Leishmania Mitochondrial Peroxiredoxin Plays a Crucial Peroxidase-Unrelated Role during Infection: Insight into Its Novel Chaperone Activity

Two-cysteine peroxiredoxins are ubiquitous peroxidases that play various functions in cells. In Leishmania and related trypanosomatids, which lack catalase and selenium-glutathione peroxidases, the discovery of this family of enzymes provided the molecular basis for peroxide removal in these organisms. In this report the functional relevance of one of such enzymes, the mitochondrial 2-Cys peroxiredoxin (mTXNPx), was investigated along the Leishmania infantum life cycle. mTXNPx null mutants (mtxnpx−) produced by a gene replacement strategy, while indistinguishable from wild type promastigotes, were found unable to thrive in a murine model of infection. Unexpectedly, however, the avirulent phenotype of mtxnpx− was not due to lack of the peroxidase activity of mTXNPx as these behaved like controls when exposed to oxidants added exogenously or generated by macrophages during phagocytosis ex vivo. In line with this, mtxnpx− were also avirulent when inoculated into murine hosts unable to mount an effective oxidative phagocyte response (B6.p47phox−/− and B6.RAG2−/− IFN-γ−/− mice). Definitive conclusion that the peroxidase activity of mTXNPx is not required for parasite survival in mice was obtained by showing that a peroxidase-inactive version of this protein was competent in rescuing the non-infective phenotype of mtxnpx−. A novel function is thus proposed for mTXNPx, that of a molecular chaperone, which may explain the impaired infectivity of the null mutants. This premise is based on the observation that the enzyme is able to suppress the thermal aggregation of citrate synthase in vitro. Also, mtxnpx− were more sensitive than controls to a temperature shift from 25°C to 37°C, a phenotype reminiscent of organisms lacking specific chaperone genes. Collectively, the findings reported here change the paradigm which regards all trypanosomatid 2-Cys peroxiredoxins as peroxide-eliminating devices. Moreover, they demonstrate, for the first time, that these 2-Cys peroxiredoxins can be determinant for pathogenicity independently of their peroxidase activity.


Introduction
The last 15 years have contributed decisively towards the dissection of the enzymatic pathways that lead to peroxide elimination in trypanosomatids, a group of organisms that includes Leishmania spp., Trypanosoma brucei and Trypanosoma cruzi, the causative agents of the different manifestations of leishmaniasis, African sleeping sickness and Chagas' disease, respectively.In these protozoan parasites, which lack the highly efficient enzymes catalase and selenium-containing glutathione peroxidases (GPXs), members of the peroxiredoxin family are regarded as key elements of the peroxide-reduction machinery [1].
Peroxiredoxins (PRXs) are ubiquitous enzymes that use a redox active cysteine residue (peroxidatic Cys) to reduce a broad spectrum of substrates, namely H 2 O 2 , organic hydroperoxides and peroxynitrite (ONOO 2 ).Upon reduction of the peroxide, the peroxidatic Cys-SH is oxidized to sulfenic acid (Cys-SOH).In PRXs harboring two active cysteines (known as 2-Cys PRXs), the sulfenic acid is reduced by another Cys residue (resolving Cys) to form a disulfide.According to the location of the resolving Cys, 2-Cys PRXs can be classified as typical or atypical [2].In trypanosomatids all PRXs characterized to date fall in the category of typical 2-Cys [3].Peroxiredoxins return to their reduced state upon reduction of the disulfide by an appropriate electron donor.In trypanosomatids such reductant is a unique oxidoreductase of the thioredoxin superfamily, known as tryparedoxin [4], which itself is reduced by these organisms' specific thiol trypanothione [N 1 ,N 8 -bis(glutathionyl)spermidine; 1,3].For that reason trypanosomatid PRXs are commonly referred to as tryparedoxin peroxidases or TXNPxs.Apart from participating in antioxidant defense, members of the 2-Cys PRX subfamily are increasingly recognized as playing a more subtle and sophisticated role as regulators of peroxide-mediated cell signaling [2].More recently, a function as molecular chaperones has also been proposed for some of these enzymes [5,6].
In trypanosomatids 2-Cys PRXs are present in the parasites' cytosol and single mitochondrion [1].Cytosolic 2-Cys PRXs (or cTXNPxs) are believed to work as general antioxidant devices that minimize the oxidative insult generated by the parasites' host.In agreement with this, overexpression of cTXNPx in L. infantum and T. cruzi was found to confer resistance to H 2 O 2 and peroxynitrite of exogenous origin [7][8][9][10][11][12], while down-regulation of the T. brucei counterpart enhanced sensitivity to bolus H 2 O 2 [13].This peroxidase activity should be particularly relevant for Leishmania and T. cruzi, which can invade and proliferate in phagocytes.Not surprisingly, cTXNPxs of these organisms were pointed out as important virulence factors [12,[14][15][16][17].
Mitochondrial TXNPxs (or mTXNPxs) are, due to their location, favorably positioned to eliminate peroxides produced endogenously, namely those formed as by-products of oxidative phosphorylation, and this was argued to be their main function [7,8,11].However, the observation that overexpression of mTXNPx confers protection towards exogenously added and macrophage-derived H 2 O 2 and peroxynitrite [7,8,10,11], suggested that these enzymes might also contribute to shield trypanosomatids from the oxidative challenge induced by their hosts.This assumption was strengthened by the observation that increased mTXNPx expression is associated with virulent T. cruzi phenotypes [17].Apart from functioning as general antioxidant devices, the Leishmania donovani mTXNPx was reported to prevent H 2 O 2induced programmed cell death [18].One additional, peroxidaserelated role suggested for mTXNPxs was regulation of kinetoplast DNA (kDNA) replication.The kDNA is a network of catenated maxi and mini DNA circles that compose the mitochondrial DNA of trypanosomatids and whose replication is initiated when the universal minicircle sequence binding protein (UMSBP) binds specific sequences on minicircle DNA [19].UMSBP binds to the DNA when it is reduced and is released when oxidized.According to the proposed model, mTXNPx would oxidize UMSBP [20].
The present study aimed at dissecting the functional relevance of mTXNPx in Leishmania infantum.Leishmania have a digenic life cycle that includes two morphologically and physiologically distinct stages: the promastigote (an extracellular form residing in the insect vector) and the amastigote (an intracellular form living inside the mammalian host).Using a homozygous knockout L. infantum line unable to express mTXNPx it was found that, while redundant in promastigotes, this mitochondrial 2-Cys PRX is essential for the establishment of a successful infection in mammals.This result establishes mTXNPx as factor determinant for Leishmania pathogenicity.Importantly, the data gathered here indicate that the essential role played by mTXNPx is not related to its peroxidase activity.Rather, the decreased infectivity of the mutants may be explained by the ability of mTXNPx to function as a chaperone, an activity disclosed here.

