TREPAR 1754 No.

of Pages 14

Review

Redox Pathways as Drug Targets

in Microaerophilic Parasites
David Leitsch,1,* Catrin F. Williams,2 and Ivan Hrdý3

The microaerophilic parasites Entamoeba histolytica, Trichomonas vaginalis, Highlights

and Giardia lamblia jointly cause hundreds of millions of infections in humans Microaerophilic parasites, such as
Entamoeba histolytica, Trichomonas
every year. Other microaerophilic parasites such as Tritrichomonas foetus and
vaginalis, and Giardia lamblia account
Spironucleus spp. pose a relevant health problem in veterinary medicine. for hundreds of millions of annual infec-
Unfortunately, vaccines against these pathogens are unavailable, but their tions worldwide.

microaerophilic lifestyle opens opportunities for specifically developed chemo- These organisms are susceptible to
therapeutics. In particular, their high sensitivity towards oxygen can be oxygen because molecular oxygen
exploited by targeting redox enzymes. This review focusses on the redox and several of its derivatives deactivate
essential proteins, such as pyruvate:fer-
pathways of microaerophilic parasites and on drugs, either already in use or redoxin oxidoreductase and ferredoxin.
currently in the state of development, which target these pathways.
In the body, microaerophilic parasites
can be exposed to molecular oxygen
The challenge of oxygen for microaerophilic protozoan parasites and reactive oxygen species. In order
Most organisms encounter oxygen which is present in habitats exposed to air. However, to prevail, microaerophilic parasites
whereas higher organisms such as humans and animals avidly utilize oxygen as a terminal employ a large array of antioxidant
electron acceptor in the respiratory chain and, consequently, depend on it for survival, they are enzymes.

inhabited by numerous microorganisms which are vulnerable to oxygen. In body parts such as Many of these enzymes are different
the intestine, the oral cavity, and the vagina oxygen concentrations are much lower than in the from their counterparts in the host and
blood or most organs. In the large intestine, oxygen concentrations are close to zero, which constitute targets for chemotherapy.
5-nitroimidazoles, including metroni-
enables the growth of microorganisms commonly termed as anaerobes, e.g. the commensal
dazole, compromise the antioxidant
gut bacteria of the genera Bacteroides, Clostridium, Bifidobacterium and others. In the defense. Several novel antiparasitic
stomach, the small intestine and the vagina, however, oxygen is present, if only in decreased drugs, including auranofin, NBDHEX,
concentrations. In air, the oxygen concentration amounts to 158 mm Hg, while only reaching and several garlic constituents seem to
have a similar mode of action.
15 mm Hg at the stomach mucosa, 38 mm Hg in the small intestine, and 8–35 mm Hg in the
vagina of humans [1]. Microorganisms inhabiting these niches have a divergent metabolism
which is often devoid of oxidative phosphorylation as found in mitochondria and aerobic
microorganisms. Instead, they are dependent on enzymes that are highly susceptible to
oxygen. Crucial steps in the breakdown of glucose, for example, are catalyzed by enzymes
which have iron-sulfur clusters (see Glossary) in their active sites. These include pyruvate: 1
Institute for Specific Prophylaxis and
ferredoxin oxidoreductase (PFOR) [2] which catalyzes the decarboxylation of pyruvate, or Tropical Medicine, Center for
hydrogenase [3] which harnesses reducing equivalents to generate hydrogen gas. Oxygen Pathophysiology, Infectiology, and
and several reactive oxygen species (ROS) derived thereof, such as superoxide radical Immunology, Medical University of
Vienna, Austria
anion (O2 ) or hydrogen peroxide (H2O2), damage iron-sulfur clusters [1] and, consequently, 2
School of Engineering, Cardiff
are highly toxic. Importantly, however, molecular oxygen is tolerated in low concentrations by University, Cardiff, Wales, United
many of these organisms and can even enhance their growth [4,5], so that the term “micro- Kingdom
3
Department of Parasitology, Charles
aerophiles” is more suitable in such cases. In terms of evolution, enzymes such as PFOR or University, Faculty of Science, Prague,
hydrogenase are typical of anaerobic bacteria, but not all anaerobic/microaerophilic micro- Czech Republic
organisms are bacteria. Humans and animals also host protists which have possibly acquired
anaerobic pathways by horizontal gene transfer from bacterial donors [6–8]. Some of these are
*Correspondence:
commensals, others are parasites. The most notable microaerophilic parasites in man are david.leitsch@meduniwien.ac.at
Entamoeba histolytica, Giardia lamblia (syn. intestinalis, duodenalis), and Trichomonas (D. Leitsch).

Trends in Parasitology, Month Year, Vol. xx, No. yy https://doi.org/10.1016/j.pt.2018.04.007 1

© 2018 Elsevier Ltd. All rights reserved.
 TREPAR 1754 No. of Pages 14

vaginalis. They are causative agents of amoebic liver abscess (E. histolytica), diarrhea and Glossary
gastrointestinal symptoms (E. histolytica and G. lamblia), and of vaginitis, cervicitis, and IC50: concentration of a given drug
urethritis (T. vaginalis), respectively. Together, these parasites are responsible for a consider- at which the activity of an enzyme or
able disease burden with a combined number of annual infections approaching the 500 million the growth of a microorganism is
reduced to 50% of the maximal rate.
mark [9]. However, other microaerophilic protists, such as Blastocystis hominis, Balantidium Iron-sulfur clusters: organometallic
coli, and Dientamoeba fragilis, also infect humans. Other related microaerophilic parasites complexes of sulfur and iron
infect cattle (Tritrichomonas foetus), reptiles (Entamoeba invadens), or fish (Spironucleus functioning as prosthetic groups in
proteins such as pyruvate:ferredoxin
vortens and Spironucleus salmonicida), to name just a few.
oxidoreductase and ferredoxin. Iron-
sulfur clusters abound in anaerobic/
As microaerophilic pathogens, these organisms do not only have to struggle with oxygen microaerophilic organisms and are
diffusing into the cell from the surrounding host tissue but also with active countermeasures of highly vulnerable to oxygen, reactive
oxygen species, and nitric oxide.
the host immune system [10] which include the production and secretion of ROS such as H2O2,
5-nitroimidazoles: derivatives of
nitric oxide (NO), and reactive nitrogen species (RNS) such as peroxynitrite (ONOO ). In imidazole with a nitro group at the
order to prevail, microaerophilic parasites deploy a large arsenal of redox pathways which are C5 position. This substance class
fascinating in terms of their diversity to biochemists and evolutionary biologists alike. Moreover, comprises metronidazole, the most
often prescribed drug against
from a medical standpoint, redox pathways constitute the Achilles’ heel of microaerophilic
infections with anaerobic/
parasites and, therefore, are attractive drug targets for chemotherapy. microaerophilic pathogens. The nitro
group is only reduced in these
Major redox pathways in microaerophilic parasites organisms, which is a prerequisite for
the compounds’ toxicity.
Redox enzymes involved in the removal of reactive oxygen species Reactive oxygen species (ROS):
Microaerophilic parasites encounter several harmful reactive species of oxygen and nitrogen in derivatives of O2 of a higher reduced
their life cycles [11]. In these organisms, molecular oxygen (O2) in itself is already toxic, causing grade and with high reactivity,
rapid oxidation of iron-sulfur clusters and other molecules with a low redox potential. Super- including radicals such as the
superoxide radical anion (O2. ) and
oxide (O2 ) and H2O2 are even more reactive and toxic in low concentrations [1,12,13]. In most non-radical species such as
microaerophilic parasites studied, O2 is removed, i.e. dismutated into H2O2 and O2, by hydrogen peroxide (H2O2).
superoxide dismutase [14–17] (Figure 1). All superoxide dismutases from microaerophilic Reactive nitrogen species (RNS):
are formed through the reaction of
parasites described so far are of the iron type and of bacterial origin. A notable exception
nitric oxide (∙ NO) and superoxide,
to this rule is G. lamblia which features a superoxide reductase [18] (Figure 1), an enzyme which and include peroxynitrite (ONOO )
employs a completely different mechanism by reducing O2 via a histidine- and cysteine- and several of its decay products
coordinated iron center using an as yet unknown electron donor, but also generating H2O2 as such as nitrogen dioxide (∙ NO2).
an end product. Hydrogen peroxide is a particularly dangerous compound because it quickly
reacts with ferrous iron (Fe2+) which is formed through reduction of ferric iron (Fe3+) by reduced
thiols [19]. Indeed, ferrous iron abounds in the reductive environment of the anaerobic/micro-
aerophilic cell, leading to the generation of hydroxyl radicals (OH ∙), which rapidly attack
proteins, membranes, and nucleic acids. Interestingly, catalase, a very effective scavenger
of H2O2, is absent from almost all microaerophilic parasites with the notable exception of
T. foetus [20]. This is indeed surprising as other trichomonadid parasites, including T. vaginalis,
do not have this enzyme. Instead, H2O2 is removed mostly by peroxiredoxins via a cysteinyl-
dependent mechanism through reduction to water [21]. Likewise, alkyl peroxides are
reduced to water and the corresponding alcohol. Peroxiredoxins have been characterized
in E. histolytica [22,23], T. vaginalis [24] and G. lamblia [25] (Figure 1). They all belong to the
standard 2-Cys-type Prx1 [21]. Peroxiredoxins of this type are recycled by thioredoxins, which
act as electron shuttling proteins and reduce the catalytic cysteines of peroxiredoxins via their
own catalytic cysteines. These, in turn, are reduced by thioredoxin reductase (TrxR), again via
catalytic cysteines [26] (Figure 1). Ultimately, the reducing power is derived from NADPH and
transferred to the catalytic site of TrxR via its flavin adenine dinucleotide (FAD) cofactor. All
TrxRs described in microaerophilic parasites so far are of the small bacterial type [24,27–29].
The large eukaryotic type of TrxR does not exist in these organisms and, consequently,
selenocysteine-dependent reduction of thioredoxin does not occur [26]. The role of the
thioredoxin-mediated redox system is not confined to the removal of H2O2 but also includes

