Trypanosomatid Diseases (Molecular Routes to Drug Discovery) || Peroxynitrite as a Cytotoxic Effector Against Trypanosoma Cruzi : Oxidative Killing and Antioxidant Resistance Mechanisms

11Peroxynitrite as a Cytotoxic Effector Against Trypanosoma cruzi :Oxidative Killing and Antioxida nt Resistanc e MechanismsMadia Trujillo, María Noel Alvarez, Lucía Piacenza, Martín Hugo,Gonzalo Peluffo, and Rafael Radi �

AbstractInside the phagosomes of activated macrophages, Trypanosoma cruzi is exposed toreactive oxygen species formed by NADPH oxidase, namely the superoxide radical(O2

�� ) and, through its dismutation, hydrogen peroxide. Moreover, induction ofnitric oxide synthase by pro-inflammatory cytokines leads to the production of nitricoxide (� NO). Neither O2

�� nor � NO are particularly cytotoxic per se, and most oftheir parasiticidal actions rely on their diffusion-controlled reaction to form pero-xynitrite – a potent oxidizing and nitrating species with a higher killing capabilitytowards microorganisms. Thus, the antioxidant mechanisms that allow the parasiteto detoxify peroxynitrite and other cytotoxic peroxides constitute an active field ofinvestigation, since they can provide clues for a rationalized drug design. Moreover,although most research has focused on the formation of reactive species and theirdetoxi fication during acute infections, Chagas is mostly a chronic disease and nodrug is currently available for its treatment at this stage, where the microorganisminfects cardiac muscle and other non-phagocytic cells. The role of peroxynitrite inT. cruzi infectivity and virulence at the chronic stage of the disease remains tobe established.

Introduction

Trypanosoma cruzi is an intracellular parasite that causes Chagas disease, alsoknown as American trypanosomiasis, endemic in 21 Latin American countries,where millions of persons are infected and thousands of people die each year of thisillness (http://www.who.int/mediacentre/factsheets/fs340/en/index.html). Macro-phage infection plays a key role during the acute phase of the disease. Inside thephagosomes of activated macrophages, the parasites are exposed to reactive oxygenspecies formed by NADPH oxidase [1,2], namely the superoxide radical (O2

��) and,through its dismutation, hydrogen peroxide (H2O2) [3]. Moreover, induction ofinducible nitric oxide synthase (iNOS) by pro-inflammatory cytokines leads to the

� Corresponding Author

Trypanosomatid Diseases: Molecular Routes to Drug Discovery, First edition. Edited by T. Jäger, O. Koch, and L. Flohé.# 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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production of nitric oxide (�NO) [4–6]. Neither O2�� nor �NO are particularly

cytotoxic, and most of their parasiticidal actions rely on their fast reaction toform peroxynitrite – an oxidizing and nitrating species with a higher capabilityto kill microorganisms [7–9] (IUPAC recommended names for peroxynitrite anion(ONOO–) and its conjugated acid, peroxynitrous acid (ONOOH), are oxoperoxoni-trate (1�) and hydrogen oxoperoxonitrate, respectively. The term peroxynitrite isused to refer to the sum of ONOO� and ONOOH). Thus, the antioxidant mecha-nisms that allow the parasite to detoxify peroxynitrite and other cytotoxic peroxidesare intensively explored, since they could provide clues for new therapeuticapproaches. Moreover, although most research has focused on the formation ofreactive species and their detoxification during acute infections, Chagas is mostly achronic disease and no drug is currently available for its treatment at this stage,where the microorganism infects cardiac muscle and other non-phagocytic cells[10–13]. The roles of reactive oxygen and nitrogen species, in particular peroxyni-trite, in T. cruzi killing and the antioxidant enzymatic mechanisms that allowparasite survival during chronic infections are only starting to be unraveled.

PeroxynitriteFormationDuringT.cruzi–MammalianHostCell Interaction

Once in the mammalian host, metacyclic trypomastigotes infect and replicateintracellularly in different types of cells, macrophages being one of the mostimportant. Phagocytic cells, such as macrophages, have an active role in microbialkilling, linking innate and adaptive immune responses. Several molecules on thehost cell surface have been described to interact with T. cruzi, such as mannose andToll-like receptors (TLRs) [14,15]. Specifically, TLR2 and TLR4 have been involvedin the recognition of T. cruzi [16,17], which interact through molecules presentsin the trypomastigote membrane. Parasite recognition leads to an upregulation ofantimicrobial activity, with the production of pro-inflammatory cytokines and apositive regulation of macrophage oxidative response [18,19].TLR-mediated phagocytosis is usually accompanied by a “respiratory burst,”

accomplished by NOX2, a specialized NADPH oxidase enzymatic complex. NOX2consists of the membrane-bound flavocytochrome b gp91phox, which transferselectrons from a cytosolic NADPH to oxygen in the phagocytic vacuole to producelarge quantities of O2

��when activated. Other components of the complex includemembrane-bound p22phox, cytosolic proteins p40phox, p47phox, and p67phox, and thesmall GTPases Rac1 and Rap1; all are translocated to the membrane in atightly regulated way [20–22]. Some pathogens, among which are some unicellularparasites, manage to evade NOX2 activation in order to survive in the phagosomeand efficiently infect the host. It has been debated whether the invasion processof T. cruzi activates NOX2 [23–25]. Results from our group and others haveshown the formation of O2

�� during T. cruzi invasion, which occurs on theluminal side of the phagosome and lasts for around 60–90min; the magnitude ofthe respiratory burst is comparable with that elicited by other phagocyticstimuli [26,27].

