Major cysteine protease (cruzipain) in Z3 sylvatic isolates of Trypanosoma cruzi from Rio de Janeiro, Brazil

Parasitol Res (2009) 105:743–749 DOI 10.1007/s00436-009-1446-5

ORIGINAL PAPER

Major cysteine protease (cruzipain) in Z3 sylvatic isolates of Trypanosoma cruzi from Rio de Janeiro, Brazil S. A. O. Gomes & D. Misael & B. A. Silva & D. Feder & C. S. Silva & T. C. M. Gonçalves & A. L. S. Santos & J. R. Santos-Mallet

Received: 10 November 2008 / Accepted: 17 April 2009 / Published online: 13 May 2009 # Springer-Verlag 2009

Abstract Trypanosoma cruzi, the etiologic agent of Chagas’ disease, is represented by a set of parasites which circulate between man, vectors, domestic and wild animals. Recently, our group isolated from Triatoma vitticeps strains of T. cruzi that were characterized as belonging to the Z3 phylogenetic lineage. Since very little is known about the biological and/or biochemical markers of sylvatic Z3 isolates, we have studied the protein and protease profiles of distinct Z3 isolates designated as SMM10, SMM53, SMM88, and SMM98. By means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, both quantitative and qualitative differences were observed in the protein profiles of these strains. All strains produced an acidic cysteine protease of 45 kDa, resembling cruzipain activity. The strain SMM10 synthesized an additional 55 kDa metalloprotease. Using Western

S. A. O. Gomes and D. Misael contributed equally to this work. S. A. O. Gomes (*) : D. Misael : C. S. Silva : T. C. M. Gonçalves : J. R. Santos-Mallet Setor de Morfologia e Ultraestrutura e Bioquímica de Artrópodes e Parasitos, Laboratório de Transmissores de Leishmanioses, Instituto Oswaldo Cruz—Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, Brazil e-mail: [email protected] B. A. Silva : A. L. S. Santos Laboratório de Estudos Integrados em Bioquímica Microbiana, Departamento de Microbiologia Geral, Bloco E-subsolo, Instituto de Microbiologia Paulo de Góes (IMPPG), Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil D. Feder Laboratório de Biologia de Insetos, Departamento de Biologia Geral, Universidade Federal Fluminense (UFF), Niterói, Rio de Janeiro, Brazil

blotting and anti-cruzipain antibody to detect cruzipain-like molecules, a 40-kDa reactive molecule was identified in all strains; in the strain SMM10, an 80-kDa protein was also reacted. Studies about cruzipain isoforms from sylvatic parasites could be valuable tools in the comprehension of the genetic variability in the pathogenesis of Chagas’ disease.

Introduction Trypanosoma cruzi, the etiologic agent of Chagas’ disease, is composed of a set of parasites that circulate between man, vectors, domestic and wild animals. T. cruzi is confined as epimastigote form in the lumen of digestive tract of invertebrate vectors and as a nonreplicative infective trypomastigote form that develops within the insect hindgut (Kollien and Schaub 2000), being transmitted by feces to the vertebrate host. Triatoma vitticeps has been found in the northern and southern States of Brazil, including Bahia (Corrêa 1986), Espírito Santo, Minas Gerais, and Rio de Janeiro (Silveira et al. 1984); in the latter, from the city of Rio de Janeiro to the north of the State (Ferreira et al. 1986). In the locality of Triunfo, 2nd District of Santa Maria Madalena, Rio de Janeiro, Gonçalves et al. (1998) reported a high incidence of this triatomine infected with T. cruzi. This triatomine may be considered as a sylvatic species by the low prevalence of human infections (Dos Santos et al. 2005, 2006a, b). However, it is beginning to colonize some peridomestic and domestic localities, which suggests a wide ecological valence that was previously low (Forattini et al. 1979; Silveira and Sakamoto 1983; Dias 1989; Dos Santos et al. 2005). The characterization of parasite populations found in nature is crucial for the elucidation of the complex transmission scenario observed in Chagas’ disease. In the

