Abstract
Extracellular proteolytic activity was detected in a Leishmania (L.) amazonensis culture supernatant and a 56-kDa protein was purified using (NH4)2SO4 precipitation followed by affinity chromatography on aprotinin–agarose. A rabbit serum obtained against the 56-kDa extracellular serine protease was used in order to analyze its location in L. (L.) amazonensis parasites. Immunocytochemistry studies revealed that the enzyme is mainly found in the flagellar pocket and cytoplasmic vesicles of promastigote forms, whereas in amastigotes, it is located in electron-dense structures resembling megasomes. These results indicate that the 56-kDa serine protease is released into the extracellular environment through the flagellar pocket; and its intracellular location suggests either a correlated enzymatic activity or intracellular trafficking.
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Introduction
Serine proteases of pathogenic parasites play a crucial role in parasite physiology and in host–parasite interactions (Salter et al. 2000; Burleigh and Woolsey 2002). These interactions include the invasion and destruction of host tissues, enabling parasite migration, dissemination, nutrition and growth. Many of these serine proteases have been purified and characterized in order to elucidate their function. Regarding their location, these enzymes have been found in various cellular compartments, are associated with the membranes and may be secreted into the extracellular environment. In trypanosomatids of the genus Leishmania, proteolytic enzymes are typically found in abundant membrane-bounded, electron-dense structures, designated as megasomes, which vary in aspect and size (Alexander and Vicherman 1975). These structures are found in the amastigote stage of species belonging to the mexicana complex; and previous studies have shown that they display lysosome-like properties (Duboise et al. 1994). Megasomes, being an important site of proteolysis, are rich in cysteine proteases, which are apparently involved in the survival of the parasite and have been investigated as a potential target for drug therapy (Coombs and Mottran 1997). In this work, we study the subcellular location of extracellular serine proteases of L. amazonensis amastigotes and promastigotes.
Materials and methods
Promastigote forms of Leishmania amazonensis (IOC 575; IFLA/BR/67/PH8) were maintained at 28 °C in brain/heart infusion medium supplemented with 4 mg% hemin, 2% heat-inactivated fetal bovine serum and 2 mg% folic acid. Cells were harvested on day 4 of cultivation and the cell-free supernatant collected for enzyme purification. The whole extract of L. amazonensis was obtained by submitting the parasites to seven cycles of freezing/thawing (−80 °C/37 °C) and the supernatant collected for further analysis. Cell viability was assessed by trypan blue cell-dye exclusion (Barankiewicz et al. 1988).
For protease purification, 2 l of cell-free culture supernatant from 8.0×1010 parasites was precipitated with 45% (NH4)2SO4, dialyzed in 10 mM Tris-HCl (pH 7.5) and then subjected to affinity chromatography using an aprotinin–agarose column (2.5 ml; Sigma). Aprotinin is a naturally occurring serine protease inhibitor that forms a reversible stoichiometric complex with the serine residue from the active sites of serine proteases (Peters and Noble 1999) and is commonly used in affinity chromatography columns specifically to purify this kind of protease (Petinate et al. 1999). The affinity column was previously balanced in 10 mM Tris-HCl, pH 7.5, 5 mM CaCl2. SDS-PAGE was performed using 12% polyacrylamide gels under reducing conditions (Laemmli 1970) and protein bands were identified by Coomassie blue staining. Both the proteolytic activity of extracellular enzymes and the effect of the serine protease inhibitor (benzamidine 1 mM) were assayed using SDS-PAGE with 0.1% gelatin incorporated in the gel at pH 7.5 (Alves et al. 1993) with or without the addition of inhibitor. In order to elucidate the type of protease which was purified, additional inhibition studies were performed, using other specific serine protease inhibitors or inhibitors displaying other protease specificities. The inhibitors were incubated with 10 μg of protein for 15 min at room temperature, using 100 mM Tris-HCl (pH 7.5) with the substrate (0.25 mmol of N-ρ-tosyl-L-arginine methyl ester; l-TAME). The reaction, taking place at a temperature of 28 °C, commenced upon the addition of a serine protease substrate and lasted for 30 min. The absorbance was monitored at 247 nm and each assay was carried out in triplicate. Control solutions lacking substrate and/or enzyme, or containing inhibitors, were obtained simultaneously and their values were subtracted as background from the experimental samples. Inhibition was expressed as a percentage of the appropriate control activity.
