Effects of serine protease inhibitors on viability and ...

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and morphology of Leishmania (Leishmania) amazonensis promastigotes. R. E. Silva-Lopez & J. A. Morgado-Díaz &. M. A. Chávez & S. Giovanni-De-Simone.
Parasitol Res DOI 10.1007/s00436-007-0706-5

ORIGINAL PAPER

Effects of serine protease inhibitors on viability and morphology of Leishmania (Leishmania) amazonensis promastigotes R. E. Silva-Lopez & J. A. Morgado-Díaz & M. A. Chávez & S. Giovanni-De-Simone

Received: 26 April 2007 / Accepted: 27 July 2007 # Springer-Verlag 2007

Abstract To investigate the importance of serine proteases in Leishmania amazonensis promastigotes, we analyzed the effects of classical serine protease inhibitors and a Kunitztype inhibitor, obtained from sea anemone Stichodactyla helianthus (ShPI-I), on the viability and morphology of parasites in culture. Classical inhibitors were selected on the basis of their ability to inhibit L. amazonensis serine proteases, previously described. The N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) inhibitors, which are potential Leishmania proteases inhibitors, in all experimental conditions reduced the parasite viability, with regard to time dependence. On the other hand, N-tosyllysine chloromethyl ketone (TLCK) did not significantly affect the parasite viability, as it was poor Leishmania enzymes inhibitor. Ultrastructural analysis demonstrated that R. E. Silva-Lopez (*) : S. Giovanni-De-Simone Laboratório de Bioquímica de Proteínas e Peptídeos, Departamento de Bioquímica e Biologia Molecular, Instituto Oswaldo Cruz, FIOCRUZ, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil e-mail: [email protected] J. A. Morgado-Díaz Divisão de Biologia Celular, Pesquisa Básica, Instituto Nacional de Câncer, Rio de Janeiro, RJ, Brazil

both Bza and TPCK induced changes in the flagellar pocket region with membrane alteration, including bleb formation. However, TPCK effects were more pronounced than those of Bza in Leishmania flagellar pocket in plasma membrane, and intracellular vesicular bodies was visualized. ShPI-I proved to be a powerful inhibitor of L. amazonensis serine proteases and the parasite viability. The ultrastructural alterations caused by ShPI-I were more dramatic than those induced by the classical inhibitors. Vesiculation of the flagellar pocket membrane, the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole, and alterations of promastigotes shape resulted. Abbreviations BHI brain heart infusion BPTI bovine pancreatic trypsin inhibitor Bza benzamidine IC inhibitor concentration EM electron microscopy MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ShPI-I Kunitz-type protease inhibitor from sea anemone Stichodactyla helianthus L- TAME N-ρ-tosyl-L-arginine methyl ester TLCK N-tosyl-lysine chloromethyl ketone TPCK N-tosyl-L-phenylalanine chloromethyl ketone

M. A. Chávez Centro de Estudio de Proteínas, Facultad de Biologia, Universidad de la Habana, Havana, Cuba

Introduction

S. Giovanni-De-Simone Departamento de Biologia Celular e Molecular, Instituto de Biologia, Universidade Federal Fluminense, Niterói, RJ, Brazil

Leishmania spp are a group of protozoan pathogens responsible for a spectrum of chronic diseases, ranging from self-healing cutaneous lesions to lethal visceral

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disorders (Herwaldt 1999). These parasites guarantee perpetuation within their hosts by triggering the expression of many molecules, which activate survival mechanisms. Among these molecules, proteases are crucial in the parasite life cycle and leishmaniasis pathogenesis (Rosenthal 1999; Sadij and McKerrow 2002). These enzymes have been implicated in a great variety of adaptation mechanisms for in-host parasite survival, which include modulation of host immune system, invasion and destruction of host tissues, parasite dissemination, and acquisition of essential nutrients that assure survival and proliferation to sustain the infection (Coombs and Mottram 1997; Rosenthal 1999; Sadij and McKerrow 2002). The existing chemotherapy of leishmaniasis, the pentavalent antimonials, was first introduced about 70 years ago and suffers from lack of safe and effective drugs which in many cases have now brought about widespread parasite drug resistance (Desai et al. 2004). However, new therapies for leishmaniasis are currently being investigated (Croft and Coombs 2003), including molecules from marine organisms (Savoia et al. 2004) and protease inhibitors (Rosenthal 1999). Thus, the biochemical characterization of Leishmania proteases provides formidable means to improve our understanding of parasite physiology and subsequently reinforce the evidence that these proteases might be potential targets for antileishmanial therapy. In previous studies, we performed the purification and biochemical characterization of three serine proteases from Leishmania (Leishmania) amazonensis promastigotes, employing similar protocols of purification. We obtained two enzymes from two different intracellular fractions: a dimeric serine protease of 110 kDa that was acquired from a detergent soluble fraction (Silva-Lopez and Giovanni De Simone 2004a) named LSPI and a monomeric enzyme of 68 kDa with high proteolytic activity purified from a watersoluble fraction, named LSPII (Silva-Lopez and Giovanni De Simone 2004b). Studies of subcellular localization using electron microscopy (EM) demonstrated that LSPII is mainly restricted to intracellular compartments resembling endocytic/exocytic elements (Morgado-Díaz et al. 2005). The third enzyme (LSPIII), a secreted 115-kDa dimeric serine protease which was purified from culture supernatant (Silva-Lopez et al. 2005) was localized in megasomes, the flagellar pocket, and structures which are morphologically similar to the compartments of mammalian endocytic/ exocytic pathways (Silva-Lopez et al. 2004). However, the location of serine proteases in the parasite was determined, their role in the Leishmania protozoa and their involvement in host-parasite interaction is unknown. Protease activity can be regulated in the cells or in the organisms by different ways, including protease inhibition through specific inhibitors (Otlewski et al. 1999;

