by propidium iodide staining. In this last methodology, cell death due to a loss in cell membrane integrity of drug-treated L. amazonensis was assessed by flow cytometry measuring the level of propidium iodide uptake into damaged promastigotes. Concen- trations of the HIV PIs capable of maintaining at least 95% of living promastigotes were used in the experiments. Experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at Universidade Federal do Rio de Janeiro and Fundac¸ a˜o Oswaldo Cruz (CEUA L-0006/07). Leishmania-macrophage interaction assay Stationary-phase promastigotes were washed with PBS, counted in a Neubauer chamber, added to the macrophage culture well plates at a parasite to cell ratio of 10:1, and incubated for 2 hours at 37uC in a 5% CO2. The free parasites were removed by successive washes with PBS. Thereafter, Leishmania-infected cells were treated for 24 hours with PIs that had a prominent effect on promastigote growth, at final concentrations of 6.25, 12.5 and 25 mM of nelfinavir and 3.125, 6.25 and 12.5 mM of amprenavir and lopinavir (concentrations of the HIV PIs capable of maintaining 95% macrophage viability). After the interaction, cells were washed with PBS at 37uC, the coverslips were fixed in methanol and stained as described below. Alternatively, parasites were pre-treated with PIs for 1 hour with sub-inhibitory drug concentrations (50 and 100 mM of nelfinavir and lopinavir; 100 and 200 mM of amprenavir), washed three times in PBS and then added to the macrophage culture in well plates. After 1 hour of interaction, cells were fixed in methanol, Giemsa-stained and dehydrated in acetone solutions progressively replaced by xylol. The assembly of the slides was done with Permount. The percentage of infected macrophages was determined by randomly counting at least 200 cells in each of duplicated cover slips. Experiments were repeated three times. The association index was obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophage. Experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at Universidade Federal do Rio de Janeiro and Fundac¸ a˜o Oswaldo Cruz (CEUA L-0006/07). Electron microscopy Control and HIV PIs-treated parasite cells were cultured as described above and promastigotes were incubated with nelfinavir or lopinavir at the IC50 concentration. The possible parasite ultrastructure modifications along different time periods of treatment with HIV PIs were performed. In this context, promastigote cells (16108) were treated for 2, 4, 6, 8, 12 and 24 hours and then fixed overnight at 4uC in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. After fixation, cells were washed in cacodylate buffer and postfixed for 1 hour in 0.1 M cacodylate buffer containning 1% osmium tetroxide, 0.8% potassium ferrocyanide and 5 mM CaCl2. The cells were then washed in the same buffer, dehydrated in acetone, and embedded in Epon. Ultrathin sections were mounted on 300-mesh grids, stained with uranyl acetate and lead citrate and observed under a Zeiss 900 transmission electron microscope (Zeiss, Oberkochen, Germany). Evaluation of peptidase expression by gelatin-SDS-PAGE Promastigotes (16108 cells) were incubated with PIs (nelfinavir, lopinavir and amprenavir) for 4 hours at 26uC at a final concentration of 100 mM. The gels were loaded with 56106 cells, which were added to SDS–PAGE sample buffer (125 mM Tris, HIV PIs on L. amazonensis pH 6.8, 4% SDS, 20% glycerol and 2% bromophenol blue). Electrophoresis was performed at a constant current of 60 mA at 4uC for 2 hours. After electrophoresis, SDS was removed by incubation with 10 volumes of 1% Triton X-100 for 1 hour. Subsequently, the gels were incubated in 50 mM sodium phosphate buffer, pH 5.5, supplemented with 2 mM DTT at 37uC. After incubation for 48 hours, the gels were washed twice with distilled water, stained for 2 hours with 0.2% Coomassie brilliant blue R-250 in methanol-acetic acid-water (50:10:40), and destained overnight in a solution containing methanol-acetic acid- water (5:10:85), to intensify the proteolytic halos. The gels were dried, photographed and the density profiles digitally processed [21]. Evaluation of peptidase expression by immunoblotting Promastigote forms treated or not with PIs, as described above, were added to SDS–PAGE sample buffer and mixed with 10% b- mercaptoethanol, followed by heating at 100uC for 5 minutes. Thereafter, protein extracts (equivalent to 56106 cells) were separated in 12% SDS–PAGE and the polypeptides electropho- retically transferred at 4uC at 100 V/300 mA for 2 hours to a nitrocellulose membrane. The membrane was blocked in 5% low- fat dried milk in PBS containing 0.5% Tween 20 (PBS/Tween) for 1 hour at room temperature. Then, membranes were washed three times (10 min each) with the blocking solution and incubated for 2 hours with the following polyclonal antibodies: anti-cpb (1:1000) raised against Leishmania mexicana cysteine peptidase B (kindly provided by Dr Mary Wilson – Department of Internal Medicine, Biochemistry, Microbiology and Epidemiology, Pro- gram in Molecular Biology, University of Iowa, USA), anti-gp63 (1:2000) raised against L. mexicana (kindly provided by Dr Peter Overath – Max-Planck-Institut f u¨r Biologie, Abteilung Membran- biochemie, Germany), and anti-a-tubulin monoclonal antibody at 1:500. The secondary antibody used was peroxidase-conjugated goat anti-rabbit IgG at 1:20000 followed by chemiluminescence immunodetection after reaction with ECL reagents [21]. The relative molecular mass of the reactive polypeptides was calculated by comparison with the mobility of SDS–PAGE standards. The X-ray films were photographed and the density profiles digitally processed [21]. Aspartyl peptidase assay The enzymatic activity over HIV-1 peptidase substrate was determined using L. amazonensis extract, which was obtained by repeated freeze-thawing cycles of cells in 10 mM Tris-HCl, pH 7.2, containing 1% CHAPS. Then, the cellular extract was incubated for 40 min at 4uC, centrifuged at 10,000 g for 30 min at 4uC, and stored at 270uC in aliquots for no longer than 4 days. Cleavage of HIV-1 peptidase substrate was monitored continuously in a spectrofluorometer (SpectraMax Gemini XPS, Molecular Devices, CA, USA) using an excitation wavelength of 340 nm and an emission wavelength of 490 nm. A 500 mM stock solution of the fluorogenic substrate sample was prepared in DMSO. The reaction was started by the addition of 2 mM substrate to the parasite extract (10 mg) in a total volume of 60 ml of 100 mM sodium acetate, 1 M sodium chloride, 1 mM EDTA, 1 mM DTT, 10% DMSO, 1 mg/ ml BSA, pH 4.7, in the presence or absence of nelfinavir, amprenavir, lopinavir or pepstatin A. The reaction mixture was incubated at 37uC for 15 min. The assays were controlled for self- liberation of the fluorophore over the same time interval. Statistical analysis All experiments were repeated at least three times, and representative images of these experiments are shown. The data PLoS ONE | www.plosone.org 3 March 2009 | Volume 4 | Issue 3 | e4918