2 Trends in Parasitology, Month Year, Vol. xx, No. yy

TREPAR 1754 No. of Pages 14

cTrxR cSOD T. vaginalis

cTrx cTrx NADH Flavin
-S-S- -SH HS- 2 O2-· H2O2 + O2 oxidase reductase

O2 H2O O2 H2O2
cTrx cTrx cPrx cPrx
-SH HS- -S-S- -SH HS- -S-S-
cPrx cPrx H2O2 2 H2O
-S-S- -SH HS-
hTrxR
hSOD
FDP
cTrx cTrx hTrx hTrx
-S-S- -SH HS-
-SH HS- -S-S- 2 O2-. H2O2 + O2
O2 H2O
Isf
Oxidized proteins hTrx hTrx hPrx hPrx
Ribonucleo de reductase -SH HS- -S-S- -SH HS- -S-S-
Methionine sulfoxide reductase O2 H2O2
hPrx hPrx H2O2 2 H2O
Transcrip on factors OsmC -S-S- -SH HS-
Rubrerythrin
Hcp hPrx
H2O2 H2O -SH HS-
H2O2 H2O
? ?

2 H2O H2O2

Prx Prx
-S-S- -SH HS-
Trx Trx
-SH HS- -S-S-
E. histolyƟca
TrxR
Oxidized proteins
Serine acetyltransferase Trx Trx
-SH HS- -S-S- SOD FDP
Transcrip on factors
Prx Prx
-
-S-S- -SH HS- 2 O2 · H2O2 + O2 O2 H2O

2 H2O H2O2

NADH NADPH
Isf
Rubrerythrin oxidase oxidoreductase

H2O2 H 2O
O2 H2O2 O2 H2O O2 H2O2

H2O2 2 H2O
?

G. lamblia
Prx1b Prx1b
-SH HS- -S-S-

SOR
TrxR Trx Trx
-SH HS- -S-S-
Trx Trx Prx
? 2O2·
-
H2O2 + O2
Prx
-S-S- ? -SH HS- -S-S- -SH HS-
NADH
FDP oxidase Diaphorase
Prx1a Prx1a
-SH HS- -S-S-
O2 H2O O2 H2O O2 H2O2
Trx Trx
-SH HS- H2O2 2 H2O
-S-S-
?
Oxidized proteins Grx
Methionine sulfoxide reductase Flavo
Transcrip on factors hemoglobin
Trx-dependent
pathways
NO + O2 NO3-

(See figure legend on the bottom of the next page.)

Trends in Parasitology, Month Year, Vol. xx, No. yy 3

 TREPAR 1754 No. of Pages 14

the maintenance and repair of oxidized proteins [26], e.g. via the reduction of oxidized
methionines by methionine sulfoxide reductase [26]. Further, it is involved in the reduction
of ribonucleotide reductase (which is absent from E. histolytica and G. lamblia, but present in
T. vaginalis), and the regulation of transcription factors. In T. vaginalis, thioredoxin also seems to
have an important role in the catabolism of excess cysteine [30]. However, TrxR also exerts a
general disulfide reductase activity independent of its major substrate thioredoxin [26]. In fact, it
has not been possible to identify a functional thioredoxin in G. lamblia as yet [31]. Several small
thioredoxin-domain-containing proteins are encoded in the G. lamblia genome, but none of
these functions as a thioredoxin. In contrast, functional thioredoxins were readily identified in
T. vaginalis [24] and E. histolytica [28].

In addition to thioredoxin-dependent peroxidases, other factors involved in the removal of H2O2

have been found in microaerophilic parasites (Figure 1). Functional rubrerythrin was detected in
T. vaginalis [32] and E. histolytica [33] in hydrogenosomal and mitosomal fractions, respectively.
This enzyme is of bacterial origin and is predicted to reduce H2O2 via its diiron and rubredoxin
domains. To this end, it depends on a reducing factor which, to date, has not been identified.
Another hydrogenosomal protein with peroxidase activity was identified just recently: OsmC,
which is a thiol-dependent peroxidase that harnesses NADH via lipoate, a disulfide containing
compound, for the reduction of H2O2 and alkyl peroxides [34]. Surprisingly, the electrons
provided by NADH in this reaction are transferred by the L and H proteins of the glycine
decarboxylase complex, which is incomplete in T. vaginalis as it lacks components P and T [34].
Indeed, providing NADH to OsmC might be the sole physiological purpose of proteins L and H,
thereby constituting a novel type of lipoate-dependent peroxide reduction.

Redox enzymes involved in the removal of molecular oxygen

In addition to ROS, O2 itself also has to be kept at low levels, and microaerophilic parasites
employ a variety of oxygen scavenging pathways to achieve this. Entamoeba histolytica [35],
T. vaginalis [36], and G. lamblia [37] each encode a flavodiiron protein (Figure 1). These proteins,

Figure 1. Redox pathways in microaerophilic parasites. Trichomonas vaginalis, upper panel, contains a vast
repertoire of redox pathways, including thioredoxin reductase (TrxR) [24], peroxiredoxins (Prx) [24], and superoxide
dismutases (SOD) [16]. Thioredoxins (Trx) fulfil a varied number of functions. Strikingly, homologs of these enzymes are
equally present in the hydrogenosome (grey ovoid) [32,112,113]. Pathways marked with the letter “c” in front of the
enzyme’s name are cytosolic, those with the letter “h” localize to the hydrogenosome. In addition, NADH oxidase and flavin
reductase remove oxygen in the cytosol [39,44]. In the hydrogenosome, two further peroxide detoxifying enzymes exist:
rubrerythrin [32] and an unusual OsmC system which depends on protein-bound lipoate and NADH. Further, oxygen is
scavenged by a flavodiirion protein (FDP) [36] and iron-sulfur flavoprotein (Isf) [47]. According to the GenBank (NIH), the T.
vaginalis genome further encodes a hybrid cluster protein (Hcp) [120], which binds one iron-sulfur cluster and one unique
“hybrid” iron-sulfur-oxygen cluster. Hybrid cluster proteins can detoxify NO, hydroxylamine or H2O2 [55], but, as shown
recently, may actually function through specific protein S-nitrosylation that counters nitrosative stress [56]. E. histolytica,
central panel, has a functional TrxR pathway, comprising TrxR [28], Trx [28], and Prx [22,23]. Importantly, Prx mostly
localizes to the cell surface [22] where it acts as virulence factor [81] and degrades H2O2 released by host cells. In addition,
E. histolytica has a FDP [35] for the removal of oxygen. Possibly (indicated by presenting the three enzymes in square
brackets), oxygen scavenging is further enhanced by a NADH oxidase [41] and NADPH oxidoreductase [42], and Isf [48].
Superoxide is removed by a functional SOD [14]. A rubrerythrin possibly localizes to the mitosome (grey ovoid). The redox
pathways in G. lamblia, lower panel, are unusual in several aspects. To date, no functional Trx was detected [31], although
the parasite has a standard bacterial type TrxR [29]. Further, G. lamblia employs a superoxide reductase (SOR) instead of a
SOD for the removal of superoxide [18]. Two functional peroxiredoxins were described in G. lamblia, of which one (Prx1a)
presumably localizes to the cytosol and the other (Prx1b) to the surface [25]. Question marks indicate that not all
components of a thioredoxin-system have been identified to date. Both a FDP [37], NADH oxidase [40], and diaphorase
[46] are employed for the removal of oxygen. G. lamblia also has a flavohemoglobin for the oxidation of NO to nitrate (NO3 )
[52,53], thereby being the first parasite described to have this pathway. The presence of a glutaredoxin (Grx) in the
mitosome (grey ovoid) is a conundrum given the fact that glutathione was not found in G. lamblia [60]. In one study, TrxR
and Prx1a were also found to localize to the mitosome [121]. Reduced proteins are presented in red, oxidized proteins in
blue. Flavoenzymes are indicated by a frame in orange.

