The proposal that no mitochondrial TXN is essential for trypanosomatid viability implies that several redox pathways that take place in this organelle cannot proceed as currently thought. Rather, an alternative reducing agent(s) in the form of a different thiol-containing protein or of low molecular mass thiol, is required to reduce, among other components, mTXNPx, nsGPX, UMSBP and 1-Cys GRX, that is, enzymes involved in processes as vital as peroxide metabolism, kinetoplast replication and iron sulfur cluster biosynthesis, respectively [1]. The observation that both E. coli and T. brucei thioredoxin reduces TXNPxs [25,33], 1-Cys GRX [8] and nsGPX [4], albeit much less efficiently than does TXN, would support thioredoxin as a candidate for TXN substitution if these are to be shown allocated to the mitochon- drion. Moreover, and even if proposing an entity for a TXN alternative is not straightforward, the genome of trypanosomatids encodes several other putative dithiol-containing proteins whose role as mitochondrial oxidoreductases is worth investigating. In an equally conceivable hypothesis the trypanosomatids’ major thiol, trypanothione, could deliver electrons directly to mTXNPxs, nsGPXs, UMSBP, and 1-Cys GRX among other proteins, overcoming the need for an intermediate oxidoreductase. However, biochemical assays only showed trypanothione-driven reduction in the case of the T. brucei 1-Cys GRX [8]. Neither the peroxidases mTXNPx [25] and nsGPX [4] nor UMSBP [34], the enzyme that initiates replication of the kinetoplast, were found to receive electrons directly from this thiol. Trypanothione might also provide a simple explanation for elimination of peroxides produced within mitochondria in the absence of an active TXN. In fact, at millimolar concentrations, trypanothione scavenges peroxides, such as hydrogen peroxide and peroxynitrite, directly and fast enough to account for effective detoxification [4,35,36]. Therefore, if such concentrations are reached in mitochondria, peroxide detoxifica- tion in this organelle could bypass the requirement for peroxidase- catalyzed reactions. Furthermore, other low molecular mass thiols (e.g. glutathione, ovothiol or free cysteine), as well as thiols exposed on protein surfaces [37] and, in the case of Leishmania, an intermembrane space located ascorbate peroxidase [38] might assist trypanothione in mitochondrial antioxidant defense. Accurate determination of the concentration of trypanothione and of other thiols in the mitochondria of the several trypanosomatids will certainly shed light on the likelihood that non-protein thiols directly reduce peroxides as well as enzymes currently considered as TXN targets. In this regard, a comparative analysis of antioxidant molecules between L. infantum lines with and without LiTXN2 may also provide relevant information. Apart from showing that trypanosomatids can thrive in the absence of an active TXN in their mitochondria, this work is novel in characterizing a member (LiTXN3) of a new class of TXNs, unique for being TA. Tail anchored proteins embrace a group of transmembrane molecules, which are increasingly recognized as critical for the normal functioning of the different organelles that harbor them. Some of these proteins include components of the translocation machinery of the mitochondria [39], as well as mitochondrial outer membrane proteins that regulate apoptosis [40] and mitochondrial morphology [41]. In the particular case of LiTXN3, the finding that this is an outer mitochondrial protein with moderate oxidoreductase activity could indicate that it is involved in redox-dependent biological processes. By analogy with thioredoxins, such redox processes could include protein folding assistance, protease activation and intracellular signaling [42,43]. In this sense, further characterization of LiTXN3 and their counterparts in Trypanosoma spp. is important to address if such oxidoreductase function exists in the context of the cell. Nevertheless, the possibility that the function of class II TXNs Mitochondrial Tryparedoxins members is independent of their redox activity cannot be excluded in a similar way to what has been previously described to a eukaryote thioredoxin [44]. In either case the essential character of class II TXNs and their amenability to specific inactivation are worth investigating. In summary, by demonstrating that trypanosomatids do not require an active tryparedoxin within their mitochondrion, the experimental work contained in this paper establishes that the redox metabolism in this organelle is not dependent on the activity of this family of enzymes. This implies reformulating the model in which a central TXN molecule supplies reducing equivalents to critical mitochondrial sulphur-containing enzymes, such as those involved in peroxide elimination and kinetoplast replication. From a different perspective, this manuscript characterized for the first time a member of a new class of TXNs that does not display classical TXN activity and whose physiological function is as yet uncovered. The chapter on tryparedoxin systems is definitely not closed. Materials and Methods 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. Parasite culture and genetic manipulations Wild type and transfected Leishmania infantum promastigostes (MHOM MA67ITMAP263) were cultured at 25uC in RPMI 1640 with GlutaMAX, (Invitrogen) supplemented with 10% inactivated fetal bovine serum (FBSi, Invitrogen), 20 mM hepes sodium salt (pH 7.4) and 50 U ml21 penicillin, 50 mg ml21 streptomycin. For growth rate determination L. infantum promastigotes previously synchronized by 4–5 daily passages at 56105 cells ml21, were cultured at 106 cells ml21 and the cell density monitored over time with a hematocytometer. Transfection of promastigotes was performed as described elsewhere [45]. Isolated clones were picked from agar plates containing selective drugs and transferred into liquid medium. Geneticin (G418; Sigma) was used at 15 mg ml21, hygromycin (Invitrogen) at 10 mg ml21 and phleomycin (Sigma) at 17.5 mg ml21. Construction of the LiTXN2 replacement vectors To assemble the HYG and the PHLEO replacement vectors, the 59 and 39 non-coding sequences flanking the LiTXN2 open reading frame were PCR amplified from L. infantum genomic DNA using the primers 59-cccaagcttTGCTCGGCATCTACACC-39, 59- cggactagtGGTAGCAAAGATGTCAGG-39, 59-cgcggatccACCA- AACACAAAGGGGTG-39 and 59-cgcgagctcGCACCGCACAA- TATACGG-39, which incorporate the HindIII, SpeI, BamHI and SacI restriction sites (clamp sequences in lower case; restriction sites underlined), respectively. Following digestion with the appropriate restriction enzymes, the PCR products were cloned into the HindIII-SpeI and BamHI-SacI sites of the HYG and PHLEO constructs used previously to target the LiTXN1 ORF [13], on both sides of the hygromycin- and phleomycin-resistance gene (HYG and PHLEO, respectively). Previous to transfection both constructs were linearized with HindIII and SacI restriction enzymes and purified from agarose gels by electroelution. PLoS ONE | www.plosone.org 9 September 2010 | Volume 5 | Issue 9 | e12607