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field, vectors can be colonized by single or mixed populations of T. cruzi belonging to different genotypes. Populations of T. cruzi can be clustered in two major phylogenetic lineages: T. cruzi I (TCI) and T. cruzi II (TCII). TCI has been mainly associated with the sylvatic transmission cycle, being also observed in domestic cycles (Brisse et al. 2000), and TCII parasites are proposed to be associated with the domestic transmission cycle (Macedo et al. 2004). Furthermore, a third group denominated zymodeme III (or Z3) was also described (Miles et al. 1978; Fernandes et al. 2001; Brandão and Fernandes 2006). However, the position of Z3 is still under review, and some isolates of T. cruzi present a hybrid profile, since they show genotypic characteristics of both TCI and TCII (Fernandes et al. 2001). Recently, T. cruzi sylvatic strains isolated from T. vitticeps collected from Santa Maria Madalena municipality were characterized as Z3 lineage by mini-exon typing using multiplex-PCR strategy (Santos-Mallet et al. 2008). Natural populations of T. cruzi are very heterogeneous in biological, biochemical, immunological, and molecular features (Miles 2003); consequently, different strains from endemic areas might be responsible for distinct clinical manifestations and chemotherapy response (Andrade 1999). In addition, the knowledge about the production of wellknown virulence markers in novel sylvatic isolates of T. cruzi could help in the understanding of the evolution of the expression of important molecules during the different steps of the parasite life cycle, promoting an update in unraveling pathological peculiarities of Chagas’ disease. Proteolytic enzymes are key molecules coupled to crucial events of parasite cells, including regular metabolism as well as in several branches of the parasite–host interaction (Vermelho et al. 2007). In this sense, T. cruzi contains a major cysteine protease named cruzipain that is expressed at variable levels in the different developmental forms of the parasite (Martínez et al. 1991; Dos Reis et al. 2006), being active at least tenfold higher in epimastigotes than in amastigote or trypomastigote forms (Campetella et al. 1990). Cruzipain is a papain-like protease, which shares biochemical characteristics with both cathepsin L and cathepsin B (Cazzulo et al. 1990b). The enzyme has been shown to be lysosomal (Bontempi et al. 1989), and it is located in an epimastigotespecific pre-lysosomal organelle called reservosome, which contains proteins that are digested during the differentiation to metacyclic trypomastigotes (Soares et al. 1992). By immunoelectron microscopy, the cruzipain molecule was also localized at the plasma membrane (Souto-Padrón et al. 1990). Although less present in amastigotes and trypomastigotes, cruzipain is an immunodominant antigen that is recognized by human patients’ sera with chronic Chagas’ disease (Martínez et al. 1991). Recently, the protease expression analysis of fresh fieldisolated strains of T. cruzi showed a heterogeneous profile

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of cysteine protease activities in TCI and TCII major phylogenetic groups (Fampa et al. 2008). In the present study, we have investigated the production of proteases, especially cruzipain, as well as the protein surface distribution in four newly sylvatic isolates of T. cruzi belonging to the Z3 genotype.

Materials and method Parasites origin T. cruzi samples were isolated from sylvatic triatomines, T. vitticeps, captured in Triunfo locality, 2nd District of Santa Maria Madalena, State of Rio de Janeiro, Brazil (Fig. 1A). The 122 specimens of T. vitticeps analyzed were captured in households of two different Triunfo areas designated as A, which is a deforested area, and B, localized in a valley with preserved vegetation (Fig. 1B; Gonçalves et al. 1998). Four isolates were used in all parts of this comparative study: SMM10, SMM88, SMM98 (from area A), and

Fig. 1 Studied area (1) and capture sites (2) of T. vitticeps in Triunfo, Santa Maria Madalena, municipal district, State of Rio de Janeiro, Brazil. A deforested area, and B localizes in a valley with preserved vegetation

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SMM53 (from area B). In addition, Dm28c strain, originally isolated from Didelphis marsupialis (TCI lineage), was also investigated.

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calculated by the comparison of the mobility of GIBCO BRL SDS-PAGE standards. Protease activity assay

Parasites were cultivated in LIT medium supplemented with 10% FBS up to 10 days at 27.3ºC. Growth was estimated by determining the cell number in a Neubauer chamber. Cellular viability was assessed by motility and trypan blue cell dye exclusion (Gomes et al. 2006). The viability of the parasites was not affected by the culture conditions employed in this work.

The proteolytic activity was assayed and characterized by 10% SDS-PAGE with 0.1% gelatin incorporated into the gel as substrate (Heussen and Dowdle 1980). The gels were loaded with 200 μg of protein from each parasite extract per slot. After electrophoresis, SDS was removed by incubation for 1 h with 2.5% Triton X-100 under constant agitation. Then, the gels were incubated in 50 mM sodium phosphate buffer, pH5.5, supplemented with 2 mM dithiothreitol (DTT) for 40 h at 37°C to promote the proteolysis. Alternatively, the gels were also incubated in the phosphate buffer described above containing one of the following proteolytic inhibitors: 1 mM 1,10-phenanthroline or 1 μM trans-epoxysuccinyl L-leucylamido-(4-guanidino) butane (E-64) (Sigma Chemical). The gels were stained for 2 h with 0.2% Coomassie 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) (Santos et al. 2005). Molecular masses of the peptidases were calculated by the comparison of the mobility of GIBCO BRL SDS-PAGE standards.

