Download Prokaryotic proteins of antioxidant defense in Trichomonas vaginalis

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
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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
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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
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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.
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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
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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
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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. We
thank Míša Marcinčiková (Charles University) for excellent technical
assistance, Petr Jedelsky (Charles University) for mass spectrometry
analyses, and Jan Mach (Charles University) for help with the figures.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
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over the last 15 years, as well as the recently completed annotation of the T. vaginalis genome (5), has shown that most
already identified hydrogenosomal proteins involved in electron generation and transport are coded by multiple and
clearly distinct genes. These include seven genes encoding
PFOR homologues, up to nine malic enzymes, seven ferredoxins, seven iron-sulfur flavoproteins, and up to nine putative
hydrogenases, of which four are equipped with a predicted
hydrogenosomal targeting peptide; many of these proteins are
transcribed, also as indicated by complex EPR spectroscopic
data (7, 16, 26–28; unpublished proteomic data). It is possible
that certain PFOR homologues preferentially use particular
ferredoxins, while others could function as electron acceptors
of complex I or in iron-sulfur cluster assembly machinery (49).
The involvement of iron-sulfur flavoproteins even in these
known reactions also cannot be excluded, as indicated by preliminary experiments (unpublished data). Other possible
sources of electrons besides pyruvate and NADH (generated
by the malic enzyme), such as amino acids or ␣-glycerophosphate (6), could also play a role. Some combinations of substrate, electron-generating enzyme, and electron-transporting
protein would likely be more efficient than others in shuttling
electrons toward TvFDP.
In summary, by fusing a hydrogenosomal targeting peptide
to a class A FDP, most likely obtained from an anaerobic
bacterium by lateral gene transfer, T. vaginalis equipped the
hydrogenosomes with a new enzyme. Functional data suggest
that this acquisition helped the parasite to cope with oxidative
stress in these oxygen-sensitive organelles.
VOL. 8, 2009
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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:
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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-
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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
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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-
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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
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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.
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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.
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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
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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.
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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
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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.
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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.
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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.
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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. After all, it has been noted that
many anaerobic bacteria possess multiple Isf paralogs, and it was
suggested that these proteins perform for anaerobes universally
important and possibly diverse functions (1). The same applies
also to T. vaginalis, which encodes seven distinct Isf paralogs and
expresses at least three of them in its hydrogenosomes. Both metronidazole and chloramphenicol (and numerous other nitro
compounds) are naturally occurring antibiotics, and reactive oxygen and nitrogen species belong to the repertoire of antimicrobial response of the immune system. The activity of Isf described
here may, therefore, represent a novel mechanism of defense
against xenobiotics that is unique in trichomonads and amoebae
but likely common among prokaryotic anaerobes.
Smutná et al.
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6.3 Publication 3: Nývltová et al., 2015
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.
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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),
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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),
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[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),
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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
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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),
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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
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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.
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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].
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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.
Acknowledgements
This work was supported by the Czech Science Foundation (13-09208J) and the Biotechnology and Biomedicine
Center of the Academy of Sciences and Charles University
(CZ.1.05/1.1.00/02.0109) from the European Regional Development
Fund. We acknowledge the use of unpublished genomic data from
N. fowleri V212 that was generously provided by Joel B. Dacks, University of Alberta, Edmonton, Canada.
9
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.molbiopara.2016.
01.006.
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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.
Unexpectedly, our phylogenetic analyses revealed the presence of genes coding for OsmC
and related Ohr proteins in some other eukaryotic organisms, mostly in protists of various
eukaryotic lineages, such as Amebozoa, Stramenopila, Ciliophora, Haptophycae,
Choanoflagellida and Excavata. Identified multicellular organisms included Basidiomycota
(Fungi), Trichuris trichiura (Nematoda) and Physcomitrella patens (Viridiplantae). Most
of these eukaryotic proteins seem to be localized in mitochondria, based on the presence of
predicted mitochondrial targeting sequence. These findings suggest that H and L proteins
of GDC (which is present in mitochondria of all known eukaryotes) could fulfill so far
unrecognized function as components of electrontransport pathway linked with OsmC/Ohr
proteins and participate in the protection of mitochondria of many eukaryotes against
peroxides.
41
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