4 Trends in Parasitology, Month Year, Vol. xx, No. yy

 TREPAR 1754 No. of Pages 14

originally designated as A-type flavoproteins, were acquired through lateral gene transfer from
bacteria and can reduce oxygen to water (prokaryotic counterparts also possess NO or mixed
NO/O2 reducing activity) [38]. The catalytic core possesses two domains, a flavin mononucle-
otide (FMN) binding domain, which constitutes the electron entry site, and a metallo-b-lacta-
mase-like domain with an active non-heme Fe-Fe center, the actual oxygen reduction site. In
addition, T. vaginalis [39] and G. lamblia [40] have a flavin-dependent NADH oxidase, which
reduces O2 by harnessing NADH as a reducing agent. The T. vaginalis enzyme has not been
characterized at the gene level but could be distinct from NADH oxidase in G. lamblia. NADH
oxidase activity was also observed in cell extracts of E. histolytica [41], but no further studies
were performed to characterize the enzyme. However, E. histolytica also has an NADPH-
dependent oxidoreductase, which reduces oxygen to H2O2 [42]. An unrelated enzyme with
identical function, termed flavin reductase, exists in T. vaginalis [39,43,44]. This enzyme rapidly
reduces oxygen to H2O2 via its FMN cofactor. Interestingly, flavin reductase constitutes the
main source of endogenously produced H2O2 in T. vaginalis [43], emphasizing the importance
of flavin reductase for oxygen scavenging in this parasite. In G. lamblia, NADPH-dependent
reduction of oxygen was also observed but the enzyme has not been identified as yet [45].
However, oxygen was shown to be reduced to H2O2 by diaphorase [46]. In addition to flavin
reductase and diaphorase, another enzyme class exists in hydrogenosomes of T. vaginalis [47]
and in E. histolytica [48]: iron-sulfur flavoprotein. This enzyme can reduce oxygen to H2O2 but
also shows high reactivity with xenobiotics. Hence, the physiological functions of iron-sulfur
flavoprotein in T. vaginalis and E. histolytica have remained unclear as yet.

Enzymes involved in the removal of NO and RNS

In addition to oxidative stress, microaerophilic parasites also encounter nitrosative stress,
mainly brought about by NO, which is produced by neutrophils and macrophages to kill off
pathogens [49]. NO exhibits a pleiotropic mode of action with multiple targets. In E. histo-
lytica, NO was shown to inhibit PFOR and to induce fragmentation of the endoplasmic
reticulum [50]. If NO reacts with O2 , then peroxynitrite (ONOO ) is formed, a RNS which also
causes a number of harmful effects, including the deamination of nucleotides and the
irreversible inhibition of metalloenzymes [51]. The pathways dedicated to defense against
nitrosative stress are less numerous than those involved in the antioxidant defense. In
G. lamblia, NO was shown to be oxidized to nitrate by a flavohemoglobin [52,53] (Figure 1),
a member of an enzyme class commonly found in bacteria and fungi. In T. vaginalis,
degradation of NO was observed in cell extracts but the responsible enzyme was not
identified [54]. It is interesting to note, however, that T. vaginalis encodes a hybrid cluster
protein (Hcp), an enzyme described to reduce multiple nitrosative and oxidant stressors,
including NO [55], and to participate in the defense against nitrosative stress through specific
protein S-nitrosylation [56]. In E. histolytica a NO degradation pathway is yet to be found. In
contrast, peroxynitrite is a substrate for peroxiredoxins, which reduce it to nitrite and water
[57]. Graphical summaries of the major redox enzyme pathways in microaerophilic parasites
can be found in Figure 1. The unusually high allele number of many redox enzymes in
T. vaginalis is discussed in Box 1.

Low molecular mass thiols as redox buffers

The redox equilibrium in a cell is not only shaped by redox enzymes but also by low molecular
mass thiols. They constitute a redox buffer which is protective against oxidative stress and
electrophilic compounds in general. In most organisms this buffer is constituted by glutathione,
a tripeptide [58] consisting of a glutamate, a cysteine and a glycine residue. Glutathione is also a
reducing substrate for glutaredoxins, which fulfill a role similar to thioredoxin-dependent
peroxiredoxins. Oxidized glutathione is subsequently recycled by the flavoenzyme glutathione

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 TREPAR 1754 No. of Pages 14

Box 1. High allele numbers of redox enzymes in T. vaginalis

The genome of T. vaginalis is not only extraordinarily large, i.e. at least 160 Mb in size [111], but it also contains an
astonishingly high number of genes (60,000). Many enzymes are encoded by multiple alleles in the T. vaginalis genome,
including superoxide dismutase, peroxiredoxin, TrxR and flavin reductase. At least 10 peroxiredoxins can be found in
the databases of which seven were demonstrated to be expressed at the protein level [32,96]. Two peroxiredoxins
(TVAG_165690, TVAG_055200) [32], two TrxRs (TVAG_348010, TVAG_125360) [112] and at least one superoxide
dismutase localize to the hydrogenosome (TVAG_049140) [113]. In total, seven alleles for superoxide dismutase exist in
the T. vaginalis genome of which five were shown to be expressed at the protein level [96,113]. The number of alleles of
iron-sulfur flavoproteins also amounts to seven. It is presently unclear why T. vaginalis employs such a large number of
alleles for these enzymes because only a small fraction of them have been assessed in functional studies [24]. More is
known about flavin reductase, which exists as seven homologs in T. vaginalis and reduces oxygen to H2O2 via its
cofactor FMN [44]. Of these seven homologs only one, FR1, has a sufficiently low Km for FMN (around 10 mM) to be of
physiological relevance, whereas the others reduce oxygen via FMN either only very slowly or require FMN concentra-
tions that are far above physiological levels. Nevertheless, several alleles of flavin reductase are expressed at a time in
many T. vaginalis strains [44]. The physiological role of these homologs has remained unclear as yet. The vast multiplicity
of functional homologs of many proteins, which also includes cytosolic metabolic enzymes such as lactate dehy-
drogenase or the electron carrier ferredoxin in the hydrogenosome, is a puzzling and fascinating trait of T. vaginalis.

disulfide reductase [58]. Depletion of glutathione can lead to formation of ROS, DNA damage
and apoptosis [59]. Interestingly, in many microaerophilic parasites cysteine, rather than
glutathione, constitutes the main cellular redox buffer as shown in G. lamblia [60], T. vaginalis
[61,62], and E. histolytica [63] (summarized in Table 1). As an exception to this rule, S. vortens
uses glutathione as its low molecular mass thiol [17]. Cysteine is an important constituent of
proteins and other molecular structures, including iron-sulfur clusters. However, it is also highly
reactive and quickly reduces metals such as Fe3+, which in turn react with H2O2 leading to the
formation of toxic hydroxyl radicals [30]. Thus, in organisms with a high intracellular concen-
tration of oxygen, and thereby of ROS, cysteine levels have to be kept low (around 200 mM),
whereas glutathione, which is less reactive, may be present in millimolar amounts. This,
however, does not apply to microaerophilic parasites, at least as long as they inhabit niches
with low oxygen concentrations. E. histolytica [64,65] and T. vaginalis [62] can both synthesize
cysteine by employing cysteine synthase but they utilize different precursors. In E. histolytica,
O-acetylserine, formed by serine acetyltransferase (SAT) is used [65,66]. In T. vaginalis, SAT is
missing and O-phosphoserine is the likely precursor of cysteine [62]. In G. lamblia, the situation
is quite puzzling. Although cysteine is the major low molecular mass thiol in this protist [60], G.
lamblia cannot synthesize cysteine by itself, and therefore is fully dependent on extracellular
cysteine sources. It does, however, encode enzymes for glutathione synthesis although glutathi-
one has not been detected among the low-molecular thiols in G. lamblia [60]. In fact, glutathione
disulfide reductase seems to be missing as well. Nevertheless, G. lamblia has a functional
glutaredoxin, which localizes to the parasite’s mitosome [67], another mitochondrion-derived

Table 1. Low molecular mass thiols in microaerophilic parasites

Parasite Thiol buffer Thiol synthesis? References

E. histolytica L-cysteine cysteine synthesis [64,66]

T. vaginalis L-cysteine cysteine synthesis [62]

G. lamblia L-cysteine glutathione synthesis pathway predicted [60]

to exist but no cysteine synthesis

S. vortens glutathione cysteine synthesis pathway predicted [17]

to exist in related S. salmonicida

T. foetus L-cysteine cysteine synthesis pathway predicted to exist [122]

Blastocystis Not known glutathione synthesis pathway predicted to exist According to

GenBank (NIH)

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 TREPAR 1754 No. of Pages 14

organelle. The implications of these findings are unclear as yet but it is currently speculated that G.
lamblia has a very low concentration of glutathione, too low to be easily detected by standard
methods, but high enough to fuel glutaredoxin in the mitosome [67].