216j 11 Peroxynitrite as a Cytotoxic Effector Against Trypanosoma cruzi: Oxidative Killing and Antioxidant

In the early stage of T. cruzi infection, natural killer and helper T (Th) cells becomeactive when they are presented to peptide antigens exposed on the surface ofantigen-presenting cells. Once activated, they secrete cytokines that regulate or assistin the active immune response. Macrophages exposed to TLR agonists and Th1cytokines (i.e., interferon (IFN)-c, tumor necrosis factor (TNF)-a), namely “classi-cally activated macrophages,” show alterations in phagocyte physiology with induc-tion of nitric oxide synthase 2 expression, responsible for the prolonged productionof large amounts of �NO [4,5] and an enhanced respiratory burst [28,29]. Microbialkilling in activated macrophages relies more heavily on the production of reactiveoxygen and nitrogen intermediates [30–35]. Under pro-inflammatory conditions,maximal and simultaneous production of O2

�� and �NOwill occur. NADPH oxidaseassembly in the phagosome membrane results in the formation of O2

�� inside thevacuole, whereas �NO, a hydrophobic radical, diffuses from the cytosol. Inside thephagosome, the diffusion-controlled reaction between �NO and O2

�� yields perox-ynitrite, a strong one- and two-electron oxidant, and a precursor of secondaryspecies, which can also lead to one-electron oxidation and nitration reactions, aswe will see below [36].Immunostimulated macrophages are involved in early parasite killing. Many

years ago, several investigators demonstrated that the ability of T. cruzi to infectand multiply inside mouse macrophages in vitro was inhibited by macrophageactivation [37–39]. Thereafter, it was suggested that the ability to produce andrelease H2O2 by activated macrophages was related to an enhanced capacity to killintracellular trypomastigotes [25,40]. Other investigators showed the importanceof �NO in infection control [41–44]. In addition, in vitro experiments revealed thatbolus peroxynitrite addition is effective in compromising T. cruzi viability [45].Moreover, coculture of the non-infective epimastigote form with resting andactivated macrophages shows that under conditions of macrophage-derivedperoxynitrite formation in the extracellular space, the oxidant was capable ofdiffusing to parasites causing intracellular probe oxidation and strongly inhibitingT. cruzi survival (around 50%) [46].Macrophage infection studies with T. cruzi trypomastigotes were performed to

evaluate the role of peroxynitrite during parasite internalization. Experiments usingoxidation of 2,7-dichlorodihydrofluorescein diacetate (DCFH2) as a probe forperoxynitrite formation indicated that the oxidant was indeed formed by immu-nostimulated macrophages (forming simultaneously O2

�� and �NO), but not bynaive macrophages (which produce only O2

��) (Figure 11.1a). The amount ofperoxynitrite inside the phagosome was sufficient to damage and kill trypomasti-gotes as evidenced by protein nitroxidative modifications detected in internalizedparasites, 60% less amastigote load into macrophages after 24 h, and ultrastructuralalterations in parasites visualized in electron microscopy studies (Figure 11.1b).Notably, overexpression of an enzyme with peroxidase and peroxynitrite reductaseactivity (cytosolic tryparedoxin peroxidase (cTXNPx), see below) significantly dimin-ished cytotoxicity [27]. The progression of Chagas disease to the chronic phasedepends on parasite persistence in tissues and on the host immune responses[47,48]. Some parasites can evade the initial assault of the immune system and infect

Peroxynitrite Formation During T. cruzi–Mammalian Host Cell Interaction j217

Figure 11.1 Peroxynitrite formation inimmunostimulated-infected macrophages:ultrastructural consequences for T. cruziinside the phagosome. (a) Detection ofperoxynitrite formed during macrophageinfection by T. cruzi trypomastigotes.Unstimulated (T. cruzi) andimmunostimulated (IFN-c/LPSþ T. cruzi)macrophages were infected withtrypomastigotes (5: 1 parasite/macrophageratio) in the presence of 30 mM DCFH2.The time course of DCFH2 oxidation wasfollowed in a fluorescence plate reader withfilters at lexc¼ 485 nm and lem¼ 520 nm, atpH 7.4 and 37 �C. Control (CTL): uninfectedmacrophages. (b) Peroxynitrite exposure of

phagocytosed T. cruzi results inultrastructural alterations as evidenced bytransmission electron microscopy.Micrographs shown unstimulated (T. cruzi)and activated (T. cruziþ IFN-c/LPS) infectedmacrophages at 1 h postinfection;peroxynitrite-producing activatedmacrophages showed marked damage ofT. cruzi organelles and membranedisorganization and disappearance. Arrowsshow normal membrane microtubularstructures in phagocytosed trypomastigotesby resting macrophages and reductions ofmembrane microtubules inside peroxynitrite-producing macrophages. n, T. cruzi nucleus;f, flagellum; r, reservosomes.


