORs in rubredoxin-containing organisms (61,181,182). The SOR represents the only enzyme detoxifying O 2- in Giardia trophozoites known to date (139). Significance of Giardia SOR should be discussed in the 26 context of metronidazole reduction under aerobic conditions, where the cytotoxic nitroradical is reoxidized by O2 to the nontoxic parent compound (“futile cycle”), thereby generating O2- (56). Lacking the catalase and glutathione peroxidase, Giardia needs another enzyme catalyzing safe reduction of peroxide evolved by SOR. Deleterious effects of H 2O2 on trophozoites are extensive and include depletion of cellular thiol pool, loss of membrane potential and cell motility (126), degradation of FDP (141) and induction of a process reminiscent of programmed cell death (including autophagy) (20). Discovery of two genes coding for peroxiredoxins (Prx1a and Prx1b) (79) outlined the possible solution as to how Giardia faces peroxides, although physiological electron donor of Giardia peroxiredoxins remains unknown (140). Nevertheless, the NADPH-dependent thioredoxin reductase (TrxR) has been previously identified in Giardia, and the protein was purified and characterized. However, attempts to measure the activity of this enzyme in crude extracts of the parasite in the presence of the putative Giardia Trx protein (encoded by the GL50803_3910 gene), produced in E. coli, or its homologues from E. histolytica or T. vaginalis, were unsuccessful (140). Beside the ROS, G. intestinalis needs to cope with NO, generated through nitrite reduction or enzymatically by NOSs that utilize arginine as a substrate. Giardia trophozoites consume arginine (54,200) for anabolic purposes (192) and as a substrate for arginine deiminase, the enzyme secreted upon interaction with the intestinal epithelial cells (179,183). Under arginine limitation NOSs produce both NO and ROS, which is leading to formation of highly toxic peroxynitrite (ONOO-) (product of reaction of NO with O2-) (220). Genome of G. intestinalis codes for a flavohemoglobin (FlavoHb), an enzyme involved in protection from nitrosative stress in bacteria and fungi, probably acquired by lateral gene transfer from a prokaryotic donor. The enzyme catalyzes reduction of NO into nontoxic nitrate NO3- under aerobic/microaerophilic conditions with O2 participating in the reaction (Fig. 9). The FlavoHb gene is up-regulated in response to NO. Likewise, it was documented that contact of Giardia trophozoites with human intestinal cells induces overexpression of FlavoHb (178,200) together with Prx1a (131). Moreover, Prx1a with FlavoHb belong to highest up-regulated genes in Giardia under the conditions of oxidative stress established by exposition to O2 and H2O2 (132). 27 Figure 9. Schematic representation of the antioxidant system in G. intestinalis. FlavoHb, flavohemoglobin; Prxs, peroxiredoxins; SOR, superoxide reductase; FDP, flavodiiron protein. Source: figure adapted from (139). 4.3 Trichomonas vaginalis Trichomonas vaginalis is a parabasalian flagellate infecting urogenital tract of approximately 3% of the world population annually (194). On rare occasions, the parasite has also been isolated from the respiratory tract of infants (144) and adults (137). It is the causative agent of the most prevalent non-viral sexually transmitted disease (STD) in humans, trichomoniasis. Majority of T. vaginalis infections proceed without apparent symptoms. The symptomatic disease is manifested by urogenital tract swelling and inflammatory discharge. The infection is associated with the elevated risk of HIV transmission, prostate and cervical cancer, decreased fertility with the risk of preterm delivery and low birth weight infants (168). Treatment of trichomoniasis is based on 5nitroimidazole derivatives such as metrinidazole and tinidazole, however, resistant strains are on the rise (111,165). The life cycle of the parasite includes free-swimming ovoid trophozoites, rapidly transforming into adherent, amoeboid form upon the contact with host tissue. These dramatic morphological changes enable adherence to the surface of urogenital tract in order to maximize the contact with the squamous epithelium (113). Trichomonas, attached 28 to the extracellular matrix (ECM), phagocytes vaginal microbiota (Staphylococcus, Lactobacillus, Enterobacter, Escherichia and Pseudomonas) (99), immune cells (lymphocytes, monocytes) (112) and erythrocytes – the prime source of fatty acids and iron (71). T. vaginalis is regarded as an anaerobic organism; however, the trophozoites in their natural habitat are confronted with fluctuating levels of oxygen during transmission and menstruation. Despite the fact that the oxygen concentrations higher than ~60μM are toxic to the parasite (59), it was observed that very low concentrations of oxygen actually boost growth of trichomonad cultures, which allows to characterize T. vaginalis as a microaerophile (164). Instead of canonical mitochondria, Trichomonas possesses mitochondrion-related organelles – hydrogenosomes. These double membrane-bounded organelles harbor the reactions of so called “extended glycolysis” (153) and provide additional ATP by substrate-level phosphorylation. Pyruvate and malate, imported to the organelles, are main substrates of hydrogenosomal metabolism generating ATP via oxygen-sensitive enzymes, such as pyruvate:ferredoxin oxidoreductase and Fe-Fe hydrogenase. These enzymes harbor iron-sulfur clusters, highly prone to irreversible damage by oxygen (89). Therefore the parasite needs efficient mechanisms to buffer oxidative stress. NADH and NADPH oxidases play an important role in scavenging oxygen permeating into the cell. Both oxidases are localized in the cytosol; NADH oxidase reduces oxygen to water and NADPH oxidase (recently characterized as flavin reductase(118)) produces hydrogen peroxide (123) (Fig. 10). Glutathione is absent and cysteine probably holds the post of main cellular antioxidant in T. vaginalis (59). This suggestion is supported by up to nine-fold increase of two cysteine synthetases during oxygen stress (78). Although NADPH oxidase and FeSOD generate hydrogen peroxide, the catalase is absent in T. vaginalis. Trichomonad genome codes for 7 paralogues of superoxide dismutase (37), some of which are localized in the hydrogenosome (171). It is presumed that hydrogen peroxide formed in hydrogenosomes is degraded by two proteins, rubrerythrin and peroxiredoxin (thiol peroxidase), that were both identified in hydrogenosomal proteome (171). Both enzymes represent bacterial proteins, apparently acquired by lateral gene transfer. Subsequent screening of T. vaginalis genome identified several rubrerythrin genes (37). 29 Peroxiredoxin is a component of thioredoxin-linked antioxidant system localized in both cytosol (45) and hydrogenosome (171). In addition to peroxiredoxin this redox system consists of thioredoxin and thioredoxin reductase (45). All five TrxR genes present in T. vaginalis genome code for low-molecular mass TrxR isoforms (Eukaryotic TrxRs belong to two distinct groups: High-molecular-mass TrxR isoforms (~55 kDa) and low-molecular-mass isoforms (~35 kDa), with different structures and catalytic mechanisms). One of these proteins is a cytosolic TrxR identified by Coombs et al. (45) while two paralogous proteins were localized in hydrogenosome (146). Another protein protecting hydrogenosomes against oxidative stress is FDP. Genome of Trichomonas encodes 4 FDP homologues, one of which is localized in hydrogenosome (TvFDP) (cf. (198) and publication 1 in this thesis). The hydrogenosomal paralogue belongs to class A FDP and is similar to the homologues from other protist parasites Entamoeba (212) and Giardia (51). Unlike many prokaryotic homologues that are able to reduce both oxygen and NO with varying efficiencies, the TvFDP is strictly specific for oxygen which is reduced to water. The remaining 3 trichomonad genes code for FDPs with distinct primary structure, belonging to the class D. They are probably localized in cytoplasm and their function was not established. TvFDP has a dimeric structure, which enables simultaneous transport of four electrons required for full reduction of oxygen. T. vaginalis does not possess rubredoxin, the electron donor of FDP in many bacteria, and the physiological redox partner of TvFDP is hydrogenosomal ferredoxin which is reduced via PFOR or rudimentary complex I (89,91). PFOR and complex I remnant represent pivotal enzymes of hydrogenosomal carbohydrate metabolism. Complex I oxidizes coenzyme NADH, reduced in oxidative decarboxylation of malate (imported into hydrogenosome) to pyruvate via malic enzyme. Subsequently, pyruvate is oxidatively decarboxylated to acetyl-CoA (which is a substrate for ATP synthesis via acetate:succinate CoA-transferase (ASCT) and succinate thiokinase (STK), (153)) and CO2 in reaction catalyzed by PFOR. Electrons released in both reactions are transferred to ferredoxin, a low-molecular weight electrontransporting protein, which serves as a reductant in hydrogenosomal processes such as formation of molecular hydrogen by Fe-Fe hydrogenase or in iron-sulfur cluster assembly (89). Ferredoxin is also a low-redox potential reductant of metronidazole, the drug effective against anaerobic microbes (90). 30 FDPs and rubrerythrin are not the only proteins of bacterial origin involved in antioxidant defense in hydrogenosomes of T. vaginalis. Another one is an iron-sulfur flavoprotein. Seven Isf genes are present in the genome, with at least three of them being expressed under basal conditions (27). One of the homologues (TvIsf3) was characterized as a functional homodimer with flavin (FMN) and iron-sulfur center in its active site ((199) and ref. 2 in this thesis). While the Isf from Methanosarcina thermophila (MtIsf) reduces oxygen and hydrogen peroxide, the trichomonad protein was found to reduce wider spectrum of electron acceptors, including the nitro-antibiotics metronidazole and chloramphenicol (46). Both proteins have the capacity to efficiently reduce oxygen, however, in contrast to MtIsf, which catalyzes the four-electron reduction of oxygen to water, the TvIsf3 apparently reduces oxygen to hydrogen peroxide utilizing two electrons. TvIsf3 accepts electrons from the coenzyme NADH or reduced ferredoxin. The ability of TvIsf3 to reduce metronidazole is of particular relevance, as this antibiotic (and related derivatives of 5-nitroimidazoles) is the only approved drug available for the treatment of trichomoniasis. Thus, the occurrence of metronidazole-resistant strains is of major concern. The therapeutic effect of nitroimidazoles is ascribed to one-electron reduction of nitro group of the antibiotic, giving rise to reactive nitroimidazole radical anion. Formation of the radical in metronidazole reduction by TvIsf3 was not detected, indicating the reduction of the drug by even number of electrons and formation of less deleterious product. However, the physiological role of TvIsf3 in the resistance of T.vaginalis to metronidazole is currently unknown. Recently, the OsmC protein (a member of OsmC/Ohr protein family) with peroxidase activity was reported in hydrogenosome of T. vaginalis ((159) and ref. 3 in this thesis). The TvOsmC protein is functionally linked with two proteins homologous with components of glycine decarboxylase complex (GDC) (152): NADH-dependent dihydrolipoamide dehydrogenase (L protein homologue) and the lipoate–bearing, hydrogen carrier protein (H protein homologue). The two other proteins that normally constitute the GDC complex (P and T proteins) are missing in T. vaginalis, making the function of L and H protein in glycine metabolism unlikely. We have shown that the L protein is able to reduce oxidized lipoamide moiety of the H protein at the expense of NADH and that reduced lipoylated H protein serves as a reductant of TvOsmC, thus constituting a novel redox chain participating in the defense of hydrogenosomes against both hydrogen peroxide and organic hydroperoxides ((159) and ref. 3 in this thesis). Unlike 31 its bacterial homologues (47,120,121), trichomonad OsmC displays higher activity with H2O2 than with organic hydroperoxides. Figure 10. Schematic representation of the antioxidant system in T. vaginalis. NADH ox., NADH oxidase; NADPH ox., NADPH oxidase; SOD, superoxide dismutase; TvIsf3, iron-sulfur flavoprotein; Prx, peroxiredoxin; Rbr, rubrerythein; OsmC, osmotically inducible protein; FDP, flavodiiron protein. Source: author’s original. 32 5. Aims The aim of this thesis was to contribute to knowledge of biochemistry of trichomonad hydrogenosomes, specifically: 1. To characterize the proteins of bacterial origin, presumably involved in ROS detoxification in the hydrogenosome, discovered during the annotation of Trichomonas vaginalis genome. 2. To elucidate their physiological functions and electrontransport pathways in which these proteins participate. 33 6. List of publications and author contribution · Smutná T., Gonçalves V.L., Saraiva L.M., Tachezy J., Teixeira M., Hrdý I. (2009). Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase. Eukaryot Cell; 8:47-55. Ø First author has major contribution to the results reported in this work, such as cloning of FDP constructs, determinations of enzymatic activities and localization of protein within the trichomonas cell; The spectroscopic analysis of TvFDP, HPLC analysis of protein cofactor and refinement of purification protocol was done in collaboration with coauthors from the Instituto de Tecnologia Química e Biológica in Oeiras, Portugal. · Smutná T., Pilařová K., Tarábek J., Tachezy J., Hrdý I. (2014) Novel functions of an iron-sulfur flavoprotein from Trichomonas vaginalis hydrogenosomes. Antimicrob Agents Chemother; 58:3224-32. Ø First author contributed by cloning of majority of constructs, expression and purification of TvIsf3, T. vaginalis cells fractionation, spectrophotometric determination of enzymatic activities, protein localization study and by the experiments focused on enzymatic reduction of recombinant protein. · Nývltová E., Smutná T., Tachezy J., Hrdý I. (2016) OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis. Mol Biochem Parasitol; in press. Ø Author’s contribution includes the cloning of TvOsmC recombinant protein and the design of expression protocol, cloning of TvOsmC-HA construct and transfection of trichomand cells, cloning and expression of H proteins, activity assays of recombinant TvOsmC protein with DTT, NADH and NADPH as electron donors. Ø In all publications the author participated on writing of manuscripts. 34 6.1 Publication 1: Smutná et al., 2009 Smutná T., Gonçalves V.L., Saraiva L.M., Tachezy J., Teixeira M., Hrdý I. (2009). Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase. Eukaryot Cell; 8:47-55. 35 EUKARYOTIC CELL, Jan. 2009, p. 47–55 1535-9778/09/$08.00⫹0 doi:10.1128/EC.00276-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved. Vol. 8, No. 1 Flavodiiron Protein from Trichomonas vaginalis Hydrogenosomes: the Terminal Oxygen Reductase䌤 Tamara Smutná,1 Vera L. Gonçalves,2 Lígia M. Saraiva,2 Jan Tachezy,1 Miguel Teixeira,2 and Ivan Hrdý1* Department of Parasitology, Charles University, Viničná 7, Prague 128 44, Czech Republic,1 and Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Av. da República, EAN, 2780-157 Oeiras, Portugal2 Received 18 August 2008/Accepted 4 November 2008 10, 22). Besides rubredoxin, roles for other iron-sulfur flavoproteins in electron transport to FDPs have been suggested in several Archaea (41); coenzyme F420H2 is the electron donor for the FDP in the methanogenic archaeon Methanothermobacter marburgensis (44). The members of other FDP classes have additional domains fused to the C terminus that participate in electron transfer from the ultimate donor molecule [NAD(P)H] to the terminal electron acceptor (41). While originally believed to be restricted solely to prokaryotes, recent progress in genome sequencing projects have revealed homologous protein sequences in the genomes of several “amitochondriate” anaerobic protists, mostly with parasitic lifestyles, such as Trichomonas, Giardia, Entamoeba, Spironucleus, and a free-living Mastigamoeba (1, 2, 33, 42). Giardia intestinalis is the only eukaryotic organism to have had data on its FDP published recently. In line with what is known for the prokaryotic homologues, the giardial protein was shown to possess high oxygen (but not NO)-reducing activity and was therefore proposed to participate in protection against oxidative stress (13). Trichomonas vaginalis is an anaerobic (or microaerophilic) protozoan parasite causing human trichomoniasis, the most common nonviral sexually transmitted infection (38), for which oxygen concentrations higher than those encountered in situ in the vagina (i.e., concentrations above ⬃60 M) are toxic (17). The glucose metabolism of T. vaginalis is compartmentalized; while the reactions of classical glycolysis producing lactate, as well as the branch resulting in the formation of glycerol (8, 48) occur in the cytosol, a substantial portion of glycolytic carbon is diverted into the hydrogenosome, a mitochondrion-related organelle where the reactions of extended glycolysis produce additional ATP by oxidative decarboxylation of pyruvate (47, 48). Typical in the trichomonad hydrogenosome is the presence of the iron-sulfur (FeS) cluster-containing enzymes pyruvate:ferredoxin oxidoreductase (PFOR), hydrogenase, and the Flavodiiron proteins (FDPs) constitute a recently established superfamily of soluble enzymes, thus far exclusively found in anaerobic and facultative aerobic organisms (2, 19, 54). Originally, the function ascribed to these proteins was the reduction of molecular oxygen to water as reported for Desulfovibrio gigas rubredoxin:oxygen oxidoreductase, the first thoroughly characterized protein of this type. This protein was found to utilize electrons derived from glycolysis for safe, fourelectron reduction of dioxygen, thus protecting the anaerobic bacterium from the deleterious effects of oxidative stress (19). Later, some of these proteins were also shown to be involved in the reduction of nitric oxide in addition to their oxygenreducing activity, thereby probably protecting the microbial organism against NO released during the immune response of the higher eukaryote host. The ratio of FDP activity toward oxygen and NO may differ substantially in various organisms; in some cases, FDP is almost exclusively reactive with oxygen, in others it is reactive with NO (20, 21, 43). FDPs are modular proteins, with flavodoxin-like and metallo--lactamase-like domains as their core modules. This twodomain structure is found in the simplest and most common members of the family, named class A FDPs. These proteins are the terminal elements of a multicomponent electron transporting chain that uses the reducing power of NAD(P)H to reduce and detoxify dioxygen and/or nitric oxide (41). Proximal electron donors to most class A FDPs are soluble electron transfer proteins. In the class A FDP rubredoxin:oxygen oxidoreductase from the sulfate-reducing bacterium Desulfovibrio gigas, the electron donor is a small protein, rubredoxin, that itself is reduced by an NADH:rubredoxin oxidoreductase (9, * Corresponding author. Mailing address: Department of Parasitology, Charles University in Prague, Viničná 7, Prague 128 44, Czech Republic. Phone: 420 221951811. Fax: 420 224919704. E-mail: [email protected]. 