Parasite cellular extracts

Western blotting analysis

Seven-day-old cultured SMM parasites, at the log growth phase, were centrifuged (500×g, 5 min, 4°C), washed three times with phosphate-buffered saline (PBS; 150 mM NaCl, 20 mM phosphate buffer, pH7.2), then resuspended in 100 μl of PBS (1×108 epimastigotes) and lysed by the addition of 1% sodium dodecyl sulfate (SDS). The cells were broken in a vortex by alternating 1-min shaking and 2-min cooling intervals, followed by centrifugation (5,000×g, 15 min, 4°C). The supernatants obtained after centrifugation corresponded to the whole parasite cellular extracts (Santos et al. 2005). The protein concentration was determined according to Lowry et al. (1951), using bovine serum albumin (BSA) as standard.

Protein extracts were separated on 12% SDS-PAGE, and the polypeptides electrophoretically transferred at 4°C at 100 V/300 mA for 2 h to a nitrocellulose membrane. The membrane was blocked in 5% low-fat dried milk in PBS containing 0.5% Tween 20 for 1 h at room temperature. Then, the membrane was washed three times (10 min each) with the blocking solution and incubated for 1 h with the anti-cruzipain polyclonal antibody (kindly provided by Dr. Juan Jose Cazzulo, Instituto de Investigaciones Biotecnologicas, Universidad Nacional de General San Martin, Buenos Aires, Argentina) diluted at 1:2,500. The secondary antibody used was peroxidase-conjugated goat anti-rabbit IgG (Sigma Chemical) at 1:5,000. Immunoblots were exposed to X-ray film after reaction with ECL reagents for chemiluminescence (Santos et al. 2006).

Parasite isolation Parasites were isolated from the digestive tracts of T. vitticeps, and then mixed with 2 ml saline (0.85% NaCl) plus 5µl of 5-fluorocytosine. The homogenates (0.5 ml) were transferred to a biphasic medium containing Novy– MacNeal–Nicolle (NNN) and liver infusion tryptose (LIT) liquid media supplemented with 10% heat-inactivated fetal bovine serum (FBS). The parasites were cryopreserved in N2. Parasites and growth condition

Protein analysis The proteins were separated on 12% SDS-polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970). Briefly, cellular parasite extracts containing the equivalent to 50 μg of protein were added to 10 μl of SDS-PAGE sample buffer (125 mM Tris, pH6.8; 4% SDS, 20% glycerol; 0.002% bromophenol blue; 5% β-mercaptoethanol), followed by heating at 100°C for 5 min. Electrophoresis was carried out at constant current of 150 V, and the gels were silver-stained (Merril 1990). Molecular masses of the polypeptides were

Biotinylation of the cell surface polypeptides For biotinylation, live parasites were centrifuged (500×g, 5 min, 4°C), washed three times with cold PBS, pH8.0, in order to remove proteins from the culture medium, diluted to 2.5×107 cells/ml in PBS and incubated in 0.1 mg/ml Sulfo-NHS-LC-biotin (Pierce Chemical) for 30 min at 4ºC. Parasites were washed three times with cold PBS, to

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remove unbound biotin (Santos et al. 2001), and processed by flow cytometry analysis.

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>200 116

Flow cytometry assay

66

Biotinylated epimastigotes were fixed in 4% paraformaldehyde in PBS, pH7.2, for 30 min followed by extensive washing in the same buffer. These fixed cells maintained their morphological integrity, as verified by light microscopic observation. They were incubated for 1 h at 30ºC with a 1:250 dilution of fluorescein isothiocyanate (FITC)-labeled avidin (Sigma Chemical). These cells were examined in an EPICS ELITE flow cytometer (Coulter Electronics, Hialeah, FL, USA) equipped with a 15-mW argon laser emitting at 488 nm. Avidin-untreated parasites were used as controls to determine autofluorescence. Each experimental population was then mapped by using a two-parameter histogram of forward-angle light scatter versus side scatter. The mapped population (n=10,000) was then analyzed for log green fluorescence by using a single-parameter histogram (Gomes et al. 2006).