Generation of mTXNPx mutants
To study the relevance of mTXNPx during the L. infantum life cycle, a mutant parasite line unable to express this enzyme was produced by homologous recombination.Since Leishmania has a diploid genome, two successive rounds of gene targeting were required to obtain homozygous knockout mutants.The first mTXNPx allele was replaced by a NEO integration cassette, while the second was disrupted with a HYG construct (Figure 1A).As confirmed by Southern blot (SB) of the double transfectants, both mTXNPx alleles were successfully targeted, thus resulting in the generation of a Dmtxnpx::NEO/Dmtxnpx::HYG mutant (hereafter referred to as mtxnpx 2 ).The mtxnpx 2 mutant was further manipulated in order to obtain a parasite line with restored mTXNPx expression.This was achieved by integrating the mTXNPx ORF into the small sub-unit rRNA locus using the pSSU-PHLEO-infantum vector (Figure 1A), which is a modified version of pSSU-NEOinfantum [21].The rescued knockout mutants, designated mtxnpx 2 / +mTXNPx (Dmtxnpx::NEO/Dmtxnpx::HYG [pSSU-PHLEO-infantum-mTXNPx]), were confirmed by PCR to have the mTXNPx ORF and the PHLEO cassette correctly integrated into the ribosomal locus (Figure 1C).Western blot and indirect immunofluorescence analysis showed that mtxnpx 2 mutants lack mTXNPx expression and that the rescued mtxnpx 2 /+mTXNPx parasites express mTXNPx in its correct subcellular compartment, the mitochondrion (Figures 1D and E).

mTXNPx is redundant in the insect stage of L. infantum
The mtxnpx 2 parasite line was generated in the promastigote stage and when grown under standard culture conditions, i.e. in serum supplemented RPMI medium at 25uC, it appeared morphologically normal (data not shown) and displayed growth rates similar to that of wild type promastigotes.Most notably, no defects in kinetoplast morphology and division were noticed (Figure 1E).These observations argue against the involvement of mTXNPx in kDNA replication, a function previously attributed to mitochondrial TXNPxs [20,22].Even though these results indicate that mTXNPx is redundant in the insect form of L. infantum, they do not discard a relevant role for this enzyme during the amastigote stage.
mTXNPx is crucial for the long term survival of L. infantum in the mammalian host The consequence of mTXNPx depletion on the survival of L. infantum amastigotes was assessed by inoculating BALB/c mice, an animal model for visceral leishmaniasis (VL), with equal numbers of stationary phase mtxnpx 2 , wild type or mtxnpx 2 /+mTXNPx promastigotes.At defined time points after infection, parasite loads in the liver and spleen (the organs preferentially infected by VL strains) were analyzed by the limiting dilution assay (LDA).Results in Figure 2 show that elimination of mTXNPx had a remarkable effect on the outcome of infection and that this aggravated over time.Indeed, whilst 2 weeks after infection mtxnpx 2 could still be recovered from both infected organs, at 4 and 8 weeks the mtxnpx 2 infection indexes were much lower than those of wild type parasites, being below the detection limit of the assay (2.7 log units) at 14 weeks.The impaired virulence of mtxnpx 2 was recovered in knockout parasites with restored mTXNPx expression (mtxnpx 2 / Author Summary Leishmania are insect-borne protozoan parasites that cause leishmaniasis, a spectrum of diseases with relevance in men and dogs and for which there are no adequate means of control.In order to successfully infect their mammalian hosts, Leishmania requires the expression of a set of key proteins.Here, we identify one mitochondrial protein of the parasite as one of such molecules.Interestingly, however, we also discovered that the reason why this enzyme is essential is not due to its well characterized peroxidase activity, which defines it as a member of the peroxiredoxin family.Instead, the importance of this protein during infection may derive from its ability to function as a chaperone, as implied here.In short, by proposing a novel, peroxidase-unrelated role for the Leishmania mitochondrial peroxiredoxin during mammals' infection, this work opens new perspectives regarding the physiologic function of this enzyme.+mTXNPx), confirming that it specifically results from depletion of this enzyme (Figure 2).Of notice, the parasitemia levels of mice infected with mtxnpx 2 /+mTXNPx were consistently below those produced by wild type parasites, a phenomenon that is frequently observed in complemented knockout mutants [23][24][25].This is likely due to the fact that in the rescued mutants mTXNPx expression is not under the control of its own untranslated regions (as occurs in wild type parasites), but rather it is regulated by the rRNA promoter of the small sub-unit rRNA and by the intergenic region of the cysteine proteinase B 2.8 gene cluster [26].
In short, these results show that mTXNPx is crucial for the long term survival of L. infantum amastigotes in the mammalian host.The impaired capacity of mTXNPx-depleted parasites to thrive in mice is not due to their failure to invade host cells (the macrophages) or to differentiate into amastigotes, as inferred from microscopic observation of monolayers of peritoneal macrophages from C57/BL6 mice infected with mtxnpx 2 parasites (Figure S1).The next question is which function, disturbed in mutant parasites, is leading to such defect in infectivity.