䌤 Published ahead of print on 14 November 2008. 47 Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 Trichomonas vaginalis is one of a few eukaryotes that have been found to encode several homologues of flavodiiron proteins (FDPs). Widespread among anaerobic prokaryotes, these proteins are believed to function as oxygen and/or nitric oxide reductases to provide protection against oxidative/nitrosative stresses and host immune responses. One of the T. vaginalis FDP homologues is equipped with a hydrogenosomal targeting sequence and is expressed in the hydrogenosomes, oxygen-sensitive organelles that participate in carbohydrate metabolism and assemble iron-sulfur clusters. The bacterial homologues characterized thus far have been dimers or tetramers; the trichomonad protein is a dimer of identical 45-kDa subunits, each noncovalently binding one flavin mononucleotide. The protein reduces dioxygen to water but is unable to utilize nitric oxide as a substrate, similarly to its closest homologue from another human parasite Giardia intestinalis and related archaebacterial proteins. T. vaginalis FDP is able to accept electrons derived from pyruvate or NADH via ferredoxin and is proposed to play a role in the protection of hydrogenosomes against oxygen. 48 SMUTNÁ ET AL. MATERIALS AND METHODS Organism. The T. vaginalis strain T1 (J.-H. Tai, Institute of Biomedical Sciences, Taipei, Taiwan) was grown in Diamond’s TYM medium without agar as previously described (35). TvFDP constructs. Several versions of recombinant TvFDP were prepared. (i) His-FDP. His-FDP is a recombinant TvFDP without the hydrogenosomal targeting sequence fused with a His6 tag at the C terminus. The PCR-amplified gene was cloned into the pQE 30 Escherichia coli expression system (Qiagen). The purified protein was used for TvFDP characterization, polyclonal antiserum preparation, spectroscopic analysis, and functional studies. (ii) TvFDPHa. TvFDP with the hydrogenosomal targeting sequence was fused with a C-terminal 2x hemagglutinin (Ha) tag. The complete TvFDP gene was cloned into the Master Neo (25) vector (kindly provided by Patricia J. Johnson, University of California, Los Angeles, CA) and used to transform trichomonad cells. This construct was used to immunolocalize the protein inside the cell. (iii) TvFDPStrep. TvFDP with the hydrogenosomal targeting sequence and fused with a streptavidin tag at the C terminus was expressed in the TagVag (12) vector (T. vaginalis expression system). TvFDPStrep protein was isolated from transformed trichomonads and used to identify the flavin cofactor. (iv) TvFDPWT. TvFDP protein with the hydrogenosomal targeting sequence was overexpressed in trichomonads (Master Neo vector) without any additional tags. Hydrogenosomes with TvFDPWT were used to determine the native molecular mass of TvFDP. Isolation of hydrogenosomes and partial purification of TvFDP. Hydrogenosomes were isolated from the T. vaginalis homogenate by differential centrifugation of sonicated cells, followed by isopycnic centrifugation of the hydrogenosome-enriched fraction on a self-forming gradient of 45% Percoll (Sigma) as previously described (49). TvFDP was identified in the T. vaginalis hydrogenosomes during the search for an NADH:ferredoxin oxidoreductase (which was eventually found to be an extremely reduced homologue of mitochondrial respiratory complex I) (25). Briefly, hydrogenosomes from 2 to 4 liters of culture were resuspended in a morpholinepropanesulfonic acid-phosphate buffer (40 mM morpholinepropanesulfonic acid, 400 mM KH2PO4, 2 mM EDTA, 10% glycerol [pH 7.0]) with protease inhibitors (2 mg of leupeptin and N-␣-tosyl-L-lysine chloromethyl ketone/ml) and then extracted by sonication (using a Vibra cell sonicator) three times for 1 min each time at an amplitude of 60, with 1-s pulses. The sonicate was centrifuged at 200,000 ⫻ g for 45 min to obtain a soluble hydrogenosomal matrix. This fraction was diluted 2.5 times with 20 mM Tris-HCl (pH 7.5) and loaded onto an SP-Sepharose FastFlow (GE Healthcare) column. Bound protein was eluted with 0 to 1 M gradient of NaCl in the same buffer. The fractions containing NADH:ferredoxin oxidoreductase activity (monitored as described in reference 25) were collected, diluted three times with 20 mM Tris-HCl (pH 9.0), and loaded onto a Reactive Blue 2 Sepharose (Sigma) column equilibrated with the same buffer. The protein was eluted with an NaCl gradient (0 to 1 M), and the active fractions were pooled and concentrated by ultrafiltration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that the sample was particularly enriched in two barely separable polypeptides with molecular masses of ⬃45 kDa. After transblotting onto a polyvinylidene difluoride membrane and Coomassie blue staining, the enriched bands were cut out and subjected to Edman degradation (performed at the Protein/DNA Technology Center, Rockefeller University, New York, NY) to determine the aminoterminal sequences of the polypeptides. The heavier polypeptide was a homologue of the 51-kDa subunit (also called NuoF in bacteria) of the electron-input module of respiratory complex I (25), while the lighter polypeptide was found to be a homologue of bacterial FDPs and named TvFDP. Edman degradation also allowed determination of the processing site where the hydrogenosomal targeting peptide is cleaved from the preprotein by a hydrogenosomal processing peptidase. Expression and purification of TvFDP. To express the His-tagged protein (His-FDP) in E. coli M15 cells, the bacteria were induced with 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside) and grown for 7 h at 28°C in LB medium supplemented with 400 M ammonium ferrous sulfate and 200 M flavin mononucleotide (FMN). The harvested cells were homogenized by passage through a French press at 18,000 lb/in2. The soluble fraction obtained by ultracentrifugation (250,000 ⫻ g, 1 h, 4°C) was applied to a Ni-NTA column (Qiagen) and eluted with a stepwise gradient of 20 mM imidazole (buffer A: 20 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol [pH 7.6]) and 400 mM imidazole (buffer B: 400 mM imidazole, 50 mM Tris-HCl, 300 mM NaCl, 10% glycerol [pH 7.6]) at a flow rate of 1 ml/min using a BioLogic HR system (Bio-Rad). HisFDP-containing fractions were dialyzed overnight against 50 mM Tris-HCl with 10% glycerol (pH 7.6), concentrated (Amicon UltraConcentrator, 30 kDa; Millipore), and analyzed by SDS-PAGE. Characterization of TvFDP. The flavin cofactor was characterized by reversedphase high-pressure liquid chromatography using a nucleosil 100-5 C18 column and a thin-layer chromatography (TLC) method (HPTLC-Alufolien; Merck). The TLC mobile phase consisted of n-butanol–acetic acid–water (6:2:4). The flavin was extracted from the protein with trichloroacetic acid at a final concentration of 10%, followed by centrifugation and supernatant neutralization with 1 M ammonium acetate (pH 7.0). The identity of the flavin was determined for recombinant His-FDP, as well as for TvFDPStrep purified from T. vaginalis hydrogenosomes according to the IBA Strep-tag protein purification protocol. FMN and FAD (Sigma) were used as standards. The amount of flavin in TvFDP was assessed after protein denaturation with 80% trichloroacetic acid, using an extinction coefficient of 12,500 M⫺1 cm⫺1 at ⫽ 450 nm (21). The iron content of TvFDP (His-FDP was used for analysis) was determined by using the 2,4,6-tripyridyl-1,3,5-triazine method (18). The native molecular mass of TvFDP was determined by gel filtration chromatography using a BioLogic HR system (Bio-Rad). The purified recombinant His-FDP, as well as the hydrogenosomal extract (prepared as described above) from FDP-overexpressing trichomonads (TvFDPWT), were run on a Superdex 75 (XK 16 column; GE Healthcare) column equilibrated with 400 mM imidazole–50 mM sodium phosphate buffer (pH 8.0) using a flow rate of 1 ml/min. The native molecular mass of the recombinant enzyme was calculated from the calibration curve determined by running the standards under the same conditions. TvFDPWT contained in the hydrogenosomal extract was determined in the elution profile by Western blotting, followed by immunodetection of TvFDP using a specific rabbit polyclonal antiserum raised against His-FDP purified on an Ni-NTA column. The rabbit antiserum was prepared at the Institute of Parasitology, Academy of Sciences of the Czech Republic, České Budějovice, according to a published protocol (53). Protein concentration was determined by the Lowry assay (34) using bovine serum albumin as a standard. Spectroscopic analysis of TvFDP. The redox titrations were performed anaerobically at 25°C under an argon atmosphere by the stepwise addition of buffered sodium dithionite (250 mM Tris-HCl [pH 9.0]), in the presence of suitable redox mediators, in 50 mM Tris-HCl–18% glycerol (pH 7.5) as previously described (51). The samples were analyzed by visible spectroscopy (Shimadzu UV-1603 spectrophotometer) or by electron paramagnetic resonance (EPR) spectroscopy Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 electron carrier ferredoxin, which are involved in the generation of molecular hydrogen using electrons released from pyruvate (36). PFOR and hydrogenase are highly oxygen-sensitive enzymes (29, 32), and it is likely that the sensitivity of trichomonads to oxygen could at least in part be due to the inactivation of these key hydrogenosomal proteins. T. vaginalis must cope with low oxygen concentrations in its natural environment and, accordingly, possesses defense mechanisms to combat oxidative damage caused by oxygen itself or by reactive oxygen species that arise either enzymatically or when the reduced prosthetic groups of enzymes such as flavins and FeS clusters come into contact with oxygen. Most eukaryotes utilize glutathione as a key redox buffer and antioxidant, but trichomonads lack this and similar thiols (17). Cysteine has been suggested as a major reducing buffer and antioxidant (17), and it is believed that the organism relies upon cytosolic NADH oxidase (reducing oxygen to water) and NADPH oxidase (reducing oxygen to hydrogen peroxide) to prevent the permeation of oxygen into the hydrogenosomes (31). Proteins of the peroxiredoxin cascade (11) are also important for cytosolic peroxide detoxification. The identified defense mechanisms of hydrogenosomes include superoxide dismutase activity (17, 30) and recently found putative peroxidases that might provide protection against peroxides (39), but the protein that was suggested long ago to be responsible for oxygen uptake and detoxification has never been identified (6). We describe here the properties of a class A FDP from T. vaginalis hydrogenosomes and suggest its role in the metabolism of oxygen and protection of the organelle. EUKARYOT. CELL VOL. 8, 2009 FLAVODIIRON PROTEIN FROM T. VAGINALIS HYDROGENOSOMES FIG. 1. Spectroscopic analysis of recombinant T. vaginalis HisFDP. UV/VIS spectrum of pure His-FDP exhibits the typical features of flavoproteins. The inset shows the results of an SDS-PAGE analysis of purified recombinant His-FDP. Hom., bacterial lysate; FDP, purified His-FDP. the strongest activity was used to reduce TvFDP in combination with T. vaginalis ferredoxin 1 that was expressed in E. coli BL21 cells and purified as described earlier (49). RESULTS Purification and properties of TvFDP. Recombinant TvFDP with a His6 tag and without the hydrogenosomal targeting sequence (His-FDP) produced in M15 E. coli cells was purified by affinity chromatography on Ni-NTA agarose under native conditions. This procedure produced an almost homogeneous protein, as determined by SDS-PAGE (Fig. 1), with an approximate yield of 10 mg of His-FDP per liter of bacterial culture. The His-FDP polypeptide migrated on SDS-PAGE as a band with a molecular mass of ⬃45 kDa, a finding in agreement with the molecular mass calculated from the amino acid sequence without the hydrogenosomal targeting peptide. To determine the native molecular mass of TvFDP, the hydrogenosomal extract from transformed T. vaginalis cells overexpressing native, nontagged TvFDPWT, as well as the recombinant His-FDP isolated from E. coli, were analyzed by gel filtration chromatography. The native molecular mass of His-FDP was ⬃92 kDa. The TvFDPWT in the elution profile was determined by SDS-PAGE and Western blot analysis with a specific antibody. TvFDPWT from the hydrogenosomal extract was eluted in the same elution volume as the purified recombinant protein (results not shown), suggesting that both the recombinant and the plasmid-encoded TvFDPWT expressed in trichomonads exist as dimers in vivo. The cofactor contained in the flavodoxin domain was identified by TLC and high-pressure liquid chromatography as noncovalently bound FMN (data not shown). This cofactor was established for both the recombinant protein and the protein containing the Strep tag (TvFDPStrep) isolated by affinity chromatography from T. vaginalis hydrogenosomes. Freshly isolated recombinant protein contained ⬃0.5 FMN and ⬃1.5 iron atoms per monomer. This stoichiometry indicates that a fully occupied protein contains one molecule of FMN and two Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 using an EMX Bruker spectrometer equipped with an Oxford Instruments ESR900 continuous flow cryostat. EPR spectra were recorded at 10 K, 9.39 GHz, 2.0 mW, and 1 mT of modulation amplitude. The electrodes (a silver/silver chloride combined electrode, or silver chloride and platinum electrodes) were calibrated against a saturated quinhydrone solution at pH 7.0, and the recorded potentials were normalized against that of the standard hydrogen electrode. Protein localization. T. vaginalis cells overexpressing TvFDPHa were used for immunodetection of TvFDP. The cells were placed on glass slides coated with 3-aminopropyltriethoxysilane (Sigma), fixed with methanol (5 min), permeabilized with acetone (5 min) (both steps at ⫺18°C), preincubated for 1 h in phosphate-buffered saline–0.25% bovine serum albumin–0.25% gelatin, and treated with antibodies as described previously (49). Anti-Ha tag monoclonal antibody (kindly provided by Patricia Johnson, University of California at Los Angeles) and hydrogenosomal malic enzyme polyclonal antiserum (15) were used as the primary antibodies. Anti-mouse immunoglobulin G (IgG) labeled with Alexa Fluor 488 (catalog no. A21202; Molecular Probes) and anti-rabbit IgG labeled with Alexa Fluor 549 (catalog no. A21207; Molecular Probes) were used as the secondary antibodies for fluorescent immunolocalization. Anti-TvFDP rabbit polyclonal serum (see above) and anti-rabbit IgG antibody conjugated with alkaline phosphatase (ICN/Cappel) were used for Western blot analysis to visualize TvFDP in T. vaginalis subcellular fractions obtained from untransformed, wild-type trichomonads and TvFDPWT-overexpressing trichomonads. Enzymatic reduction of TvFDP. TvFDP spectra were recorded on a Shimadzu UV-1601 spectrophotometer. The enzymatic reduction of TvFDP (His-FDP construct, 30 to 150 nmol) was monitored in stoppered cuvettes with a silicone septum in 860 l of phosphate buffer (100 mM KH2PO4/KOH, 150 mM NaCl, 10% glycerol [pH 7.4]) using 44 mM pyruvate, 0.25 mM coenzyme A (CoA), ⬃40 g of T. vaginalis ferredoxin 1 (52), and ⬃10 g of T. vaginalis PFOR as elements of the reduction pathway. Alternatively, another enzymatic system consisting of ⬃10 g of T. vaginalis complex I homologue purified from hydrogenosomes (25), ⬃40 g of T. vaginalis ferredoxin 1 (52), and 220 M NADH in the same buffer as described above was used, but the spectra obtained with this system were less informative due to partially overlapping absorptions of NADH and TvFDP. Anaerobiosis of the system was achieved by degassing the buffer followed by flushing with N2 and the addition of glucose oxidase (28 U), catalase (104 U), and glucose (3 mM) into the reaction mixture (50). This enzymatic oxygen- and hydrogen peroxide-scavenging system was omitted from experiments aimed to identify the product of oxygen reduction by TvFDP. H2O2 was determined by the FOX method using xylenol orange as an indicator in a spectrophotometric assay at 560 nm. This assay is able to reliably detect 100 pmol of hydrogen peroxide in a 50-l sample (55). Approximately 150 nmol of TvFDP was reduced by the enzymatic system (see above) and briefly exposed to air to oxidize TvFDP; 50 l of the reaction mixture was mixed with 950 l of FOX1 reagent (100 M xylenol orange, 250 M ammonium ferrous sulfate, 100 mM sorbitol, 25 mM H2SO4) (55). Hydrogen peroxide (0 to 250 pmol) was used to construct the calibration curve and as a positive control. DPTA-NONOate (Alexis Biochemicals, Switzerland) was used as a nitric oxide donor to prepare solutions of 12 M to 1 mM of nitric oxide in the reaction mixture (37). The presence of chemically generated NO in the reaction mixture was verified by using a membrane-type NO-sensitive electrode (ISO-NOP, World Precision Instruments) connected to an ISO-NO Mark II meter (World Precision Instruments). Possible inactivation of the enzymatic reduction system by NO was excluded by checking for persistent TvFDP-reducing activity after anaerobic exposure to NO followed by reoxidation of TvFDP with air and resealing of the cuvette. Purification of PFOR. In order to obtain a homologous enzymatic system capable of reducing TvFDP, T. vaginalis PFOR was purified as follows: hydrogenosomes from 4 liters of culture were treated with 2% octylglucoside (MP Biomedicals) in 10 mM KH2PO4 (pH 6.8) on ice for 60 min and then centrifuged at 120,000 ⫻ g for 30 min. The resulting membrane pellet containing PFOR was extracted with 5 ml of degassed and nitrogen-equilibrated 1% deoxycholate (Sigma) in 10 mM KH2PO4–2 mM dithiothreitol (pH 7.4) for 60 min, followed by centrifugation at 120,000 ⫻ g for 30 min. The supernatant fraction was mixed with 1 ml of Ni-NTA agarose (Qiagen), followed by incubation for 60 min at 4°C. The mixture was loaded onto a disposable column and the flowthrough fraction containing PFOR plus minor contaminants was collected. This step was found to selectively remove the malic enzyme, the dominant hydrogenosomal protein with a native molecular mass close to that of PFOR (240,000 Da), due to specific binding of the malic enzyme to Ni-NTA agarose. The PFOR-enriched eluate was concentrated and loaded at a rate of 0.5 ml/min onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with degassed 10 mM KH2PO4–2 mM dithiothreitol–1% deoxycholate (pH 7.4). Fractions (750 l) were collected, analyzed by SDS-PAGE, and checked for PFOR activity (24). The fraction with 49 50 SMUTNÁ ET AL. EUKARYOT. CELL FIG. 2. Amino acid sequence of TvFDP (XP001583562, TVAG_036010). The residues binding the diiron ligands are marked with an asterisk (ⴱ), and the conserved flavodoxin signature motif is shaded. The position of the hydrogenosomal processing peptidase cleavage site is marked with an arrow. FMNSQ/FMNred forms (Fig. 5). However, during the course of the enzymatic reduction by PFOR, a small band appeared at 398 nm during the early stages of the reduction (Fig. 6). Such a peak has not been observed in any of the other FDPs studied thus far, and its origin remains to be clarified. Redox titration, followed by EPR spectroscopy, allowed us to both observe the signature of the diiron center and to determine its reduction potentials (Fig. 7 and Fig. 8). The diiron center had resonances at g ⫽ 1.955, 1.805, and 1.57, which is characteristic of the mixed valence [Fe(III)-Fe(II)] state of a diiron site. By measuring the change in amplitude of the resonance at g ⫽ 1.8, the complete redox titration was constructed (Fig. 8) and analyzed with a Nernstian system of two consecutive one-electron transitions, yielding reduction potentials for the Fe(III)-Fe(III)/Fe(III)-Fe(II) and Fe(III)Fe(II)/Fe(II)-Fe(II) transitions of 50 ⫾ 20 and 190 ⫾ 20 mV. Function. In order to establish the physiological function of TvFDP, we studied the recombinant, His-tagged protein (HisFDP), which could be obtained in large quantities and displayed properties (i.e., FMN and iron cofactor, UV/VIS spectrum, and dimeric structure) similar to those of known FDPs. Studies of flavoproteins often utilize sodium dithionite as a FIG. 3. Subcellular localization of TvFDP. (A) SDS-PAGE of subcellular fractions of transformed T. vaginalis line overexpressing TvFDP (TvFDPWT); (B) SDS-PAGE of hydrogenosomes of untransformed T. vaginalis (TvT1); (C and D) Western blots probed with anti-TvFDP polyclonal antiserum. Molecular mass standards are indicated in kilodaltons. hom., homogenate; cyt., cytosol; hydr., hydrogenosomes. Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 iron atoms per monomer; the incomplete occupancy is probably due to cofactor loss during purification. Pure, concentrated His-FDP was dark yellow and displayed a typical FDP UV/VIS spectrum (Fig. 1) dominated by the features of the flavin moiety, as the diiron center has a very low absorption. Sequence analysis. TvFDP displayed homology with other described FDPs from both prokaryotic and eukaryotic sources. The residues implicated in binding the binuclear iron center (His94, His161, His240, Glu96, Asp98, and Asp180), as well as the conservative flavodoxin-like signature motif starting at position 271 (Val271) (Fig. 2) (41, 54), were all conserved in the T. vaginalis protein. Soluble hydrogenosomal proteins are synthesized in the cytoplasm and typically contain a short amino acid presequence with a targeting function on their amino termini (3, 14). These targeting signals are cleaved by a specific hydrogenosomal processing peptidase (4) upon translocation of the protein into the organelle. TvFDP also possesses such a signal. The processing peptidase cleavage site was determined by amino acid sequencing via Edman degradation of mature TvFDP partially purified from hydrogenosomes; the cleavage site was found to be located between serine 11 and alanine 12 (Fig. 2). The conserved arginine residue typically located in the ⫺2 position relative to the cleavage site in hydrogenosomal presequences was in an unusual ⫺4 position in this case. Subcellular localization. His-FDP was used to raise a specific antiserum that was subsequently utilized to determine the localization of native, nuclear-encoded TvFDP, as well as overexpressed TvFDPWT within trichomonad cells. Western blot analysis of T. vaginalis subcellular fractions showed that the TvFDP was expressed and specifically localized to the hydrogenosomal fraction (Fig. 3). To further verify the localization of TvFDP protein by immunofluorescence microscopy, T. vaginalis cells overexpressing TvFDPHa with an Ha tag at the C terminus were used. The protein was colocalized with the malic enzyme (hydrogenosomal marker enzyme) in the hydrogenosomal compartment (Fig. 4). Spectroscopic studies. The visible spectrum of TvFDP was characteristic of a flavoprotein, with the main absorption bands at about 455 and 350 nm. Upon chemical reduction with sodium dithionite, there was no clear indication of the formation of a semiquinone radical, either of the blue or the red type, suggesting that the semiquinone form is not stabilized. Indeed, redox titration monitored by visible spectroscopy yielded a curve that could be fitted with two identical reduction potentials of approximately ⫹25 mV for the FMNox/FMNSQ and the VOL. 8, 2009 FLAVODIIRON PROTEIN FROM T. VAGINALIS HYDROGENOSOMES reductant of the flavin moiety. This approach, however, is of little use when studying the physiological function of a protein whose presumed electron acceptor is oxygen. To reduce HisFDP enzymatically, we used a system consisting of pyruvate, CoA, PFOR purified from T. vaginalis hydrogenosomes, and purified recombinant T. vaginalis ferredoxin 1 (52). The primary electron source was pyruvate in a CoA-dependent PFOR reaction that oxidatively decarboxylates pyruvate, transfers electrons to ferredoxin, and releases acetyl-CoA. The reduced ferredoxin then served as a reductant for His-FDP in an anaerobic spectrophotometric assay. Catalytic amounts of PFOR and ferredoxin 1 fully reduced His-FDP in minutes. After complete reduction of His-FDP, the absorbance peak at 455 nm was totally bleached (Fig. 6). Upon introduction of air into the reaction mixture by opening the cuvette, His-FDP was FIG. 5. Redox titration of the flavin cofactor monitored by visible spectroscopy at 460 nm. The full line was calculated for two consecutive one-electron redox processes, using molar absorptivities for the oxidized and semiquinone forms of 14,400 and 4,500, respectively, and with E1 ⫽ E2 ⫽ 25 mV. FIG. 6. Enzymatic reduction of TvFDP. Spectra of gradual reduction of His-FDP were obtained in an anaerobic system consisting of 44 mM pyruvate, 0.25 mM CoA, T.vaginalis PFOR (10 g), and recombinant T. vaginalis ferredoxin (40 g) in a phosphate buffer (100 mM KH2PO4/KOH, 150 mM NaCl, 10% glycerol [pH 7.4]). The dashed spectrum indicates reoxidized His-FDP after introduction of air into the cuvette. The recording time for one spectrum was 38 s. immediately reoxidized (as documented by the regression of its spectral features to those of the native protein [Fig. 6]), indicating that an electron acceptor of TvFDP is indeed oxygen that is reduced with high affinity. When the cuvette was resealed again, the reduction of His-FDP resumed, indicating that the enzymatic system was not damaged by the short exposure to oxygen. To determine the product of the oxygen reduction, approximately 150 nmol of His-FDP was first fully enzymatically reduced as described above and then reoxidized with air. The mixture was subsequently analyzed by using the FOX method (55) for the presence of hydrogen peroxide. No hydrogen peroxide was detected, indicating that the product of oxygen reduction by TvFDP is water. Since the ability of certain bacterial FDPs to reduce nitric FIG. 7. EPR spectrum of as-purified TvFDP at 10 K. Microwave frequency, 9.39 GHz; microwave power, 2.0 mW; modulation amplitude, 1 mT. Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 FIG. 4. Immunodetection of TvFDP in T. vaginalis cells. (A) Nomarski differential contrast; (B) visualization of malic enzyme, hydrogenosomal marker; (C) TvFDP labeling; (D) merge of color channels showing the presence of TvFDP in the hydrogenosomes with DAPI (4⬘,6⬘-diamidino-2-phenylindole) staining for nuclei. 51 52 SMUTNÁ ET AL. oxide has been well documented (21, 40, 45, 46), we tested the NO-reducing activity of His-FDP; NO was chemically generated by using DPTA-NONOate. The presence of NO in the reaction mixture was verified by a NO-specific electrode connected to an ISO-NO Mark II meter. Approximately 30 nmol of His-FDP was first enzymatically reduced and then combined with DPTA-NONOate to obtain 12 M to 1 mM NO in an anaerobic assay mixture. No change in the spectrum of the reduced protein was observed with any concentration of NO tested over a period of 15 min (data not shown), indicating that TvFDP has negligible, if any, reactivity toward nitric oxide. DISCUSSION T. vaginalis hydrogenosomes harbor oxygen-sensitive proteins and are accordingly equipped with defensive enzymes to neutralize oxidative stress. These protective proteins include superoxide dismutase (17) and putative peroxidases such as thiol-dependent peroxidase and rubrerythrin (39). However, the enzyme that is logically first in the line of defense, oxygen reductase, has remained elusive since its presence was first proposed in the 1970s (6). In the present study, we describe the properties of an FDP from T. vaginalis (TvFDP) that functions as a true oxygen reductase. This protein, first identified by proteomic methods (see Materials and Methods) during our search for other hydrogenosomal activities, localized to hydrogenosomes as demonstrated by immunoblotting with specific antibodies. The subcellular localization of TvFDP was further confirmed by immunofluorescence microscopy of transformed trichomonads expressing a tagged version of the enzyme. The organellar localization is consistent with the presence of a cleavable hydrogenosomal targeting peptide with a characteristic amino acid composition on the amino terminus of the conceptually translated protein (Fig. 2). Analysis of cofactor content suggests that TvFDP binds two iron atoms and one FMN per monomer as in other homologues. Like most of its counterparts that have already been studied in this respect, native TvFDP is an ⬃92-kDa ho- modimer of identical 45-kDa subunits. This dimeric quaternary structure is essential for efficient electron transfer from the flavin of one monomer to the diiron center of the other monomer; these regions come into close contact due to the headto-tail organization of each monomer, a feature common to all FDPs whose structures have been determined thus far. Within a single monomer, the redox centers are too far apart to allow electron transfer at a physiologically relevant rate (50). The reduction potentials determined here for the FMN and the diiron center are higher than those observed for the E. coli enzyme, the only FDP extensively characterized in this respect thus far (50, 51). Another remarkable characteristic was observed in the reduction potentials of the flavin transitions, which were almost identical; this has not been reported for any other FDP thus far. This precludes the formation of a stable flavin semiquinone form as observed in other FDPs and favors an almost two-electron oxidation of the flavin. Nevertheless, the measured reduction potentials are suitable for either NO or O2 reduction, and further studies in other FDPs are needed to determine whether these differences are enzymatically relevant. Due to the high reduction potentials of either substrate, the specificity toward these small molecules cannot be related to their redox parameters. The UV-visible and EPR spectroscopic data are similar to those of other FDPs characterized thus far. The data for the diiron center showed that the iron ions were antiferromagnetically coupled in the half-reduced state, yielding a total spin of S ⫽ 1/2. For the oxidized and reduced forms, it was not possible to obtain any EPR spectral signature; in the oxidized state the total spin state is most probably S ⫽ 0, while in the reduced form it could be either zero or S ⫽ 4 as observed for the E. coli enzyme. In order to determine the activity and substrate specificity of TvFDP, we needed to reconstruct in vitro the electron transfer chain from an electron donor to the terminal acceptor, oxygen and/or nitric oxide. This was a nontrivial task, since T. vaginalis does not possess rubredoxin, the physiological reductant of some FDPs, and analysis of the T. vaginalis genome did not provide any clue about potential redox partners for TvFDP. Nevertheless, T. vaginalis encodes a large number of [2Fe-2S] ferredoxins, small electron carrier proteins linked to PFOR, hydrogenase, and a remnant of complex I (28). In addition, the genome also encodes several predicted hydrogenosomal ironsulfur flavoproteins with unknown function but presumably involved in electron transfer (5). We have found that the enzymatic system consisting of PFOR purified from T. vaginalis hydrogenosomes and a homogeneous preparation of recombinant T. vaginalis ferredoxin 1 could effectively reduce TvFDP at the expense of electrons derived from pyruvate by the activity of PFOR. Strictly anaerobic conditions had to be used to follow the reduction of the flavin cofactor of TvFDP and also to prevent autooxidation of ferredoxin in the spectrophotometric assay. Alternatively, another redox system could be used to reduce TvFDP; this system consists of a purified hydrogenosomal remnant of complex I and recombinant T. vaginalis ferredoxin 1. In this case, the electron donor is NADH, but since its absorbance partially interfered with the absorbance of oxidized FMN of TvFDP in a spectrophotometric assay, the PFOR-based system was preferentially used. Under these experimental conditions, full reduction of TvFDP, monitored as disappearance of the absorbance of FMN, was achieved within Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 FIG. 8. Redox titration of the diiron center monitored by EPR spectroscopy, following the changes in EPR intensity at the gmed value of the diiron center. The full line was also calculated for two oneelectron reduction processes and corresponds to the formation and disappearance of the EPR resonance of the Fe(III)-Fe(II) mixed valence state, with reduction potentials of 190 and 50 mV. EUKARYOT. CELL VOL. 8, 2009 FLAVODIIRON PROTEIN FROM T. VAGINALIS HYDROGENOSOMES quired by certain unicellular eukaryotes via lateral gene transfer from prokaryotic sources (1). Eukaryotic FDP homologues have thus far been invariably found in the genomes of anaerobic fermentative protists, most with parasitic lifestyles. This suggests that the role of FDPs in these eukaryotes is similar to that in prokarytes, i.e., protection against oxidative/nitrosative stress. However, the only eukaryotic homologue studied to date is the FDP from G. intestinalis (13). Giardia (a member of Diplomonadida) is believed to share a common ancestor with trichomonads (23), and the T. vaginalis protein described in the present study is the closest relative to the giardial homologue (which, however, is natively a tetramer) (1), suggesting that this particular gene entered this eukaryotic lineage before the divergence of diplomonads and trichomonads. Notably, the sister group to diplomonad/trichomonad FDPs consists of the branch of archaebacterial FDPs of methanogens (1), some of which have been shown to utilize only oxygen and not nitric oxide (44), like the homologues from both human parasites. Class A FDP is not the only FDP present in T. vaginalis. In fact, four genes encoding FDP homologues could be identified in the genome of this parasite (5) (http://www.tigr.org/tdb/e2k1/tvg; locus numbers TVAG_036010, TVAG_129610, TVAG_049830, and TVAG_263800). One of these proteins is the subject of the present study; the remaining three are highly similar homologues that belong to a strongly separated clade and form a group with other eukaryotic sequences from Entamoeba and Mastigamoeba and with eubacterial sequences from Clostridium sp. (1). Unlike hydrogenosomal TvFDP, the genes of these three trichomonad proteins do not encode a clear amino-terminal extension with an organellar targeting function (and were not detected in the proteome of hydrogenosomes, unpublished data); thus, these putative proteins are likely localized to the cytoplasm. Because of their novel primary structure, these trichomonad homologues were proposed to constitute a fourth, D class of FDPs, together with clostridial proteins, that have an NADH:rubredoxin oxidoreductase and rubredoxin module fused to the flavodiiron core and could probably directly utilize NAD(P)H as an electron donor (50). In this respect, it should be mentioned that nitric oxide reducing activity has been detected in intact as well as lysed T. vaginalis cells; this activity has been ascribed to FDP partly because a cross-reactive band was visualized in a T. vaginalis cell lysate using a heterologous antibody against E. coli flavorubredoxin (42). Since the molecular size of the denatured candidate protein (⬃60 kDa) was far from the predicted size of a class D FDP monomer (⬃95 kDa), its identity is doubtful. Nevertheless, if these class D FDP homologues are indeed expressed, the observed nitric oxide reducing activity (42) might be due to their presence in the T. vaginalis cytosol, where they could provide protection against the host immune response as well as against oxygen. Finally, it should be stated that despite the evidence collected in vitro using a functional electron transport chain consisting of the T. vaginalis hydrogenosomal proteins PFOR, ferredoxin 1, and TvFDP (or complex I, ferredoxin 1, and TvFDP), the identity of the actual physiological components of the presumed pathway remains uncertain. This is largely due to the complexity of the predicted electron transporting pathways of hydrogenosomes. The core electron-generating reactions utilizing malate and pyruvate as the carbon and electron source, as coined by Müller (36), are still valid, but research Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 a few minutes. The reaction time depended on the concentration of PFOR and ferredoxin in the assay (Fig. 6). Upon introduction of air into the cuvette, TvFDP immediately reoxidized as documented by the regression of the spectral curve to the original, oxidized pattern (Fig. 6). The immediate reoxidation of the flavin cofactor indicates a fast reaction with a high affinity for oxygen; however, the kinetic parameters of the electron transfer could not be determined due to the autooxidative nature of the trichomonad ferredoxin, which precluded a continuous aerobic spectrophotometric assay. To function as a protective, O2 scavenging enzyme, TvFDP should reduce oxygen by four electrons to water. The product of oxygen reduction was determined by the FOX assay with enzymatically reduced His-TvFDP that was reoxidized by short exposure to air. No traces of hydrogen peroxide were detected, indicating that TvFDP indeed reduces dioxygen to water using four molecules of the one-electron carrier ferredoxin per cycle. It should be noted that FDPs do not reduce hydrogen peroxide (M. Teixeira, unpublished data). Experiments designed to verify the potential reactivity of enzymatically reduced TvFDP with nitric oxide, the known substrate of several members of the flavodiiron superfamily, did not show any such activity for the trichomonad protein, despite the broad range of NO concentrations and prolonged reaction times tested. This is similar to what was found for the only other eukaryotic FDP characterized, the protein from G. intestinalis, which is also virtually unreactive with NO (13). Analysis of the primary structure of TvFDP shows that the enzyme belongs to the class A family of FDPs. Members of this family are the simplest representatives of the superfamily, consisting of only the core flavodiiron module, which is formed by a metallo -lactamase-like domain in the amino-terminal half of the protein and by a flavodoxin-like domain forming the rest of the polypeptide chain (41). The mature (without the hydrogenosomal targeting peptide) trichomonad protein is colinear over its entire length with its homologues, with conservation of all residues implicated in binding the binuclear iron center and a well-conserved flavodoxin signature motif at the start of the flavodoxin-like domain (Fig. 2). Attempts have been made to attribute the selective specificity of some FDPs for oxygen (and not NO) to particular amino acid residues (44). This issue has been discussed in the light of data recently obtained for Giardia FDP (13), and we can only conclude that the results concerning both the Giardia (13) and the Trichomonas FDPs (the present study) invalidate the original hypothesis (44) and leave the question of the substrate specificity of FDPs unsettled. In addition, tryptophan 361 (W347 in reference 44), which is missing in the FDP from M. marburgensis but conserved in rubredoxin-specific FDPs, has been proposed to play a role in electron shuttling between rubredoxin and the FMN of rubredoxin-specific FDPs (44). However, neither Trichomonas nor Giardia FDPs, which have W361 conserved, have rubredoxins as electron donors, since there are no genes encoding these proteins (http://www.trichdb .org/trichdb/; http://www.giardiadb.org/giardiadb/). The absence of this tryptophan in Methanothermobacter FDP might be related to the nature of the proposed electron donor to FDPs from methanogens, enabling easy access to F420H2. FDPs have already been subjected to a phylogenetic study, as particularly good candidates for genes that have been ac- 53 54 SMUTNÁ ET AL. EUKARYOT. CELL ACKNOWLEDGMENTS This study was supported by the Czech Science Foundation grant 204/06/0944 to I.H. and by Ministry of Education, Youth, and Sports of the Czech Republic grant MSM0021620858. Further support was provided by the Fundação para a Ciência e Tecnologia–Portugal, projects PTDC/BIA-PRO/67263/2006, and Structure, Dynamics and Functions of Proteins (REEQ/336/BIO/2005). V.L.G. is the recipient of a Ph.D. grant from Fundação para a Ciência e Tecnologia–Portugal (SFRH/ BD/29428/2006). We acknowledge the help of J. Vicente (Instituto de Tecnologia Química e Biológica) with the EPR-monitored redox titration. 