non-phagocytic cells in organs such as the heart and the digestive tract. Indeed,chagasic cardiomyopathy is characterized by the presence of amastigote nests in thecardiac fiber [49]. T. cruzi invasion to cardiomyocytes triggers the production ofinflammatory mediators (TNF-a, interleukin (IL)-1b) and the induction of iNOSwithin the cells [50,51]. They can also be exposed to �NO diffusing from inflamma-tory cells. Experiments using knockout mice for IFN-c, IL-12, and NOS isoformsclearly demonstrated a key role of the IL-12/IFN-c/iNOS axis in the control ofT. cruzi infection and for the outcome in the disease [52,53]. Moreover, cardiomyo-cytes and other non-phagocytic cells possess sources of O2

�� such as the mitochon-drial electron transport chain or uncoupled nitric oxide synthases [54,55]. Thesuperoxide radical could also be formed inside the parasite from its own electrontransport chain or other metabolic processes as well as by redox cycling of anti-chagasic drugs [56–58]. It is therefore tempting to speculate that peroxynitrite couldbe formed also in infected non-phagocytic cells or even inside the parasite and that itcould be implicated in the control of the infection. Host-nitrated proteins have beenidentified in heart lesions in a mouse model of Chagas disease [59,60]. However,formation of peroxynitrite within or reaching the parasite from external sources andits possible role in controlling proliferation during the chronic stage of the disease isstill to be demonstrated.

Peroxynitrite Diffusion and Reactivity with T. cruzi Targets

Peroxynitrous acid has a pKa of 6.5–6.8 [61–63] and therefore the anionic form ismore abundant than its conjugated acid at physiological pH. Homolysis of theperoxo bond (k¼ 0.9 s�1, pH 7.4, 37 �C) makes peroxynitrous acid decay, giving riseto hydroxyl (�OH) and nitrogen dioxide (�NO2) radicals at approximately 30% yields[64,65]. Both radicals may participate in secondary, indirect reactions, resulting inthe oxidation, hydroxylation, or nitration of different targets. In biological systems,however, most peroxynitrite will react directly with different biomolecules beforedecomposing. Low-molecular-weight and protein thiols, metal centers, and carbondioxide (CO2) constitute themain targets for peroxynitrite in vivo (for a recent review,see [66]). The direct reaction with thiols involves a two-electron oxidation of thedeprotonated form of the thiol by peroxynitrous acid, yielding nitrite and a sulfenicacid intermediate that, in the presence of another accessible thiol group, results indisulfide formation [67,68]. In the case of metal centers, such as those in hemeproteins or other metal-containing proteins, mechanisms of reaction with perox-ynitrite include one- and two-electron oxidations as well as metal-catalyzed isomeri-zation to nitrate [69–73]. Metal oxidation may result in the formation of strongoxidative species such as compound 1 or 2 of heme peroxidases or other oxo-metalcomplexes, which may participate in nitro-oxidative target modifications [74].Moreover, the peroxynitrite anion reacts with CO2 (k¼ 4.6� 104M�1 s�1, pH 7.4,37 �C) forming a transient intermediate that homolyzes to �NO2 and carbonateradical (CO3

��) in around 35% yields [75–77]; radicals that in turn can participate inindirect oxidation and/or nitration reactions [74].

Peroxynitrite Diffusion and Reactivity with T. cruzi Targets j219

The main low-molecular-weight thiol in trypanosomatids, trypanothione (theterm trypanothione (T(SH)2) is used for bis(glutathionyl) spermidine and itsdisulfide is defined as trypanothione disulfide (TS2) [78]) is oxidized by peroxyni-trite with a rate constant of 7200M�1 s�1, pH 7.4, 37 �C [79,80], as expected for alow-molecular-weight dithiol with a pKa of 7.4 [81,82]. It also reacts with differentthiol-containing proteins of the parasite, including the fast-reacting peroxidaticthiols in peroxiredoxins (Prxs) [83] (see below), and with metal-containing proteinssuch as ascorbate peroxidase and iron-dependent superoxide dismutases (SODs) Aand B (Hugo et al., unpublished andMartinez et al., unpublished results). Expositionto peroxynitrite also leads to the nitration of parasite proteins, which is higher whenintracellular thiol content decrease [84]. It must be taken into account that, at leastduring acute infections, most peroxynitrite is formed outside the parasite, inside thephagosome of activated macrophages, and that it must diffuse to and through theparasite plasma membrane to reach intracellular targets and exert its cytotoxicactions. While peroxynitrous acid can passively diffuse through membranes, theanionic species requires the presence of a 4,40-diisothiocyanodihydrostilbene-2,20-disulfonic acid (H2DIDS)-sensitive Cl�/HCO3