A polyclonal antibody was raised in a rabbit by subcutaneous injection of heat-inactivated 56-kDa protease. The purified protein (0.1 mg) was emulsified with incomplete Freud’s adjuvant (first, second boosters), blood was collected 7 days after the fourth injection and the serum specificity was evaluated by Western blotting (Alves et al. 2000).
In order to determine the subcellular location of the extracellular proteases, electron microscopy studies were performed using amastigote forms (obtained from lesion fragments of infected animals) and axenic promastigotes. The specimens were fixed (4% paraformaldehyde, 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2), dehydrated in methanol and embedded in Lowicryl K4M resin. Thin slices were incubated in phosphate-buffered saline (pH 8.0) containing 1.5% bovine serum albumin and 0.01% Tween 20, then incubated with the rabbit antiserum against a 56-kDa protease (1:50) and finally incubated with anti-rabbit antibody (1:200) labeled with 10-nm gold particles. The sections were stained with aqueous uranyl acetate and lead citrate and observed in a Zeiss EM transmission electron microscope.
Results and discussion
A combination of ammonium sulfate precipitation and affinity chromatography was carried out to purify an extracellular protease from Leishmania amazonensis. The proteinaceous material from chromatography was analyzed by SDS-PAGE under reducing conditions and a major protein band of 56 kDa was detected (Fig. 1a, lane 1), showing its highest hydrolytic activity at pH 7.5 (Fig. 1a, lane 2). However, some proteolytic activity was found in proteins above 90 kDa. The better characterized proteases, such as lysosomal cathepsins, fit the traditional concept of small and monomeric proteins, while others, many of which are polymeric, had subunit molecular masses of 50–100 kDa (Salter et al. 2000). The serine protease of Acantamoeba healyi is a small secreted enzyme of 33 kDa (Kong et al. 2000), while the Trypanosoma cruzi serine oligopeptidase is bigger, at 120 kDa (Burleigh and Woolsey 2002). The type of protease was determined using specific inhibitors for known protease classes. Preliminary inhibition studies, using SDS-PAGE gelatin, suggested that the isolated protease in this work belongs to the serine protease class because of its complete inhibition of gelatinolytic activity using benzamidine, a serine protease inhibitor (Fig. 1a, lane 3). In order to confirm the result obtained by SDS-PAGE-gelatin, additional inhibition-colorimetric assays were performed. Using aprotinin (0.38 μM), the enzymatic activity was completely abolished; and N-tosyl-L-phenylalanine chloromethyl ketone (100 μM), another serine protease inhibitor, caused approximately 60% inhibition of the enzymatic activity. In contrast, L-trans-epoxysuccinyleucylamido-(4-guanidino)-butane (10 μM), pepstatin (1 mM) and ethylenediaminetetracetic acid (10 μM), known inhibitors of cystein, aspartic and metalloproteases, did not affect the ability of the enzyme in the hydrolysis of l-TAME. These results indicated that the purified protease in L. amazonensis supernatant belongs to a serine protease class.
Electrophoretic and immunoblotting analysis of extracellular serine proteases. Molecular mass values of standards proteins (kDa) are shown on the left and right sides of the gels. Material from the supernatant was precipitated, dialyzed and then subjected to affinity chromatography, using an aprotinin–agarose column. a The serine protease was eluted from the column and the fractions were pooled and analyzed by 12% SDS-PAGE (lane 1) and 12% gelatin/SDS-PAGE (lane 2). The asterisk indicates a protein of 56 kDa with the highest proteolytic activity. In order to verify the effect of a serine protease inhibitor on protease activity, the 12% gelatin/SDS-PAGE was incubated with 1 mM benzamidine (lane 3). b The protein from a soluble whole extract of Leishmania amazonensis (80 μg) was resolved by SDS-PAGE, transferred to a nitrocellulose membrane and the reactivity of Leishmania antigens was analyzed using an anti-56 kDa antibody. The asterisks indicate that the antibody recognized not only a 56-kDa protein, but also another at 115 kDa. Electrophoresis was run on 12% SDS-PAGE under reducing conditions
The specificity of the polyclonal antiserum to 56-kDa serine protease was evaluated by immunoblotting, using soluble whole extracts from axenic promastigotes of L. amazonensis. As observed in Fig. 1b, the antiserum reacted against Leishmania proteins, supporting the conjecture that the purified serine protease from L. amazonensis supernatant is present in internal structures of the parasite. Similar results were observed in other kinds of proteases, such as cysteine proteases (Monteiro et al. 2001). Furthermore, the antibody to the 56-kDa serine protease not only recognized a protein of 56 kDa, but also another with a molecular mass of about 115 kDa. This result could indicate that the 56-kDa protein showed immunological similarities to another intracellular protein of 115 kDa, possibly a zymogen.
