In recent years, the introduction of anti-retroviral drugs, particularly aspartyl peptidase inhibitors (PIs) used in the chemotherapy of HIV, into medical practice has led to a marked improvement in the life expectancy of AIDS sufferers by the fall of HIV viremia and by restoring the immune responses with an increase in the number of CD4+ T lymphocytes [4–7]. In addition, the functional upgrading of two critical components of innate antimicrobial immunity, such as neutrophils and monocytes, may contribute to the improved cell-mediated immune responses against opportunistic infections in highly active anti-retroviral therapy (HAART)-treated patients [5]. Indeed, after the introduction of PIs in the antiretroviral therapy for HIV, the number of coinfected cases reported in European Leishmania-endemic countries fell sharply. Nevertheless, the disease still constitutes an issue in patients who have AIDS, being the third most frequent parasitic opportunistic infection in Europe [8]. The drastic reduction in the incidence, morbidity and mortality of AIDS coinfections after the introduction of PIs in the antiretroviral therapy was the first line of evidence that these compounds could exert a direct effect on opportunistic pathogens [9], which was demonstrated in several fungi, such as Cryptococcus neoformans [10], Candida albicans [11] and Fonsecaea pedrosoi [12], and also in the protozoa Plasmodium falciparum [13]. Finally, it was recently demonstrated that two PIs (indinavir and saquinavir) have a direct effect on Leishmania infantum and Leishmania major promasti- gotes in vitro, which are responsible for visceral and cutaneous manifestations, respectively [14]. Leishmania species are dimorphic protozoa existing as promas- tigote and amastigote forms, which survive in the lumen of the digestive system of the sand fly and within the phagolysosomal compartment of mammalian macrophages, respectively. In the mammalian host, these protozoa are obligate intracellular parasites of cells from the macrophage-dendritic cell lineages [15]. Since Leishmania and HIV infect the same target cells, it is believed that complex interactions between these pathogens take place. Peptidases participate in several physiological and pathological processes in different cell types. In Leishmania, this class of hydrolytic enzymes directly acts in different steps of the microorganism-host interplay, being considered as virulence factors [16–18]. Considering all these facts together, we have conducted a study to investigate the direct effect of five different HIV PIs (indinavir, saquinavir, nelfinavir, lopinavir and ampre- navir), commonly used in HAART, on L. amazonensis growth ability, ultrastructure, interaction of this human pathogen with mouse peritoneal macrophages in vitro, and expression of peptidases, which are classical leishmanial virulence factors. Methods Chemicals Saquinavir and nelfinavir were obtained from Hoffmann-La Roche AG (Grenzach-Wyhlen, Germany), indinavir from Merck Sharp & Dohme GmbH (Haar, Germany), lopinavir from Abbott Laboratories (Abbott Park, IL, USA) and amprenavir from GlaxoSmithKline (NC, USA). Anti-a-tubulin monoclonal anti- body, bovine serum albumin (BSA), dimethylsulfoxide (DMSO), heat-inactivated fetal bovine serum (FBS), 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide dye (MTT), dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA), propidium iodide, Schneider’s insect medium, 3-[(3-cholamidopropyl)-di- methylammonio]-1-propanesulfonate-Chaps (CHAPS) and HIV- 1 peptidase substrate [Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile- Val-Gln-Lys(DABCYL)-Arg] were purchased from Sigma Chem- HIV PIs on L. amazonensis ical Co. (St Louis, USA). Media constituents, buffer components, reagents used in electrophoresis and immunoblotting were purchased from GE Life Care (Little Chalfont, UK). All other reagents were analytical grade or superior. Parasite culture Leishmania amazonensis (MHOM/BR/77/LTB0016 strain) was obtained from Leishmania Type Culture Collection (Fundac¸ a˜o Oswaldo Cruz, Rio de Janeiro, RJ, Brazil). Promastigote forms were maintained by weekly transfers in 25-cm2 culture flasks with Schneider’s insect medium, pH 7.0, supplemented with 10% FBS at 26uC. Multiplication inhibition assay The effects of five distinct HIV aspartyl PIs (amprenavir, indinavir, lopinavir, nelfinavir and saquinavir) on promastigote forms of L. amazonensis were assessed by a method similar to that described previously [19]. Promastigotes were counted using a haemocytometer and re-suspended in fresh medium to a final concentration of 56107 viable promastigotes/ml. Viability was assessed by mobility and lack of staining after challenge with trypan blue. Each inhibitor compound was added to the culture at final concentrations ranging from 15 mM to 500 mM (starting from a 20 mM solution in DMSO that was serially diluted in culture medium). Alternatively, promastigotes were also treated with different concentrations (25, 50 and 100 mM) of pepstatin A (a classic aspartyl peptidase inhibitor). Dilutions of DMSO corre- sponding to those used to prepare the drug solutions were assessed in parallel. After 24, 48, 72 and 96 hours of incubation at 26uC, the number of viable motile promastigotes was quantified by counting the flagellates in a Neubauer chamber. The 50% inhibitory concentration (IC50), i.e. the minimum drug concen- tration that caused a 50% reduction in survival/viability in comparison with that in identical cultures without the compound, was evaluated after 48 hours for the most effective PIs. These values were determined by linear regression analysis by plotting the number of viable promastigotes versus log drug concentration using Origin Pro 7.5 computer software. Viability assays The effect of the HIV PIs on the murine macrophage viability was evaluated by an MTT micromethod described previously [20]. First, peritoneal macrophages from female BALB/c mice (6– 8 weeks old) were collected in cold phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH 7.2) and 56105 cells were allowed to adhere in 96-well tissue culture plates for 1 hour at 37uC, in a 5% CO2 atmosphere. Non-adherent cells were removed by washes with PBS and the wells refilled with DMEM medium supplemented with 10% FBS. Macrophages were then incubated with increasing concentration of PIs (1.56 to 25 mM) in triplicate. After 24 hours, the medium was discharged and the formation of formazan was measured by adding MTT (5 mg/ml in PBS, 50 mg/well) and incubating the wells for 3 hours in the dark at 37uC. The plates were subsequently centrifuged at 1100 rpm for 7 minutes, the supernatant was removed, the pellet was dissolved in 200 ml of DMSO and the absorbance measured in an ELISA reader at 490 nm (Bio-Tek Instruments). Concen- trations of the HIV PIs capable of maintaining 95% macrophage viability were used in the interaction assays. To determinate the promastigotes viability, parasites were resuspended in Schneider’s insect medium supplemented with 10% FBS in 96-well plates (16107 promastigotes/well), and incubated at 26uC with increasing concentrations of HIV PIs (50– 250 mM) in triplicate. After 1–4 hours, viable cells were estimated PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4918