Mitochondrial Tryparedoxins Figure 2. Depletion of LiTXN2 does not affect survival of L. infantum throughout its life cycle. A. Proliferation of promastigotes, either txn22 (black circles) or wild type (grey squares), monitored throughout 5 days of culture. Data are expressed as the percentage of parasite density relative to the highest value recorded at each experiment. Values represent mean and standard deviation of three independent growth curves. B. Survival of L. infantum intracellular amastigotes measured as the parasite burden in the livers (left) and spleens (right) of BALB/c mice. Parasite burdens were determined at different times after inoculation with either txn22 (black circles) or wild type (grey squares) parasites. No statistically significant differences were found between animals infected with txn22 and wild type parasites. Data represent mean and standard deviation of 2 independent experiments (involving a total of 21 animals infected with txn22). doi:10.1371/journal.pone.0012607.g002 In the classical Holmgren assay [24], 6HisLiDTXN3 proved to be an active oxidoreductase. Indeed, when incubated with DTT, LiDTXN3 was capable of reducing insulin (Figure 5A), a reaction that results in aggregation of the insoluble insulin B chain and that can be monitored spectrophotometrically at 650 nm. When DTT was replaced by trypanothione, LiDTXN3 could still reduce insulin, albeit at a lower rate (Figure 5A), whereas dihydrolipoa- mide, another mitochondrial reductant, could only marginally support the activity of LiDTXN3 on insulin (data not shown). Neither thioredoxin nor glutathione served as reductants for LiDTXN3 in this assay (data not shown). To get an insight into the efficiency of trypanothione catalyzed LiDTXN3 reduction, NADPH consumption was monitored (at 340 nm) in the presence of trypanothione reductase (TR), trypanothione, recombinant TXN and insulin (Figure 5B). Since Figure 3. The cytosolic LiTXN1 enzyme is not present in the mitochondrion of txn22 promastigotes. Protein samples resulting from the permeabilization of txn2`u promastigotes with increasing concentrations of digitonin in the presence of proteinase K were analyzed by western blot using anti-LiTXN1 sera. Anti-LicTXNPx2 and anti-LimTXNPx were employed to detect cytosolic and mitochondrial compartments, respectively. doi:10.1371/journal.pone.0012607.g003 reduction of TXN by trypanothione is the limiting step in this cascade [10], the slopes of NADPH consumption reflect the velocity of this reaction. As depicted in Figure 5B, when compared with LiTXN2, LiDTXN3 was found to be poorly reduced by trypanothione. Finally, when tested in the routine assay for reduction of TXN- dependent peroxidases [2] LiDTXN3 exhibited negligible activity (Figure 5C). The peroxidases assayed were the mitochondrial 2- Cys PRX (LimTXNPx) [25] and two of its cytosolic isoforms (LicTXNPx1 and LicTXNPx2) [10], as well as a nsGPX (LinsGPxA1; Gene ID LinJ26_V3.0770). The full-length 6Hi- sLiTXN3 was also tested in this assay towards LicTXNPx1, LicTXNPx2 and LimTXNPx, with the same results (data not shown). In short, by showing that LiTXN3 was unable to reduce TXN- dependent peroxidases these results confirm what the amino acid substitutions in the LiTXN3 sequence (Figure 4) suggested, that this enzyme does not function as a classical TXN. Also in accordance with primary structure predictions, the reaction between LiTXN3 and trypanothione is considerably impaired but not hindered. Evidence is also provided that LiTXN3 retains oxidoreductase activity, making it reasonable to speculate that, although unable to reduce 2-Cys PRXs and nsGPXs, it could still sustain the activity of mitochondrial enzymes such as UMSBP and 1-Cys GRX. For this, however, LiTXN3 would have to share with these enzymes the same subcellular localization. LiTXN3 localizes to the mitochondrion To address the subcellular localization of LiTXN3, immunolo- calization studies were carried out using a transgenic L. infantum line expressing LiTXN3 as an N-terminal His-tagged chimera (see Materials and Methods) and subsequent detection with a specific anti-histidine antibody (Figure 6A). Immunofluorescence analysis of 6HisLiTXN3-expressing parasites revealed that the subcellular distribution of this protein was similar to that of the mitochondrial PLoS ONE | www.plosone.org 4 September 2010 | Volume 5 | Issue 9 | e12607