� exchanger (the presence of aH2DIDS-sensitive Cl– channel that contributes to pH homeostasis has beendescribed in Leishmania major promastigotes [78]; the Cl–/HCO3

– exchanger wasalso reported to be expressed in different T. cruzi stages [85]) [86,87]. Our group hasdemonstrated that even in the presence of physiological concentrations of CO2, thediffusion process outcompetes the decay by extracellular reaction with CO2

if diffusion distances are less than 5mm [46]. That is particularly so in the caseof the intraphagosomal actions of peroxynitrite as diffusion distances are less than0.1mm (Figure 11.1b) [27]. Once inside the parasite, the main target for directperoxynitrite reactivity will be dictated by the rate constants of reactions times targetconcentrations. Due to the limited capability of diffusion of peroxynitrite anion, andits rapid consumption by intracellular targets, compartmentalization should also betaken into account. Using available data, mostly related to thiols in the parasite, thefractional amount of peroxynitrite that would react with different targets can beestimated. Concerning the cytosol of T. cruzi epimastigotes, if only the thiol-containing compounds GSH (the reactivity of peroxynitrite with other relativelyabundant low-molecular-weight thiols such as glutathionyl spermidine and ovothiolA has not been investigated so far), T(SH)2, cytosolic tryparedoxin (TXN) I andcTXNPx are considered, and in spite of a much higher rate constant for the reactionwith cTXNPx, an important fraction of peroxynitrite is expected to react also with thelow-molecular-weight thiols, mostly T(SH)2, due to their much higher concentra-tion. In the presence of physiological concentrations of CO2, most peroxynitrite isexpected to react with it to formCO3

�� and �NO2, resulting in one-electron oxidationand nitration reactions (Table 11.1). The mitochondria of the parasite can also beexposed to peroxynitrite, either formed endogenously, from the reaction between thediffusible �NO and respiratory chain-derived O2

��, or diffusing from externalsources. The reported mTXNPx concentration inside epimastigote whole homoge-nates is 1.35 mM, which taking into account that the single mitochondrion occupies12% of the cell volume in normal epimastigotes [97] would translate into a


mitochondrial concentration of 11.3mM. Thus, the product of reactivity withperoxynitrite (1.8� 107M�1 s�1, pH 7.4 [83]) times enzyme concentration in themitochondrion of epimastigotes will be around 20 s�1. The cellular distribution oftrypanothione in T. cruzi has not been investigated so far, precluding comparisonswith low-molecular-weight thiol-mediated peroxynitrite consumption inside themitochondria. Kinetic considerations indicate that CO2 constitutes a major targetfor peroxynitrite reactivity also in this compartment. The scenario changes in theinfective forms of the parasite (Table 11.1), since during metacyclogenesis theamount of GSH and T(SH)2 decreases [92,93,98], and the expression of bothcytosolic and mitochondrial TXNPxs increases in different parasite strains (around6- and 2-fold for cTXNPx and mTXNPx, respectively, in Y strain [95]), which wouldallow the peroxidases to compete better for peroxynitrite and limit cytotoxicity [99].The reactivity of peroxynitrite with other potential targets in T. cruzi other than thiol-containing compounds has been less studied. Indeed, the kinetics of the reactionsbetween peroxynitrite and parasitic metal-containing proteins of antioxidant func-tions, namely ascorbate peroxidase and Fe-SOD A and B, are currently underinvestigation in our lab.Exogenous or endogenous exposure of T. cruzi to peroxynitrite leads to the

disruption of different physiological processes, such as cellular respiration, ener-getic metabolism and calcium homeostasis, cell motility and growth, and eventually

Table 11.1 Comparing the reactivity of peroxynitrite with thiol-containing cytosolic targetsin T. cruzi.

T(SH)2 GSH cTXNPxepimastigotes/trypomastigotes

TXNI CO2

ka) (M�1 s�1) 7200 1350 1.5� 0.5� 106 3500 4.6� 104

Concentration (mM) 1100b) 700b) 5.6c)/33.6d) 300–500e) 1300k�C (s�1) 7,9 0.95 8.4/50.4 �10�3 60

a) pH-dependent rate constants measured at pH 7.4. Note, since normal acidification of the phag-osomes is not inhibited by the parasite, the pH of phagosomes harboring T. cruzi is acidic, with valuesin the 5–7 range [88,89] Nevertheless, amastigotes, and trypomastigotes are able to maintain a near-neutral pH over a wide range of external pHs by mechanisms that depend on plasmamembrane Hþ-ATPase [90].