Depletion of mTXNPx has no impact on the parasite antioxidant capacity
The L. infantum mTXNPx is an efficient reductase of hydroperoxides [27] as well as of peroxynitrite (the apparent second order rate for peroxynitrite reduction by mTXNPx is 1.6610 6 M 21 s 21 , at pH 7.4 and 37uC; Romao S, Radi R and Toma ´s AM, unpublished results).As suggested previously [7,8,11], the most likely function for mTXNPxs is the elimination of peroxides generated as a consequence of the parasite's aerobic metabolism.Accordingly, mtxnpx 2 mutants were assessed for their sensitivity to antimycin A (AA), an inhibitor of the mitochondrial electron transport chain that leads to the local production of reactive oxygen species [28].However, the observation that AA had the same growth inhibitory effect in mtxnpx 2 and wild type promastigotes (Figure 3A) suggests that mTXNPx is not critical for the elimination of peroxides generated within the mitochondrion.A different explanation for the impaired infectivity of mtxnpx 2 could be their inability to deal with host-derived oxidants [11].However, this premise found no support in the observation that susceptibility of mtxnpx 2 promastigotes to H 2 O 2 (added as bolus) or to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) was similar to that of wild type parasites (Figures 3B  and C).This hypothesis was explored further by exposing mtxnpx 2 to host-derived oxidants.As shown in the NBT assay in Figure 3D (inset), phagocytosis of L. infantum promastigotes by murine peritoneal macrophages triggers the generation of superoxide anion (O 2 .2 ), a precursor of H 2 O 2 .The absence of mTXNPx did not render promastigotes more sensitive to such oxidative burst, as deduced from the comparison of the infection indexes of macrophages inoculated with mtxnpx 2 and control parasites (wild type and mtxnpx 2 /+mTXNPx) (Figure 3D).Additional evidence that mTXNPx is not implicated in Leishmania protection against hostderived oxidants was obtained in an in vivo infection experiment using mutant mice with impaired pro-oxidant capacity, namely B6.p47 phox2/2 and B6.RAG2 2/2 IFN-c 2/2 mice.B6.p47 phox2/2 mice carry a targeted disruption of the p47 subunit of the phagocyte NADPH oxidase (phox) complex that is responsible for phagocytosis-induced production of O 2 .

2
. B6.RAG2 2/2 IFN-c 2/2 mice lack B and T cells and do not express interferon gamma (IFN-c), i.e. a pro-inflammatory cytokine predominantly secreted by type-1 T cells that activates the inducible nitric oxide synthase (iNOS) and up-regulates phox, thus allowing for peroxynitrite generation in macrophages.Mutants and control mice of the same genetic background (C57BL/6) were infected with equal numbers of mtxnpx 2 and of control parasites (wild type and mtxnpx 2 /+mTXNPx) and 5 weeks later the parasite burden was analyzed.The results, plotted in Figure 3E, show that the number of mtxnpx 2 parasites recovered from the organs of infected mice was either consistently below that of control parasites or undetectable irrespective of the mouse strain serving as host.In other words, even when inoculated in hosts that are unable to mount an efficient oxidative response, mtxnpx 2 do not recover infectivity.This observation reinforces the idea that the essential function of mTXNPx in amastigotes is not to provide protection against peroxides of exogenous origin.Additionally, it indicates that the avirulent phenotype of mtxnpx 2 is not due to any microbicidal component induced by the host adaptive immune system, namely by the T lymphocytes.Rather, the inability of mtxnpx 2 to thrive in mammalian hosts appears to be a consequence of factors inherent to the parasite.

The critical function of mTXNPx is independent of its peroxidase activity
Results in the previous section suggested that the crucial function played by mTXNPx during infection is not that of an antioxidant device.To definitively conclude about the contribution of mTXNPx peroxidase activity to Leishmania infectivity, a peroxidase-inactive variant of the enzyme (mTXNPxC81S) was tested for its ability to rescue the avirulent phenotype of the knockouts.Cys81 is the peroxidatic Cys of mTXNPx and its replacement by a serine should abolish peroxidase activity, as observed previously [29,30].Loss of peroxidase activity of the C81S variant was confirmed in vitro using a recombinant enzyme lacking the first 26 amino acids that compose the mitochondrial targeting peptide (DmTXNPxC81S) to mimic the mature protein in the parasite's mitochondrion [8].As expected from previous mutational analysis [29,30], when assayed for TXNPx activity, the DmTXNPxC81S mutein was unable to catalyze H 2 O 2 reduction, even when present at a 10-fold higher concentration than the control wild type DmTXNPx enzyme (Figure 4A).Of notice, on the basis of circular dichroism spectra, substitution of the peroxidatic Cys to a Ser did not alter the secondary structure of the mutein relative to the wild type enzyme, the estimation of a-helix and b-strand contents for both enzymes being of 35% and 29%, respectively (data not shown).
To test whether expression of mTXNPxC81S could restore infectivity of mTXNPx knockouts, mtxnpx 2 /+mTXNPxC81S mutants were generated by transfecting mtxnpx 2 with pSSU-PHLEOinfantum-mTXNPxC81S.Confirmation for the correct integration of the construct was obtained by PCR (Figure 4B) and the exact subcellular location verified by indirect immunofluorescence analysis (Figure 4C).The mtxnpx 2 /+mTXNPxC81S transfectants were then inoculated into BALB/c mice and the parasite burden in livers and spleens evaluated at 4 and 8 weeks post infection.The results, depicted in Figure 4D, show that virulence was recovered in mtxnpx 2 complemented with mTXNPxC81S and that the parasite load produced by this line was similar to that of mtxnpx 2 / +mTXNPx.These observations thus provide solid evidence that the essential role played by mTXNPx during the infective stage of Leishmania is independent of its peroxidase activity.