Redox pathways of microaerophilic parasites and their interactions with the

host
Most microaerophilic parasites do not invade host cells and are too big to be phagocytized by
macrophages. Thus, they are not subjected to respiratory bursts within macrophages. Never-
theless, the amounts of NO and ROS being released by macrophages and epithelial cells into
the gut lumen and other environments seem to play a role in the control of infections with
microaerophilic parasites [68–70]. Moreover, host cells are not the only source of these
molecules. Lactobacilli inhabiting the vaginal epithelium also release H2O2 in order to outcom-
pete other microorganisms [71]. All this necessitates functional antioxidant and antinitrosative
pathways. Also other defense measures such as the inhibition of NO production by scavenging
arginine, the precursor of NO in many cells, are in place in G. lamblia [69], T. vaginalis [72], and
E. histolytica [73]. In addition, NO production can also be inhibited by modulating signal
pathways in immune cells [74].

However, irrespective of defense measures undertaken by the immune system, microaerophilic

parasites must be capable of dealing with oxidative stress. They either transgress into tissues
that are well oxygenated, e.g. E. histolytica when it causes abscesses in the liver [75], or they
are confronted with varying oxygen concentrations, such as T. vaginalis during the menstrual
cycle [76] and G. lamblia through digestive activity of the small intestine [77]. Given the reductive
environment of the anaerobic cell, intracellular oxygen is quickly reduced to ROS. Moreover,
some oxygen scavenging reactions in these organisms, e.g. as catalyzed by flavin reductase in
T. vaginalis, result in the formation of H2O2 [44]. Thus, microaerophilic parasites robustly
upregulate antioxidant enzymes upon contact with oxygen [78–80].

A striking and well-studied example of the importance of antioxidant enzymes for survival and
virulence is peroxiredoxin in E. histolytica. This enzyme is expressed to a greater extent in
virulent strains than in avirulent ones [81] and in the closely related but nonpathogenic relative
Entamoeba dispar [82]. Strikingly, enforced expression of peroxiredoxins in an avirulent strain
increased resistance to H2O2 and partially reestablished the capability to cause liver abscesses
in hamsters [81]. Accordingly, downregulation of peroxiredoxins by antisense RNA inhibition
impaired virulence [83].

Redox pathways of parasites as targets for chemotherapy

Effective vaccines against parasites are practically unavailable and, consequently, chemother-
apy constitutes the mainstay of disease management. Therefore, redox pathways in parasites,
including those in Plasmodium falciparum [84] and trypanosomatids [85] are being intensely
studied and evaluated as drug targets. Redox pathways in parasites are seen as effective
targets because, in most cases, the parasites’ enzymes differ strongly from their functional
counterparts in humans and animals. Trypanosomatids, for example, do not possess a TrxR or
a glutathione disulfide reductase but instead feature an alternative redox system based on
trypanothione [85], a low molecular mass thiol formed from glutathione and spermidine.
Trypanothione is reduced by trypanothione reductase and functions similarly to glutathione
[86]. In microaerophilic parasites such an extravagant development has not taken place, but
most redox enzymes are of bacterial origin [6,24,27,42] and are, therefore, clearly distinct from
redox enzymes of their hosts. This opens opportunities for the development of drugs that
specifically target the redox enzymes of these parasites.

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 TREPAR 1754 No. of Pages 14

TrxR as a drug target

An obvious candidate enzyme is TrxR, which functions as a central redox regulator that
controls numerous functions, probably the most important being the reduction of peroxir-
edoxin via thioredoxin [26]. As peroxiredoxin is likely of essential importance for the parasite’s
survival in the host [81–83], the loss of its function would terminate the infection. In addition,
other thioredoxin-dependent pathways, such as ribonucleotide reduction and redox repair of
oxidized proteins through reduction of methionine sulfoxide and oxidized cysteines would
also be inhibited. Several compounds have been identified as inhibitors of TrxR in micro-
aerophilic parasites, such as organotellurium compounds [87] and auranofin [88–90]. The
latter was originally approved for the treatment of rheumatoid arthritis and has proved to be an
antiparasitic drug with a very broad spectrum [91]. It effectively inhibits the activity of purified
TrxR of E. histolytica [88], T. vaginalis [90] and G. lamblia [89] in vitro via an as yet elusive
mechanism [92] and also inhibits the growth of these parasites in vitro and in vivo [88–90].
Further, several garlic-derived compounds such as allicin and ajoene strongly inhibit TrxR, as
shown in the fish parasite S. vortens [93]. However, in this context the 5-nitroimidazoles
constitute the most important drug class, which includes the gold standard drug for the
treatment of anaerobic/microaerophilic infections, metronidazole [94]. 5-nitroimidazoles are
exclusively toxic in anaerobic/microaerophilic organisms because a strongly reductive envi-
ronment is required to reduce the drug’s nitro group, which constitutes a prerequisite for
toxicity. Reduced 5-nitroimidazoles, i.e. reactive nitroimidazole intermediates, thereupon
react with numerous targets in the cell [94]. Indeed, TrxR was identified as a target of
metronidazole and other 5-nitroimidazoles such as tinidazole and ornidazole in E. histolytica
[95], T. vaginalis [96], G. lamblia [97] and S. vortens [93]. 5-nitroimidazoles can form covalent
adducts with cysteines in TrxR and inhibit the enzyme’s disulfide reductase activity [95,96].
Interestingly, other proteins associated with the thioredoxin redox system are similarly
affected, such as thioredoxin in E. histolytica [95] and ribonucleotide reductase in T. vaginalis
[96]. There might be several reasons for this. First, proteins that depend on reduction by TrxR
or thioredoxin contain reactive cysteines which are easily accessible to reactive 5-nitro-
imidazole intermediates. Second, TrxR not only exerts disulfide reductase activity but also
reduces oxygen and nitro compounds, including 5-nitroimidazoles, as side substrates [95–
97]. Thus, TrxR is a source of reactive 5-nitroimidazole intermediates and proteins in the
proximity of TrxR might therefore be more prone to adduct formation. A recently described
antigiardial nitro compound, NBDHEX, might display a similar mode of action as it is reduced
by TrxR and also forms covalent adducts with the enzyme resulting in TrxR inhibition [98].
Finally, it is important to note that redox enzymes and metronidazole resistance in T. vaginalis
are densely interwoven (as described further in Box 2).