b) Epimastigotes of T. cruzi (Silvio strain and Y strain) in the presence of 100 mM putrescine, latelogarithmic phase. The concentration of free GSH increases and that of T(SH)2 decreases whenepimastigotes are cultured in polyamine-free media [91]. It is also lower in trypomastigotes than inepimastigotes, in Y, and some other strains [92,93].

c) Epimastigotes of T. cruzi Dm28-c and Ystrain [94]. A value of 2.8 mMwas reported for cTXNPx usingwhole homogenates without taking into account compartmentalization, so we can safely assume thatin the cytosol of the parasite the concentration of the enzyme will be at least twice as high.

d) The concentration of cTXNPx was reported to increase (around 6-fold) in trypomastigotes of T. cruzi Ystrain [95].

e) Estimated from a report indicating that TXNI constitutes around 3% of the total soluble proteincontent within epimastigotes, late logarithmic phase of growth [96]. Reactivity with CO2 is also shownfor comparative purposes.

Peroxynitrite Diffusion and Reactivity with T. cruzi Targets j221

causes parasite death [45,100,101]. The inhibitory effect of peroxynitrite on theenergetic metabolism of the parasite depends on the inhibition of succinatedehydrogenase and NADH-fumarate reductase, which play a key role in theseparasites since the NADH-dependent segment of the respiratory chain is inactiveand electrons from NADH must be transferred to fumarate to produce succinatethrough fumarate reductase to enter the respiratory chain [102]. Inactivation of bothenzymes was reverted by treatment with dithiothreitol, which together with the factthat cysteine residue(s) were critical for activity and the protective actions ofcompetitive inhibitors of the enzymes, indicated that active-site thiol(s) oxidationwas involved in peroxynitrite-mediated inactivation of these enzymes [100]. Theprotective action of methionine 5 mM (a scavenger of peroxynitrite andperoxynitrite-derived species [103]) and uric acid 2 mM (mostly a radical scavengeror repairing agent that reacts quite slowly with peroxynitrite [104,105]), together withthe minimal effect of physiological concentrations of CO2, indicated that mostprobably the effect of peroxynitrite on critical thiols was at least partially dependenton peroxynitrite-derived radicals, even in the absence of effect of classical hydroxylradical scavengers [100]. Peroxynitrite dose-dependently inhibited non-mitochon-drial Ca2þ uptake systems from T. cruzi epimastigotes, probably through theinactivation of a P-type Ca2þ -ATPase [101]. The oxidant also caused the parasiteenergetic pool to decrease, but the drop in ATP levels was delayed with respect to thedecrease of the ATP-dependent Ca 2þ transport. Moreover, peroxynitrite inhibitedmitochondrial calcium uptake systems of the parasite, an effect that was associatedwith a decrease in the membrane potential, but was independent of effects on theelectron transport chain, and has been proposed to be mediated by lipid and proteinoxidation [101], which have been demonstrated to occur when the parasites areexposed to this oxidant [106]. In T. cruzi , calcium homeostasis participates in keycellular functions such as multiplication, differentiation, programmed cell death,and host cell invasion [107– 109], processes that could therefore be altered byexposition to peroxynitrite.

Peroxynitrite Detoxification Systems

In 2000, Bryk et al. re port ed for t h e fi rst time the peroxynitrit e r eductase ac tivit y ofa t hiol-depe nd ent peroxidase of t he Prx type [110]. Since then, i t has been we llestab lished that me mb ers of diff erent s ubgroups of Prxs catalyz e th e tw o-e lectronreduction of peroxynitrite to nitrite [111]. T. cruzi expresses a mitochondrial and acytosolic Prx that have been named TXNPxs since they catalyze the reduction ofdifferent peroxides including peroxynitrite using the small protein TXN asreducing substrate [80,83,112,113]. Ac c ording to the Per oxiRedox in Classi fi cationIndEX (h ttp://csb.wf u.ed u/p rex /i n dex.p hp) bot h mitochondrial a nd cytosolicTXNPxs (mTXNPx and cTXNPx, respectively) belong to the AhpC/Prx1 subfamily,which is mechanistically classified as typical 2-Cys Prxs and includes TXNperoxidases, Arabidopsis thaliana 2-Cys Prx, barley Bas1, Saccharomyces cerevisiaeTSA1 and TSA2, bacterial alkyl hydroperoxide reductase C (AhpC), and


mammalian PRDXs 1–4. All Prxs possess a peroxidatic cysteine residue (CP), whichrapidly reduces peroxides such as hydrogen peroxide (H2O2), fatty acid hydroper-oxides, and other organic hydroperoxides (ROOH) as well as peroxynitrite to water,alcohols, and nitrite, respectively, with different selectivity [114,115]. In the process, CP

gets oxidized to sulfenic acid. In 2-Cys Prxs, which require a second cysteine residuefor catalytic activity, this sulfenic acid forms a disulfide bridge with a resolving cysteine(CR), which in typical 2-Cys Prxs is located in the C-terminus of another subunit toform an intermolecular disulfide bridge. The latter is then reduced by the reducingsubstrate, which for the AhpC/Prx1 subfamily is usually thioredoxin or a thioredoxin-related protein, such as TXNs in trypanosomatids [114,116] (Figure 11.2). The crystalstructure of recombinant cytosolic TcTXNPx (containing a histidine tail) underreduced state has been determined at 2.8-A