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Results and discussion T. cruzi is highly heterogeneous in terms of genetics and biological properties. To explore the diversity of T. cruzi strains belonging to the Z3 phylogenetic group, we focused our study in two biological markers: (1) protein expression and (2) cruzipain production. These biological and/or biochemical characteristics are involved in key events of the parasite life cycle. The separation of soluble whole proteins of the Z3 strains revealed complex and distinct polypeptide profiles, containing approximately 20–30 bands ranging from 25 to >200 kDa (Fig. 2). There were generally minor qualitative differences in the whole protein profile among the strains, such as the 80-kDa polypeptide that was expressed only in SMM88 and Dm28c; the 55 kDa polypeptide that was detected in both SMM10 and SMM88, but not in the other strains; and a 50-kDa component observed exclusively in SMM53 and SMM98 strains. However, polypeptides presenting similar molecular masses were detected in all the Z3 strains studied, including the 45 and >200 kDa components (Fig. 2). The surface polypeptides play a wide variety of physiological roles, including transmembrane receptors involved with signal transduction, cell surface protection, as well as adhesive molecules implicated in processes such as cell–cell and cell–substrate interactions. Surface proteins are also important antigens that elicit humoral and cell-mediated immune responses (De Souza 1995). In order to analyze the relative amount of surface proteins expressed in the four Z3

100 80 55 50 45

35 28

M M

10 88 53 98 28c M M M M M M Dm S SM SM S

Fig. 2 Polypeptide profiles observed in the Z3 sylvatic isolates of T. cruzi. Protein analysis was carried out on 12% SDS-PAGE, and the gel was silver-stained. Numbers on the left indicate the relative molecular mass markers (MM), expressed in kilodaltons. The double arrows indicate polypeptides with similar molecular mass that are observed in all strains analyzed (SMM10, SMM53, SMM88, SMM98, and Dm28c), while arrowheads show polypeptides just detected in some of these strains. The numbers on the right indicate the molecular masses of these highlighted polypeptides

sylvatic isolates of T. cruzi, we used a membraneimpermeable derivate of biotin, Sulfo-NHS-LC-biotin, which has provided a convenient alternative to radioiodination for the labeling of eukaryotic cell surface polypeptides (Hurley et al. 1985), including trypanosomatids (Santos et al. 2001; Gomes et al. 2006; Matteoli et al. 2009). By phase contrast microscopy, parasites remained intact and motile after the biotin labeling (data not shown). As demonstrated in Fig. 3, the SMM10 strain showed a relatively lower biotin binding in relation to SMM53, SMM88, and SMM98 strains. These latter three SMM strains presented similar fluorescence intensity. As it is well-known, cruzipain is a key molecule that participates in several steps of T. cruzi life cycle, including development and virulence (Cazzulo et al. 2001). Our results demonstrated a major protease of apparent molecular mass of 45 kDa in all the SMM strains (Fig. 4), which was completely inhibited by E-64, a powerful cysteine protease inhibitor (data not shown). In addition, the strain SMM10 produced an additional proteolytic activity of 55 kDa (Fig. 4) that was blocked by 1,10-phenanthroline, a metalloprotease inhibitor (data not shown). Similarly, Dm28c yielded an extra metalloprotease of 65 kDa (Fig. 4). In order to confirm that the ubiquitous cysteine protease detected in these Z3 sylvatic strains presents similarities with the major cysteine protease (cruzipain) produced by T. cruzi, a Western blotting assay was performed using an

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747

116

Cell Count

66

45

35

Fluorescence Intensity Fig. 3 Analysis of the surface polypeptide profiles of T. cruzi sylvatic isolates. Parasites (SMM10, SMM53, SMM88, SMM98, and Dm28c) grown for 7 days were harvested, fixed in paraformaldehyde, and sequentially incubated in absence (control; autofluorescence) or in the presence of Sulfo-NHS-LC-biotin and then with avidin-FITC. For simplicity, only the autofluorescence of SMM10 is shown, since the other strains are similar (data not shown)

anti-cruzipain antibody. In this set of experiment, a strong reactive band with apparent molecular mass between 40 and 50 kDa was observed in Dm28c as well as in all the sylvatic isolates analyzed (Fig. 5). Interestingly, an additional 80 kDa polypeptide was also evidenced by the anticruzipain antibody in both Dm28c and SMM10, probably a cruzipain isoform (Fig. 5). Cruzipain is a high mannosetype glycoprotein containing about 10% carbohydrate (Cazzulo et al. 1990b). The molecular weight of cruzipain can be estimated from its sequence and considering two