Ironically, metronidazole, introduced more than 50 years ago [94] without any previous studies
on its mode of action, seems to fulfill the requirements of an ideal antiparasitic drug: it is
specifically toxic to the targeted parasites due to their anaerobic/microaerophilic metabolism
and inhibits an essential enzyme activity, i.e. that of TrxR. However, 5-nitroimidazoles do
actually have pleiotropic mode of action as they also damage DNA [99] and bind to non-protein
thiols, i.e. cysteine in most microaerophilic parasites [93,95–97], thereby compromising the
redox status of the cell. It is, therefore, impossible to exactly gauge the contribution of TrxR
inhibition on the overall toxic effect of 5-nitroimidazoles. The same can be said for auranofin and
garlic-derived compounds. In G. lamblia, a tenfold overexpression of TrxR had no effect on the
IC50 of auranofin [100]. Further, auranofin obviously indiscriminately reacts with cysteine
because supplementation of growth media for G. lamblia and T. vaginalis with cysteine reduced
the toxicity of auranofin to these parasites up to 100-fold [101]. Moreover, auranofin was also
shown to inhibit human TrxR [102]. Ajoene and diallyl disulfide, on the other hand, effectively

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 TREPAR 1754 No. of Pages 14

Box 2. The redox system and metronidazole resistance

A clear connection between metronidazole resistance and redox enzymes has been reported for E. histolytica and T.
vaginalis. In a metronidazole-resistant E. histolytica cell line, TrxR was found down-regulated, whereas peroxiredoxin
and superoxide dismutase were upregulated [114]. In another study by the same workgroup, overexpression of
superoxide dismutase was reported to increase tolerance to metronidazole in the presence of oxygen [115]. In T.
vaginalis, superoxide dismutase [96,116] and peroxiredoxins [96] were also found to be upregulated in metronidazole-
resistant laboratory strains. In contrast, TrxR was inactive due to a lack of the FAD cofactor [96] and flavin reductase,
which scavenges oxygen, was not expressed [44]. The latter enzyme is similarly affected in clinical T. vaginalis isolates,
which leads to higher intracellular oxygen concentrations and a slower activation of the metronidazole prodrug [44,117].
Importantly, total insensitivity to metronidazole can be rapidly induced in T. vaginalis by treatment with diphenylene
iodonium (DPI), which binds to reduced flavins and, thereby, inhibits all flavoenzymes in the parasite, including TrxR
[118]. This is accompanied by a strong upregulation of superoxide dismutase and peroxiredoxin [118]. Furthermore,
central metabolic pathways such as PFOR are deactivated in DPI-treated cells, as previously observed in highly
metronidazole resistant T. vaginalis strains [119]. Importantly, PFOR is also capable of reducing metronidazole [119].
These observations suggest that metronidazole resistance, at least when induced in vitro, is mainly caused by a
disturbance of flavin-dependent redox pathways. This is accompanied by an upregulation of antioxidant enzymes to
counter the ensuing oxidative stress caused by the loss of flavoenzyme activities such as TrxR. The expression of iron-
sulfur flavoprotein, a hydrogenosomal factor that can reduce metronidazole [47] is also possibly affected by DPI. In G.
lamblia, fewer interconnections between metronidazole resistance and redox enzymes were described. However, an
enzyme activity analogous to flavin reductase is repressed in metronidazole-resistant cell lines [45].

deplete non-protein thiol pools in S. vortens [93], and even act in a synergistic fashion with
metronidazole [103]. This is indicative of ajoene and diallyl disulfide having also other targets.
Thus, to date no specific inhibitor of TrxR in microaerophilic parasites has been identified. This,
in fact, also applies for peroxiredoxins [21]. A lack of specificity, however, does not necessarily
restrict the potential of auranofin and other alternative treatment options. Auranofin is already an
approved drug and only needs to be reevaluated for use in antiparasitic therapy [104] and garlic
is an all-time favorite in the world’s most exquisite cuisines. As such, garlic and its constituents
are not considered as drugs but as food products and health supplements.

Inhibition of cysteine synthesis

Another avenue of research is the identification of inhibitors of cysteine synthesis in micro-
aerophilic parasites. In E. histolytica and T. vaginalis, cysteine is synthesized by cysteine
synthase [62,64], whereas humans do not have this enzyme but synthesize cysteine through
the trans-sulfuration pathway with cystathionine as a precursor [62]. The inhibition of cysteine
synthase would, therefore, not harm the human host but deprive the parasites of cysteine. As
an analogy, the specific inhibition of trypanothione synthetase in trypanosomatids has been a
long standing goal in the field [85]. In E. histolytica, the situation is complicated by the fact that,
at least in in vitro culture, cysteine synthase mainly produces S-methylcysteine rather than
cysteine itself and that the parasite is dependent on an exogenous cysteine source despite
having cysteine synthase [105]. This is independent of cysteine’s antioxidant role in growth
media for microaerophilic parasites, because E. histolytica requires exogenous cysteine even
under perfect anaerobiosis [101]. However, this notwithstanding, downregulation of the three
cysteine synthase homologues in E. histolytica inhibits growth of the parasite [48], suggesting
that cysteine synthesis is an essential pathway and a valid drug target. In T. vaginalis, cysteine in
the growth medium is dispensable under anaerobiosis [101], confirming that endogenous
cysteine synthesis is of importance for this parasite. In the search for cysteine synthase
inhibitors, which could eventually substitute for metronidazole in the treatment of E. histolytica
and T. vaginalis infections, a number of studies have been conducted [106–108], resulting in the
identification of several potential lead compounds featuring a naphtoquinone moeity, including
kerriamycin C and deoxyfrenolicin [107]. In E. histolytica, also inhibition studies with SAT have
been conducted [109]. This enzyme, which is likewise missing from humans, synthesizes the
precursor of cysteine, O-acetylserine. Only one of the three SATs encoded in the parasite’s

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 TREPAR 1754 No. of Pages 14

Metronidazole Auranofin Ajoene Diallyl sulfide

Allicin NBDHEX Deoxyfrenolicin Kerriamycin C

Figure 2. Structures of the antiparasitic compounds discussed in the text. All images were taken from the PubChem database (https://pubchem.ncbi.nlm.
nih.gov).

Table 2. Redox enzymes as (potential) targets for chemotherapy

Enzyme Crystal structure (PDB ID) Target validation Refs.

Thioredoxin reductase G. lamblia (5M5J) In G. lamblia not a good target under culture conditions [100], but [88–90,101]
E. histolytica (4A5L, 4CCR, 4CW9, auranofin-treated G. lamblia [89], E. histolytica [88], and T. vaginalis
4CCQ, 4CBQ, 4A65) [90] are less pathogenic in mice.

Cysteine synthase E. histolytica (2PQM) Silencing of all three cysteine synthase genes in E. histolytica inhibits [48]
growth.

Serine acetyltransferase E. histolytica (3Q1X, 3P1B, 3P47) Silencing of serine acetyltransferase 3 in E. histolytica inhibits growth. [48]

Peroxiredoxin Not available for either parasite Down-regulation of peroxiredoxin renders E. histolytica less [83]
pathogenic.

Flavodiiron protein G. lamblia (2Q9U) No data available yet

genome seems to have an essential function [48], but further research is needed to elucidate
SAT function in E. histolytica during infection.

The structures of the compounds discussed in this chapter are given in Figure 2. The redox
enzymes currently considered as (potential) targets for chemotherapy in microaerophilic
protists are summarized in Table 2.

10 Trends in Parasitology, Month Year, Vol. xx, No. yy

 TREPAR 1754 No. of Pages 14

Conclusion Outstanding Questions

Redox pathways in microaerophilic parasites constitute the Achilles’ heel of these organisms and Microaerophilic parasites have several
can be exploited for the development of effective chemotherapeutics. In fact, metronidazole, the redox enzymes that are highly unusual,
such as flavodiiron proteins. However,
most widely used drug against virtually all microaerophilic and anaerobic microorganisms, targets research has so far focused on stan-
redox pathways, if in a fairly indiscriminate manner, which also results in disagreeable side-effects dard enzymes such as thioredoxin
for the host during treatment (see Outstanding Questions). Thus, the development of more specific reductase. Will there be more diversi-
fied research in the foreseeable future?
and customized drugs targeting redox enzymes in microaerophilic parasites would be highly
desirable. By far the best studied example is TrxR for which several inhibitors, such as auranofin,
To date, a truly specific inhibitor for any
NBDHEX and ajoene have been described. Unfortunately, the mechanisms by which inhibition of redox enzyme in a microaerophilic par-
TrxR is achieved are either still poorly understood, as with auranofin [92] and NBDHEX [110] or asite is still lacking. Will such an inhibi-
even completely unknown, as with ajoene and metronidazole. This makes the search for improved tor be developed? This would be highly
desirable as metronidazole and other
and more effective TrxR inhibitors difficult. In the years to come, however, more intense research
drugs in use have side effects due to a
efforts should not only focus on TrxR inhibition. Other redox enzymes in microaerophilic parasites, pleiotropic mode of action.
such as FDP, should also be systematically probed for their potential as drug targets. This would
open new opportunities for urgently needed alternative treatment options. The emergence of resistance can be a
problem with specific inhibitors. Will it
be possible to avoid fast emergence of
Acknowledgements
resistance in microaerophilic
CW holds a Sêr Cymru II Fellowship part-funded by the European regional Development Fund through the Welsh
parasites?
Government. IH acknowledges the support by the programs NPU II (LQ1604) provided by the Ministry of Education,
Youth and Sport of the Czech Republic, project BIOCEV (CZ.1.05/1.1.00/02.0109), and project CePaViP (CZ.02.1.01/
0.0/0.0/16_019/0000759) supported by ERD Funds.