Is mTXNPx functioning in vivo as a molecular chaperone?
In addition to acting as peroxidases, 2-Cys PRXs may in some cases exhibit chaperone activity [5,6], an attribute that, to date, has never been described for any TXNPx.The possibility of mTXNPx acting as a chaperone was therefore investigated as an The chaperone activity of purified recombinant DmTXNPx was assessed in vitro by testing its ability to suppress the aggregation of thermally denatured citrate synthase (CS).When incubated at 43uC citrate synthase unfolds, leading to significant aggregation that can be monitored by measuring light scattering in a spectrofluorometer [31].Addition of DmTXNPx to the reaction at a 10-fold molar excess completely suppressed the thermal aggregation of CS (Figure 5A).This effect was much less pronounced when DmTXNPx was added at 5-fold molar excess.Importantly, the DmTXNPxC81S mutein was also capable of preventing CS aggregation, exhibiting the same behavior as the wild type enzyme (Figure 5A).
To gain insight into the physiological significance of the chaperone activity of mTXNPx a thermotolerance assay was conducted.The experiment consisted in monitoring cell growth of mtxnpx 2 and control parasites (wild type, mtxnpx 2 /+mTXNPx, and mtxnpx 2 /+mTXNPxC81S) at both 25uC and 37uC, which are the temperatures encountered by the parasite in the insect vector and in the mammalian host, respectively.The cell growth curves depicted in Figure 5B show that, while at 25uC the proliferation rates of all parasite lines were indistinguishable, at 37uC the growth rate of mtxnpx 2 was significantly lower than that of wild type parasites.The thermo-sensitive phenotype observed for mtxnpx 2 was recovered in the rescued mtxnpx 2 /+mTXNPx and mtxnpx 2 /+mTXNPxC81S mutants.These observations show that expression of mTXNPx The data is expressed as the percentage of promastigote replication relative to control cultures without any exogenous agent.Graphs represent means and standard deviation of three or more experiments (each performed in duplicate).D. Monolayers of peritoneal macrophages from C57BL/6 mice were infected with stationary phase wild type (light grey bars), mtxnpx 2 (black bars) or mtxnpx 2 /+mTXNPx (dark grey bars) promastigotes and 24 hrs later the infection indexes determined.The inset shows light microscopy photographs of macrophages incubated with NBT in the absence (''w/out L.i.'') or presence (''w/L.i.'') of parasites.Deposits of dark blue insoluble formazan resulting from NBT reaction with superoxide anion were visible in macrophages 30 min after contact with L. infantum promastigotes (right panel), but not in macrophages to which no parasites were added (left panel).Images were acquired at 1006 magnification.E. Mice from different strains (C57BL/6, B6.p47 phox2/2 and B6.RAG2 2/2 IFN-c 2/2 ) were inoculated intraperitoneally with wild type (light grey bars), mtxnpx 2 (black bars) or mtxnpx 2 /+mTXNPx (dark grey bars) stationary phase promastigotes.Five weeks after infection parasite burden (PB) was determined in the livers and spleens of mice by the limiting dilution assay.The Y axis represents log 10 PB.Data represent mean and standard error of the mean of 3 independent experiments involving a total of 72 animals.ND, not detected.Differences between mtxnpx 2 and controls are statistically significant.Statistical analysis of these results is in TextS1 and Table S3.doi:10.1371/journal.ppat.1002325.g003renders promastigotes more permissive to 37uC, i.e. this enzyme confers thermotolerance to L. infantum irrespective of its peroxidase activity.It is thus reasonable to speculate that the crucial role played by mTXNPx during amastigote development in the mammalian host is that of a molecular chaperone.

Discussion
In parasites of the family Trypanosomatidae the peroxidase activity of 2-Cys PRXs (which in these organisms are designated TXNPxs) is accepted as the basis of their physiologic functions [1,3].Data presented in this report demonstrate that this activity is not crucial in the case of mTXNPx, even though the protein itself is essential for L. infantum amastigote survival.Instead, experimental evidence is provided suggesting that it may be the activity of mTXNPx as a molecular chaperone, unraveled here, that is determinant for parasite viability.
The finding that a peroxidase-inactive version of mTXNPx is competent to ensure amastigote survival excludes per se a crucial role for the peroxidase-related functions previously attributed to mTXNPxs, namely elimination of peroxides and regulation of H 2 O 2 -induced apoptosis [7,8,11,18].Accordingly, these activities must be taken over by other means.Detoxification of exogenouslyderived peroxides (such as those generated by the host immune response) might be efficiently fulfilled by cytosolic 2-Cys PRXs and non-selenium glutathione peroxidase-like enzymes (nsGPX) [7,8,11,[32][33][34].As for reduction of peroxides of mitochondrial origin, different scenarios can be envisaged.One possibility is that mitochondrial nsGPX carries out this antioxidant function.This hypothesis requires that the substrate specificities of nsGPX and mTXNPx overlap [33][34][35], and implies the existence of an efficient nsGPX reductant in the mitochondrion, which is still to be found [36].Of notice, in the particular case of H 2 O 2 , this could diffuse and be reduced by cytosolic peroxidases.In a different perspective, elimination of peroxides may occur via non-catalytic pathways.In this situation, low molecular weight thiols, such as trypanothione (the trypanosomatids' specific thiol), glutathione, ovothiol, free cysteines and even thiol groups exposed on protein surfaces would directly reduce peroxides [37][38][39].In either circumstance, an ascorbate peroxidase, located in the intermembrane space of the Leishmania mitochondrion [40] (gene LinJ.34.0070 in the L. infantum GeneDB), may also contribute to peroxide elimination in this organelle.
A different role previously attributed to mTXNPxs is the regulation of kDNA replication.As proposed by Sela et al. [20], mTXNPx would affect kDNA replication by oxidizing UMSBP and, consequently, turning ''off'' the binding of this protein to the minicircle origin of replication.However, in this report no evidence could be found linking mTXNPx depletion to defects in replication rate or in kDNA morphology of L. infantum promastigotes.Even though these analyses could not be extended to amastigotes, kDNA replication is highly conserved in trypanosomatids [19], making it difficult to accept that mTXNPx would regulate kDNA replication only in this life stage.Moreover, the proposed model for kDNA replication established a fundamental role for a mitochondrial TXN as reductant of UMSBP and it was shown previously that such enzyme is not essential in Leishmania and does not exist in trypanosomes [36].The mechanism underlying regulation of kDNA replication involving mTXNPxs (and a mitochondrial TXN) must, therefore, be re-evaluated.
This work has also, for the first time, uncovered that trypanosomatid 2-Cys PRXs can function as molecular chaperones and that this activity might be relevant for parasite survival in vivo.Several pieces of evidence point into this direction.i) Purified recombinant mTXNPx was shown to prevent the thermal aggregation of CS, a well-known chaperone substrate [31].This activity does not depend on the peroxidatic capacity of mTXNPx, as the mTXNPxC81S mutein was also active in this assay, thus providing a rationale for the recovery of virulence observed for mtxnpx 2 /+mTXNPxC81S.This is in line with observations reported by Jang et al. [5].ii) The chaperone activity of 2-Cys PRXs is known to depend on their quaternary structure and, as shown before, mTXNPx is capable of associating into decameric ring-like structures [27], which are the minimal oligomeric arrangements required for such function [41].iii) Gain of chaperone function and loss of peroxidase activity of 2-Cys PRXs are, in most  S4.doi:10.1371/journal.ppat.1002325.g004organisms, accompanied with the overoxidized form of the protein [2] and mTXNPx undergoes overoxidation when promastigotes are exposed to 37uC (Figure S2).Since Leishmania do not possess sulfiredoxins to enzymatically regenerate overoxidized 2-Cys PRXs [1] as occurs in higher eukaryotes, such peroxidase-inactive form of mTXNPx is likely to persist until the protein is synthesized de novo.iv) Additional support consubstantiating a role for mTXNPx as a chaperone came from the observation that lack of mTXNPx expression rendered promastigotes more sensitive to 37uC, the temperature encountered in the mammalian host.Such thermo-sensitive phenotype is reminiscent of some chaperonedeficient mutants [42][43][44].Accepting that mTXNPx is a molecular chaperone in vivo, one can only speculate how this function could impact on parasite survival under the conditions met in the host.In mitochondria, molecular chaperones are associated with a multitude of functions.They participate in the biogenesis of new mitochondrial peptides by assisting their transmembrane transport and their refolding to the native conformation inside the matrix.In addition, they are part of the mitochondrial protein quality control system that repairs damaged or misfolded proteins or mediates their removal by proteolysis [45].Under situations of cellular stress (including elevated temperatures), the activity of mitochondrial chaperones becomes even more imperative for protein homeostasis [45].Accordingly, it is reasonable to assume that mTXNPx functions as a molecular chaperone that ensures integrity of mitochondrial functions, its activity being particularly relevant when parasites reside in vertebrate host.
Another important outcome of this report is the identification of mTXNPx as a new factor critical for Leishmania infectivity.This finding may have impact on the development of new strategies to control leishmaniasis as the mtxnpx 2 parasite line produced here can be regarded as the basis of a live attenuated vaccine.In this context, the observation that mtxnpx 2 cannot give rise to a productive infection even in immunocompromised mice, suggests that this strategy would not pose major safety issues.
In conclusion, the work presented in this manuscript sheds lights on the functional relevance of mTXNPx by showing that, even though this molecule is crucial for L. infantum survival during infection of the vertebrate host, its peroxidase activity is superfluous.A novel peroxidase-unrelated function, as a molecular chaperone, is proposed for this enzyme, which could be critical for mitochondrial functionality under the conditions encountered by the parasite in the mammalian host.This report thus constitutes a turning point into the current state of knowledge regarding the physiologic role of peroxiredoxins in trypanosomatids.

