unequivocally demonstrated for L. infantum [13] and T. brucei [14,15]. Since the mitochondria of trypanosomatids contain several enzymes that are specifically reduced by TXN, it is a common belief that these organisms also possess a mitochondrial TXN [1,16,17]. Still, the presence of a TXN within the mitochondrion has only been convincingly shown for the L. infantum TXN2 enzyme [10]. Such mitochondrial TXN is anticipated to be vital because many of these mitochondrial TXN- dependent molecules are essential to the parasite, namely mTXNPx (Castro H, Toma´s AM, unpublished data), UMSBP [18], 1-Cys GRX [19] and possibly, as suggested by Schlecker et al. [20], nsGPX. In this manuscript the actual need for an active mitochondrial TXN for normal parasite metabolism was investigated. For this, a L. infantum line unable to express the mitochondrial LiTXN2 enzyme was produced by gene targeting. The observation that these mutants behaved as wild type parasites throughout the entire Leishmania life cycle, raised the hypothesis that a second mitochondrial TXN, different from LiTXN2, would exist. To investigate this premise the genome of L. infantum was surveyed for additional TXN open reading frames (ORFs) and one candidate was found, LiTXN3. Biochemical characterization and subcellular localization studies of LiTXN3, nevertheless, revealed that this molecule was not capable of acting as a fully active TXN in the mitochondrion of L. infantum. All together, the data presented here demonstrates that, contrary to the accepted view, Leishmania can survive without an active TXN in their mitochondrion. Impor- tantly, comparison of TXN sequences of Leishmania with those of Trypanosoma, along with an exhaustive phylogenetic analysis, strongly suggest that the non-requirement for TXN activity within the mitochondrion is also a feature of Trypanosoma. The consequences of this finding in what concerns the functioning of several mitochondrial processes are discussed. Results Generation of LiTXN2 mutants To investigate the relevance of the mitochondrial LiTXN2 enzyme for L. infantum survival a parasite line unable to express this molecule was produced by homologous recombination. Since Leishmania have a diploid genome, two successive rounds of gene targeting were required to obtain LiTXN2 homozygous knockout mutants. Accordingly, two linear DNA fragments containing either the HYG or the PHLEO genes, flanked by part of the upstream and downstream regions of LiTXN2, were introduced successively into parasites by electroporation (Figure 1A). Southern blot analysis of the resulting hygromycin- and phleomycin-resistant parasites confirmed the successful replacement of both LiTXN2 alleles (Figure 1B). The double targeted mutants produced in this way (Dtxn2::HYG/Dtxn2::PHLEO, hereon referred to as txn22) were further confirmed to lack LiTXN2 expression by western blot and indirect immunofluorescence analysis (Figure 1C–D). LiTXN2 is not essential throughout the parasite life cycle During its life cycle Leishmania proceed through two morpho- logically and physiologically distinct stages, the promastigote (an extracellular form residing in the insect vector) and the amastigote (an intracellular form that parasitises mammalian host macro- phages). LiTXN2 homozygous knockout mutants generated in the promastigote stage showed no obvious morphological alterations when compared to wild type parasites, not even in the kinetoplast (Figure 1D), that is, the structure that in trypanosomatids contains the mitochondrial DNA (kDNA). Moreover, under standard culture conditions, the growth rate of txn22 was similar to that Mitochondrial Tryparedoxins of promastigotes expressing wild type levels of LiTXN2 (Figure 2A). These observations are particularly relevant because they argue against the participation of LiTXN2 in kDNA replication (via reduction of UMSBP), a function previously attributed to mitochondrial TXNs [7,17]. To assess the consequences of LiTXN2 depletion on amastigote survival, txn22 mutants were inoculated into BALB/c mice, an animal model for Leishmania infection. Since L. infantum is an agent of visceral leishmaniasis, at defined time points after infection, parasite burdens were evaluated in the liver and spleen of infected mice by the limiting dilution assay. As shown in Figure 2B, elimination of LiTXN2 did not affect the ability of L. infantum to replicate and give rise to a productive infection in a mammalian host. Indeed, the parasitemia produced by txn22 was not significantly different from that observed with wild type parasites. The demonstration that LiTXN2 is not essential throughout the L. infantum life cycle suggested that, in addition to LiTXN2, a second TXN would be active within the L. infantum mitochondrion. LiTXN1 is restricted to the cytosol In biochemical assays mitochondrial thiol-containing molecules, such as LimTXNPx, can be reduced by the cytosolic enzyme LiTXN1 [10]. It is nevertheless unlikely that LiTXN1 interacts physiologically with such proteins owing to their distinct subcellular compartmentalization. Using parasites lacking LiTXN2 expression and a digitonin/proteinase K assay, LiTXN1 was confirmed to be absent from the mitochondrion. Based on the differential sterol content of their membranes, the various subcellular compartments can be selectively permeabilized by digitonin, the mitochondrial membrane being more resistant to this detergent than the cell membrane. When proteinase K (PK) and digitonin are added to the cells simultaneously, PK will only gain access to, and hence digest, the protein content of permeated organelles. Western blot analysis of samples resulting from treatment of txn22 mutants with digitonin/PK (Figure 3) showed that LiTXN1 is readily digested at digitonin concentrations that are too low to permeabilize mitochondrial membranes (controlled by the anti-LimTXNPx antibody). In other words, and in agreement with bioinformatic predictions (MitoProtII, Predotar, PlasMit), LiTXN1 is not present in the mitochondrion of txn22 parasites. Therefore, if L. infantum does harbor a second TXN in its mitochondrion this is not LiTXN1. Screening of the L. infantum genome uncovers LiTXN3, a candidate substitute for LiTXN2 To investigate the existence of alternative mitochondrial TXNs the L. infantum genome was screened for TXN coding sequences and found to contain, apart from the well characterized LiTXN2 and LiTXN1 genes, five additional sequences, designated here as LiTXN3, LiTXN4, LiTXN5, LiTXN6 and LiTXN7 (Gene IDs: LinJ31_V3.2000, LinJ31_V3.2010, LinJ29_V3.1230, LinJ29_ V3.1220 and LinJ35_V3.0380, respectively, according to the TriTrypDB). However, with the exception of LiTXN3, all these ORFs encode theoretical proteins with amino acid substitutions that preclude them from acting as TXNs (for additional information refer to Text S1 and Figure S1). These include alterations in the TXN active site motif, as well as in the residues that form the hydrogen bond network required for normal TXN activity [21]. Accordingly, only LiTXN3 can be regarded as a candidate substitute for LiTXN2 and, as such, its characterization was pursued here. LiTXN3 encodes a protein with a theoretical molecular weight of 21.6 kDa and a pI of 6.82. Figure 4 shows the LiTXN3 predicted amino acid sequence and compares it to TXNs known PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e12607