�resolution (Protein Data Bank ID:

1UUL) [117]. The quaternary structure of TccTXNPx is an assembly of five symmetrichomodimers that associate into a decamer. The CP (Cys52) is located in a first VCPmotif in the N-terminal portion of helixa1, and buried inside the active-site pocket that

Figure 11.2 Catalytic cycle of T. cruzi TXNPxsusing peroxynitrite as oxidizing substrate.Both mitochondrial and cytosolic TXNPxsfrom T. cruzi catalyze the two-electronreduction of peroxynitrous acid to nitrite(NO2

�). These enzymes, which belong to theAhpC/Prx1 subfamily of Prxs, display a ping-pong mechanism of reaction. During theoxidizing part of the catalytic cycle thethiolate in CP of reduced cytosolic ormitochondrial TcTXNPxs (SP

�) is oxidized tosulfenic acid (SPOH), with rate constants of1–2� 106 and 1.8� 107M�1 s�1, respectively,at pH 7.4 and 25 �C. The resulting sulfenicacid reacts with the thiol group of CR locatedin another subunit and an intermolecular

disulfide is formed. Reduction of the oxidizedforms of the enzymes by the sequentialaction of TXN, trypanothione (T(SH)2),trypanothione reductase (TR), and NADPHhas been reported in vitro. TXNI has alsobeen proved to be the natural reducingsubstrate for the cytosolic TcTXNPx in vivo(shown in gray). However, the identity of thereducing substrate for the mitochondrialisoform is still a matter of debate. Theperoxynitrite reductase activity of T. cruziTXN-dependent peroxidases of theglutathione peroxidase type has not beeninvestigated so far. (Adapted from [115] withkind permission by Elsevier).

Peroxynitrite Detoxification Systems j223

contains Pro45 and Thr49, residues that are highly conserved among Prxs[118,119]. The thiol in CP forms a hydrogen bond with Oc of Thr49 and Ne1 ofArg128 – interactions that are essential for catalytic activity in the homologousenzyme from L. donovani [117,118]. In turn, CR (Cys173), from the secondmolecule of the homodimer, is again located in a VCP motif, in a hydrophobicsolvent-excluded environment that is also important for activity [117]. Based on thecrystal structure of the enzyme, molecular dynamics simulations combined withelectrostatics analysis on monomeric and different oligomeric forms of TccTXNPxwere recently performed. The calculations indicate that the pKa values of CP arehighly dependent on the oligomeric state: a CP pKa comparable to that of freecysteine was calculated for the monomer and also for the subunits of the dimerwhen averaged over time, while all subunits in the decamer show a lowered CP pKa.The effect of these CP pKa shifts on enzymatic activity of the different oligomericforms of TccTXNPx has not been experimentally addressed yet. It is important tonote that, even though the mechanism of reaction between peroxides and thiolsinvolves the protonated form of the peroxide and thiolates as reactive species, thefractional amount of thiolate available for a CP (usually pKa< 6.4 [110,120,121])compared to free cysteine at physiological pH would only increase by a factor ofaround 10 and cannot explain the 103–107 acceleration in rate constants of reactionof CP in Prxs with respect to the uncatalyzed reaction [122] Therefore, destabiliza-tion of the decameric form of these enzymes would be expected to lead to anaround 10-fold decrease in enzymatic activity, if only the effects on the pKa of CP

were considered. Active-site microenvironment factors contributing to the catalyticmechanism of these enzymes are only starting to be identified [122–125], and therelationship between these factors and oligomerization is still unknown.mTXNPx from T. cruzi shows an important sequence similarity with the

cytosolic form – it possesses a mitochondrial import sequence at its N-terminusand the CR is located at a IPC motif. To date, the crystal structure of trypano-somatid mTXNPxs has not been resolved, but light scattering and high-resolutionelectron microscopy of mTXNPx from L. infantum indicated that the mitochon-drial enzyme also adopts a decameric, ring-like quaternary structure [126]. Bothcytosolic and mitochondrial TcTXNPx rapidly reduced peroxynitrite to nitrite, withrate constants of 1–2� 106 and 1.8� 107M�1 s�1, pH 7.4, 25 �C [83] (Figure 11.3).They also reduced other peroxides such as H2O2 and artificial organic hydro-peroxides such as tert-butyl hydroperoxide [83,112,113]. Moreover, the oxidizedform of TccTXNPx is reduced by cytosolic TXNI from T. brucei with a rate constantof 1.3� 106M�1 s�1 [83]. Although the mitochondrial isoform could also bereduced by cytosolic TXNI from T. brucei in vitro [83], it is obviously an artificialsituation that does not take into account compartmentalization and the naturalreducing substrate for TcmTXNPx in vivo is still a matter of debate. A recent reportindicated that, according to sequence homology, the second TXN coding sequencein T. cruzi (TcTXN2) groups together with TXN3 from Leishmania, which isanchored at the mitochondrial outer membrane with the redox-active domainfacing the cytosol [127]. The localization of TcTXN2 still requires experimentalconfirmation. In addition, reduction of TXN by trypanothione requires the latter