Ethics statement
The experimental animal procedures were approved by the Local Animal Ethics Committee of Institute for Molecular and Cell Biology, University of Porto, Portugal and licensed by DGV (General Directory of Veterinary, Ministry of Agriculture, Rural Development and Fishing, Govt. of Portugal), in May 18, 2006 with reference 520/000/000/2006.All animals were handled in strict accordance with good animal practice as defined by national authorities (DGV, Law nu1005/92 from 23rd October) and European legislation EEC/86/609.

Generation of knockout and rescue constructs
Primers used to generate the constructs are summarized in Table S1.The accuracy of all assembled constructs was verified by sequencing.To produce the NEO replacement construct, sections of the 59 and 39 non-coding sequences flanking the mTXNPx ORF Figure 5. Evidences for the chaperone activity of mTXNPx.A. Chaperone activity of purified recombinant DmTXNPx and DmTXNPxC81S was analyzed in vitro by assessing the enzymes' ability to suppress the thermal aggregation of citrate synthase (CS).Aggregation of CS was induced at 43uC and monitored by measuring light scattering at 500 nm in the absence (open circles) or presence of 5:1 (grey-filled squares) or 10:1 (black squares) ratios of DmTXNPx monomers to CS monomers.Alternatively, the mutein DTXNPxC81S was added to the reaction at 5:1 (grey-filled triangles) or 10:1 (black triangles) ratios.B. Wild type (light grey squares), mtxnpx 2 (black triangles) mtxnpx 2 /+mTXNPx (dark grey diamonds) and mtxnpx 2 /+mTXNPxC81S (dark grey circles and dashed line) L. infantum promastigotes were seeded at 10 6 ml 21 in complete RPMI medium and incubated at 25uC (left) and 37uC (right).The Y axis represents ln (or log e ) of cell densities recorded throughout 5 days.Values represent mean and standard error of the mean of 4 to 8 independent curves.Also indicated are the statistically significant differences (i.e.p,0.05) between mtxnpx 2 and each of the control parasite lines, i.e. wild type (*), mtxnpx 2 /+mTXNPx ( N ) and mtxnpx 2 /+mTXNPxC81S (w).doi:10.1371/journal.ppat.1002325.g005 were PCR-amplified from a cosmid clone [8] using primers P1/P2 and P3/P4, respectively.Following digestion with the appropriate restriction enzymes, PCR products were cloned into the BamHI-XhoI and KpnI sites of the pTEX-NEO plasmid [46], on both sides of the neomycin phosphotransferase (NEO) gene.To assemble the HYG disruption construct, fragments containing part of the 59 and 39 untranslated regions and of the mTXNPx coding sequence were amplified by PCR from genomic DNA of the NEO-targeted mutants, using primers P5/P6 and P7/P8.Upon digestion with the appropriate restriction enzymes, PCR products were cloned into BamHI-EcoRV and KpnI sites of the pTEX-HYG plasmid, a version of pTEX-NEO wherein the NEO gene had been replaced by the hygromycin phosphotransferase (HYG) open reading frame.To generate the rescue construct, the mTXNPx ORF was introduced into the XmaI and the Klenow-treated BamHI restriction sites of the pSSU-NEO-infantum plasmid [21].This plasmid is a modified version of the pSSU-int [26], wherein the HYG gene had been replaced by NEO and the 59 flanking sequence of the small sub-unit rRNA (18S rRNA) gene of Leishmania mexicana substituted by the homologous sequence of L. infantum.Upon cloning of the mTXNPx coding sequence into pSSU-NEO-infantum, the NEO ORF was replaced by the bleomycin hydrolase (PHLEO) gene.One additional rescue construct (pSSU-NEO-infantum-mTXNPxC81S), containing a mutated version of mTXNPx, was generated by site directed mutagenesis (see below).Before transfection of L. infantum promastigotes, the NEO, HYG and PHLEO constructs were linearized by digestion with HincII, BamHI-SacI, and NdeI-PmeI, respectively, and purified from agarose gels by electroelution.