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Chem. 280:34599–34608. Vidakovic, M. S., G. Fraczkiewicz, and J. P. Germanas. 1996. Expression and spectroscopic characterization of the hydrogenosomal [2Fe-2S] ferredoxin from the protozoan Trichomonas vaginalis. J. Biol. Chem. 271:14734–14739. Vondruskova, E., J. van den Burg, A. Zikova, N. L. Ernst, K. Stuart, R. Benne, and J. Lukes. 2005. RNA interference analyses suggest a transcriptspecific regulatory role for mitochondrial RNA-binding proteins MRP1 and MRP2 in RNA editing and other RNA processing in Trypanosoma brucei. J. Biol. Chem. 280:2429–2438. Wasserfallen, A., S. Ragettli, Y. Jouanneau, and T. Leisinger. 1998. A family of flavoproteins in the domains Archaea and Bacteria. Eur. J. Biochem. 254:325–332. Wolff, S. P. 1994. Ferrous ion oxidation in presence of ferric ion indicator xylenol orange for measurement of hydroperoxides. Methods Enzymol. 233: 182–189. Downloaded from ec.asm.org at PRIRODOVEDECKA FAKULTA UK on January 6, 2009 34. FLAVODIIRON PROTEIN FROM T. VAGINALIS HYDROGENOSOMES 6.2 Publication 2: Smutná et al., 2014 Smutná T., Pilařová K., Tarábek J., Tachezy J., Hrdý I. (2014) Novel functions of an ironsulfur flavoprotein from Trichomonas vaginalis hydrogenosomes. Antimicrob Agents Chemother; 58:3224-32. 36 Novel Functions of an Iron-Sulfur Flavoprotein from Trichomonas vaginalis Hydrogenosomes Tamara Smutná, Katerina Pilarová, Ján Tarábek, Jan Tachezy and Ivan Hrdý Antimicrob. Agents Chemother. 2014, 58(6):3224. DOI: 10.1128/AAC.02320-13. Published Ahead of Print 24 March 2014. These include: REFERENCES CONTENT ALERTS This article cites 49 articles, 31 of which can be accessed free at: http://aac.asm.org/content/58/6/3224#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ Downloaded from http://aac.asm.org/ on May 15, 2014 by guest Updated information and services can be found at: http://aac.asm.org/content/58/6/3224 Novel Functions of an Iron-Sulfur Flavoprotein from Trichomonas vaginalis Hydrogenosomes Tamara Smutná,a Katerina Pilarová,a Ján Tarábek,b Jan Tachezy,a Ivan Hrdýa Department of Parasitology, Charles University in Prague, Prague, Czech Republica; NMR Spectroscopy Unit, Institute of Organic Chemistry and Biochemistry, Czech Academy of Sciences, Prague, Czech Republicb I ron-sulfur flavoproteins (Isf) constitute a relatively recently recognized family of proteins commonly present in diverse representatives of the Bacteria and Archaea domains, particularly in species inhabiting anaerobic environments (1). The only eukaryotic species found to possess an Isf homolog in its genome was the anaerobic intestinal pathogen Entamoeba histolytica (1). These proteins have subunits of approximately 20 kDa, harbor a noncovalently bound flavin mononucleotide (FMN) cofactor, and contain a conserved, compact, four-cysteine motif responsible for coordinating the low-redox-potential 4Fe-4S redox center (2, 3). Given the presence and properties of these redox cofactors, it has been suggested that Isf play a role in electron transport, possibly functioning as a one- to two-electron switch (2). The prototype Isf from Methanosarcina thermophila (MtIsf), a strictly anaerobic methane-producing thermophile (1), has been characterized as a functional homodimer interacting with ferredoxin as a physiological electron donor (4, 5). The protein was eventually found to catalyze the reduction of dioxygen and hydrogen peroxide to water, and its role in combating oxidative stress in strictly anaerobic prokaryotes had been proposed (6). Despite the broad distribution of Isf proteins in bacteria (including human pathogens), to date, the purported physiological role of Isf proteins has been explored only in the case of MtIsf. The anaerobic eukaryotic parasite Trichomonas vaginalis is a causative agent of human vaginitis and urethritis, a widespread infection affecting approximately 170 million people per year (7, 8). The disease is treated with derivatives of 5=-nitroimidazoles, such as metronidazole (MTZ) and tinidazole. These drugs are reductively activated within the susceptible cells and are highly effective against many anaerobic microorganisms, though the emergence of resistant isolates has been reported (9). Trichomonads are flagellated protists belonging to the supergroup Excavata. One of their distinctive features is the absence of cristate oxygenrespiring mitochondria. Instead, trichomonads possess hydrogenosomes, organelles of mitochondrial ancestry that produce molecular hydrogen and ATP in the reactions of extended glycolysis (10). The enzymes involved in hydrogen formation, pyruvate: ferredoxin oxidoreductase (PFO), which oxidatively decarboxy- 3224 aac.asm.org lates pyruvate and forms acetyl coenzyme A (acetyl-CoA; subsequently utilized in the substrate-level synthesis of ATP), and Fe-Fe hydrogenase (the terminal oxidoreductase which forms hydrogen using pyruvate-derived electrons transferred by 2Fe-2S ferredoxin), are particularly oxygen-sensitive proteins (11). Nevertheless, T. vaginalis is exposed to oxygen concentrations of up to 60 M in its natural environment (12) and must be able to effectively scavenge oxygen and reactive oxygen species (ROS) to protect its vital proteins from oxidative damage. Indeed, trichomonads are equipped with a number of oxygen and ROS detoxification systems, including oxygen-reducing NADH oxidase (13), superoxide-removing superoxide dismutase (12, 14), rubrerythrin (15) and the thioredoxin system (16) aimed at peroxides, and flavodiiron protein (FDP), which functions as a ferredoxin-dependent oxygen reductase and forms water (17). Annotation of the T. vaginalis genome (18) confirmed the presence of genes coding for the above-mentioned enzymes and identified several other candidate proteins possibly involved in ROS or xenobiotic detoxification. Among these were seven distinct paralogs of Isf; thus, T. vaginalis is the second eukaryote (in addition to Entamoeba histolytica) identified to possess otherwise strictly prokaryotic Isf proteins. The results of subsequent proteomic studies showed that three Isf paralogs are expressed in T. vaginalis hydrogenosomes under standard in vitro cultivation conditions (19, 20). In this communication, we report the characterization and novel catalytic properties of Isf from T. vaginalis hydrogenosomes and suggest that this protein plays a general role in the defense against xenobiotics. Antimicrobial Agents and Chemotherapy Received 25 October 2013 Returned for modification 23 November 2013 Accepted 18 March 2014 Published ahead of print 24 March 2014 Address correspondence to Ivan Hrdý, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02320-13 p. 3224 –3232 June 2014 Volume 58 Number 6 Downloaded from http://aac.asm.org/ on May 15, 2014 by guest Iron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function. Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics. Iron-Sulfur Flavoprotein from T. vaginalis MATERIALS AND METHODS June 2014 Volume 58 Number 6 tec) were used as the primary antibodies. Anti-mouse IgG labeled with Alexa Fluor 488 (Molecular Probes) and anti-rabbit IgG labeled with Alexa Fluor 594 (Molecular Probes) were used as the secondary antibodies for fluorescent immunolocalization. Anti-TvIsf3 rabbit polyclonal serum (Eurogentec) and anti-rabbit IgG conjugated with horseradish peroxidase were used for Western blotting to visualize TvIsf3 in T. vaginalis subcellular fractions obtained from untransformed wild-type trichomonads. Activity assays. The ability of various compounds to accept electrons from chemically reduced TvIsf3 was initially assayed by recording the visible spectra using a Shimadzu UV-1601 spectrophotometer and 1-ml stoppered cuvettes with a silicone septum in phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, 10% glycerol [pH 8.0]) (assay buffer) at 25°C. The anaerobiosis of the system was achieved by degassing the buffer by evacuation, followed by extensive flushing with N2 and the addition of oxygen- and hydrogen peroxide-scavenging system consisting of glucose oxidase (28 U), catalase (104 U), and glucose (3 mM) to the reaction mixture (27). Prior to initiation of the reaction by the addition of potential electron acceptors, TvIsf3 (20 to 30 nmol) was reduced by the stepwise addition of Tris-buffered 30 mM sodium dithionite (pH 9.0); particular attention was paid during this step not to overtitrate the protein with an excess of dithionite. The enzymatic oxygen- and hydrogen peroxide-scavenging system was omitted from experiments aimed at identifying the reaction products of TvIsf3 with oxygen. Hydrogen peroxide was determined using the FOX assay as described previously (28). The assays with dipropylenetriamine NONOate (DPTA-NONOate; Alexis Biochemicals) used as a nitric oxide donor (12 M to 1 mM final concentration) and hydrogen peroxide (35 M final concentration) were prepared in the assay buffer at a pH of 5.5; this lower pH allowed for a faster evolution of NO from DPTA-NONOate and prevented the decomposition of hydrogen peroxide. The assay mixture with hydrogen peroxide as a substrate was supplemented with glucose oxidase (28 U) and glucose (3 mM) to eliminate the oxygen generated during the decomposition of hydrogen peroxide. Reoxidation of TvIsf3 with NaNO3, NaNO2, and hydroxylamine was tested with final concentrations of acceptors up to 1 mM. Assays with metronidazole and chloramphenicol were performed using acceptor concentrations of up to 200 M. All the acceptors used in the assays were rendered anaerobic by flushing the stock solutions in stoppered vials with nitrogen. Activity determinations under continuous, turnover conditions were performed with 220 M NADH as the electron donor in spectrophotometric assays at 340 nm and 37°C. In addition to DPTA-NONOate, NOsaturated water (obtained by purging degassed water with pure NO to yield approximately 2 mM NO solution) was used as the NO source for assays at pH 8.0. Otherwise, the conditions were identical to those used to determine the activity of dithionite-reduced TvIsf3 as described above. Kinetic parameters were calculated using Lineweaver-Burk plots and UVProbe software (Shimadzu). Enzymatic reduction of TvIsf3. The experiment aimed at determining whether hydrogenosomal ferredoxin is capable of reducing TvIsf3 was performed using the anaerobic spectrophotometric assay system described above with 20 to 30 nmol of TvIsf3, 5 mM pyruvate, 0.25 mM coenzyme A (CoA), ⬃40 g of recombinant T. vaginalis ferredoxin 1 (29), and ⬃10 g of T. vaginalis PFO purified from hydrogenosomes (17) as the components of the reducing system. Determination of metronidazole radical. The electron paramagnetic resonance (EPR) spectra were recorded using an EMXplus-10/12 CW (continuous wave) spectrometer (Bruker) equipped with the Premium X-band microwave bridge. The experiment was performed under turnover conditions using 5 mM NADH and 3 mM metronidazole in 1 ml of anaerobic assay buffer. The reaction was started by the addition of ⬃20 nmol of TvIsf3. The mixture was drawn into the quartz flat EPR cell (ER160FC-Q; Bruker), which was immediately closed and inserted into the standard rectangular cavity (ER 4102003ST; Bruker). Prior to insertion, the EPR cell was flushed with gaseous N2 to minimize the influence of oxygen in the air on the sample. The positive control giving rise to aac.asm.org 3225 Downloaded from http://aac.asm.org/ on May 15, 2014 by guest Organism. The T. vaginalis strain T1 (J.-H. Tai, Institute of Biomedical Sciences, Taipei, Taiwan) was grown in Diamond’s TYM (Trypticaseyeast extract-maltose) medium without agar, as previously described (21). The T. vaginalis cell line used for immunolocalization of the Isf paralog (in this work named TvIsf3) by fluorescence microscopy was prepared by transforming T. vaginalis T1 with a TagVag vector (22) carrying the complete TvIsf3 gene (TVAG_154730) fused with a C-terminal 2⫻ hemagglutinin (HA) tag. T. vaginalis cell fractionation. The T. vaginalis cell fractions used in the Western blot analysis were isolated from the cell homogenate obtained by sonication. The lysate was fractionated using differential centrifugation, followed by isopycnic centrifugation of the hydrogenosome-enriched fraction on a self-forming gradient of 45% Percoll (Sigma), as previously described (23). Expression and purification of TvISf3. The TvIsf3 gene was amplified and cloned into the pET-42b vector (Novagen). Recombinant TvIsf3 with a 6⫻ His tag at the C terminus and without an amino-terminal hydrogenosomal targeting presequence was expressed in Escherichia coli BL21(DE3) cells grown anaerobically in 1-liter screwcap bottles in LB medium supplemented with 2 mM NaNO3 as an electron acceptor. The bacterial culture was induced with 0.25 mM isopropyl-D-thiogalactopyranoside (IPTG), and the cells were grown for 12 h at 18°C in LB medium supplemented with 400 M ammonium ferrous sulfate, 200 M flavin mononucleotide (FMN), and 250 M cysteine. The bacteria (1- to 3-liter cultures) were harvested by centrifugation, washed with buffer containing 20 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and 10% glycerol (pH 8.0), and homogenized by passage through a French press at 18,000 lb/in2. The soluble fraction (approximately 25 ml) obtained after ultracentrifugation (250,000 ⫻ g, 45 min, and 4°C) of the cell lysate was applied onto a nickel-nitrilotriacetic acid (Ni-NTA) Superflow agarose column (Qiagen) and eluted with a stepwise gradient of 20 mM to 400 mM imidazole in buffer containing 50 mM NaH2PO4, 300 mM NaCl, and 10% glycerol (pH 8.0) at a flow rate of 1 ml min⫺1 using a BioLogic HR system (Bio-Rad). The TvIsf3 was specifically released from the column with 250 mM imidazole. The purity of the isolated TvIsf3 was verified by SDS-PAGE. Characterization of TvISf3. The flavin cofactor extracted from recombinant TvIsf3 was characterized by thin-layer chromatography (TLC) (HPTLC-Alufolien; Merck) as previously described (17), and the recombinant protein isolated from bacteria grown in LB medium without addition of FMN was used for the flavin determination. The quantity of flavin in TvIsf3 was determined after protein denaturation with 80% trichloroacetic acid using an extinction coefficient of 12,500 M⫺1c⫺1 at a of 450 nm (24). The iron content of TvIsf3 was determined using the 2,4,6tripyridyl-1,3,5-triazine method (25). The native molecular mass of TvIsf3 was determined by gel filtration chromatography using a BioLogic HR system (Bio-Rad). The purified recombinant TvIsf3 was applied onto Superdex 75 10/300 GL and Superdex 200 10/300 GL columns (GE Healthcare) equilibrated with sodium phosphate buffer (50 mM NaH2PO4, 150 mM NaCl, 10% glycerol [pH 8.0]) using a flow rate of 0.5 ml min⫺1. The native molecular mass of TvIsf3 was calculated from the calibration curves determined by running appropriate protein standards under the same conditions. The protein concentrations were determined by the Lowry assay (26) using bovine serum albumin as a standard. Protein localization. T. vaginalis cells overexpressing TvIsf3 fused with a 2⫻ HA tag were used for the immunodetection of the protein within the cell. The cells were allowed to adhere to glass slides coated with 3-aminopropyltriethoxysilane (Sigma) and then fixed with methanol (5 min) and permeabilized with acetone (5 min) (both steps at ⫺18°C). The slides were preincubated for 1 h in phosphate-buffered saline with 0.25% bovine serum albumin and 0.25% gelatin and then treated with antibodies as described previously (23). An anti-HA tag monoclonal antibody (Exbio) and hydrogenosomal malic enzyme polyclonal antiserum (Eurogen- Smutná et al. FIG 1 Amino acid sequence alignment for comparison of Isf homologs from Trichomonas vaginalis (GenBank accession no. XP_001313900.1; TVAG_154730), metronidazole nitroradical anion consisted of the hydrogenosomes (⬃400 g) supplemented with the components of the PFO reaction and metronidazole and/or of the ferredoxin-reducing NADH dehydrogenase module of complex 1 isolated from Trichomonas hydrogenosomes, NADH, T. vaginalis ferredoxin 1, and metronidazole as previously described (30). The following parameters were used to record the EPR spectra: sweep width of 20 mT, power of 5 mW, modulation amplitude of 0.05 mT (or 0.4 mT), time constant of 10.24 ms, conversion time of 20 ms, and resolution of 0.01 mT. The experimental temperature was 25°C. RESULTS The TvIsf paralog herein named TvIsf3 was selected for our study because its presence in hydrogenosomes was confirmed by two independent proteomic studies (19, 20) and because our previous analysis revealed that the mRNA of this gene was produced at the highest level of all seven TvIsf paralogs (unpublished data). The polypeptide derived from TvIsf3 (TVAG_154730) consists of 223 amino acids and has a calculated molecular mass of 25.4 kDa. The amino acid sequence contains the typical conserved motif CX2CX2CX10C (Cys56, Cys59, Cys62, and Cys73) of iron-sulfur flavoproteins shown to coordinate the 4Fe-4S cluster (2, 3); however, the number of amino acids between Cys62 and Cys73 is higher (10 residues) in TvIsf3 than in many prokaryotic homologs (usually 4 to 7 amino acid residues) (2). TvIsf3 is colinear with and highly similar to its prokaryotic counterparts, displaying approximately 16% pairwise identity with the prototype MtIsf, 31% with C. acetobutylicum, and 22% with A. fulgidus homologs. However, the trichomonad protein contains a short extension, of 8 amino acids, at its amino terminus that is not present in the prokaryotic homologs (Fig. 1). Similar presequences identified in a number of hydrogenosomal proteins are implicated in protein targeting and in the translocation of proteins into the