In order to determine the subcellular location of the serine protease in L. amazonensis, electron microscopy immunolocalization studies were performed. The immunoreactivity was assessed using axenic promastigotes and amastigotes obtained from cutaneous lesions induced in mice. It was possible to observe that the antibody reacted poorly with the parasite surface and moderately with internal structures in most samples (about 95%) of both forms of the parasite (Fig. 2). In promastigotes, gold particle labeling showed the serine protease to be predominantly located in the flagellar pocket and in vesicular structures which are morphologically similar to the compartments that found in mammalian endocytic/exocytic pathways (Fig. 2a,b). In amastigotes, the enzyme was detected not only in subcellular structures similar to those of promastigotes, such as the flagellar pocket and cytoplasmic vesicles (Fig. 2), but also in electron-dense structures corresponding to megasomes (Fig. 2c,d). It is known that the flagellar pocket membrane is the only part of the cell surface that supports exocytosis and endocytic traffic, because of its lack of attached microtubules. However, the flagellar pocket membrane is an obligatory intermediary station for membrane-bound molecules trafficking between intracellular membranes and the cell surface and vice-versa (Overath et al. 1997). Although only a few cases have been examined, both membrane-bound and secreted proteins appear on the cell surface, underscoring the role of this membrane in the delivery of proteins to the cell surface and exterior (Landfear and Ignatushchenko 2001). The processing and trafficking of cysteine protease, the best studied lysosomal Leishmania protease, has been reported in L. mexicana and is targeted to megasomes via the flagellar pocket (Brooks et al. 2000). It was also shown that megasomes are the main sites of proteolytic activity in Leishmania, which results in differentiation process participation and in parasite intracellular survival (Ueda-Nakamura et al. 2002).
Subcellular location of serine protease of L. amazonensis. F Flagellum, K kinetoplast, M megasome, N nucleus, P flagellar pocket; a, c bars 2.0 μm; b, d bars 3.2 μm. a Promastigote forms showed immunolabeling at the flagellar pocket. In both forms, the cell surface was poorly labeled (arrowheads). c Amastigotes displayed moderate labeling in the flagellar pocket, cytoplasmic vesicles (arrows) and megasomes. b Promastigote anterior region and d amastigote at high magnification showed immunolabeling at cytoplasmic vesicles (arrows) that subtended the flagellar pocket in both forms of L. amazonensis
In conclusion, L. amazonensis secretes a 56-kDa serine protease into the culture supernatant through the flagellar pocket with the participation of different components that resemble mammalian endocytic/exocytic organelles. Furthermore, the fact that this enzyme is located in megasomes, where cysteine proteases are also found, might suggest that the amastigote serine protease in this organelle contributes, in association with the cysteine proteases, to maintain the parasite life cycle and disease pathogenesis. However, further studies should be conducted to demonstrate this possibility.
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Acknowledgements
This work was supported by grants from the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ), the Fundação Oswaldo Cruz (FIOCRUZ, PAPES) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We are grateful indebted to Mr. G. Alves for technical assistance on Leishmania cultures.
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Silva-Lopez, R.E., Morgado-Díaz, J.A., Alves, C.R. et al. Subcellular localization of an extracellular serine protease in Leishmania (Leishmania) amazonensis . Parasitol Res 93, 328–331 (2004). https://doi.org/10.1007/s00436-004-1144-2
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DOI: https://doi.org/10.1007/s00436-004-1144-2