Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14

Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14 Vanessa D. Ataydea, Ivan Neiraa, Mauro Corteza, Daniele Ferreiraa, Edna Freymüllerb, Nobuko Yoshidaa,* aDepartamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862-6º̄ andar, São Paulo, SP 04023-062, Brazil bCentro de Microscopia Eletrônica, Escola Paulista de Medicina, Universidade Federal de São Paulo, R. Botucatu, 862-6º̄ andar, São Paulo, SP 04023-062, Brazil Received 12 January 2004; received in revised form 5 March 2004; accepted 5 March 2004 Abstract We investigated the properties of metacyclic trypomastigotes of non-virulent Trypanosoma cruzi clone CL-14, as compared to the parental isolate CL. In contrast to the CL isolate, which produces high parasitemias in mice, metacyclic forms of clone CL-14 failed to produce patent infection. In vitro, the number of clone CL-14 parasites that entered epithelial HeLa cells, after 1 h incubation, was approximately four-fold lower than that of the CL isolate and at 72 h post-infection intracellular replication was not apparent whereas cells infected with the CL isolate contained large number of parasites replicating as amastigotes. CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. Cysteine proteinase cruzipain and trans-sialidase activities were lower in CL isolate than in clone CL-14. The surface profile was similar, except that the expression of gp82, the stage-specific glycoprotein that promotes CL isolate mucosal infection in vivo and host cell invasion in vitro, was greatly reduced on the surface of clone CL-14 metacyclic forms. Genistein, a specific inhibitor of protein tyrosine kinase, which is activated in CL isolate by binding of gp82 to its host cell receptor, did not affect host cell entry of clone CL- 14. In contrast with CL isolate, the infectivity of clone CL-14 was not affected by phospholipase C inhibitor U73122 but was diminished by a combination of ionomycin plus NH4Cl, which releases Ca 2þ from acidic vacuoles. Internalisation of clone CL-14, but not of CL isolate, was significantly increased by treating parasites with neuraminidase, which removes sialic acid from the mucin-like surface molecule gp35/50. Taken together, our data suggest an association between the non-virulence of clone CL-14 metacyclic forms and the reduced expression of gp82, which precludes the activation of signal transduction pathways leading to effective host cell invasion. q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Trypanosoma cruzi; Clone CL-14; Gp82; Metacyclic trypomastigotes; Cell invasion; Signal transduction 1. Introduction The virulence of Trypanosoma cruzi, the protozoan parasite that causes Chagas’ disease, is associated with its ability to invade host cells and to replicate. Invasion of mammalian cells by T. cruzi is a multi-step process that requires the interaction of parasite and target cell molecules and activation of signal transduction pathways in both cells. Metacyclic trypomastigotes, the developmental forms responsible for initiating T. cruzi infection in the mamma- lian host, engage the stage-specific surface glycoprotein gp82 to establish the initial parasite–host cell interaction and promote invasion (Ramirez et al., 1993; Ruiz et al., 1998). Binding of gp82 triggers the signaling cascades in the parasite as well as in the target cell, leading to Ca2þ mobilisation (Ruiz et al., 1998; Yoshida et al., 2000), which is an essential requirement for parasite internalisation (Moreno et al., 1994; Tardieux et al., 1994; Dorta et al., 1995). Experiments in mice have indicated that metacyclic trypomastigotes have the uniquely specialised properties of gastric mucosal invasion upon oral challenge (Hoft, 1996; Hoft et al., 1996), a route to which is attributed the microepidemics responsible for more than half of the acute cases of Chagas’ disease recorded between 1968 and 2000 in Brazilian Amazon (Coura et al., 2002). Recent studies have shown that stage-specific gp82 plays a central role in 0020-7519/$30.00 q 2004 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2004.03.003 International Journal for Parasitology 34 (2004) 851–860 www.parasitology-online.com * Corresponding author. Tel.: þ55-11-5576-4532; fax: þ55-11-5571- 1095. E-mail address: [email protected] (N. Yoshida). http://www.elsevier.com/locate/PARA mucosal invasion of metacyclic forms, leading to systemic infection upon oral inoculation (Neira et al., 2003). T. cruzi isolate CL, which is highly infective in vitro and in vivo (Yoshida, 1983; Ruiz et al., 1998; Neira et al., 2003) has been used in experiments to establish the role of gp82 in host cell invasion (Ruiz et al., 1998; Favoreto et al., 1998; Yoshida et al., 2000). What is intriguing is that CL-14, a clone derived