Production of the mTXNPxC81S mutein
A site directed mutagenesis strategy was employed to introduce the Cys81 to Ser (C81S) mutation in the mTXNPx ORF cloned into pSSU-PHLEO-infantum (detailed above) and pET28c (detailed below).For this, the full-length plasmids were PCR-amplified with sense and antisense mutated primers (P9 and P10 in Table S1) using Pfu polymerase (Stratagene).Upon digestion of parental (wild type) DNA with 10 units of DpnI (New England Biolabs) for 1 h at 37uC, the reaction (4 ml) was used directly to transform Escherichia coli DH5a strain.Mutant colonies were confirmed to carry the site-directed mutation by sequencing both strands.

Transfection of L. infantum and isolation of mutants
Promastigotes in the logatithmic phase of growth were electroporated at 450 V and 350-400 mF with 1 to 5 mg of DNA as described elsewhere [47].Parasites were allowed to recover in 10 ml of culture medium without selective drugs for 24 hours.Drugs were then added to 5 ml of the liquid culture and the remaining 5 ml were pelleted and plated onto agar plates containing the same drug(s).Geneticin (G418; Sigma) was used at 15 mg ml 21 , hygromycin (Invitrogen) at 10 mg ml 21 and bleomycin (Sigma) at 17.5 mg ml 21 .Upon 2 to 3 weeks of growth on agar, colonies were picked up and transferred into liquid medium.

Drug sensitivity assays
L. infantum promastigotes in the logarithmic phase of growth were seeded at 10 6 cells ml 21 in 24-well plates containing increasing concentrations (in duplicate) of either antimycin A (AA), H 2 O 2 or 3-morpholinosydnonimine hydrochloride (SIN-1) (all from Sigma).Parasites were allowed to grow for 3-4 days and cell densities were measured in a spectrophotometer at 600 nm.

Animals and ethics statement
BALB/c, C57BL/6 and National Marine Research Institute (NMRI) mice were purchased from Charles River (Madrid, Spain).Mice with a targeted disruption of the p47 subunit of the NADPH-oxidase complex on a C57BL/6 background (B6.p47 phox2/2 ) were purchased from Taconic (Lille Skensved, Denmark).Until the day of infection with L. infantum, and as prophylatic treatment against bacterial infection, trimethoprimsulfamethoxazole (Bactrim; 600 mg l 21 ) was administered in the drinking water to B6.p47 phox2/2 mice.Mice double deficient in gamma interferon (IFN-c) and in recombinase activating gene-2 (RAG-2) were obtained by crossing single knock out strains on C57BL/6 background.All mice were raised in specific pathogenfree conditions.Euthanasia was performed in a 20% isofluorane atmosphere and all efforts were made to minimize suffering.

Infection of monolayers of murine macrophages with promastigotes
Macrophages obtained by peritoneal lavage of C57/BL6 mice were seeded at 4610 5 cells per well in DMEM Glutamax complemented with 10% FBSi, 50 U ml 21 penicillin and 50 mg ml 21 streptomycin (all from Gibco), and allowed to adhere for 2 hrs at 37uC, 5% CO 2 .Upon washing with Hanks balanced salt solution (HBSS, Gibco), macrophages were incubated with opsonized promastigotes (in the stationary phase of growth) at a parasite:macrophage ratio of 10:1.For detection of superoxide anion (O 2 ? 2 ), a freshly prepared solution of 1 mg ml 21 nitroblue tetrazolium (NBT), prepared in Dulbecco's PBS was added to macrophages.After 30 min of incubation at 37uC, cells were washed with HBSS, fixed with methanol and deposition of formazan observed with an optical microscope Olympus CX31.For determination of infection indexes, infection with promastigotes was allowed to proceed for 3 hrs, after which noninternalized parasites were removed with HBSS.Cells were cultured for additional 24 hrs and then washed, fixed with 4% paraformaldehyde (w/v) in PBS, permeabilized with 0.1% (v/v) Triton X-100, and incubated with the anti-cTXNPx1 [8] antibody and propidium idodide (PI).Secondary antibody was Alexa Fluor 488 anti-rabbit IgG (Molecular Probes).Slides were mounted in Vectashield (Vector Laboratories).Images were acquired with an Axiocam MR ver.3.0 camera (Carl Zeiss, Germany) coupled to an AxioImager Z1 microscope (Carl Zeiss, Germany).A minimum of 3000 macrophages was counted in each experiment using specifically designed software (unpublished data) and the infection index was obtained by multiplying the percentage of infected macrophages by the average number of intracellular amastigotes per macrophage.

Determination of parasite burden by the limiting dilution assay
Parasites of all lines were passaged through NMRI mice at least twice prior to infection experiments.Next, 10 8 L. infantum stationary phase promastigotes, were inoculated intraperitoneally into 6-to 9-weeks old male BALB/c mice.At defined time points, mice were sacrificed and their livers and spleens excised, weighed and homogenized in Schneider's medium (Sigma) supplemented with 10% FBSi, 100 U ml 21 penicillin, 100 mg ml 21 streptomycin (all from Gibco), 5 mM hepes sodium salt pH 7.4 (Sigma), 5 mg ml 21 phenol-red (Sigma) and 2% sterile human urine.Homogenates were then diluted to 10 mg ml 21 and these cell suspensions titrated in quadruplicate across a 96-well plate in serial four-fold dilutions (four titrations per organ).After two weeks of growth at 25uC, the last dilution containing promastigotes was recorded and the number of parasites per gram of organ (parasite burden) calculated as described by Buffet et al. [48].The detection limit of this method is 500 parasites g 21 (i.e.2.7 log units).