hydrogenosomal matrix; these presequences are cleaved (usually with the conserved arginine residue in the ⫺2 position relative to the processing site) upon maturation of the cytosolically translated protein within the hydrogenosome (31, 32). Production, purification, and biochemical characterization. The TvIsf3 without the predicted hydrogenosomal targeting sequence (Fig. 1) was overproduced in E. coli. The protein with the 6⫻ His tag at the C terminus was affinity purified close to homo- 3226 aac.asm.org geneity, as confirmed by SDS-PAGE in which the protein migrated as a polypeptide with an apparent molecular mass of approximately 26 kDa. Gel filtration chromatography used to determine the native molecular mass of TvIsf3 recovered the protein in a dominant peak corresponding to a size of approximately 51 kDa, indicating that TvIsf3 forms dimers; studies of Isf from Methanosarcina thermophila and three other prokaryotic species also showed the existence of a dimeric structure for these proteins (1, 2, 5). However, the major peak of the TvIsf3 dimer was preceded by a small shoulder with a retention time corresponding to a protein of approximately 90 kDa, indicating that a minor portion (estimated at less than 5%) of TvIsf3 was eluted from the column as a tetramer (data not shown). The UV-visible (UV-vis) spectrum of purified TvIsf3 displayed a complex pattern similar to that of other previously characterized Isf proteins (1, 5), with absorbance maxima at 490, 460, 435, 385, and 278 nm (Fig. 2 and 3). The mild acidification of the protein sample destroyed the ironsulfur cluster and resulted in spectra typical of flavins, with maxima at 373 and 447 nm (Fig. 2, inset). The TLC analysis of the flavin extracted from TvIsf3 purified from bacteria grown without flavin supplementation identified the cofactor as FMN (data not shown). The flavin quantification showed that there were approximately 0.57 mol of FMN per mol of Isf monomer, suggesting that 2 FMN molecules are bound to each homodimer. The amount of nonheme iron in the different protein preparations ranged from 2.4 to 3.4 mol per mol of monomer, indicating the coordination of the 3Fe-4S or 4Fe-4S cluster in the active center of the protein. However, lower-than-stoichiometric levels of both FMN and iron indicated some cofactor loss, most likely during the purification procedure. Subcellular localization of TvIsf3. Subcellular fractions of wild-type trichomonads prepared by differential centrifugation were analyzed by Western blotting using polyclonal antibodies against TvIsf3. As expected, TvIsf3 was found in the hydrogenosomal fraction (Fig. 4). In addition, the hydrogenosomal localization of TvIsf3 was further confirmed by immunofluorescence microscopy using T. vaginalis cells transformed with a plasmid carrying the complete TvIsf3 gene with the N-terminal hydrogenosomal targeting presequence and the hemagglutinin tag at the Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on May 15, 2014 by guest Clostridium acetobutylicum (NP_349133.1), Methanosarcina thermophila (Q50562.2), and Archaeoglobus fulgidus (NP_070721.1). The cleavable hydrogenosomal targeting sequence of TvIsf3 is underlined. The conserved cysteines binding the iron-sulfur cluster are shaded. The amino acid residues implicated in FMN binding in M. thermophila and A. fulgidus based on the crystal structures (2) are highlighted with a black background. Iron-Sulfur Flavoprotein from T. vaginalis visible spectrum of TvIsf3 exhibits the characteristic features of iron-sulfur flavoproteins. The lower trace represents the partial spectrum obtained after the reduction of TvIsf3 with sodium dithionite. The dashed line shows TvIsf3 reoxidized by air. The inset shows a typical flavin spectrum obtained after iron-sulfur cluster degradation by acidification of the protein sample. C terminus. TvIsf3 was colocalized with the hydrogenosomal marker protein malic enzyme (Fig. 5). Function of TvIsf3. The recombinant, affinity-purified, Histagged version of TvIsf3 (Fig. 3) was used in the experiments aimed at determining the potential electron acceptors that could function as physiological substrates. Under anaerobic conditions, with caution taken not to overtitrate the protein with an excess of reductant, the protein in a sealable spectrophotometric cuvette was almost completely stepwise reduced with sodium dithionite. Following reduction, the ability of a variety of electron acceptors to reoxidize the TvIsf3 was assessed. Reoxidation of the protein was monitored as a regression of its UV-vis spectrum to the original oxidized state. When the reduced protein was exposed to air by opening the cuvette, the spectrum immediately reassumed an oxidized pattern (Fig. 2). This observation shows that oxygen is reduced readily and is in agreement with the previous finding that Isf from Methanosarcina thermophila reduces oxygen (6). FIG 3 Complete UV-visible spectrum of affinity-purified TvIsf3. The partial representative spectrum in inset A shows the reoxidation (dashed line) of sodium dithionite-reduced (lower trace) TvIsf3 (approximately 25 nmol; upper trace) with several electron acceptors (H2O2, NO, metronidazole, and chloramphenicol). Details of the experimental setup are described in Materials and Methods. Inset B shows SDS-PAGE of the lysate of TvIsf3-expressing E. coli and the purified TvIsf3. June 2014 Volume 58 Number 6 FIG 5 Immunodetection of TvIsf3 in T. vaginalis cells. (A) Nomarski differential contrast; (B) visualization of malic enzyme, a hydrogenosomal marker; (C) TvIsf3 labeling; (D) merged image of color channels showing the localization of TvIsf3 within the hydrogenosome with DAPI (4=,6=-diamidino-2-phenylindole) staining for nuclei. aac.asm.org 3227 Downloaded from http://aac.asm.org/ on May 15, 2014 by guest FIG 2 UV-visible spectroscopic analysis of recombinant TvIsf3. The UV- FIG 4 Subcellular localization of TvIsf3. (A) SDS-PAGE analysis of subcellular fractions of T. vaginalis. (B) Western blot probed with TvIsf3 polyclonal antiserum. The molecular mass standards are indicated in kilodaltons. hom, homogenate; cyt, cytosol; hyd, hydrogenosomes. Smutná et al. demonstrate that both NADH and a substrate are required to observe the turnover activity of TvIsf3. The anaerobic spectrophotometric assays were performed in an assay buffer with air-saturated water, metronidazole, or chloramphenicol as a substrate and NADH as a reductant. The details are described in Materials and Methods. Hydrogen peroxide, the other substrate shown to be reduced by MtIsf (6), was then tested. The reaction mixture (pH 5.5) was supplemented with an oxygen-scavenging and H2O2-regenerating system consisting of glucose oxidase and glucose to prevent oxygen from affecting the results. TvIsf3 was readily reoxidized by hydrogen peroxide (Fig. 3). The ability of T. vaginalis lysates to reduce NO has been documented (33), but no enzyme responsible for this ability has been identified thus far. Therefore, we also tested the ability of NO to reoxidize the reduced TvIsf3. Approximately 25 nmol of dithionite-reduced TvIsf3 was exposed to 40 nmol of chemically generated NO, with the pH of the buffer (5.5) facilitating the rapid evolution of NO from DPTA-NONOate. The reduced protein was immediately reoxidized by NO (Fig. 3). Another potential electron acceptor we tested was the antitrichomonad drug metronidazole. When 30 nmol (0.5 l of 10 mg ml⫺1 of anaerobic MTZ solution) of metronidazole was added to approximately 20 nmol of chemically reduced protein, the spectrum of TvIsf3 immediately returned to its original oxidized state, indicating that MTZ can serve as an acceptor for TvIsf3 (Fig. 3). Chloramphenicol, another antibiotic with a nitro group (NO2), was also able to serve as a reducible acceptor. Approximately 20 nmol of almost completely dithionite-reduced TvIsf3 3228 aac.asm.org TABLE 1 Apparent kinetic parameters of recombinant, affinity-purified TvIsf3 with 220 M NADH as an electron donora Substrate kcat (s⫺1) Km (M) kcat/Km Chloramphenicol Metronidazole Oxygen 130 ⫾ 13 56 ⫾ 7 10 ⫾ 1 77.63 ⫾ 3.55 27.28 ⫾ 2.83 5.64 ⫾ 0.69 1.67 2.05 1.77 a The means were calculated from at least three separate determinations. Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on May 15, 2014 by guest FIG 6 NADH-dependent reduction of acceptors by TvIsf3. Panels A and B was instantly reoxidized by equimolar amounts of an anaerobic solution of the drug (Fig. 3). However, other nitrogen compounds, such as hydroxylamine, NaNO2, and NaNO3, were unable to reoxidize the dithionite-reduced TvIsf3 protein. Unexpectedly, and in contrast to the published data on MtIsf (6), TvIsf3 was found to utilize the pyridine nucleotide coenzyme NADH (but not NADPH) as an electron donor. This enabled the direct determination of kinetic parameters of TvIsf3 under turnover conditions with the electron acceptors identified in previous experiments with dithionite-reduced protein (Fig. 6). Due to higher concentrations of reactants, the turnover assays also allowed for a more reliable identification of reaction end products and, thus, the stoichiometry of electron transfer. This way, the oxygen reduction by TvIsf3 was first assayed. Upon consumption of most of the NADH, the cuvette content (an aerobic reaction mixture without O2-scavenging enzymes) was immediately probed for the presence of hydrogen peroxide using the FOX assay (28) to determine the final product of O2 reduction. In contrast to the results previously reported for MtIsf (6), the assay did indicate the presence of hydrogen peroxide. To corroborate this result, we determined the ratio of oxygen reduced per NADH oxidized using anaerobic conditions and known amounts of air-saturated water (the oxygen concentration in water at 37°C was taken as 199 M) as a substrate. One mole of reduced oxygen resulted in 1 mol of oxidized NADH, confirming the two-electron transfer from NADH to oxygen and the formation of hydrogen peroxide (data not shown). The two nitro-antibiotics, metronidazole and chloramphenicol, were reduced by TvIsf3 with NADH as a reductant as well (Fig. 6). The kinetic data of TvIsf3 with oxygen, metronidazole, and chloramphenicol are summarized in Table 1. The observed values seem to fall within the physiologically relevant range. Using EPR spectroscopy and high concentrations of NADH and metronidazole in a turnover assay, we attempted to determine whether the product of MTZ reduction by TvIsf3 was a nitroradical anion, the species most suspected of being responsible for the toxic effect of MTZ on susceptible organisms (34, 35). We were unable to detect the typical signal of MTZ radical anion, indicating that MTZ is likely reduced by an even number of electrons. The control consisting of hydrogenosomes, metronidazole, and reactants of the PFO reaction as well as the single-enzyme system consisting of a purified, ferredoxin-reducing NADH dehydrogenase module of complex 1 and T. vaginalis ferredoxin 1 (30) produced an easily detectable signal (data not shown). Virtually no activity under turnover conditions was observed with hydrogen peroxide and nitric oxide, although both substrates were able to immediately reoxidize the dithionite-reduced protein. To exclude the negative effect of low pH in the reaction of TvIsf3 with DPTA-NONOate, the assay was repeated in pH 8.0 assay buffer with NO-saturated water, with the same result. Iron-Sulfur Flavoprotein from T. vaginalis onstrate the continuous reduction of TvIsf3 (approximately 20 M) by recombinant T. vaginalis ferredoxin 1 (⬃40 g) in an anaerobic system consisting of 0.25 mM CoA, 5 mM pyruvate, and T. vaginalis PFO (⬃10 g) in an assay buffer supplemented with oxygen-scavenging enzymes. The recording time for one spectrum was 38 s. Ferredoxin-mediated reduction. Isf protein from Methanosarcina thermophila was proposed to interact with ferredoxin as a physiological electron donor (4). To determine whether ferredoxin can also function as a reductant of TvIsf3, we reconstructed in vitro the anaerobic reaction system consisting of PFO purified from T. vaginalis hydrogenosomes (17), recombinant hydrogenosomal ferredoxin 1 (29), recombinant TvIsf3, and pyruvate and CoA as substrates. Electrons released from pyruvate were transferred to ferredoxin 1 by the activity of PFO and subsequently to TvIsf3, as documented by the continuous regression of the TvIsf3 UV-vis spectrum to the reduced state (Fig. 7). In contrast, the control reaction without ferredoxin did not show any appreciable reduction of TvIsf3 over a period of 20 min. When air was introduced into the cuvette, the TvIsf3 spectrum immediately returned to its oxidized pattern; when the cuvette was sealed again, the reduction of TvIsf3 resumed, confirming the turnover and thus the functionality of the components of the enzymatic system. This multicomponent setup nevertheless could not be used to determine the parameters of the electron transfer from ferredoxin to TvIsf3 because reduced ferredoxin rapidly oxidized in the presence of all electron acceptors tested that at the same time were capable of reoxidation of TvIsf3. DISCUSSION In this study, we characterized the iron-sulfur flavoprotein (TvIsf3) of T. vaginalis, the only eukaryote in addition to members of the Entamoeba genus in which such proteins could be detected using searches of sequence databases. In contrast, Isf homologs are June 2014 Volume 58 Number 6 aac.asm.org 3229 Downloaded from http://aac.asm.org/ on May 15, 2014 by guest FIG 7 Ferredoxin-mediated reduction of TvIsf3. The UV-visible spectra dem- common in the genomes of diverse anaerobic Bacteria and Archaea. Nonetheless, despite the broad distribution among prokaryotes, the Isf from the methanoarchaeon Methanosarcina thermophila has been the only Isf studied in terms of function to date. Because of its activity as an oxygen and hydrogen peroxide reductase, the general function of MtIsf and its homologs in oxidative stress protection has been proposed (6). The phylogenetic relationships of T. vaginalis Isf were not studied in detail, but the protein, most likely acquired through horizontal gene transfer, is a close homolog of its counterparts from both the Archaea and Bacteria domains. In T. vaginalis, the protein is localized in the hydrogenosomes, and this localization is consistent with the presence of an amino-terminal hydrogenosomal targeting sequence that is absent in otherwise highly similar prokaryotic homologs. TvIsf3 binds one molecule of FMN per subunit, and the complex pattern of the UV-visible spectrum (which is sensitive to acidification), the conserved four-cysteine motif, and the ⬃3.5 mol of nonheme iron per subunit of the affinity-purified protein are indicative of the presence of the 4Fe-4S redox cluster. The same flavin and FeS cluster were identified in MtIsf (4). Gel filtration chromatography showed that the overwhelming majority of the enzymatically active TvIsf3 protein had a dimeric composition, in agreement with an original report on several bacterial Isf proteins (1). However, in the present work, a small portion of TvIsf3 was also recovered from the column in the form of a tetramer, an observation that may have been overlooked if not for a more recent structural study of MtIsf and Isf from Archaeoglobus fulgidus showing that these proteins formed tetramers (2). Thus, it may be possible that a relatively dilute protein tends to disintegrate into stable dimers under the dynamic conditions of column chromatography and potentially suboptimal buffer composition, whereas the native quaternary structure is tetrameric under physiological conditions. The activity of TvIsf3 was first assessed by the ability of various substrates to reoxidize the dithionite-reduced protein. Using this approach, oxygen, hydrogen peroxide, nitric oxide, and the broad-spectrum nitro-antibiotics metronidazole and chloramphenicol were identified as electron acceptors of TvIsf3. Subsequently and rather surprisingly, we found that TvIsf3 can accept electrons from the coenzyme NADH. This observation was unexpected because the sequences of Isf proteins, including T. vaginalis paralogs, do not contain a recognizable Rossmann fold with a glycine-rich, NAD(P)-binding motif, and the prototype MtIsf was reported to utilize ferredoxin rather than pyridine nucleotide coenzyme as an electron donor (6). The activity of TvIsf3 with NADH enabled the continuous spectrophotometric assays and the determination of the kinetic parameters of the enzyme. With its capacity to reduce oxygen, TvIsf3 is similar to MtIsf (6). However, in contrast to MtIsf, which was reported to reduce oxygen with four electrons and form water (6), the trichomonad protein reduced oxygen to hydrogen peroxide using two electrons. The kinetic parameters of the reaction seem physiologically relevant; thus, the activity of TvIsf3 may account for the formation of toxic hydrogen peroxide within hydrogenosomes under aerobic conditions. Nevertheless, the organelles are equipped with defense mechanisms to cope with peroxidative stress (15, 16). Hydrogen peroxide, the other substrate shown to interact with MtIsf, was reduced by TvIsf3 only at a very low rate in an NADHdependent continuous assay, although it immediately reoxidized Smutná et al. 3230 aac.asm.org FDP homologs, including the one from T. vaginalis hydrogenosomes, lack NO reductase activity (17, 41, 42). Other proteins thus should be present that afford the protection against nitrosative stress. In addition to TvIsf3 and two other Isf paralogs present in the hydrogenosomes (19, 20), among those to be considered are three other putative flavodiiron protein homologs, all lacking hydrogenosomal targeting signals (18). However, no experimental data on these proteins are currently available. The results of the experiment showing the possible activity of TvIsf3 with NO prompted us to test the ability of other nitrogen compounds to function as potential electron acceptors, including metronidazole, a 5=-nitroimidazole drug used to treat anaerobic infections, including trichomoniasis. Indeed, dithionite-reduced TvIsf3 was immediately reoxidized upon the addition of an anaerobic metronidazole solution, showing that metronidazole is reduced by TvIsf3. This observation was confirmed in a continuous assay with NADH as the electron donor. The nitroimidazole radical anion arising from the ferredoxin-mediated, one-electron reduction of the nitro group is formed in the cell lysates of trichomonads as well as in isolated hydrogenosomes and is considered to be the most toxic form of the drug (30, 43). The characteristic complex signal of the metronidazole radical anion is generally detectable by EPR spectroscopy (30), and upon completing the reaction, we probed the mixture originally containing TvIsf3, NADH, and metronidazole for the presence of such a radical anion. No such signal was observed, indicating that metronidazole was reduced by an even number of electrons, thus obviating the formation of the radical and giving rise to a more reduced, less toxic product. Reductive inactivation of metronidazole to a nontoxic amine derivative by nitroimidazole reductases (Nim proteins) has been associated with 5=-nitroimidazole resistance in certain bacterial strains (44, 45). It is noteworthy that bacterial Nim protein homologs are encoded by T. vaginalis, along with several nitroreductases (18) that, on the other hand, are responsible for