116

metalloprotease

66

metalloprotease 45

cysteine protease

35

M SM

10

M SM

53

M SM

88

M SM

98

c 28 Dm

Fig. 4 Gelatin-SDS-PAGE showing the proteolytic activity profiles of T. cruzi sylvatic isolates. Parasites (SMM10, SMM53, SMM88, SMM98, and Dm28c) grown for 7 days were harvested and lysed by SDS. The gel was incubated in 50 mM sodium phosphate buffer, pH5.5, supplemented with 2 mM DTT for 40 h at 37°C. Numbers on the left indicate the relative molecular mass markers, expressed in kilodaltons

M SM

10

M SM

53

M SM

88

M SM

98

c 28 Dm

Fig. 5 Western blotting showing the reactivity of cellular polypeptides of T. cruzi sylvatic isolates with the anti-cruzipain polyclonal antibody. Numbers on the left indicate the relative molecular mass markers, expressed in kilodaltons

high-mannose oligosaccharide chains as about 40 kDa; however, this enzyme presents an anomalous behavior in SDS-PAGE, yielding apparent molecular mass values ranging from 35 to 80 kDa, depending on the experimental conditions (Martínez and Cazzulo, 1992). Fampa et al. (2008) performed a comparative study using TCI and TCII strains isolated from different sylvatic mammalian hosts. In that study, TCI isolates presented a homogeneous protease profile consisting of two proteases of 66 and 45 kDa, while TCII isolates exhibited heterogeneous patterns with two to four protease activities, suggesting a more diversity of protease expression in TCII than TCI strains. Furthermore, single or double polypeptides around 50-kDa were detected, irrespective of the phylogenetic TCI or TCII background, when Western blotting was employed using the anti-cruzipain antibody (Fampa et al. 2008). Interestingly, dendrogram analyses based on presence/absence matrices of proteases and cruzipain evidenced a TCI separation from the TCII group with 50–60% similarity, suggesting that the cysteine protease diversification contributes to differential host infection between TCI and TCII genotypes and are good markers for separating parasites belonging to the two phylogenetic groups (Fampa et al. 2008). On the other hand, Z3 sylvatic isolates presented only a major cysteine protease, presenting biochemical and immunological similarities with cruzipain. Cysteine proteases were already detected in many species belonging to the Trypanosomatidae family (Mottram et al. 1998; Santos et al. 2005; Vermelho et al. 2007) and are regarded as essential for survival of several parasitic protozoa (Sajid and McKerrow 2002). The cysteine proteases from parasites, among them T. cruzi, have shown to be

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valuable targets for chemotherapy. Due to the biologic relevance of cruzipain in the life cycle of T. cruzi, many studies aim to develop specific inhibitors against the active core of this multifunctional enzyme in order to obtain new drugs capable of protecting human against T. cruzi infection (Cazzulo et al. 1990a, b). For instance, Cazzulo et al. (2001) showed effective rescue from lethal mice infection using cysteine protease inhibitors, such as N-piperazine-FhF-vinyl sulfone phenyl. Previous data from our laboratory pointed out to different results when a murine model infected with the T. cruzi Z3 sylvatic isolates SMM53 and SMM98 was used. Parasitemia of SMM53-infected mice became negative by day8 postinfection and remained until 10 days after the beginning of the experiment, with high level of mortality, being the index of survival of the treated group by approximately 20%. However, mice infected with SMM98 did not succumb until 60 days, showing low levels of mortality. These data can be related with different Chagas’ disease manifestations, since that differences in histological studies have revealed an increase of inflammatory infiltrates with high numbers of mononuclear cells in animals inoculated with SMM98 (Silva et al., personal communication). As cruzipain is associated with parasite virulence (Alexander et al. 1998), the presence of this protease in these sylvatic isolates might contribute for mice mortality. In this work, we confirm previous data (Martínez et al. 1991; Lima et al. 2001) that showed that cruzipain is the principal cysteine protease produced by T. cruzi. The results presented herein showed the occurrence of this major cysteine protease also in T. cruzi sylvatic isolates belonging to the Z3 genotype. Based on this data, complementary studies about cruzipain isoforms will be done in order to know the specific cruzipain expressed by sylvatic strains of T. cruzi. Acknowledgements We thank Luciana Reboredo de Oliveira and Simone Caldas Teves from FIOCRUZ for their excellent technical assistance. The authors wish to thank Dr. Marta Helena Branquinha for the useful critical English reviewing as well as for valuable suggestions on the manuscript. This study was supported by grants from Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Fundação Universitária José Bonifácio (FUJB), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação Oswaldo Cruz (FIOCRUZ), Rio de Janeiro.

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