Krowarsch et al. 2003). These inhibitors are valuable tools for investigation of the biochemical properties and the biological functions of proteases (McKerrow et al. 1999). In addition, invasion blockage of many parasites, including Plasmodium falciparum (Roggwille et al. 1996) and Toxoplasma gondii (Conseil et al. 1999) has been observed due to the use of specific serine protease inhibitors. To investigate the importance of serine proteases in L. amazonensis promastigotes survival, we analyzed the influence of inhibitors of these enzymes considering two criteria: (1) cell viability and (2) parasite morphological alterations. Inhibitor choice was based on capacity to impede Leishmania serine proteases (Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005). Therefore, we employed three classical low-molecularweight serine protease inhibitor, which reacts with the active site of protease: benzamidine (Bza), which reacts with Asp residue of the serine protease’s active sites and is able to competitively inhibit, with high specificity, trypsinlike and chymotrypsin-like serine proteases (Sturzebecher et al. 1992), N-tosyl-lysine chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanine chloromethyl ketone (TPCK). TPCK and TLCK are irreversible inhibitors that have Phe and Lys residues, respectively, in chloromethyl ketone linkage and react with His of the active site of the active enzyme (Powers et al. 2002). We have previously reported that Bza completely inhibited the LSPI activity (SilvaLopez and Giovanni De Simone 2004a) and 80% LSPIII activity (Silva-Lopez et al. 2005) but did not affect the LSPII (Silva-Lopez and Giovanni De Simone 2004b), while TPCK inhibited all enzymes in different degrees, TLCK had a moderate influence on the LSP II and LSP III activity but did not affect the detergent-soluble Leishmania serine protease (Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005). Furthermore, we also analyzed the effect of a high molecular weight inhibitor from sea anemone Stichodactyla helianthus (ShPI-I), which is a Kunitz-type inhibitor of 6,110.6 Da that exhibits a broad specificity for serine, cysteine, and aspartic proteases (Delfin et al. 1996). Regardless of the fact that ShPI-I inhibits other peptidases, it was demonstrated that it has a great specificity for serine proteases due to the lowest Ki values obtained for serine in comparison to the Ki of cysteine and aspartic proteases (Delfin et al. 1996). Furthermore, the higher specificity of ShPI-I observed for serine proteases might also be explained by the fact that this inhibitor demonstrated a nearly identical molecular architecture to the bovine pancreatic trypsin inhibitor (BPTI), an important mammalian serine protease inhibitor. Finally, ShPI-I is usually purified by affinity chromatography using trypsin, a known pancreatic serine protease immobilized in Sepharose matrix (Antuch et al. 1993).

