Construction of the 6HisLiTXN3 expression vector For construction of pTEX6HisLiTXN3 the LiTXN3 ORF was PCR amplified with PWO polymerase (Roche) using the primers 59-cgcgcacatATGGAGCCCAACTTCTTCAAC-39 and 59-caccg- ctcgagTCACAAGCTGCGGCTGAT-39 (clamp sequences in low- er case; restriction sites underlined) and cloned into the NdeI-XhoI restriction sites of pET28a (Novagen). The 6HisLiTXN3 chimera (with an N-terminal 6-His tag) was then excised from pET28a with BglII/XhoI and cloned into the corresponding sites of pTEX [46]. Determination of parasitemia indexes by the limiting dilution assay BALB/c and National Marine Research Institute (NMRI) mice were purchased from Charles River (Lyon, France). Mice were raised in specific pathogen-free conditions at the animal house facility of the Institute for Molecular and Cell Biology (IBMC). Prior to the infection experiments, all promastigote lines were passaged in NMRI mice. Next, 108 L. infantum stationary phase promastigotes, were inoculated intraperitoneally into 6 to 9 weeks old male BALB/c mice. At defined time points after infection, mice were sacrificed and their livers and spleens excised, weighed and homogenized in Schneider’s medium (Sigma) supplemented with 10% FBSi, 5 mM hepes sodium salt (pH 7.4), 100 U ml21 penicillin, 100 mg ml21 streptomycin, 5 mg ml21 phenol-red and 2% sterile human urine. Homogenates were then diluted to 10 mg ml21 and these cell suspensions titrated across a 96-well plate in serial four-fold dilutions (four titrations per organ) in quadrupli- cate. 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 elsewhere [47]. Indirect immunofluorescence assay Immunofluorescence assays were performed according to Castro et al. [26]. Briefly, recombinant parasites were fixed with 4% paraformaldehyde (w/v) in 0.1 M Na2HPO4, 0.1 M NaH2PO4, 0.15 M NaCl pH 7.2 (PBS), permeabilized with 0.1% (v/v) Triton X-100 (prepared in PBS), spotted onto polylysine-coated microscope slides and incubated with appropri- ate antibodies. Primary antibodies were anti-LiTXN2 [10], anti- LimTXNPx [26] and anti-His (Abcam). Secondary antibodies were Alexa Fluor 488 anti-mouse IgGs, and Alexa Fluor 488 and 568 anti-rabbit (Molecular Probes). Slides were mounted in Vectashield (Vector Laboratories) and examined with an AxioI- mager Z1 microscope, equipped with an Axiocam MR ver.3.0 camera and the Axiovision 4.7 software (all from Carl Zeiss). Digitonin/proteinase K assay Twelve and a half million promastigotes (about 25 mg of total protein) suspended in 18.75 ml DIG buffer (25 mM Tris, pH 7.5, 1 mM EDTA, 0.6 M sucrose) containing 50 mg ml21 proteinase K (PK), were permeabilized by addition of 6.25 ml of prediluted digitonin (Calbiochem) to final concentrations of 0–2 mg of digitonin per mg of cellular protein. Samples were mixed by vortex and incubated for 10 min at 37uC. To terminate the PK reaction, 100 mM phenylmethylsulfonyl fluoride (PMSF) was added to all samples. Proteins were collected by trichloroacetic acid (TCA) precipitation and analyzed by western blot. Alkaline carbonate extraction of membrane proteins Aliquots containing equal amounts of L. infantum promastigotes (56108) were pelleted and suspended in 2 ml of either 10 mM Tris-HCl pH 7.4, 10 mM Tris-HCl 0.5 M NaCl pH 7.4, or Mitochondrial Tryparedoxins 0.1 M Na2CO3 pH 11.5 (a cocktail of protease inhibitors was added to all samples). Parasite suspensions were