References
1. Lloyd, D. et al. (2015) Avoid excessive oxygen levels in experi-
ments with organisms, tissues and cells. Adv. Microb. Physiol.
67, 293–314
2. Narikawa, S. et al. (1987) Differentiation of obligate anaerobes
by assay of pyruvate:ferredoxin oxidoreductase activity. Eur. J.
Clin. Microbiol. 6, 74–75
3. Adams, M.W. et al. (1980) Hydrogenase. Biochim. Biophys.
Acta 594, 105–176
4. Paget, T.A. et al. (1990) Trichomonas vaginalis requires traces of
oxygen and high concentrations of carbon dioxide for optimal
growth. Mol. Biochem. Parasitol. 41, 65–72
5. Baughn, A.D. et al. (2004) The strict anaerobe Bacteroides
fragilis grows in and benefits from nanomolar concentrations
of oxygen. Nature 427, 441–444
6. Nixon, J.E. et al. (2002) Evidence for lateral transfer of genes
encoding ferredoxins, nitroreductases, NADH oxidase, and
alcohol dehydrogenase 3 from anaerobic prokaryotes to Giardia
lamblia and Entamoeba histolytica. Eukaryot. Cell 1, 181–190
7. Alsmark, U.C. et al. (2009) Horizontal gene transfer in eukaryotic
parasites: a case study of Entamoeba histolytica and Trichomo-
nas vaginalis. Methods Mol. Biol. 532, 489–500
8. Andersson, J.O. et al. (2006) Evolution of four gene families with
patchy phylogenetic distributions: influx of genes into protist
genomes. BMC Evol. Biol. 6, 27103
9. Torgerson, P.R. et al. (2015) World Health Organization Esti-
mates of the Global and Regional Disease Burden of 11 Food-
borne Parasitic Diseases, 2010: A Data Synthesis. PLoS Med.
12, e1001920
10. Thomas, D.C. (2017) The phagocyte respiratory burst: Historical
perspectives and recent advances. Immunol. Lett. 192, 88–96
11. Lloyd, D. et al. (2004) The plasma membrane of microaerophilic
protists: oxidative and nitrosative stress. Microbiology 150,
1183–1190
12. Gu, M. et al. (2013) Superoxide poisons mononuclear iron
enzymes by causing mismetallation. Mol. Microbiol. 89, 123–134
13. Lloyd, D. et al. (2000) The microaerophilic flagellate Giardia
intestinalis: oxygen and its reaction products collapse
membrane potential and cause cytotoxicity. Microbiology
146, 3109–3118
14. Tannich, E. et al. (1991) Pathogenic and nonpathogenic Ent-
amoeba histolytica: identification and molecular cloning of an
iron-containing superoxide dismutase. Mol. Biochem. Parasitol.
49, 61–71
15. Lindmark, D.G. et al. (1974) Superoxide dismutase in the anaer-
obic flagellates, Tritrichomonas foetus and Monocercomonas
sp. J. Biol Chem. 249, 4634–4637
16. Viscogliosi, E. et al. (1996) Phylogenetic implication of iron-
containing superoxide dismutase genes from trichomonad spe-
cies. Mol. Biochem. Parasitol. 80, 209–214
17. Williams, C.F. et al. (2014) Antioxidant defences of Spironucleus
vortens: Glutathione is the major non-protein thiol. Mol. Bio-
chem. Parasitol 196, 45–52
18. Testa, F. et al. (2011) The superoxide reductase from the early
diverging eukaryote Giardia intestinalis. Free Radic. Biol. Med
51, 1567–1574
19. Park, S. et al. (2003) High levels of intracellular cysteine promote
oxidative DNA damage by driving the fenton reaction. J. Bac-
teriol 185, 1942–1950
20. Müller, M. (1973) Biochemical cytology of trichomonad flagel-
lates I. Subcellular localization of hydrolases, dehydrogenases,
and catalase in Tritrichomonas foetus. J. Cell Biol. 57, 453–474
21. Angelucci, F. et al. (2016) Typical 2-Cys peroxiredoxins in
human parasites: several physiological roles for a potential che-
motherapy target. Mol. Biochem. Parasitol 206, 2–12
22. Poole, L.B. et al. (1997) Peroxidase activity of a TSA-like antioxi-
dant protein from a pathogenic amoeba. Free Radic. Biol. Med
23, 955–959
23. Bruchhaus, I. et al. (1997) Removal of hydrogen peroxide by
the 29 kDa protein of Entamoeba histolytica. Biochem. J. 326,
785–789
24. Coombs, G.H. et al. (2004) The amitochondriate eukaryote
Trichomonas vaginalis contains a divergent thioredoxin-linked
peroxiredoxin antioxidant system. J. Biol. Chem. 279, 5249–
5256