reductive activation of nitromidazole drugs in microaerophilic bacteria such as Helicobacter pylori (46). However, the activities of these proteins in T. vaginalis remain to be studied, and no relationship between the transcription levels of corresponding mRNAs and metronidazole sensitivity was found in either T. vaginalis or E. histolytica that also possesses such genes (47). We also tested chloramphenicol, a broad-spectrum antibiotic with a nitro group effective against both Gram-positive and Gram-negative bacteria (including anaerobes) (48), for its ability to function as an electron acceptor. We found that chloramphenicol was able to accept electrons from TvIsf3 in the same rapid manner as the other active acceptors we assayed, and similar to oxygen and metronidazole, it supported the oxidation of NADH under turnover conditions. It is noteworthy that several chloramphenicol reduction products that have been tested for antibiotic activity do not appear to have appreciable microbicidal effects (49). Moreover, the reductive inactivation of chloramphenicol has been shown to occur in actively growing cultures of Clostridium acetobutylicum, an anaerobic bacterium that possesses Isf homologs, and the ferredoxin-dependent, pyruvate-stimulated reduction of chloramphenicol and other aryl-nitro compounds by cell extracts of Clostridium has been described (50). It is possible that the mechanism described here, that is, the reduction of various acceptors by Isf using the electrons released from pyruvate by PFO and transferred by ferredoxin, is responsible for this decadesold observation. Antimicrobial Agents and Chemotherapy Downloaded from http://aac.asm.org/ on May 15, 2014 by guest the dithionite-reduced protein. The same observation was made also for nitric oxide. While NO rapidly reoxidized the chemically reduced TvIsf, it could not serve as a substrate that would effectively support the continuous oxidation of NADH under turnover conditions. The easiest explanation of this discrepancy is that hydrogen peroxide and nitric oxide, both strong oxidants, in a thermodynamically favorable reaction simply reoxidize the reduced FMN in the TvIsf3. However, the structurally related flavodiiron proteins also use oxygen and nitric oxide as electron acceptors; some representatives reduce both substrates, while others display strong substrate specificity (24, 36, 37). For example, the flavodiiron protein from T. vaginalis hydrogenosomes is strictly specific for oxygen, and nitric oxide could not reoxidize its FMN cofactor (17). Exactly which structural features of the flavodiiron proteins affect the substrate specificity is currently unclear. Therefore, the possibility exists that the reactivity of reduced TvIsf3 with hydrogen peroxide and NO actually reflects the genuine protein specificity. It cannot be excluded that while NADH does not serve as an electron donor for the reduction of hydrogen peroxide and NO, ferredoxin could. If this is indeed the case, then an intriguing question arises as to what is the physiological electron donor to TvIsf3. It would seem logical that if the protein utilizes NADH, this would be its natural redox partner. Trichomonad hydrogenosomes contain several NAD-dependent enzymes, and the most abundant one, the malic enzyme, provides NADH during oxidative decarboxylation of malate, one of the catabolic substrates of hydrogenosomes (10). On the other hand, ferredoxin and not the pyridine nucleotide coenzyme was identified as an electron donor to Isf from Methanosarcina for the reduction of oxygen and hydrogen peroxide (6). T. vaginalis encodes seven paralogs of ferredoxin (18), and most of them, if not all, are expressed in the hydrogenosomes (19, 20), where they serve as one-electron carriers that link the crucial reactions of hydrogenosomal metabolism (10). Ferredoxin is an electron donor to the oxygen-reducing and water-forming flavodiiron protein (17); in this work, we show that upon in vitro reduction by hydrogenosomal PFO, ferredoxin can also transfer electrons to TvIsf3. A comparison of the kinetic parameters of TvIsf3 with NADH versus ferredoxin would help to assess which of the donors is the more likely redox partner; however, this seemingly straightforward experiment was not possible because trichomonad ferredoxin 1 rapidly oxidized in the presence of all active acceptors used in this study. If under in vivo conditions TvIsf3 is indeed capable of NO reduction, this activity would be physiologically relevant. NO is a microbicidal molecule that is released by macrophages during inflammation (38), and its toxic effect on T. vaginalis under microaerobic conditions has been described previously (39). Nevertheless, trichomonads infesting the vaginal epithelium cause chronic, non-self-limiting infections (40), and the parasite must therefore be able to effectively resist the immune response of the host. It has been shown that T. vaginalis can degrade NO (33), although it remained unclear which protein was responsible for this capability. As mentioned above, the proteins of the flavodiiron superfamily, which are similarly widespread in anaerobic bacteria as Isf and are present in certain anaerobic eukaryotic parasites, including T. vaginalis (17), function as oxygen and NO reductases with variable affinities for these acceptors (24, 36, 37). It was originally suggested that the flavodiiron protein could be responsible for the NO reduction observed in T. vaginalis lysates (33); however, subsequent experiments showed that eukaryotic Iron-Sulfur Flavoprotein from T. vaginalis ACKNOWLEDGMENTS This work was supported by grant GC13-09208J of the Czech Science Foundation to J. Tachezy, by Charles University grant GAUK 407911 to K.P., and by Charles University grant UNCE 204017 to T.S. The kind support of the Institute of Organic Chemistry and Biochemistry (RVO: 61388963) is acknowledged by J. Tarábek. REFERENCES 1. Zhao T, Cruz F, Ferry JG. 2001. 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Chem. 10:21–31. http: //dx.doi.org/10.1016/0009-8981(64)90210-4. aac.asm.org 3231 Downloaded from http://aac.asm.org/ on May 15, 2014 by guest In summary, we found that the bacterial-type Isf protein present in T. vaginalis hydrogenosomes could reduce a variety of substrates, all of them being molecules toxic for anaerobic microbes. While the chloramphenicol-reducing activity of TvIsf3 is probably an inherited trait of a bacterial protein with no adaptive function in trichomonads, the relevance of reductive inactivation of metronidazole by trichomonad Isf paralogs for the drug resistance occurring in the field and induced in in vitro cultures remains to be explored. With the exception of oxygen and hydrogen peroxide reduction, the Isf activities described in this report have not been observed in prokaryotic Isf proteins; regardless, the ability of bacterial Isf homologs to fulfill these other roles seems likely and should be verified experimentally. 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(2016) OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis. Mol Biochem Parasitol; in press. 37 G Model ARTICLE IN PRESS MOLBIO-10951; No. of Pages 10 Molecular & Biochemical Parasitology xxx (2016) xxx–xxx Contents lists available at ScienceDirect Molecular & Biochemical Parasitology OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis Eva Nývltová 1 , Tamara Smutná 1 , Jan Tachezy, Ivan Hrdý ∗ Department of Parasitology, Charles University in Prague, Faculty of Science, Prague, Czech Republic a r t i c l e i n f o Article history: Received 4 September 2015 Received in revised form 5 January 2016 Accepted 12 January 2016 Available online xxx Keywords: Trichomonas Hydrogenosomes OsmC Glycine decarboxylase complex Peroxide Lipoate a b s t r a c t Osmotically inducible protein (OsmC) and organic hydroperoxide resistance protein (Ohr) are small, thiol-dependent peroxidases that comprise a family of prokaryotic protective proteins central to the defense against deleterious effects of organic hydroperoxides, which are reactive molecules that are formed during interactions between the host immune system and pathogens. Trichomonas vaginalis, a sexually transmitted parasite of humans, possesses OsmC homologues in its hydrogenosomes, anaerobic mitochondrial organelles that harbor enzymes and pathways that are sensitive to oxidative damage. The glycine decarboxylase complex (GDC), which consists of four proteins (i.e., L, H, P and T), is in eukaryotes exclusively mitochondrial enzymatic system that catalyzes oxidative decarboxylation and deamination of glycine. However, trichomonad hydrogenosomes contain only the L and H proteins, whose physiological functions are unknown. Here, we found that the hydrogenosomal L and H proteins constitute a lipoate-dependent redox system that delivers electrons from reduced nicotinamide adenine dinucleotide (NADH) to OsmC for the reductive detoxification of peroxides. Our searches of genome databases revealed that, in addition to prokaryotes, homologues of OsmC/Ohr family proteins with predicted mitochondrial localization are present in various eukaryotic lineages. Therefore, we propose that the novel OsmC-GDC-based redox system may not be limited to T. vaginalis. © 2016 Elsevier B.V. All rights reserved. 1. Introduction Reactive oxygen species (ROS) such as singlet oxygen, superoxide anion, hydroxyl radical and hydrogen peroxide commonly form within living cells and are generally regarded as unwanted byproducts of aerobic metabolism that have the potential to damage DNA, lipids and proteins [1,2]. In addition, upon reaction with cellular components, ROS give rise to oxidatively modified molecules such as lipid hydroperoxides, which further promote cellular damage [3]. Therefore, various protective mechanisms against ROS are universally present in all domains of life [2]. However, ROS can also play positive physiological roles. At very low concentrations, some ROS participate in signaling or in cell proliferation and differentiation pathways [4]; at higher levels, ROS are microbicidal molecules that are produced by the host immune system to combat bacterial ∗ Corresponding author at: Charles University in Prague, Faculty of Science, Department of Parasitology, Viničná 7, Prague 128 44, Czech Republic. E-mail address: [email protected] (I. Hrdý). 1 These authors contributed equally to the work. and parasitic infections [5,6]. Thus, for pathogenic microorganisms, effectively coping with host-mounted non-specific responses that include ROS is vitally important. Whereas no enzymatic protection exists against singlet oxygen and hydroxyl radical, superoxide-removing superoxide dismutases (SODs) are almost ubiquitous, and catalase and various peroxidases that detoxify hydrogen peroxide are very common in both non-pathogenic and pathogenic organisms [7,8]. The first enzyme discovered to specifically reduce and detoxify organic hydroperoxides in bacteria was a two-component, NADH-dependent AhpC/AhpF alkyl hydroperoxide reductase from pathogenic Salmonella typhimurium [9]. In 1998, a novel, small, cysteine-based protein that conferred protection against hydroperoxides, organic hydroperoxide resistance protein (Ohr), was described from a phytopathogenic gamma proteobacterium, Xanthomonas campestris pv. phaseoli [10]; subsequently, it was recognized that the protein, along with its homologue, i.e., osmotically inducible protein (OsmC) [11], is widely distributed in the bacterial world [12]. The involvement of OsmC in defense against oxidative stress was proposed in a study investigating the survival of Escherichia coli cells in media of different NaCl concentrations [13], and the hydroperoxidase activity of http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 0166-6851/© 2016 Elsevier B.V. All rights reserved. Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 2 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx OsmC has subsequently been repeatedly demonstrated [14–17]. The peroxide-reducing activity of Ohr/OsmC depends on dithiols as the source of electrons for the reduction of the protein’s disulfide group that forms upon catalytic reduction of organic hydroperoxide [18]; thus, the enzymes qualify as thiol-dependent peroxidases (EC 1.11.1.15). The physiological electron donor was unknown, although dihydrolipoate has been suggested as a possible donor [14,19]. It has been shown that Ohr from another plant pathogen, Xylella fastidiosa, can effectively reduce hydroperoxides using electrons from lipoylated proteins, such as the E2 subunit of ɑketoglutarate dehydrogenase or pyruvate dehydrogenase complex that is reduced by dihydrolipoamide dehydrogenase (E3 subunit) at the expense of NADH [20]. This observation has been replicated for Mycobacterium smegmatis [21], and similar results have been obtained for recombinant OsmC from E. coli, fostering the proposed functional relatedness of Ohr and OsmC homologues [14,20]. Anaerobic parasitic protists need effective antioxidant defense because they have to deal with fluctuating oxygen levels in their natural environment as well as with ROS formed by host immune cells. Such parasite is also Trichomonas vaginalis [22–25], a flagellated protist that causes widespread sexually transmitted disease in humans [26–28]. Trichomonads, like many other anaerobic protists of diverse evolutionary affinities, do not possess classical oxygenrespiring mitochondria. Instead, they harbor modified, anaerobic forms of this organelle in trichomonads (and several other protists) called hydrogenosomes because of their hydrogen-producing enzymatic machinery linked to carbohydrate metabolism [29]. Initial biochemical work [30,31], later supplemented with genomic data and proteomic studies [32–35], allowed for the construction of a biochemical map of the hydrogenosome, which is characterized by the presence of oxygen-sensitive enzymes, such as pyruvate:ferredoxin oxidoreductase, hydrogenase or proteins of iron–sulfur cluster assembly machinery [36]. Hydrogenosomal enzymes are protected from oxidative damage and xenobiotics by a relatively wide array of organellar defense proteins. Some of these proteins are almost ubiquitous (SOD or the thioredoxin system); some proteins are typical in anaerobic bacteria rather than in eukaryotes and were likely acquired through horizontal gene transfer (rubrerythrin, flavodiiron protein, iron–sulfur flavoprotein) [37–39]. In addition, the genome sequence analysis predicted and the proteomic data confirmed [32,35] the presence of OsmC homologues in hydrogenosomes, demonstrating that the occurrence of OsmC proteins is not limited to prokaryotes, as was generally believed. One of the unexpected results of the T. vaginalis genome project annotation [33] was the identification of L and H proteins of the four-protein glycine decarboxylase complex (GDC; also known as the glycine cleavage system, EC 1.4.4.2) [40]. Subsequently, the presence of both proteins in hydrogenosomes was verified in independent proteomic studies [32,35]. GDC catalyzes reversible, NAD- and tetrahydrofolate-dependent oxidative decarboxylation and deamination of glycine into CO2 and NH3 , and it produces NADH and methylenetetrahydrofolate, the one-carbon unit donor molecule that participates in a number of crucial biosynthetic pathways. In eukaryotes, it is an exclusively mitochondrial protein complex consisting of the P protein, which contains pyridoxal phosphate; the lipoate-binding, hydrogen-transferring H protein (analogous to E2 protein of ɑ-ketoacid dehydrogenase complexes); the tetrahydrofolate-binding T protein; and the L protein, the NADdependent dihydrolipoamide dehydrogenase that is analogous to the E3 protein of ɑ-ketoacid dehydrogenase complexes [41,42]. The presence of the L and H proteins in T. vaginalis has been interpreted as evidence for the as yet unknown role of hydrogenosomes in amino acid metabolism, particularly when a serine hydroxymethyltransferase (SHMT), which is usually functionally linked with GDC through the methylenetetrahydrofolate pool, was also identified in both the genome and the organelles [43]. However, dihydrofolate reductase, which is necessary for the formation of tetrahydrofolate (the active coenzyme of both GDC and SHMT), is apparently absent from the T. vaginalis genome (http://trichdb.org/trichdb/). Moreover, the remaining two proteins of GDC, the P and T proteins, could not be found in the T. vaginalis genome, and because the L and H proteins alone cannot function as glycine decarboxylase, their physiological roles, along with the proposed involvement of hydrogenosomes in amino acid metabolism, have remained unresolved. Here, we report a new and unexpected role of the two redox-active proteins of incomplete hydrogenosomal GDC in the reduction of the OsmC protein and suggest that this system functions in antiperoxide defense in T. vaginalis hydrogenosomes. Moreover, we show that OsmC homologues are present in several eukaryotic lineages, most likely in the mitochondria, which suggests that lipoate- and OsmC-based peroxide detoxification might be a presently unrecognized feature of mitochondria in many eukaryotes. 2. Materials and methods 2.1. Phylogenetic analysis The sequences of OsmC and Ohr proteins were downloaded from GenBank release 208.0 (originally 267 sequences) and were aligned to the T. vaginalis sequences (TVAG 410350, TVAG 125860, TVAG 412560, TVAG 210030) retrieved from the T. vaginalis genome database (http://trichdb.org/trichdb/) by MAFFT [44] using the L-INS-i algorithm in the Geneious 7.0.5 platform (Biomatters Ltd.). Sequences with >99% sequence identity to another sequence were removed, and the alignment was manually trimmed in the Geneious 7.0.5 platform, which resulted in 167 sequences (Table S1) with 134 aligned positions. The Le and Gascuel (LG) substitution model and the gamma model of rate heterogeneity were selected for construction of the phylogenetic tree using the maximum likelihood method in RAxML version 7.2.8 [45] in the Geneious 7.0.5 platform. The substitution model was selected based on a ProTest 2.4 server test (http://darwin.uvigo.es/). The statistical support was assessed by bootstrapping with 500 repetitions in RAxML. Bayesian posterior probabilities were calculated in MrBayes 3.2.1 [46] with four Monte Carlo Markov Chains, each with 100,000 generations with a sampling frequency every 200 generations. The cleavage site of hydrogenosomal/mitochondrial processing peptidase was predicted using the PSORT II program (http://psort.hgc.jp/cgi-bin/ runpsort.pl). 2.2. Cultivation of trichomonads T. vaginalis strain T1 (J.H. Tai, Institute of Biomedical Sciences, Taipei, Taiwan) was maintained axenically in Diamond’s Trypticase-Yeast-extract-Maltose (TYM medium) supplemented with 10% heat-inactivated horse serum without agar at pH 6.2 and 37 ◦ C [47]. 2.3. Cloning of genes The genes for the L (TVAG 272760) and H proteins (TVAG 177600) of the T. vaginalis GDC fragment, OsmC homologue (TvOsmC, TVAG 410350), thioredoxin reductase (TVAG 281360) and thioredoxin (TVAG 385350) were amplified without hydrogenosomal targeting peptides (Table S2) by PCR from T. vaginalis genomic DNA and inserted into the pET42b+ vector (Novagen) with 6x histidine (His) tags fused to the C-termini of the constructs. T. vaginalis expresses two paralogues of H protein [35]. and both appear to function as substrates for the L protein Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model ARTICLE IN PRESS MOLBIO-10951; No. of Pages 10 E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx [40]; the H2 paralogue was selected in this study because of its high activity with the L protein [40]. TvOsmC-HA construct for T. vaginalis transformation and localization of the gene product within trichomonad cells was inserted into the TagVag2 plasmid [48] with 2× hemagglutinin (HA) tag at the C-terminus. The cells were transfected by electroporation with 30 g of circular plasmid and selected with 200 g.ml−1 of Geneticin. 