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Materials and methods Parasites and culture conditions Leishmania (Leishmania) amazonensis promastigotes (IOC 575; IFLA/BR/67/PH8) were maintained at 28°C in brain heart infusion medium (BHI; Difco, Detroit, USA) supplemented with 10% (v/v) heat-inactivated fetal-calf serum, 0.5 mg of hemin, and 0.5 mg of folic acid per liter. All experiments were performed with parasite cultures in logarithmic phase of growth. Cell growth was estimated by counting the promastigotes in a Neubauer chamber, and the cell viability was assessed through trypan blue dye exclusion (Barankiewicz et al. 1988). Effect of ShPI-I on enzymatic activity Enzymes employed in this study (LSPI, LSPII, and LSPIII) were purified in accordance with the protocols previously described (Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005). The effect of ShPI-I (10−5, 10−6, and 10−7 M) on the LSPI, LSPII, and LSPIII enzymatic activities was determined using L-TAME (N-ρ-tosyl-Larginine methyl ester) as substrate. Briefly, the inhibitor was preincubated with proteases (0.03 nM, active concentration) for 15 min at room temperature. The reaction commenced upon addition of the substrate (0.25 mM) at 28°C for 30 min, and the residual activity was measured as previously described (Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005). Substrate was digested in Tris–HCl 100 mM, pH 7.0 for LSPI, pH 8.0 for LSPII, and pH 7.5 for LSPIII, and the digestion was monitored by measuring the absorbance increase at 247 nm. Appropriate controls were carried out in parallel using the same enzyme solutions free of inhibitor. Inhibition was expressed as the percentage of the appropriate control activity (100%). The Kunitz type inhibitor was purified from sea anemone Stichodactyla helianthus as described previously (Delfin et al. 1996).

scribed previously (Kano et al. 2003). The conversion of MTT to the formazan product by mitochondrial electron transporter chain is an indicator of cell viability, and a decrease in the amount of MTT converted indicates toxicity to the parasite (Paris et al. 2004). ShPI-I was employed at 10−5, 10−6, and 10−7 M, and the incubation times were 2, 4, 8 and 16 h. Briefly, after inhibitor incubation, MTT (2 mg/ ml in PBS) was added to each well until reaching a final dye concentration of 200 μg/ml. Plates were then returned to the incubator for 2 h. The purple formazan product was subsequently dissolved by adding 250 μl acidified isopropanol (95% isopropanol; 5% 2 N HCl), and 200 μl aliquots were collected. The absorbance of each sample was analyzed at 590 nm using the Inter-med model NJ-2300 Microplate Reader. Parasite viability (%) was calculated regarding the controls (0 and 100% of viability). Electron microscopy For EM analysis, promastigotes were cultured as described above and incubated for 6 h with the inhibitors: Bza (10−3 M), TLCK, and TPCK (10−4 M); ShPI-I (10−5 M). Afterwards, the parasites were washed in PBS and fixed for 1 h in a solution containing 2.5% glutaraldehyde, 1% paraformaldehyde, 0.8% sucrose, and 2 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.4. Post fixation was carried out in 1% osmium tetroxide in cacodylate buffer, containing 0.8% potassium ferrocyanide and 5 mM CaCl2. Subsequently, the cells were dehydrated with acetone and embedded in Epon (Silva-Lopez et al. 2004). Ultrathin sections were collected, stained with uranyl acetate and lead citrate then observed in a Zeiss CEM-900 transmission electron microscope. Control cultures were processed without inhibitor addition.

Results Effect of ShPI-I on Leishmania amazonensis serine proteases

Viability measurements by colorimetric MTT assay Promastigotes, harvested in the exponential growth phase, were resuspended in fresh medium to achieve 4×105 cells/ ml and were then seeded in 96-well culture plates, the fresh inhibitors added in triplicate at different final concentrations (Bza at 10−3 M, TPCK and TLCK at 10−4 M). The inhibitor concentration was established according to previous studies (Silva-Lopez and Giovanni De Simone 2004a, b; SilvaLopez et al. 2005). The plates were incubated at 28°C for 4, 8, 16 or 32 h, and promastigotes viability was evaluated by the quantitative colorimetric MTT ([3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide]) assay, as de-

The enzymatic studies using the ShPI-I demonstrated an important inhibitory effect on the L. amazonensis serine protease activities (Table 1). This inhibitor, at molar concentration of 10−7, did not affect the enzyme, but at 10−6 M, it inhibited the enzymatic activity from 45 to 68% and at 10−5 M completely inhibited the activity of all assayed proteases (Table 1). Viability assays The viability assay using MTT as a marker was first performed incubating promastigotes with the inhibitors

Parasitol Res Table 1 Effects of concentration range of ShPI-I on L. amazonensis promastigote serine proteases residual activity (%) Enzyme

ShPI-I (10−5 M)

ShPI-I (10−6 M)

ShPI-I (10−7 M)

LSPI LSPII LSPIII

0±0% 0±0% 0±2%

39±3% 32±5% 55±12%

100±8% 100±4% 100±10%

The percentage of the remaining activity of protease on L-TAME as compared with a control reaction without preincubation. The values refer to the mean and standard deviation of four separate experiments carried out in duplicate.