Trends in Parasitology, Month Year, Vol. xx, No. yy 11

 TREPAR 1754 No. of Pages 14
25. Mastronicola, D. et al. (2014) Functional characterization of
peroxiredoxins from the human protozoan parasite Giardia
intestinalis. PLoS Negl. Trop. Dis 8, e2631
26. Lu, J. et al. (2014) The thioredoxin antioxidant system. Free
Radic. Biol. Med. 66, 75–87
27. Bruchhaus, I. et al. (1995) Identification of an Entamoeba his-
tolytica gene encoding a protein homologous to prokaryotic
disulphide oxidoreductases. Mol. Biochem. Parasitol. 70,
187–191
28. Arias, D.G. et al. (2007) Thioredoxin-linked metabolism in Ent-
amoeba histolytica. Free Radic. Biol. Med. 42, 1496–1505
29. Brown, D.M. et al. (1996) A thioredoxin reductase-class of
disulphide reductase in the protozoan parasite Giardia duode-
nalis. Mol. Biochem. Parasitol. 83, 211–220
30. Westrop, G.D. et al. (2009) The mercaptopyruvate sulfurtransferase
of Trichomonas vaginalis links cysteine catabolism to the production
of thioredoxin persulfide. J. Biol. Chem. 284, 33485–33494
31. Leitsch, D. et al. (2017) Giardia lamblia: missing evidence for a
canonical thioredoxin system. Parasitol. Open 3, e13
32. Pütz, S. et al. (2005) Rubrerythrin and peroxiredoxin: two novel
putative peroxidases in the hydrogenosomes of the microaer-
ophilic protozoon Trichomonas vaginalis. Mol. Biochem. Para-
sitol. 142, 212–223
33. Maraliková, B. et al. (2010) Bacterial-type oxygen detoxification
and iron-sulfur cluster assembly in amoebal relict mitochondria.
Cell. Microbiol. 12, 331–342
34. Nývltová, E. et al. (2016) OsmC and incomplete glycine decar-
boxylase complex mediate reductive detoxification of peroxides
in hydrogenosomes of Trichomonas vaginalis. Mol. Biochem.
Parasitol. 206, 29–38
35. Vicente, J.B. et al. (2012) A detoxifying oxygen reductase in the
anaerobic protozoan Entamoeba histolytica. Eukaryot. Cell 11,
1112–1118
36. Smutná, T. et al. (2009) Flavodiiron protein from Trichomonas
vaginalis hydrogenosomes: the terminal oxygen reductase.
Eukaryot. Cell 8, 47–55
37. Di Matteo, A. et al. (2008) The O2-scavenging flavodiiron protein
in the human parasite Giardia intestinalis. J. Biol. Chem. 283,
4061-8.
38. Romao, C.V. et al. (2016) Structure of Escherichia coli Flavo-
diiron Nitric Oxide Reductase. J. Mol. Biol. 428, 4686–4707
39. Linstead, D.J. et al. (1988) The purification and properties of two
soluble reduced nicotinamide:acceptor oxidoreductases from
Trichomonas vaginalis. Mol. Biochem. Parasitol. 27, 125–133
40. Brown, D.M. et al. (1996) A H2O-producing NADH oxidase from
the protozoan parasite Giardia duodenalis. Eur. J. Biochem.
241, 155–161
41. Brown, D.M. et al. (1995) Free radical detoxification in Giardia
duodenalis. Mol. Biochem. Parasitol. 72, 47–56
42. Jeelani, G. et al. (2010) Two atypical L-cysteine-regulated
NADPH-dependent oxidoreductases involved in redox mainte-
nance, L-cystine and iron reduction, and metronidazole activa-
tion in the enteric protozoan Entamoeba histolytica. J. Biol.
Chem. 285, 26889–26899
43. Chapman, A. et al. (1999) Hydrogen peroxide is a product of
oxygen consumption by Trichomonas vaginalis. J. Biosci. 24,
339–344
44. Leitsch, D. et al. (2014) Trichomonas vaginalis flavin reductase 1
and its role in metronidazole resistance. Mol. Microbiol. 91, 198–
208
45. Leitsch, D. et al. (2011) Pyruvate:ferredoxin oxidoreductase and
thioredoxin reductase are involved in 5-nitroimidazole activation
while flavin metabolism is linked to 5-nitroimidazole resistance in
Giardia lamblia. J. Antimicrob. Chemother. 66, 1756–1765
46. Li, L. et al. (2006) A likely molecular basis of the susceptibility of
Giardia lamblia towards oxygen. Mol. Microbiol. 59, 202–211
47. Smutná, T. et al. (2014) Novel functions of an iron-sulfur flavo-
protein from Trichomonas vaginalis hydrogenosomes. Antimi-
crob. Agents Chemother. 58, 3224–3232
12 Trends in Parasitology, Month Year, Vol. xx, No. yy
48. Jeelani, G. et al. (2017) Genetic, metabolomic and transcrip-
tomic analyses of the de novo L-cysteine biosynthetic pathway
in the enteric protozoan parasite Entamoeba histolytica. Sci.
Rep. 7, 15649
49. Thomas, D.D. et al. (2008) The chemical biology of nitric
oxide: implications in cellular signaling. Free Radic. Biol. Med.
45, 18–31
50. Santi-Rocca, J. et al. (2012) Endoplasmic reticulum stress-
sensing mechanism is activated in Entamoeba histolytica upon
treatment with nitric oxide. PLoS One 7, e31777
51. Radi, R. (2013) Peroxynitrite, a stealthy biological oxidant. J.
Biol. Chem. 288, 26464–26472
52. Mastronicola, D. et al. (2010) Flavohemoglobin and nitric oxide
detoxification in the human protozoan parasite Giardia intesti-
nalis. Biochem. Biophys. Res. Commun 399, 654–658
53. Rafferty, S. et al. (2010) Giardia lamblia encodes a functional
flavohemoglobin. Biochem. Biophys. Res. Commun 399, 347–
351
54. Sarti, P. et al. (2004) Trichomonas vaginalis degrades nitric oxide
and expresses a flavorubredoxin-like protein: a new pathogenic
mechanism? Cell Mol. Life Sci. 61, 618–623
55. Wang, J. et al. (2016) The roles of the hybrid cluster protein, Hcp
and its reductase, Hcr, in high affinity nitric oxide reduction that
protects anaerobic cultures of Escherichia coli against nitro-
sative stress. Mol. Microbiol. 100, 877–892
56. Seth, D. et al. (2018) A multiplex enzymatic machinery for cellular
protein S-nitrosylation. Mol. Cell. 69, 451–464
57. Bryk, R. et al. (2000) Peroxynitrite reductase activity of bacterial
peroxiredoxins. Nature 407, 211–215
58. Krauth-Siegel, R.L. et al. (2012) Low-molecular-mass antioxi-
dants in parasites. Antioxid. Redox Signal. 17, 583–607
59. Armstrong, J.S. et al. (2002) Role of glutathione depletion and
reactive oxygen species generation in apoptotic signaling in a
human B lymphoma cell line. Cell Death Differ 9, 252–263
60. Brown, D.M. et al. (1993) Cysteine is the major low-molecular
weight thiol in Giardia duodenalis. Mol. Biochem. Parasitol 61,
155–158
61. Ellis, J.E. et al. (1994) Antioxidant defences in the microaero-
philic protozoan Trichomonas vaginalis: comparison of metro-
nidazole-resistant and sensitive strains. Microbiology 140,
2489–2494
62. Westrop, G.D. et al. (2006) Cysteine biosynthesis in Trichomo-
nas vaginalis involves cysteine synthase utilizing O-phosphoser-
ine. J. Biol. Chem 281, 25062–25075
63. Fahey, R.C. et al. (1984) Entamoeba histolytica: a eukaryote
without glutathione metabolism. Science 224, 70–72
64. Nozaki, T. et al. (1998) Molecular cloning and characterization of
the genes encoding two isoforms of cysteine synthase in the
enteric protozoan parasite Entamoeba histolytica. Mol. Bio-
chem. Parasitol. 97, 33–44
65. Jeelani, G. et al. (2016) Entamoeba thiol-based redox metabo-
lism: A potential target for drug development. Mol. Biochem.
Parasitol. 206, 39–45
66. Nozaki, T. et al. (1999) Characterization of the gene encoding
serine acetyltransferase, a regulated enzyme of cysteine biosyn-
thesis from the protist parasites Entamoeba histolytica and
Entamoeba dispar. Regulation and possible function of the
cysteine biosynthetic pathway in Entamoeba. J. Biol. Chem.
274, 32445-52.
67. Rada, P. et al. (2009) The monothiol single-domain glutaredoxin
is conserved in the highly reduced mitochondria of Giardia
intestinalis. Eukaryot. Cell 8, 1584–1591
68. Lin, J.Y. et al. (1992) Macrophage cytotoxicity against Ent-
amoeba histolytica trophozoites is mediated by nitric oxide from
L-arginine. J. Immunol. 148, 3999–4005
69. Eckmann, L. et al. (2000) Nitric oxide production by human
intestinal epithelial cells and competition for arginine as potential
determinants of host defense against the lumen-dwelling path-
ogen Giardia lamblia. J. Immunol. 164, 1478–1487
 TREPAR 1754 No. of Pages 14
70. Lloyd, D. et al. (2003) Nitrosative stress induced cytotoxicity in
Giardia intestinalis. J. Appl. Microbiol. 95, 576–583
71. Hawes, S.E. et al. (1996) Hydrogen peroxide-producing lactobacilli
and acquisition of vaginal infections. J. Infect. Dis. 174, 1058–1063
72. Margarita, V. et al. (2016) Symbiotic Association with Myco-
plasma hominis can influence growth Rate, ATP production,
cytolysis and inflammatory response of Trichomonas vaginalis.
Front Microbiol. 2016 (7), 953
73. Elnekave, K. et al. (2003) Consumption of L-arginine mediated
by Entamoeba histolytica L-arginase (EhArg) inhibits amoebici-
dal activity and nitric oxide production by activated macro-
phages. Parasite Immunol. 25, 597–608
74. Wang, W. et al. (1994) Entamoeba histolytica modulates the
nitric oxide synthase gene and nitric oxide production by macro-
phages for cytotoxicity against amoebae and tumour cells.
Immunology 83, 601–610
75. Ralston, K.S. et al. (2011) Tissue destruction and invasion by
Entamoeba histolytica. Trends Parasitol. 27, 254–263
76. Ellis, J.E. et al. (1992) Influence of oxygen on the fermentative
metabolism of metronidazole-sensitive and resistant strains of