to be maintained at reduced state by the flavoenzyme trypanothione reductase atthe expense of NADPH, but information regarding the occurrence of trypano-thione reductase in mitochondria is rather contradictory [128,129]. Alternatively,dihydrolipoamide dehydrogenase/lipoamide was reported to be able to reduceintramitochondrial TXN2 from L. infantum [130]. T. cruzi expresses other thiol-dependent peroxidases, which show sequence similarity with glutathione peroxi-dases although TXN is a much more efficient reducing substrate [131]. Theseenzymes are presented in detail elsewhere in this book. To date, their capacity tocatalyze peroxynitrite reduction has not been investigated in vitro or in cellularsystems. In a recent collaborative work we have demonstrated that a related thiol-dependent glutathione peroxidase from poplar, GPx 5, reduces peroxynitrite witha rate constant of 1.4� 106M�1 s�1, pH 7.4, 25 �C [132]. Further studies arerequired to find out whether thiol-dependent glutathione peroxidase-like enzymesparticipate in peroxynitrite detoxification in T. cruzi and other trypanosomatids.

Figure 11.3 Kinetics of peroxynitrite reductionby cTXNPx from T. cruzi. The rate constant ofperoxynitrite reduction by TccTXNPx wasmeasured by a direct approach, followingperoxynitrite decay at 310 nm(e¼ 1600M�1 cm�1). Peroxynitrite in dilutedsodium hydroxide was mixed with 100mMsodium phosphate plus 100mM dtpa without(a), with wild-type TccTXNPx (32.2mM) (b), orwith TccTXNPx mutated at the peroxidaticcysteine residue (37.5mM) (c). Measured finalpH was 7.4. The inset shows a plot of initial rateof peroxynitrite decay versus TccTXNPxconcentration. From this plot a rate constant

of peroxynitrite reduction by the enzyme wascalculated as 1� 106M�1 s�1. The kinetic of thereaction was also measured by two indirect,competition approaches (with horseradishperoxidase oxidation to compound I and withmanganese(III)-meso-tetrakis)N-methylpyridinium-3-yl) porphyrin oxidation)with similar results. From these experiments,a mean rate constant value of1.5� 0.5� 106M�1 s�1 for peroxynitritereduction by TccTXNPx at pH 7.4 wasdetermined. (Adapted from [83] with kindpermission by Elsevier).

Peroxynitrite Detoxification Systems j225

Likewise, the capacity of the heme-dependent peroxidase ascorbate peroxidasefrom T. cruzi to reduce peroxynitrite in vitro remains to be investigated. In anycase, the fact that parasites transformed to overexpress this enzyme were moreresistant to hydrogen peroxide treatment, but not to peroxynitrite, argues against arole of ascorbate peroxidase in peroxynitrite detoxification [84].

T. cruzi Antioxidant Enzymes as Virulence Mediators

The role of peroxynitrite detoxification by T. cruzi antioxidant enzymes in theoutcome of the infection has been demonstrated in both cellular and animal modelsof the disease. At a cellular level, phagocyted wild-type trypomastigotes loadedwith dihydrorhodamine showed a much higher probe oxidation than TccTXNPx-overexpressing trypomastigotes when infecting IFN-c/lipopolysaccharide (LPS)-activatedmacrophages, in agreement with the enhanced capacity of these geneticallymodified cells to detoxify peroxynitrite (Figure 11.4a). Moreover, studies usingparasites preloaded with [5,6-3H]uridine as a probe of membrane integrity showedthat macrophages activated with IFN-c and LPS were toxic to wild-type trypomas-tigotes, while TccTXNPx-overexpressing cells showed resistance (Figure 11.4b),indicating that the simultaneous generation of O2