TLCK, TPCK, and Bza for 4, 8, 16 and 32 h (Fig. 1). It is important to point out that the choice of the molar concentration of TLCK, TPCK, and Bza was in accordance with the values stipulated in literature (Roggwille et al. 1996; Shaw et al. 2002; Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005). TPCK and Bza, which proved to be important inhibitors of the L. amazonensis serine protease activities, were more effective in reducing the parasite viability, their effects being time dependent. TPCK, in the same concentration of TLCK, reduced the viability by about 63% after 8 h of incubation, and after 16 and 32 h, its effect was even more pronounced, diminishing the parasite viability to 82 and 93%, respectively. The effect of Bza in the first 4 h was not so dramatic as the effect of TPCK because the viability only decreased about 20%. The effect of Bza after 16 and 32 h of incubation was a bit more notable than the TPCK effect and reduced the Leishmania viability about 84 and 98%, respectively. The effect of both TPCK and Bza on the parasite viability was similar. As expected, TLCK did not significantly affect the viability of the parasites. Therefore, the viability decreased merely about 13% in relation to the control group only after 32 h of incubation. To evaluate the effect of ShPI-I on Leishmania viability, we applied the same molar concentration used for the enzymatic assays. As displayed in Fig. 2a–d, ShPI-I at 10−7 M did not affect the viability of parasites, but at 10−6 and 10−5 M, the inhibitor significantly decreased the viability of Leishmania in all conditions of incubation, however, the effect was time- and dose-dependent. The maximal influence of ShPI-I on parasite viability was observed after 16 h of exposure to 10−5 M of the inhibitor. Approximately only 5% of viable parasites were observed after this treatment. Morphological studies by EM To determine ultrastructural changes induced by the classical serine protease inhibitors in L. amazonensis promastigotes, the parasites were treated with Bza

(10−3 M), TLCK, and TPCK (10−4 M) and processed for routine EM. As observed in Fig. 3a–f, both inhibitors induced similar alterations in the promastigote morphology. Parasites incubated in the presence of Bza displayed significant morphological alterations in the region of the flagellar pocket (about 95% of observed promastigotes; Fig. 3b and c), compared to the morphology of the untreated analyzed parasites (Fig. 3a). The membrane that covers the flagellar pocket in these parasites was altered and the formation of membrane blebs was also perceived. No significant modification was noticed in any of the other cellular structures of the parasites treated with this inhibitor. When the culture was exposed to TPCK, similar alterations in the flagellar pocket could be observed, but the effects of this inhibitor were more pronounced than the Bza effects (Fig. 3d and e). Furthermore, we also noticed the presence of a great number of intracellular vesicular bodies in the parasites (Fig. 3f). These results revealed that TPCK induced more deleterious effects and more significant morphological changes in the L. amazonensis promastigotes in comparison with Bza. Similar studies were performed using TLCK, but this inhibitor did not induce alterations in the parasite morphology (data not shown). To analyze morphological alterations caused by ShPI-I in Leishmania, promastigotes were incubated with 10−5 M of this inhibitor and also processed for EM. It was possible to detect more dramatic changes induced by ShPI-I (Fig. 4b and c) than those resulting from the classical serine protease inhibitors TPCK and Bza. ShPI-I induced ultrastructural changes in all parts of the parasite body. At the flagellar pocket level, we were able to perceive the appearance of vesicles, which were accompanied by bleb formations (Fig. 4b). In the cytoplasm, the presence of vesicles that resemble autophagic vacuoles was also noted (Fig. 4b and c). This phenomenon was not encountered in control cells. Furthermore, all parasites exhibited shape alterations, and promastigote anomalies appeared (Fig. 4b and c).

Discussion Only recently have Leishmania serine peptidases been purified and characterized (Ribeiro de Andrade et al. 1998, Silva-Lopez and Giovanni De Simone 2004a, b; Silva-Lopez et al. 2005), and their function in the parasite physiology is still under investigation. It is known that these enzymes are encountered in megasomes, the flagellar pocket and structures which are morphologically similar to the compartments of mammalian endocytic/exocytic pathways (Silva-Lopez et al. 2004; Morgado-Díaz et al. 2005). Inhibitors of L. amazonensis serine protease were employed to study the influence of these enzymes in the viability of promastigotes in culture. Both TPCK and Bza

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Fig. 1 MTT assay for the viability of L. amazonensis treated with classical serine protease inhibitors. Promastigotes (4×104 cells/100 μl) were treated with inhibitors Bza (10−3 M), TPCK, and TLCK (10−4 M) for 4 h (a), 8 h (b), 16 h (c), or 32 h (d). The data are the

mean of three independent experiments, carried out in triplicate, and standard deviation is represented by error bars. Statistical significance (p