of HIV PIs in vitro were observed at substantial PI concentrations (mM range), much higher than those needed for HIV peptidase inhibition (on the nM order) [33]. This probably reflects a much lower affinity of these drugs for a yet unidentified target in Leishmania compared with their high affinity for HIV peptidase [33]. Parasitic protozoa of the genus Leishmania are a biologically diverse group of microorganisms. Taxonomic studies of field Leishmania isolates indicate a tremendous diversity within this genus [34], and genetic diversity may have a direct impact on relevant properties of the organisms, including distinct clinical manifesta- tions and drug susceptibility [35]. So, it is not surprising that the effect of HIV PIs on Leishmania species responsible for visceral or mucocutaneous leishmaniasis seems to be very distinct. Savoia and colleagues [14] reported the IC50 for L. major promastigote in the 10 mM range for indinavir and saquinavir after 24 hours of growth, while L. infantum, responsible for visceral leishmaniasis, was inhibited only partially (about 30%) at the highest concen- tration tested (50 mM) and only after 72 hours of growth [14]. In the course of this manuscript preparation, Trudel and colleagues [36] reported the effects of nelfinavir, ritonavir and saquinavir on the extracellular growth of L. infantum promastigotes. No significant alteration in parasite growth was reported in drug concentrations up to 25 mM, after 72 hours of incubation [36]. Our results showed an IC50 for L. amazonensis in the 15 mM range for nelfinavir or lopinavir after 24 hours of growth. The effectiveness of HIV PIs in treating parasitic infections may be associated to their capacity to modulate or block the cell proteasome or to promote apoptosis [9]. Alternatively, it could act directly on aspartyl peptidases produced by protozoa. This proteolytic class has already been described in L. amazonensis [37] and L. mexicana [38]. In the latter, it was also shown that diazo- acetyl-norleucinemethylester (DAN), an aspartyl peptidase inhib- itor, significantly reduces promastigote growth. Moreover, in the present report, we demonstrated that nelfinavir and lopinavir powerfully inhibited the hydrolysis of HIV-1 peptidase substrate, at 10 and 1 mM, respectively. Although it seems reasonable to assume that the HIV PIs target Leishmania aspartyl peptidase and/ or proteasome, the possibility of unrelated effects of inhibitors on L. amazonensis should also be considered, such as nonspecific or generally toxic effects on parasite cells. In this context, electron- micrographic examination of protozoan cells exposed to nelfinavir or lopinavir revealed some peculiar alterations in vital cellular structures, such as cytoplasmic membrane and internal cellular structures, suggesting irreversible metabolic injuries that culminate in the parasite cell death. Some of the ultrastructural alterations observed in L. amazo- nensis, such as increase in the number of vesicles and wrapping of the nucleus by the endoplasmic reticulum, are suggestive of autophagy. In this sense, recent data have provided evidence for the existence of different forms of programmed cell death. In addition to the long time established existence of apoptosis, autophagy has been increasingly described in eukaryotic cells [39]. Autophagy is a normal degradative process that exists in all eukaryotic cells and is stimulated in response to a variety of environmental stresses, which necessitate the use of autophagic mechanisms to enable cellular survival [40,41]. Autophagy involves the sequestration of cytosol or cytoplasmic organelles within double membranes, thus creating autophagosomes (also called autophagic vacuoles). Autophagosomes subsequently fuse with endosomes and eventually with lysosomes, thereby creating autophagolysosomes or autolysosomes. In the lumen of these latter structures, lysosomal enzymes operating at low pH then catabolize the autophagic material [42,43]. Low levels of autophagy are HIV PIs on L. amazonensis considered to be responsible for better survival, while abnormally high levels of autophagy might be responsible for cell death [43]. Up to now, the deregulation events responsible for uncontrolled autophagic activity have not been well characterized [44]. Additionally, in contrast to mammalian cells, the autophagic process in unicellular organisms is less understood. Finally, the accumulation of autophagic vacuoles can precede apoptotic cell death [44]. Indeed, Leishmania cells grown in the presence of lopinavir for 24 hours displayed a chromatin condensation, which is one of the hallmarks of apoptosis. Therefore, it would be interesting to further explore the mechanism of cell death induced by HIV PIs in Leishmania. In the opportunistic fungal pathogen Cryptococcus neoformans, indinavir selectively inhibits the production of some virulence factors, such as urease and peptidase. Additionally, this inhibitor also interfered with polysaccharide capsule formation, the major virulence factor produced by this microorganism [45]. In this context, we aimed to assess if the PIs would induce any change in the peptidase expression by L. amazonensis. Over the past few years it has become clear that peptidases from the pathogenic trypanosomatids play an important role in several steps of the host infection: adhesion, penetration, intracellular survival, replication, differentiation, infectivity, immune evasion and nutrition. Trypanosomatids present a large and varied array of intracellular and/or extracellular peptidases whose regulated expression entails specific functions in the parasite life-cycle stages [18]. Leishmania spp. contain multiple, highly active intracellular cysteine peptidase activities and an abundant intracellular or surface-associated metallopeptidase, named gp63 or leishmanoly- sin [16-18]. The majority of the studies so far developed have dealt with only three types of cysteine peptidases, designated CPA, CPB and CPC [16]. The generation of mutants for CPA, CPB and CPC genes in L. mexicana has provided the first genetic support for the key role of leishmanial cysteine peptidases in parasite virulence, and hence their validation as drug targets [16]. Accordingly, it has been demonstrated that cysteine peptidases are preferentially expressed in virulent, as opposed to avirulent L. amazonensis promastigotes [22]. Leishmanolysin is also involved in Leishmania virulence and pathogenicity. The expression of the enzyme is increased in metacyclic promastigotes and it may be a ligand involved in the interaction of the parasite with defensive systems of the host, including components of the complement system and the macrophage surface. This enzyme can also play a role in intracellular amastigote survival within macrophage phagolysosomes. Nevertheless, data from gp63 knockouts are conflicting [17]. In this context, contrary to the view that HIV PIs would lead to a reduction in peptidase levels, as described for virulence factors in fungi, our data revealed a significant increase in the expression of these molecules when parasites were subjected to PIs before protein extraction. Curiously, a similar effect was described when L. amazonensis was treated with parthenolide, which is a sesquiterpene lactone purified from the hydroalcoholic extract of aerial parts of Tanacetum parthenium [46]. One hypothesis could be that the HIV PIs are inhibiting an aspartyl peptidase that should be otherwise degrading some of the gp63 and CPB peptidases. Accordingly, the degradation of the HIV-1 peptidase substrate was substantially inhibited by HIV PIs. An alternative hypothesis could be that the HIV PIs are exerting stress, or some other non-specific effect, on the promastigotes that leads to changes in parasite gene expression. Indeed, stresses such as heat shock are important to the differentiation of L. amazonensis into axenic amastigotes, whereup- on cysteine peptidase gene expression, and hence activity, is upregulated [37,47]. PLoS ONE | www.plosone.org 9 March 2009 | Volume 4 | Issue 3 | e4918