2.4. Expression of recombinant proteins Recombinant, His-tagged L and H proteins of GDC, TvOsmC protein, thioredoxin reductase and thioredoxin were expressed in E. coli BL21 (DE3) cells. Bacterial cultures were grown in LB medium at 22 ◦ C to OD A600 = 0.7, and the induction of protein expression was initiated by the addition of 0.5 mM isopropyl d-1-thiogalactopyranoside (IPTG). To assure the lipoylation of H protein, 0.1 mM ␣-lipoic acid (Sigma–Aldrich) was added to the medium. The induction was performed for 6 h. The proteins were purified from the soluble fraction of bacterial lysates obtained by sonication and centrifugation (150,000 × g for 30 min) by affinity chromatography on a Ni-NTA agarose column under native conditions, according to the manufacturer’s protocol (Qiagen). The purified recombinant TvOsmC protein was used as an antigen for rat immunization. 2.5. Activity assays of recombinant proteins The capacity d of recombinant T. vaginalis OsmC protein to reduce peroxides and to accept electrons from the redox system consisting of recombinant T. vaginalis L and H proteins was assayed as the continuous oxidation of NADH at 340 nm (ε = 3220 M−1 cm−1 ) and 25 ◦ C using a Shimadzu UV 2600 spectrophotometer. The inorganic peroxide H2 O2 and two organic peroxides, cumene hydroperoxide and tert-butyl hydroperoxide (Sigma–Aldrich), were used as final electron acceptors of the reaction. The assay mixture contained 2 ml of 50 mM Tris–HCl buffer, pH 8.0, approximately 2 M L protein, approximately 8 M H protein, up to approximately 2.5 M TvOsmC, 180–200 M NADH and varying concentrations of peroxides (0.05–0.5 mM). The capacity of up to 0.5 mM free ␣-lipoic acid to replace the lipoylated H protein was tested using 50 mM stock solution of lipoic acid in 40% ethanol. The kinetic parameters of TvOsmC were calculated using double-reciprocal plots in the Shimadzu UV–vis software package. The interaction of recombinant T. vaginalis thioredoxin reductase (∼2.5 M) and thioredoxin (∼4 M) with TvOsmC was assayed spectrophotometrically in 50 mM Tris–HCl, pH 8.0, in the presence of either NADPH or NADH and 0.5 mM H2 O2 under aerobic and anaerobic conditions. Up to 2 mM dithiothreitol (DTT) and 2mercaptoethanol were tested as sole potential electron donors to TvOsmC. In this case, a ferrous oxidation–xylenol orange (FOX) assay [49] was used to determine the initial and final concentrations of hydrogen peroxide. 2.6. Activity determination in hydrogenosomes Peroxide-reducing activity was also assayed in hydrogenosomes obtained by fractionation of the T. vaginalis homogenate (see below). The assay mixture consisted of 2 ml of 50 mM Tris–HCl buffer, pH 8.0, 180–200 M NADH or NADPH, 0.5 mM peroxide (H2 O2 , cumene hydroperoxide or tert-butyl hydroperoxide), 0.1% Triton X-100 and hydrogenosomal sample. The spectrophotometric assay at 340 nm and 25 ◦ C was performed under anaerobic conditions using nitrogen-purged buffer in a stoppered cuvette. All calculations were based on at least three independent measurements. 3 2.7. TvOsmC native molecular mass determination The native molecular mass of TvOsmC was determined by gel filtration chromatography using a BioLogic HR system (Bio-Rad). The affinity-purified recombinant TvOsmC was applied onto a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated with sodium/potassium phosphate buffer (5.0 mM Na2 HPO4 , 1.5 mM KH2 PO4 , 150 mM NaCl, pH 7.4) using a flow rate of 0.75 ml min−1 . The native molecular mass of TvOsmC was calculated from the calibration curve determined by running appropriate protein standards under the same conditions. 2.8. Fractionation of T. vaginalis cells All steps were performed at 4 ◦ C and in the presence of protease inhibitors (Complete Mini EDTA-free cocktail tablets, Roche). To separate subcellular fractions, cells (1–2 l of culture) were collected by centrifugation at 1500 × g for 15 min, washed and resuspended in ST buffer (0.25 M sucrose, 10 mM Tris, 0.5 mM KCl, pH 7.2). The cells were lysed by sonication on ice, and the sonicate was centrifuged at 800 × g for 10 min. The supernatant was centrifuged at 9000 × g for 20 min to separate crude cytosolic and large granular fractions. The crude cytosolic fraction was further centrifuged at 150,000 × g for 30 min to obtain the final cytosol. The large granular fraction was fractionated on a self-forming gradient of 45% Percoll to obtain purified hydrogenosomes as described in [50]. Subcellular fractions were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting for the presence of TvOsmC and hydrogenosomal marker protein malic enzyme [51] using polyclonal rat and rabbit antibodies, respectively. To further examine the localization (surface vs. matrix) of TvOsmC within hydrogenosomes, a protease protection assay was used. Isolated hydrogenosomes were incubated for 30 min at 37 ◦ C in ST buffer supplemented with 200 g ml−1 trypsin (Sigma–Aldrich) or trypsin with 0.1% Triton X-100. The incubation was terminated upon the addition of 5 mg ml−1 soybean trypsin inhibitor followed by incubation on ice for 5 min. Proteins were precipitated with cold acetone for 1 h at −20 ◦ C and analyzed by SDS-PAGE followed by Western blotting. Purified hydrogenosomes were also used in biochemical experiments to assess the in situ peroxide-reducing activity. The processed amino terminus of affinity-purified TvOsmC was determined by Edman degradation, as described in [38]. 2.9. Immunofluorescence microscopy To visualize the HA-tagged TvOsmC protein within trichomonad cells, transfected trichomonads were fixed for 30 min by the addition of formaldehyde (final 1%) directly into the culture in TYM medium. The cells were collected and carefully washed with PBS by centrifugation at 800 × g for 5 min, allowed to adhere to glass slides coated with 3-aminopropyltriethoxysilane (Sigma–Aldrich) for 30 min, washed with PBS and permeabilized with 1% Triton X100 for 10 min. Fixed cells were incubated with polyclonal rabbit anti-malic enzyme antiserum (Eurogentec) to label the hydrogenosomal marker protein malate dehydrogenase (decarboxylating) (malic enzyme) [51] and with mouse anti-HA monoclonal antibody (Exbio) as primary antibodies. Secondary antibodies were Alexa Fluor 594 donkey anti-rabbit and Alexa Fluor 488 donkey anti-mouse antibodies (Life Technologies). Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx 4 Fig. 1. Phylogeny of the OsmC/Ohr protein family. Maximum-likelihood (ML) tree of OsmC/Ohr proteins (167 taxa and 134 sites). Bootstrap support (BP) and posterior probability (PP) values were calculated for each branch using RAxML and MrBayes, respectively. Only BV and PP values greater than 50% and 0.5, respectively, are shown. Branches with maximum support (BV = 100%; PP = 1.0) are depicted using black circles. Prokaryotic and eukaryotic clades are shown in black and in color, respectively. 3. Results 3.1. Phylogenetic analysis The amino acid sequence of the putative OsmC protein (TVAG 410350, OsmC-1 in [32]) was used as a query for a BLAST search in the TrichoDB database (http://trichdb.org/trichdb/ ), which resulted in the identification of three additional homologous genes with sequence identities of 19–28%, all annotated as conserved hypothetical proteins. The lengths of the proteins were in the range of 139–146 amino acid residues. Unexpectedly, subsequent searches for OsmC/Ohr homologues in the NCBI database revealed, in addition to prokaryotic homologues, a large set of eukaryotic sequences. The highest sequence identity to T. vaginalis OsmC (TVAG 410350) belonged to the sequence from the nematode Trichuris trichiura (17%). To determine to which gene subfamily the trichomonad and other eukaryotic proteins belonged, we performed phylogenetic analysis with 167 OsmC/Ohr orthologues. The sequences clustered with high statistical support into two major groups corresponding to OsmC and Ohr protein subfamilies; however, the branching order within these two groups was not resolved (Fig. 1). All trichomonad sequences clustered to a common branch within the OsmC group, which indicated that the trichomonad proteins represent OsmC paralogues. The OsmC subfamily also contained members of mainly unicellular eukaryotes from the groups Amoebozoa, Stramenopila, Ciliophora, Chromerida, Haptophyta, and Choanoflagellida and two members of Viridiplantae. In addition to T. vaginalis, Naegleria gruberi and Naegleria fowleri (Joel B. Dacks, University of Alberta, Edmonton, Canada, personal communication of unpublished data) were the only other members of the excavata group with an identified OsmC homologue. T. trichiura was the only metazoan found to possess OsmC. Interestingly, fungi of the Basidiomycota and Ascomycota groups possess both OsmC and Ohr homologues. Another eukaryotic gene for Ohr-like protein was found in a bryophyte Physcomitrella patens (Fig. 1). 3.2. Localization of TvOsmC in T. vaginalis cells All T. vaginalis OsmC paralogues possess short amino terminal extensions with predicted cleavage sites for mitochondrial (hydrogenosomal) processing peptidase [33,52] that resemble hydrogenosomal targeting sequences [53] (Fig. 2). The protein processing within hydrogenosomes was confirmed experimentally for TvOsmC TVAG 410350, the subject of this study. Affinity isolation of the TvOsmC protein from hydrogenosomes followed by Edman sequencing [38] revealed the absence of the first 11 amino acids compared with the conceptually translated protein, which corresponded to its cleavage exactly at the predicted site (Fig. 2). Specific subcellular localization of the TvOsmC protein was further investigated by immunofluorescence microscopy of Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx 5 Fig. 2. Comparative protein sequence alignment of T. vaginalis OsmC. TvOsmC paralogues (TvOsmC1, TVAG 410350; TvOsmC2, TVAG 125860; TvOsmC3, TVAG 210030; TvOsmC4, TVAG 412560) were aligned with OsmC and Ohr sequences of Pseudomonas putida (PpOsmC, Q88RP0; PpOhr, Q88LR9) and Xanthomonas campestris (XcOsmC, Q8P768; XcOhr, Q8PDS1). An asterisk (*) indicates two conserved cysteine residues that constitute the active site for peroxide detoxification. The predicted amino terminal hydrogenosomal targeting sequences are underlined, and the experimentally verified cleavage site of hydrogenosomal processing peptidase is marked by an arrow. The cleavage site of hydrogenosomal processing peptidase was predicted using the PSORT II program (http://psort.hgc.jp/cgi-bin/runpsort.pl). T. vaginalis cells expressing the tagged version of TvOsmC (Fig. 3A) and by Western blot analysis of T. vaginalis subcellular fractions using antibodies against both tagged and native versions of the protein (Fig. 3B). All of these data showed that TvOsmC localized exclusively in hydrogenosomes. Specifically, the protein was present inside hydrogenosomes (as expected for soluble protein with processed organellar targeting presequence) and was not associated with the hydrogenosomal surface, as it was protected against proteolytic degradation unless the hydrogenosomes were permeabilized with detergent (Fig. 3C). The double-band present in fractions obtained from the cells expressing the HA-tagged TvOsmC (Fig. 3B,C, top panel) likely indicates incomplete processing of the tagged protein. Importantly, bioinformatics analysis of other eukaryotic OsmC/Ohr homologues predicted the presence of amino-terminal mitochondrial targeting sequences in most proteins, indicating high probability of their mitochondrial localization, which is consistent with localization of trichomonad OsmC in hydrogenosomes (Table S1). 3.3. Activity of TvOsmC depends on L and H proteins It has been shown that OsmC/Ohr proteins could accept electrons for the reduction of hydroperoxide substrates from lipoylated proteins such as E2 components of ɑ-ketoglutarate dehydrogenase or pyruvate dehydrogenase complex [14,20,21]. Therefore, we decided to investigate the possibility that the lipoate-bearing protein homologous to the H component of GDC, known to be present in T. vaginalis hydrogenosomes [32,35,40], functions in a similar way. It is relevant to mention that T. vaginalis encodes putative lipoate–protein ligase (http://trichdb.org/trichdb/, TVAG 094820), the enzyme indispensable for joining the lipoyl prosthetic group with its acceptor protein. We presumed that the hydrogenosomal homologue of L protein with the activity of dihydrolipoamide dehydrogenase [40] would use NADH for the reduction of the oxidized lipoamide moiety of H protein to dihydrolipoamide that, in turn, could serve as a reductant for TvOsmC, a Cys-based peroxidase. Indeed, when all three affinity-purified, recombinant proteins (L, H and TvOsmC; Fig. 4A, inset; with the amount of TvOsmC being the limiting factor in the reaction mixture) were combined in a spectrophotometric cuvette, the oxidation of NADH upon the addition of peroxide could be readily observed. The activity was dependent on the presence of all components of the system. Low background activity (up to approximately 10% of activity of the complete sys- Table 1 Kinetic parameters of recombinant T. vaginalis OsmC. NADH and recombinant L and H proteins constituted the electron-donating system to TvOsmC. The experimental conditions are specified in Section 2. Substrate Km [M] kcat [s−1 ] kcat /Km [M−1 s−1 ] H2 O2 2.042 ± 0.096 343.2 ± 9.26 1.68 × 108 ± 0.071 × 108 Cumene hydroperoxide 4.303 ± 0.032 216.8 ± 7.52 5.04 × 107 ± 0.142 × 107 tert-Butyl hydroperoxide 3.935 ± 0.064 234.1 ± 7.36 5.95 × 107 ± 0.169 × 107 tem) (Fig. 4) of L protein alone (a flavoprotein) with NADH and without peroxide substrate under aerobic conditions was likely caused by turnover reaction due to oxidation of L protein’s FAD cofactor. The peroxides assayed as substrates of TvOsmC were the organic hydroperoxides cumene hydroperoxide and tert-butyl hydroperoxide and H2 O2 . The kinetic parameters of recombinant TvOsmC are presented in Table 1. Unlike its bacterial homologues from both protein subfamilies that strongly preferred organic hydroperoxides over H2 O2 [14,20,21,54], T. vaginalis OsmC displayed similar activities with all three peroxides tested, with hydrogen peroxide being the best substrate (Table 1). In its quaternary structure, TvOsmC resembles its prokaryotic homologues [14,16,55] and natively forms approximately 28 kDa dimers, as determined by gelfiltration chromatography using affinity-purified TvOsmC (Fig. S1). It has been reported previously that the T. vaginalis L protein is able to reduce free lipoamide, albeit with Km approximately 10 times higher for lipoamide than for its presumed natural electron acceptor, the lipoylated H protein [40]. We replaced the H protein with lipoamide in our assay system and indeed observed the reduction of peroxides (an approximately 50 times higher concentration of lipoamide over H protein was needed), demonstrating that T. vaginalis OsmC could use free dihydrolipoamide in place of the lipoylated H protein as a reductant. NADH-dependent peroxidase activity was also assayed in purified hydrogenosomes of wild-type, untransformed trichomonads. We observed low activity with all three peroxides tested (approximately 48 ± 4, 18 ± 5 and 23 ± 4 nanomol mg−1 min−1 for H2 O2 , cumene hydroperoxide and tert-butyl hydroperoxide, respectively). To assess whether TvOsmC could also accept electrons from other reducing systems or is specific only for its presumed electrondonating system consisting of NADH and the L and H proteins, we prepared two recombinant proteins of the hydrogenosomal Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 6 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx Fig. 3. Localization of TvOsmC in T. vaginalis cells. A, immunofluorescence microscopy. T. vaginalis OsmC fused at the C-terminus with an HA tag co-localized with hydrogenosomal marker protein malic enzyme. Proteins were visualized using rabbit polyclonal antibody against T. vaginalis malic enzyme and mouse monoclonal antibody against the HA tag. Donkey Alexa Fluor 488 ␣-rabbit and donkey Alexa Fluor 594 ␣-mouse were used as secondary antibodies. B, Western blot analysis of T. vaginalis cellular fractions. Cell lysate (Lys) was separated into cytosolic (Cyto) fraction and hydrogenosomes (Hydr) using differential and isopycnic centrifugation. The fractions of TvOsmC-overexpressing cells were probed with anti-HA tag antibody (top panel). The fractions of wild-type trichomonads were probed with antibody against native TvOsmC (bottom panel). C, Western blot of the protease protection assay of T. vaginalis hydrogenosomes. Hydr, hydrogenosomes without treatment; Hydr + trypsin, hydrogenosomes treated with 200 g ml−1 trypsin; Hydr + trypsin + TX-100, hydrogenosomes treated with trypsin and Triton X-100. The transblotted hydrogenosomes were probed with an anti-HA tag antibody labeling TvOsmC and with an antibody against the hydrogenosomal marker protein malic enzyme (ME). thioredoxin system, the T. vaginalis thioredoxin reductase and thioredoxin, both of which have been identified in hydrogenosomes and which, together with the effector thioredoxin peroxidase, constitute a near-ubiquitous system for peroxide detoxification [37,56]. We could not observe any activity of TvOsmC with the above proteins and with neither NADH nor NADPH as reductants Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx 7 Fig. 4. Representative figure showing the NADH-dependent reduction of peroxides by TvOsmC mediated by L and H proteins. The figure demonstrates that all components are necessary for TvOsmC peroxidase activity. A, B, and C, initiation of the reaction by the addition of TvOsmC, H protein and peroxide, respectively, into the otherwise complete reaction mixture (see Section 2). Inset, SDS-PAGE of purified recombinant TvOsmC, H and L proteins. Approximately 2–3 g of affinity-purified proteins were loaded on a 15% gel and visualized by Coomassie staining. Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx 8 and with hydrogen peroxide as substrate in the spectrophotometric assay. DTT, mercaptoethanol, NADH or NADPH alone did not function as immediate electron donors of T. vaginalis OsmC. 4. Discussion In this communication, we report the presence of the OsmC protein, a peroxidase effective in reductive detoxification of hydrogen peroxide and organic hydroperoxides, in the hydrogenosomes of the anaerobic human pathogen, T. vaginalis. Remarkably, the TvOsmC protein is functionally linked with a GDC-derived reducing system that consists of NADH-dependent dihydrolipoamide dehydrogenase (L protein homologue) and the lipoate-bearing, hydrogen carrier protein (H protein homologue). This novel function of L and H proteins of GDC provides an explanation for their presence in T. vaginalis hydrogenosomes [40] and points to a currently unrecognized mechanism of peroxide detoxification in mitochondria of other eukaryotes. The incomplete GDC that consists of only H and L proteins was unexpectedly identified during the annotation of the T. vaginalis genome project [33], and the presence of these two proteins in hydrogenosomes was considered evidence for the unknown role of these organelles in amino acid metabolism [40]. However, because the two other obligatory components of GDC, the P and T proteins, are missing in the T. vaginalis genome [33], it was proposed that the incomplete complex cannot function as glycine decarboxylase, and the real functions of the two GDC protein homologues remained unknown [36]. Our investigations demonstrated that the dihydrolipoamide dehydrogenase activity of L protein can use NADH for the reduction of oxidized lipoamide moiety of H protein that, in turn, serves as a proximal reductant of trichomonad OsmC. This mechanism is similar to that observed by Cussiol et al. [20], who found that both bacterial Ohr and OsmC proteins could be reduced by lipoylated E2 subunits of pyruvate dehydrogenase and ɑ-ketoglutarate dehydrogenase complexes. Trichomonad OsmC extends the list of peroxide-detoxifying proteins already known to reside in hydrogenosomes, i.e., rubrerythrin and thioredoxin peroxidase [37,56]. The presence of rubrerythrin and a complete thioredoxin system in hydrogenosomes has been demonstrated by proteomic methods, but the proof of actual peroxide-detoxifying activity in hydrogenosomes is lacking. Functional characterization of TvOsmC and detection of corresponding protein and activity in hydrogenosomes shows that the organelles are indeed capable of coping with peroxides. Hydrogen peroxide, an oxidant and precursor of highly toxic hydroxyl radicals arising via the Fenton reaction, could be formed within hydrogenosomes by the activity of SOD [37,57], by autoxidation of FeS cluster- and flavin-containing proteins, and by the activity of the recently described FeS flavoprotein [39]. The activity of TvOsmC and other peroxidases is thus likely of vital importance for a parasite that relies on oxygen-sensitive hydrogenosomal enzymes [33,58–60] but that inhabits an environment that is naturally not completely oxygen free [25], is under the control of the host’s immune system, which has the capacity to form ROS and organic hydroperoxides as reaction intermediates in respiratory burst [61–63], and is populated with bacterial flora that produces hydrogen peroxide [24,64]. Several recent studies have conducted transcriptomic [23,24] or proteomic [32] analyses of T. vaginalis cells exposed to challenging conditions such as increased oxygen levels and contact with human vaginal epithelia [23] and glucose [24] or iron [32] limitation. Along with the responses of other specifically affected genes, a common response of T. vaginalis to these different stimuli, i.e., the upregulation of genes/proteins involved in protection against oxidative stress, can be observed in all of these studies. Although TvOsmC did not show the profound changes exhibited by, e.g., superoxide dismutase, thioredoxin and thioredoxin peroxidase or rubrerythrin, it was consistently among the significantly upregulated proteins. Of the four OsmC paralogues coded by T. vaginalis, only the TvOsmC TVAG 410350, the subject of this study, has been identified in all the above-mentioned studies as well as studies of the hydrogenosomal proteome [35] and the transformation of T. vaginalis cells to the amoeboid, cell-adherent form [65], indicating that this is the dominantly expressed paralogue. Low-level transcription (increased in response to glucose limitation) has been noted for TvOsmC TVAG 412560 and TVAG 210030 [24], and the former paralogue has also been identified in the proteomic study of iron-limited trichomonads [32]. In comparison with bacterial Ohr and OsmC homologues [14,20,21,54] that strongly prefer organic hydroperoxides, the kinetic parameters of the trichomonad OsmC are comparable for all peroxides tested, with hydrogen peroxide being the preferred substrate. Similar activity with both inorganic and organic peroxides was also observed for OsmC from Thermus thermophilus [16]. In absolute numbers, TvOsmC activity was approximately 15 times higher with organic hydroperoxides and up to four orders of magnitude higher with hydrogen peroxide than were the activities of the characterized bacterial homologues. In addition, the apparent Km value of trichomonad protein was approximately one order of magnitude lower for organic hydroperoxides and more than two orders of magnitude lower for hydrogen peroxide (Table 1) than were the values of bacterial proteins [14,20,54], indicating that TvOsmC was the more efficient catalyst. Similar to its prokaryotic counterparts, trichomonad protein apparently cannot accept electrons from thioredoxin, a redox dithiol protein that is linked to NADPH-dependent thioredoxin reductase and is present in hydrogenosomes as a component of the thioredoxin antioxidant system [37,56]. In contrast to bacterial OsmC/Ohr proteins, the trichomonad homologue could not be reduced even by millimolar concentrations of DTT. The only active reductant we found was dihydrolipoate, either free or attached to the H protein homologue. The GDC is present in mitochondria of all known eukaryotes, and mitochondrial L proteins share a common ancestry with their ␣-proteobacterial homologs [40]. However, previous phylogenetic analysis of L protein from T. vaginalis hydrogenosomes revealed its independent origin, most likely by horizontal gene transfer from firmicute bacteria, whereas the origin of H protein was not resolved [40]. Although our phylogenetic analysis clustered the OsmC and Ohr orthologues to well-supported branches, low statistical support of branching order within the OsmC subtree did not allow resolution of the origin of T. vaginalis OsmC paralogues. Lateral gene transfer is the most plausible explanation for the isolated occurrences of OsmC proteins in T. vaginalis, N. gruberi and N. fowleri within the large excavata supergroup together with their unstable branching with various bacterial groups. It is thus appealing to speculate that the two proteins homologous to GDC components now present in T. vaginalis were in fact never part of the glycine decarboxylase complex, but rather functioned in a bacterium as electron donors to OsmC, and that the whole system was acquired altogether by a T. vaginalis predecessor through lateral gene transfer. Interestingly, the situation in T. vaginalis is mirrored in the firmicute bacterium Enterococcus faecalis, which lacks P and T proteins and possesses L, H and OsmC homologues [42]. Nevertheless, the interpretation of the phylogenetic reconstruction requires caution. We cannot exclude the possibility that the OsmC topology is affected by factors such as mutation saturation, different substitution rates and codon usage preferences that may result in the false grouping of analyzed orthologues. However, the patchy distribution of OsmC genes in eukaryotes favors the inference of lateral gene acquisition [66]. Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 G Model MOLBIO-10951; No. of Pages 10 ARTICLE IN PRESS E. Nývltová et al. / Molecular & Biochemical Parasitology xxx (2016) xxx–xxx Unexpectedly, we found that the genes for OsmC and Ohr proteins are present in some other eukaryotic organisms. Moreover, most of these eukaryotic proteins seem to be localized in mitochondria based on the presence of short amino terminal extensions with predicted cleavage sites for mitochondrial processing peptidase. Indeed, OsmC-like protein is listed among mitochondrial proteins in the proteomic study of Tetrahymena thermophila [67]. However, to our knowledge there has been no functional study of OsmC/Ohrlike proteins in a eukaryote, except for this work. Based on our discovery of a novel function of L and H proteins in the reduction of hydrogenosomal TvOsmC and the predicted (or even confirmed) presence of OsmC/Ohr proteins together with GDC in mitochondria of many eukaryotes, it is likely that, in addition to their known functions, H and L components of mitochondrial GDC (or analogous E2 and E3 subunits of ␣-ketoacid dehydrogenase complexes) may also interact with OsmC/Ohr proteins and thus participate in the protection of mitochondria against peroxidative stress. Further studies are required to test this exciting hypothesis. Interestingly, OsmC/Ohr proteins are not generally present in eukaryotes; rather, they have a quite peculiar distribution. They are present mostly in protists of various eukaryotic lineages, such as Amoebozoa, Stramenopila, Ciliophora, Haptophycae, Choanoflagellida and Excavata, while identified multicellular organisms include only Basidiomycota (Fungi), T. trichiura (Nematoda) and P. patens (Viridiplantae). We also noticed that OsmC/Ohr homologues are present in most representatives of Ascomycota, Basidiomycota, Stramenopila and Amoebozoa (except Entamoeba histolytica) that we tested, while occurrences of these proteins in other lineages (Metazoa, Viridiplantae, Excavata) are rare. For example, the parasitic nematode T. trichiura is the only metazoan with an annotated OsmC-like protein. Therefore, it might be of interest to assess the OsmC/Ohr proteins of eukaryotic pathogens (including N. fowleri, which causes typically fatal meningoencephalitis) as drug targets and to test whether the described peroxide-detoxifying system in T. vaginalis is essential for the viability of this parasite. Regrettably, gene silencing in T. vaginalis is still not amenable, and inactivation of the four OsmC paralogues present in the trichomonad genome would be hardly feasible. However, the deletion of a primary electron donor for peroxide reduction by TvOsmC, the hydrogenosomal L protein homologue, which is coded by a single gene (http://trichdb.org/trichdb/), could be a more realistic task in the effort to explore the physiological role of TvOsmC and its GDC-like redox system. In conclusion, we have demonstrated the capacity of hydrogenosomal L and H proteins, for which no physiological functions were known, to serve as an NADH-dependent reducing system for TvOsmC peroxidase activity. We propose that a similar functional connection that involves redox-active protein complexes with lipoylated components (GDC, ␣-ketoacid dehydrogenase complexes) may exist in the mitochondria of those eukaryotes that possess OsmC/Ohr-like proteins. 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Immun. 80 (2012) 3900–3911, http://dx.doi. org/10.1128/IAI.00611-12. [66] J.O. Andersson, R.P. Hirt, P.G. Foster, A.J. Roger, Evolution of four gene families with patchy phylogenetic distributions: influx of genes into protist genomes, BMC Evol. Biol. 6 (2006) 27, http://dx.doi.org/10.1186/1471-2148-6-27. [67] D.G. Smith, R.M. Gawryluk, D.F. Spencer, R.E. Pearlman, K.W. Siu, M.W. Gray, Exploring the mitochondrial proteome of the ciliate protozoon Tetrahymena thermophila: direct analysis by tandem mass spectrometry, J. Mol. Biol. 374 (2007) 837–863, http://dx.doi.org/10.1016/j.jmb.2007.09.051. Please cite this article in press as: E. Nývltová, et al., OsmC and incomplete glycine decarboxylase complex mediate reductive detoxification of peroxides in hydrogenosomes of Trichomonas vaginalis, Mol Biochem Parasitol (2016), http://dx.doi.org/10.1016/j.molbiopara.2016.01.006 7. Conclusions Hydrogenosomes of T. vaginalis belong to best characterized mitochondrion related organelles (MRO). These organelles harbor oxygen sensitive enzymes of ATP generating pathways. Trichomonas is considered an anaerobe, but it inhabits the environment of vaginal mucosa where it is challenged with oxidative stress caused by ROS generated by host immune cells and fluctuating levels of oxygen during menstruation cycle. Adverse conditions of trichomonads’ natural niche require the presence of effective enzymatic mechanisms to cope with oxidative stress and to protect vulnerable enzymes in trichomonad hydrogenosomes. Current state of knowledge shows that this protection of hydrogenosomes is provided by superoxide dismutase and putative peroxidases such as thioredoxin peroxidase and rubrerythrin. In this work, we have identified and characterized new enzymes that protect hydrogenosomes against oxidative damage and xenobiotics. 7.1 TvFDP Annotation of T. vaginalis genome revealed the presence of a number of bacterial proteins, probably acquired by lateral gene transfer. One of these proteins is a flavodiiron protein (TvFDP), a member of a broad flavodiiron protein family. This protein was first identified by proteomic methods during the research for other hydrogenosomal enzymes. Hydrogenosomal localization of the protein was subsequently confirmed by immunofluorescent microscopy of HA-tagged protein construct expressed in trichomonads and by immunoblotting with specific antibodies. Native protein was determined to be a ~92 kDa dimer, binding two iron atoms and one FMN moiety per monomer. This dimeric quaternary structure with head-to-tail conformation of monomers is essential for protein function, because it enables efficient electron transfer from the flavin of one monomer to diiron centre of the other one. Enzymatic activity of homologous bacterial proteins was established as O2 and/or NO reduction dependent on protein electron carrier such as rubredoxin. Although rubredoxin is not coded in T. vaginalis genome, trichomonads code for several copies of [2Fe-2S] ferredoxin, small electrontransport protein linked to key enzymes of hydrogenosomal metabolism such as PFOR, hydrogenase and Complex I remnant. We have found that enzymatic system consisting of ferredoxin 1 and PFOR or 38 ferredoxin 1 and Complex I can effectively reduce recombinant TvFDP via reduced ferredoxin 1 at the expense of electrons derived from pyruvate (PFOR activity) or NADH (Complex I activity). TvFDP, reduced by either system, was immediately reoxidized by oxygen. The mechanism of oxygen reduction involves four electrons, resulting in full reduction of O2 to water. Although bacterial FDP homologues are able to reduce NO, this activity could not be detected in TvFDP. The TvFDP specificity solely for oxygen is in agreement with the data reported for the FDPs characterized from other parasitic protists E. histolytica and G. intestinalis. 7.2 TvIsf3 Recent proteomic studies of hydrogenosomes revealed the presence of three paralogues of iron-sulfur flavoprotein (Isf). The protein named TvIsf3 was the one with highest level of mRNA transcription, in cells grown under basal conditions, out of all seven Isf paralogues present in T. vaginalis genome. Localization of TvIsf3 in hydrogenosomes was confirmed by immunofluorescence microscopy of T. vaginalis cells transformed with plasmid bearing complete TvIsf3 gene with N-terminal hydrogenosomal targeting presequence as well as by Western blot analysis of purified trichomonad cell fractions. The recombinant TvIsf3 with 6x His tag was expressed in E. coli and purified to apparent homogeneity. The protein was characterized as homodimer with native molecular mass of approximately 51 kDa. Although the majority of enzymatically active TvIsf3 protein was purified in a dimeric composition, in accordance with the data reported for several bacterial Isf proteins, small portion of TvIsf3 was eluted from gel filtration chromatography column as a tetramer. This finding is in agreement with structural reports on Isf proteins from Methanosarcina thermophila (MtIsf) and Archaeoglobus fulgidus (AfIsf) and it thus seems possible that native TvIsf3 forms tetramers which disintegrate into stabile dimers under potentially suboptimal conditions of column chromatography during purification. TvIsf3 was found to bind one FMN and 4Fe-4S active center as indicates pattern of the UV-visible spectrum. TvIsf3 was found to accept electrons from NADH, however the protein sequence does not contain a recognizable Rossmann fold (nor the others characterized Isfs do) with a glycine rich motif responsible for binding of pyridine nucleotide coenzyme and bacterial homologue MtIsf was referred to preferentially utilize ferredoxin as electron donor. 39 TvIsf3 similar to MtIsf is able to reduce oxygen, although trichomonad protein enables two-electron reduction of oxygen to hydrogen peroxide in contrast to MtIsf fourelectron reduction to water. Despite the assumption that TvIsf3 could reduce hydrogen peroxide in the manner of MtIsf, we could observe only marginal reaction rate with this substrate in NADH dependent continuous assays. We required the same results with NO, however both substrates readily reoxidized chemically reduced TvIsf3. It could be a consequence of oxidation of reduced FMN in TvIsf3 by strong oxidants, such NO and hydrogen peroxide, in a thermodynamically favorable reaction. Another explanation could be that TvIsf3 displays substrate specifity similar to substrate specifity described for Fdps or the physiological reaction with NO/H2O2 requires different electron donor, such as ferredoxin, as it was described for enzymatic activity of MtIsf. Metronidazole (5-nitroimidazole) is used in treatment of trichomoniasis and other infections caused by anaerobic pathogens. Reactive nitro radical anion, resulting from oneelectron reduction of the drug within the susceptible cell, is believed to exert the cytotoxic effect. However, TvIsf3 reduces metronidazole with even number of electrons, precluding the formation of free radical and producing less toxic product instead. Potential role of TvIsf3 in resistance to metronidazole requires further study. Chloramphenicol, a broad-spectrum antibiotic with a nitro-group, can also be reduced by TvIsf3. Remarkably, reductive inactivation of chloramphenicol was described in cultures of anaerobic bacterium Clostridium acetobutylicum that possesses Isf homologues. Detoxification of chloramphenicol and other aryl-nitro compounds was mediated by bacterial cell extracts in ferredoxin-dependent and pyruvate-stimulated reaction by means of unknown mechanism. We can speculate that the described antibioticreducing capacity of C. acetobutylicum extracts depends on Isf activity which uses electrons derived from pyruvate by PFOR and transferred by ferredoxin, and thus represents a novel mechanism of nitro antibiotic resistance. However, such suggestion needs to be experimentally verified. 7.3 TvOsmC TvOsmC represents a hydrogenosomal thiol-dependent peroxidase effective in reductive detoxification of both hydrogen peroxide and organic hydroperoxides. This peroxidase, originally believed to occur exclusively in prokaryotes, is functionally linked with two proteins homologous with components of (normally four-protein) glycine 40 decarboxylase complex (GDC), the H and L proteins, revealed during the annotation of T. vaginalis genome. Experiments in this work provided explanation for the presence and function of H and L proteins of incomplete GDC in T. vaginalis hydrogenosomes. Incomplete GDC cannot function as glycine decarboxylase in aminoacid metabolism and physiological role of H and L proteins in hydrogenosomes was unknown. We revealed that the lipoate-bearing, hydrogen carrier protein (H protein homologue) and NADH-dependent dihydrolipoamide dehydrogenase (L protein homologue) reduce TvOsmC in NADHdependent reaction thus constituting a novel system providing protection against peroxides. 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