��and �NO is involved in thecytotoxic effect of macrophages on internalized parasites at this time point of theinfection (2 h) (Figure 11.4b). Previous reports have shown that the concentration offree thiols (GSH, GSH-Spd, and T(SH)2) varies between the different parasite stages(epimastigotes, trypomastigotes, and amastigotes) and also between differentT. cruzi strains [93]. Moreover, several proteomic analyses have suggested theupregulation of members of the T. cruzi antioxidant network (trypanothionesynthetase (TS), mTXNPx; TXN, iron SOD A (FeSODA), and ascorbate peroxidase)in the infective metacyclic trypomastigote compared with the non-infective epimas-tigote stage [133,134]. Due to the fact that T. cruzi is not a clonal population our grouphas searched for the upregulation of different components of the antioxidantnetwork in several strains belonging to the major phylogenetic groups duringthe differentiation process to the infective metacyclic trypomastigotes. Our resultsshow for all the analyzed strains, upregulation of cTXNPx, mTXNPx, and TS proteincontent during metacyclogenesis, making this a general preadaptation process thatallows T. cruzi to deal with the nitroxidative environment of the vertebrate hostfound during invasion [95]. Most importantly, a positive association betweenvirulence of different T. cruzi strains and cTXNPx, mTXNPx, and TS levels wasfound, highlighting the interplay between the antioxidant enzyme network and hostoxidative defense mechanisms [95]. In accordance, strains of augmented virulenceshowed an abundant heart inflammatory infiltrate with high parasitemias in theacute phase of Chagas disease. Moreover, overexpression of TccTXNPx on trypo-mastigotes increases virulence as evidenced by a 3-fold increase in parasitemia withthe presence of severe inflammatory infiltrates in heart and skeletal muscle whencompared with the wild strain. These data reinforce the concept that success ofinfection and progression to the chronic phase depend on parasite antioxidant


Figure 11.4 T. cruzi killing by intraphagosomalperoxynitrite and protective effect of TccTXNPx.(a) Wild-type (CL-Brener) or TccTXNPx-overexpressing trypomastigotes preloaded withdihydrorhodamine (DHR) were exposed tounstimulated (T. cruzi) or immunostimulatedinfected (IFN-c/LPSþ T. cruzi) macrophages.Dihydrorhodamine oxidation after 2 h ofinfection was determined in a fluorescence

plate reader. (b) Macrophages were infectedwith wild-type or TccTXNPx-overexpressingtrypomastigotes preloaded with [5,6-3H]uridine.After incubation (2 h) supernatants werecollected and radioactivity measured in a liquidscintillation counter. Data are mean� standarderror of three independent experiments. (Takenfrom [27]).

T. cruzi Antioxidant Enzymes as Virulence Mediators j227

enzyme levels. T. cruzi contains a repertoire of four FeSODs that detoxify O2��

generated in the cytosol (TcSODB1), glycosomes (TcSODB1-2), and mitochondria(TcSODA and C) [135]. SODs readily eliminate O2

�� at the site of production (e.g.,cytosolic redox cycling of drugs, mitochondrial electron transport chain) and maycontribute to parasite immune evasion by (i) protecting from direct cytotoxic effectsof O2

��, such as aconitase inhibition [136], by its dismutation toH2O2, (ii) regulatingthe O2

��-mediated induction of parasite programmed cell death [137], and(iii) inhibiting the endogenous formation of peroxynitrite. Whether FeSODs con-stitute virulence factors for infections caused by T. cruzi remains to be established.

Conclusions

Macrophages constitute key host cells during the acute infection by T. cruzi. Insidethe phagosome of immunostimulated macrophages, simultaneous production of�NO and O2

�� leads to peroxynitrite formation. Peroxynitrite can diffuse to theparasite where, through oxidation and/or nitration of different targets, it causes thedisruption of different physiological processes such as energetic metabolism,calcium homeostasis, cell motility, and growth, and eventually causes parasitedeath. T. cruzi contains a battery of antioxidant enzymes that catalyze the detoxifi-cation of the reactive species it encounters. Among them, cytosolic and mitochon-drial TXNPxs have been demonstrated to rapidly reduce peroxynitrite as well asother peroxides. Parasites overexpressing these enzymes were more resistant toperoxynitrite-mediated toxicity, either added in bolus or formed by activated macro-phages. Interestingly, we tested the content of both cTXNPx and mTXNPx indifferent T. cruzi strains, and found a much higher expression in trypomastigotescompared with epimastigotes, indicating a higher capability of peroxide detoxifica-tion. Moreover, when analyzing different naturally occurring T. cruzi strains, thecontent of both enzymes correlated with virulence in an animal model of disease.Also, genetically engineered parasites overexpressing TccTXNPx recapitulated thehigh parasitemia and tissue damage observed in the virulent strains [27]. Altogether,these data support that parasitic TXNPxs could constitute drug targets for arationalized drug development. A potential role of peroxynitrite in controllingT. cruzi infection during the chronic stage of Chagas disease, where non-phagocyticcells such as myocytes are the main host cells and can control T. cruzi infection by�NO-dependent processes [50,51], deserves further investigation.

Acknowledgments

This work was supported by grants from Comisi�on Sectorial de Investigaci�onCientífica, Universidad de la Rep�ublica and National Institutes of Health(2R01Hl063119-05 and 1R01AI095173-01). Financial support was also providedto the authors by the Programa de Desarrollo de Ciencias B�asicas (PEDECIBA,Uruguay). M.H. was partially supported by a fellowship from Agencia Nacional deInvestigaci�on e Innovaci�on.


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