disrupted by sonication, incubated for 30 min at 4uC and sedimented by ultracentrifugation for 60 min at 100,0006g. Soluble and insoluble fractions obtained in both procedures were analyzed by western blot. Expression and purification of recombinant LiDTXN3 LiTXN3 ORF lacking the sequence that encodes the C-terminal hydrophobic tail (LiDTXN3), was PCR-amplified with PWO polymerase (Roche) using the primers 59-cgcgcacatATGGAGCC- CAACTTCTTCAAC-39 and caccgctcgagTCAACGGGTGTA- GTCCAAA-39 (clamp sequences in lower case; restriction sites underlined). The PCR product was cloned into the NdeI-XhoI restriction sites of the pET28a expression vector (Novagen), so that LiDTXN3 could be expressed as a fusion protein carrying an N- terminal tail of six histidines in Escherichia coli BL21 strain. Protein expression was induced for 3 h at 37uC in the presence of 50 mg ml21 kanamycin and 0.1 mM isopropyl-b-D-thiogalactopyrano- side (IPTG) following the protocol detailed before [25]. Bacteria were then pelleted, suspended in 500 mM NaCl, 20 mM Tris-HCl pH 7.6, disrupted by sonication and centrifuged at 10,0006g 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 2.5 ml min21. Fractions confirmed to contain the protein by SDS-PAGE were pooled, applied to PD-10 columns (Amersham) and eluted with PBS. Enzymatic and kinetics assays For the insulin reduction assay, a 12.5 mg ml21 insulin solution was freshly prepared in 100 mM potassium phosphate, 2 mM EDTA, pH 7.0. Reaction mixtures were prepared in 100 mM potassium phosphate, 2 mM EDTA, pH 7.0, containing 125 mM insulin, 5 mM of purified recombinant TXN and different reducing systems: i) 330 mM dithiothreitol (DTT), ii) 300 mM NADPH, 1 U ml21 of T. cruzi trypanothione reductase (TR), 100 mM trypanothione disulfide (TS2, Bachem), iii) 300 mM NADPH, 2 U ml21 of yeast glutathione reductase (Sigma), 500 mM glutathione (Sigma), iv) 300 mM NADPH, 2 U ml21 of E. coli thioredoxin reductase (Sigma), 5 mM E. coli thioredoxin (Sigma) and v) 200 mM NADH, 0.5 U ml21 porcine lipoamide dehydrogenase (Sigma), 100 mM dihydrolipoamide (Sigma). The increase in turbidity due to the formation of insoluble insulin B chain was followed at 650 nm. For assaying LiDTXN3 reduction by trypanothione disulfide, several reaction mixtures were prepared in 100 mM potassium phosphate, 2 mM EDTA, pH 7.0, which contained 300 mM NADPH, 1 U ml21 T. cruzi TR, increasing concentrations of TS2 (from 25 to 100 mM) and 5 mM LiDTXN3. The assay mixtures were preincubated until a constant baseline was obtained and the reaction was started by adding 125 mM insulin. The absorption decrease was followed at 340 nm. Routine determination of peroxidase reduction by TXN was performed according to Nogoceke et al. [2]. Briefly, reaction mixtures were prepared in a total volume of 300 ml of 50 mM Tris-HCl, 1 mM EDTA, pH 7.6, containing 300 mM NADPH, 1 U ml21 T. cruzi TR, 100 mM trypanothione disulfide (TS2, Bachem), 5 mM TXN and 5 mM of N-terminal His tagged L. infantum peroxidases: i) LimTXNPx (Gene ID LinJ23_V3.0050) [25], ii) LicTXNPx1 (Gene ID LinJ15_V3.1140) [10], iii) LicTXNPx2 (Gene ID LinJ15_V3.1120) [10], and (iv) LinsGPXA1 (Gene ID LinJ26_V3.0770; Teixeira F, Castro H, Toma´s AM, unpublished). After a 10 min preincubation period reactions were started by addition of 70 mM hydrogen peroxide (H2O2, Sigma) PLoS ONE | www.plosone.org 10 September 2010 | Volume 5 | Issue 9 | e12607