Trichomonas vaginalis. Mol. Biochem. Parasitol. 56, 79–88
77. Mastronicola, D. et al. (2016) Antioxidant defence systems in the
protozoan pathogen Giardia intestinalis. Mol. Biochem. Para-
sitol. 206, 56–66
78. Vicente, J.B. et al. (2009) Entamoeba histolytica modulates a
complex repertoire of novel genes in response to oxidative and
nitrosative stresses: implications for amebic pathogenesis. Cell.
Microbiol. 11, 51–69
79. Ma’ayeh, S.Y. et al. (2015) Transcriptional profiling of Giardia
intestinalis in response to oxidative stress. Int. J. Parasitol. 45,
925–938
80. Gould, S.B. et al. (2013) Trichomonas vaginalis during the early
infection of vaginal epithelial cells and amoeboid transition. Int. J.
Parasitol. 43, 707–719
81. Davis, P.H. et al. (2006) Comparative proteomic analysis of two
Entamoeba histolytica strains with different virulence pheno-
types identifies peroxiredoxin as an important component of
amoebic virulence. Mol. Microbiol. 61, 1523–1532
82. Choi, M.H. et al. (2005) An unusual surface peroxiredoxin pro-
tects invasive Entamoeba histolytica from oxidant attack. Mol.
Biochem. Parasitol. 143, 80–89
83. Sen, A. et al. (2007) The 29-kilodalton thiol-dependent peroxi-
dase of Entamoeba histolytica is a factor involved in pathogen-
esis and survival of the parasite during oxidative stress.
Eukaryot. Cell 6, 664–673
84. Jortzik, E. et al. (2012) Thioredoxin and glutathione systems in
Plasmodium falciparum. Int. J. Med. Microbiol. 302, 187–194
85. Leroux, A.E. et al. (2016) Thiol redox biology of trypanosomatids
and potential targets for chemotherapy. Mol. Biochem. Para-
sitol. 206, 67–74
86. Fairlamb, A.H. et al. (1985) Trypanothione: a novel bis(gluta-
thionyl)spermidine cofactor for glutathione reductase in trypa-
nosomatids. Science 227, 1485–1487
87. McMillan, P.J. et al. (2009) Differential inhibition of high and low
Mr thioredoxin reductases of parasites by organotelluriums sup-
ports the concept that low Mr thioredoxin reductases are good
drug targets. Parasitology 136, 27–33
88. Debnath, A. et al. (2012) A high-throughput drug screen for
Entamoeba histolytica identifies a new lead and target. Nat.
Med. 18, 956–960
89. Tejman-Yarden, N. et al. (2013) Auranofin, a reprofiled drug, is
effective against metronidazole-resistant Giardia lamblia. Anti-
microb. Agents Chemother 57, 2029–2035
90. Hopper, M. et al. (2016) Auranofin inactivates Trichomonas vag-
inalis thioredoxin reductase and is effective against trichomonads
in vitro and in vivo. Int. J. Antimicrob. Agents 48, 690–694
91. Debnath, A. et al. (2013) Reprofiled drug targets ancient pro-
tozoans: drug discovery for parasitic diarrheal diseases. Gut
Microbes 4, 66–71
92. Parsonage, D. et al. (2016) X-ray structures of thioredoxin and
thioredoxin reductase from Entamoeba histolytica and prevailing
hypothesis of the mechanism of auranofin action. J. Struct. Biol.
194, 180–190
93. Williams, C.F. et al. (2012) Disrupted intracellular redox balance
of the diplomonad fish parasite Spironucleus vortens by 5-nitro-
imidazoles and garlic-derived compounds. Vet. Parasitol. 190,
62–73
94. Leitsch, D. (2017) A review on metronidazole: an old warhorse in
antimicrobial chemotherapy. Parasitol. 23 (Nov), 1–12
95. Leitsch, D. et al. (2007) Nitroimidazole action in Entamoeba
histolytica: a central role for thioredoxin reductase. PLoS Biol.
5, 1820–1834
96. Leitsch, D. et al. (2009) Trichomonas vaginalis: metronidazole
and other nitroimidazole drugs are reduced by the flavin enzyme
thioredoxin reductase and disrupt the cellular redox system.
Implications for nitroimidazole toxicity and resistance. Mol.
Microbiol. 72, 518–536
97. Leitsch, D. et al. (2012) Nitroimidazole drugs vary in their mode
of action in the human parasite Giardia lamblia. Int. J. Parasitol.
Drugs Drug Resis. 2, 166–170
98. Camerini, S. et al. (2017) Proteomic and functional analyses
reveal pleiotropic action of the anti-tumoral compound NBDHEX
in Giardia duodenalis. Int. J. Parasitol. Drugs Drug Resist 7, 147–
158
99. Uzlikova, M. et al. (2014) The effect of metronidazole on the cell
cycle and DNA in metronidazole-susceptible and resistant
Giardia cell lines. Mol. Biochem. Parasitol 198, 75–81
100. Leitsch, D. et al. (2016) Evaluation of Giardia lamblia thioredoxin
reductase as drug activating enzyme and as drug target. Int. J.
Parasitol. Drugs Drug Resist 6, 148–153
101. Leitsch, D. (2017) Drug susceptibility testing in microaerophilic
parasites: cysteine strongly affects the effectivities of metroni-
dazole and auranofin, a novel and promising antimicrobial. Int. J.
Parasitol. Drugs Drug Resis. 7, 321–327
102. Gromer, S. et al. (1998) Human placenta thioredoxin reductase
Isolation of the selenoenzyme, steady state kinetics, and inhibi-
tion by therapeutic gold compounds. J. Biol. Chem. 273,
20096–20101
103. Williams, C.F. et al. (2016) The redox-active drug metronidazole
and thiol-depleting garlic compounds act synergistically in the
protest parasite Spironucleus vortens. Mol. Biochem. Parasitol
206, 20–28
104. Caparelli, E.V. et al. (2016) Phase I clinical trial results of aur-
anofin, a novel antiparasitic agent. Antimicrob. Agents Chemo-
ther 61, e01947-16
105. Husain, A. et al. (2010) Metabolome analysis revealed increase in
S-methylcysteine and phosphatidylisopropanolamine synthesis
upon L-cysteine deprivation in the anaerobic protozoan parasite
Entamoeba histolytica. J. Biol. Chem. 285, 39160–39170
106. Nagpal, I. et al. (2012) Virtual screening, identification and in vitro
testing of novel inhibitors of O-acetyl-L-serine sulfhydrylase of
Entamoeba histolytica. PLoS One 7, e30305
107. Mori, M. et al. (2015) Identification of natural inhibitors of Ent-
amoeba histolytica cysteine synthase from microbial secondary
metabolites. Front. Microbiol 6, 962
108. Singh, S. et al. (2013) Molecular dynamic simulation and inhibitor
prediction of cysteine synthase structured model as a potential
drug target for trichomoniasis. Biomed. Res. Int. 2013, 390920
109. Agarwal, S.M. et al. (2008) Inhibitors of Escherichia coli serine
acetyltransferase block proliferation of Entamoeba histolytica
trophozoites. Int. J. Parasitol. 38, 137–141
110. Brogi, S. et al. (2017) Structural characterization of Giardia
duodenalis thioredoxin reductase (gTrxR) and computational
analysis of its interaction with NBDHEX. Eur. J Med. Chem.
135, 479–490
111. Carlton, J.M. et al. (2007) Draft genome sequence of the sexu-
ally transmitted pathogen Trichomonas vaginalis. Science 315,
207–212
Trends in Parasitology, Month Year, Vol. xx, No. yy 13
 TREPAR 1754 No. of Pages 14
112. Mentel, M. et al. (2008) Protein import into hydrogenosomes of
Trichomonas vaginalis involves both N-terminal and internal
targeting signals: a case study of thioredoxin reductases. Eukar-
yot. Cell 7, 1750–1757
113. Beltrán, N.C. et al. (2013) Iron-induced changes in the proteome of
Trichomonas vaginalis hydrogenosomes. PLoS One 8, e65148
114. Wassmann, C. et al. (1999) Metronidazole resistance in the
protozoan parasite Entamoeba histolytica is associated with
increased expression of iron-containing superoxide dismutase
and peroxiredoxin and decreased expression of ferredoxin 1
and flavin reductase. J. Biol. Chem. 274, 26051–26056
115. Wassmann, C. et al. (2000) Superoxide dismutase reduces
susceptibility to metronidazole of the pathogenic protozoan
Entamoeba histolytica under microaerophilic but not under
anaerobic conditions. Arch. Biochem. Biophys. 376, 236–238
116. Rasoloson, D. et al. (2001) Metronidazole-resistant strains of
Trichomonas vaginalis display increased susceptibility to oxy-
gen. Parasitology 123, 45–56
117. Yarlett, N. et al. (1986) Metronidazole-resistant clinical isolates of
Trichomonas vaginalis have lowered oxygen affinities. Mol. Bio-
chem. Parasitol. 19, 111–116
14 Trends in Parasitology, Month Year, Vol. xx, No. yy
118. Leitsch, D. et al. (2010) The flavin inhibitor diphenyleneiodonium
renders Trichomonas vaginalis resistant to metronidazole, inhib-
its thioredoxin reductase and flavin reductase, and shuts off
hydrogenosomal enzymatic pathways. Mol. Biochem. Parasitol.
171, 17–24
119. Rasoloson, D. et al. (2002) Mechanisms of in vitro development
of resistance to metronidazole in Trichomonas vaginalis. Micro-
biology 148, 2467–2477
120. Schneider, R.E. et al. (2011) The Trichomonas vaginalis hydro-
genosome proteome is highly reduced relative to mitochondria,
yet complex compared with mitosomes. Int. J. Parasitol. 41,
1421–1434
121. Jedelský, P.L. et al. (2011) The minimal proteome in the reduced
mitochondrion of the parasitic protist Giardia intestinalis. PLoS
One 6, e17285
122. Westrop, G.D. et al. (2017) Metabolomic profiling and stable
isotope labelling of Trichomonas vaginalis and Tritrichomonas
foetus reveal major differences in amino acid metabolism includ-
ing the production of 2-hydroxyisocaproic acid, cystathionine
and S-methylcysteine. PLoS One 12, e0189072