Heterologous expression and purification of DmTXNPx and DmTXNPxC81S
To mimic the mature mitochondrial protein, truncated wild type mTXNPx (DmTXNPx), i.e. lacking the first 26 amino acids that compose the mitochondrial targeting peptide [8], was produced in the E. coli strain BL21 (DE3) Tuner upon transformation a pET28c (Novagen) construct containing the DmTXNPx gene fragment (PCR-amplified with primers P11/P12 in Table S1).To produce the mutant DmTXNPxC81S protein, the pET28c-DmTXNPxC81S was generated by site directed mutagenesis (detailed above) and used to transform the same E. coli strain.Both enzymes were expressed in bacteria as fusion proteins carrying an N-terminal six histidine tag.Upon induction for 3 hrs at 30uC in the presence of 50 mg ml 21 kanamycin and 0.1 mM isopropyl-b-D-thiogalactopyranoside (IPTG), bacteria were pelleted, suspended in 500 mM NaCl, 20 mM Tris-HCl pH 7.6, disrupted by sonication and centrifuged at 30,0006g for 30 min at 4uC.The supernatant was applied to a His Bind resin (Novagen) column and the recombinant protein eluted with an imidazole gradient (5 to 1000 mM) at a flow rate of 1 ml min 21 .Fractions confirmed to contain the protein by SDS-PAGE were pooled, applied to PD-10 columns (Amersham) and eluted in 50 mM sodium phosphate buffer pH 8.0.For removal of the His tag, enzymes were first digested with biotinylated thrombin (Novagen), and then incubated with immobilized streptavidin for removal of the protease.Proteins were concentrated by ultrafiltration, recovered in 40 mM hepes pH 7.5 and quantified by the bicinchoninic acid (BCA) protein assay (Pierce), using bovine serum albumin (BSA) as standard.

Determination of peroxidase activity
Routine determination of TXNPx activity was performed according to Nogoceke et al. [4].Briefly, reaction mixtures were prepared in a total volume of 300 ml of 50 mM Tris-HCl, 1 mM EDTA, pH 8.0, containing 200 mM NADPH, 0.5 U ml 21 L. infantum TR, 50 mM trypanothione disulfide (TS 2 , Bachem), 2.5 mM L. infantum TXN2 [49] and varying concentrations of DmTXNPx or DmTXNPxC81S.Reactions were started by addition of 70 mM hydrogen peroxide (H 2 O 2 , Sigma) and NADPH consumption was followed at 340 nm.All reactions were performed at 25uC and monitored with a Shimadzu UV-2401 PC spectrophotometer (Shimadzu Corporation).

Determination of chaperone activity
The chaperone activity of DmTXNPx and DmTXNPxC81S was measured using citrate synthase (CS) from porcine heart (Sigma) as substrate, as described before [31].Each enzyme was diluted in 40 mM hepes sodium salt pH 7.5 to reach final concentrations of 0.75 or 1.5 mM.A cocktail of protease inhibitors and 100 mM DTT were added to the reaction mixture to prevent degradation of CS and disulfide cross-linking between the peroxiredoxin and CS molecules, respectively, both of which could result in an apparent chaperone-like activity.Samples were pre-incubated at 43uC for 5 min and the reaction started by addition of CS to a final concentration of 0.15 mM.Light scattering due to CS aggregation at 43uC was monitored using a FluoroMax-4 spectrofluorometer, with excitation and emission wavelengths set to 500 nm and excitation and emission slits set to 2 nm.Data were recorded for 20 min.

Thermotolerance assays
L. infantum promastigotes of all lines, previously synchronized by 4-5 daily passages of 5610 5 cells ml 21 , were seeded at 10 6 ml 21 and allowed to grow for 4 days at either 25uC or 37uC.Every 24 hours cell densities were determined with a Neubauer-counting chamber for growth curve determination.

Statistical analysis
Comparisons between the parasite lines were carried out using analysis of variance.When normality or homogeneity of variances was not observed the Kruskal-Wallis non parametric test was used.In this case, multiple comparisons were carried using the Mann-Whitney test, with the Bonferroni correction.In order to investigate whether the distribution of the negative versus positive organs (in the LDA) was independent of the parasite line, the Chisquare and the Fisher's exact tests were employed (see Text S1).Statistical significance was assessed for p,0.05.The analysis was carried out using IBM SPSS Statistics 19.

Supporting Information
Figure S1 mTXNPx depletion has no impact on the capacity of L. infantum to invade macrophages and differentiate into amastigotes.Fluorescence microscopy images showing monolayers of peritoneal macrophages from C57BL/6 mice 24 hrs after infection with mtxnpx 2 (upper panels) and with wild type L. infantum (lower panels).For parasite detection, the anti-cTXNPx1 antibody [8], which specifically recognizes Leishmania antigens, was used (left).Merging with propidium iodide (PI) is also shown (right).Arrows point to intracellular parasites.Images were acquired at a 2006 magnification.(PDF) Table S2 Distribution of organs testing negative and positive in the limiting dilution assays used to generate the plot in Figure 2 of the main text.

(PDF)
Table S3 Distribution of organs testing negative and positive in limiting dilution assays used to generate the plot in Figure 3E of the main text.(PDF) Table S4 Distribution of organs testing negative and positive in limiting dilution assays used to generate the plot in Figure 4D of the main text.(PDF) Text S1 Statistical analysis of parasite burden.(PDF)

Figure 1 .
Figure 1.Generation of mTXNPx mutants.A. Genomic organization of the mTXNPx locus with its flanking b-propeller (b-prop) and cyclophilin (cyclo) genes in i) wild type and in ii) NEO-and iii) HYG-targeted alleles.The HYG disruption construct eliminated 144 nt of the mTXNPx coding sequence, leaving intact the first 292 and the last 245 nt (light grey boxes).SacI (S) restriction sites are indicated.Scheme iv) represents the small subunit 18S rRNA (SSU) locus of mtxnpx 2 /+mTXNPx mutants with the integrated PHLEO construct harboring the mTXNPx coding sequence.Dark grey boxes represent the 59 and 39 coding regions of SSU, of 5S rRNA and of the large subunit 28S rRNA (LSU).Thick lines represent the integrated DNA constructs.Arrowheads show the location of primers used in PCR analysis (in C.) to diagnose for the correct integration of the PHLEO cassette into the ribosomal locus.The number in between arrowheads refers to the expected size of the corresponding PCR product.B. Southern blot analysis of SacIdigested genomic DNA of wild type and mtxnpx 2 mutants, hybridized with mTXNPx and NEO ORFs.In mtxnpx 2 , the 5.6 kb band revealed by the mTXNPx probe corresponds to the gene disrupted by the HYG cassette.C. PCR analysis of genomic DNA from wild type, mtxnpx 2 and mtxnpx 2 / +mTXNPx parasites, using specific primers to amplify mTXNPx ORF (681 bp) or to diagnose for the correct integration of the PHLEO cassette into the ribosomal locus (1095 bp; primer location represented in A.). D. Western blot analysis of wild type, mtxnpx 2 and mtxnpx 2 /+mTXNPx promastigotes, incubated with the anti-mTXNPx antibody and, upon stripping, with anti-gp63 antibody (control for loading).The band above 37 kDa in the upper panel is dimeric mTXNPx.E. Indirect immunofluorescence of parasites lines as above, incubated with anti-mTXNPx antibody (green), merged with DAPI (blue).10006 magnification.The kDNA is indicated by arrows.doi:10.1371/journal.ppat.1002325.g001

Figure 2 .
Figure 2. Depletion of mTXNPx impairs Leishmania virulence.Wild type (light grey squares), mtxnpx 2 (black triangles) and mtxnpx 2 /+mTXNPx (dark grey diamonds) promastigotes were inoculated intraperitoneally into BALB/c mice.At different time points after infection, parasite burden (PB) was determined by the limiting dilution assay, as the number of parasites per gram of liver (left) or spleen (right).The Y axis indicates log 10 PB.Data represent mean and standard error of the mean of 6 independent experiments (involving a total of 85 animals infected with mtxnpx 2 ), except for mtxnpx 2 /+mTXNPx-infected mice, which refer only to 4 experiments.ND, not detected.From week 4 onward, differences between mtxnpx 2 and controls are statistically significant.Statistical analysis of these results is in TextS1 and Table S2.doi:10.1371/journal.ppat.1002325.g002

Figure 3 .
Figure 3. Depletion of mTXNPx has no impact on the antioxidant capacity of promastigotes. A. B. C. Wild type (light grey squares) and mtxnpx 2 (black triangles) promastigotes were cultured in the presence of increasing concentrations of (A) antimycin A (AA), (B) H 2 O 2 or (C) the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1).Four days later cell densities were measured in a spectrophotometer at 600 nm.The data is expressed as the percentage of promastigote replication relative to control cultures without any exogenous agent.Graphs represent means and standard deviation of three or more experiments (each performed in duplicate).D. Monolayers of peritoneal macrophages from C57BL/6 mice were infected with stationary phase wild type (light grey bars), mtxnpx 2 (black bars) or mtxnpx 2 /+mTXNPx (dark grey bars) promastigotes and 24 hrs later the infection indexes determined.The inset shows light microscopy photographs of macrophages incubated with NBT in the absence (''w/out L.i.'') or presence (''w/L.i.'') of parasites.Deposits of dark blue insoluble formazan resulting from NBT reaction with superoxide anion were visible in macrophages 30 min after contact with L. infantum promastigotes (right panel), but not in macrophages to which no parasites were added (left panel).Images were acquired at 1006 magnification.E. Mice from different strains (C57BL/6, B6.p47 phox2/2 and B6.RAG2 2/2 IFN-c 2/2 ) were inoculated intraperitoneally with wild type (light grey bars), mtxnpx 2 (black bars) or mtxnpx 2 /+mTXNPx (dark grey bars) stationary phase promastigotes.Five weeks after infection parasite burden (PB) was determined in the livers and spleens of mice by the limiting dilution assay.The Y axis represents log 10 PB.Data represent mean and standard error of the mean of 3 independent experiments involving a total of 72 animals.ND, not detected.Differences between mtxnpx 2 and controls are statistically significant.Statistical analysis of these results is in TextS1 and TableS3.doi:10.1371/journal.ppat.1002325.g003

Figure 4 .
Figure 4.The peroxidase activity of mTXNPx is not a critical determinant of L. infantum virulence.A. Classical assay for TXNPx enzymatic activity.Reaction mixtures contained 200 mM NADPH, 0.5 U ml 21 LiTR, 50 mM TS 2 , 2.5 mM LiTXN2 and recombinant TXNPx.DmTXNPx was added to a final concentration of 0.5 mM (filled squares) and DmTXNPxC81S to 5 mM (filled triangles).Reactions were started by addition of 70 mM H 2 O 2 at 60 sec and peroxidase activity followed by monitoring NADPH consumption at 340 nm.The negative control contains no TXNPx (open circles).B. PCR analysis of genomic DNA from mtxnpx 2 /+mTXNPxC81S parasites using specific primers to amplify the mTXNPx ORF (681 bp) or to diagnose for the correct integration of the PHLEO cassette into the ribosomal locus (1095 bp; location of primers in Figure 1A).Controls, performed with genomic DNA from wild type and mtxnpx 2 parasites, are also included.C. Indirect immunofluorescence of mtxnpx 2 /+mTXNPxC81S parasites incubated with the anti-mTXNPx antibody (green labeling), merged with DAPI (blue labeling).Parasites were photographed at 10006 magnification.D. Parasite burden (PB) in liver and spleen of BALB/c mice, determined by the limiting dilution assay, 4 and 8 weeks after infection with mtxnpx 2 (black bars), mtxnpx 2 /+mTXNPx (dark grey bars) or mtxnpx 2 /+mTXNPxC81S (open bars) stationary phase promastigotes.The Y axis represents log 10 PB.Data indicate mean and standard error of the mean of 2 independent experiments involving a total of 54 animals.ND, not detected.Differences between mtxnpx 2 and controls are statistically significant.Statistical analysis of these results is in TextS1 and Table S4.doi:10.1371/journal.ppat.1002325.g004

Figure
Figure S2 Susceptibility of mTXNPx to overoxidation in promastigotes exposed to 376C.Leishmania infantum promastigotes grown for 4 days at either 25uC or 37uC (as in Figure 5B) were collected (1.4610 7 cells) and lysed in the presence of 200 mM N-ethylmaleimide (NEM) (left).Alternatively, cell lysates were pre-treated with H 2 O 2 (50 mM H 2 O 2 for 30 min, followed by a further addition of 50 mM H 2 O 2 ) prior to fixation with NEM (right).Extracts were examined by western blot with the anti-mTXNPx antibody.The dimer corresponds to the oxidized form of mTXNPx, whereas the monomer corresponds to either the reduced or the overoxidized protein.In cell lysates of parasites grown at 37uC the monomeric form of mTXNPx persists even upon H 2 O 2 pre-treatment, indicating that this is the overoxidized (peroxidase inactive) form of the enzyme.This western blot is representative of two independent experiments.Protein loading was controlled by Ponceau staining.(PDF)Table S1 List of oligonucleotides employed to generate DNA constructs.(PDF)