from the CL isolate, is unable to produce a patent infection even when injected into new-born mice (Lima et al., 1990), negative parasitism resulting from extensive histopathological analysis of mouse tissues and organs, at different times after intraperitoneal or intravenous injection of metacyclic forms (Lima et al., 1995). As different T. cruzi isolates may differentially engage their repertoire of surface molecules to interact with host cells, activating distinct signal transduction pathways that deter- mine the efficiency of parasite internalisation (Ruiz et al., 1998; Neira et al., 2002), the deficient infectivity of clone CL-14 could be associated with its surface profile. Investigating the molecular basis of lack of virulence of clone CL-14 may be important to further clarify the complex process of T. cruzi infection. To this end, we analysed in this study the properties of metacyclic forms of clone CL-14, as compared to those of CL isolate, focusing on the profile of surface molecules, gp82 in particular; the activities of cruzipain and trans-sialidase, enzymes that have been implicated in target cell penetration (Meirelles et al., 1992; Schenkman et al., 1991); and the ability to invade epithelial HeLa cells upon treatment of parasites with drugs that interfere with signal transduction and intracellular Ca2þ mobilisation. 2. Materials and methods 2.1. Trypanosoma cruzi, mammalian cells and cell invasion assay The T. cruzi isolate CL (Brener and Chiari, 1963) was maintained cyclically in mice and in axenic culture in liver infusion tryptose (LIT) medium and the clone CL-14 (Chiari, 1981) was grown in LIT medium throughout this study. To obtain cultures enriched in metacyclic trypomas- tigotes, Grace’s medium (Life Technologies) was also used. Metacyclic forms from cultures at the stationary growth phase were purified by passage through DEAE-cellulose column, as described (Teixeira and Yoshida, 1986). HeLa cells, the human carcinoma-derived epithelial cells, were grown at 37 8C in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal calf serum, streptomycin (100 mg/ml) and penicillin (100 U/ml) in a humidified 5% CO2 atmosphere. Host cell invasion assays were carried out as detailed elsewhere (Yoshida et al., 1989), by seeding the parasites onto each well of 24-well plates containing 13-mm diameter round glass coverslips coated with 1.5 £ 105 HeLa cells. After varying periods of time, depending on the experiment, the duplicate coverslips were washed in PBS and stained with Giemsa. The number of intracellular parasites was counted in at least 500 Giemsa stained cells. 2.2. Oral infection of mice with T. cruzi Four-week-old female Balb/c mice were inoculated orally with purified metacyclic trypomastigotes. The parasites were introduced by intrapharyngeal route through a plastic tube adapted to a 1 ml plastic syringe. Starting on day 13 p.i., parasitemia was monitored by examining 5 ml peripheral blood samples under phase contrast microscope, twice a week. 2.3. Flow cytometry Live metacyclic trypomastigotes (4 £ 107) were incu- bated for 1 h on ice, with monoclonal antibodies (MAbs) directed to different T. cruzi surface molecules, or with unrelated MAb 1C3 directed to Leishmania amazonensis gp63 (Barbiéri et al., 1993). After washings in PBS, the parasites were fixed with 2% paraformaldehyde in PBS for 30 min. The fixative was washed out, and the parasites were incubated with fluorescein-labeled goat anti-mouse IgG for 1 h at room temperature. Following two more washes, the number of fluorescent parasites was estimated with a Becton Dickinson FACscan cytometer. Assays with fixed and permeabilised parasites were carried out as follows: fixation with 2% paraformaldehyde, washings in PBS, treatment with 0.1% saponin in PBS at room temperature for 30 min, washings in PBS, incubation with MAb 3F6 for 1 h at room temperature, washes in PBS, and incubation with fluor- escein-conjugated antibody as described above. 2.4. TEM and immunocytochemistry Purified metacyclic trypomastigotes (5 £ 107) were washed in PBS and fixed with a solution containing 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.2. After 1 h at room temperature and washings in cacodylate buffer, the parasites were treated with PBS containing 0.1 M glycine, for 30 min at room temperature, to block free aldehyde groups and then washed in PBS. The parasites were blocked with PBS containing 5% BSA for 1 h at room temperature and incubated with MAb 3F6 or with unrelated antibody, diluted 1:10 in blocking solution, for 2 h at room temperature. Following washings in PBS containing 1% BSA, the parasites were incubated for 2 h with anti-mouse IgG coupled to 10 nm colloidal gold particles diluted 1:40 in blocking solution. After inclusion in 2% agarose and treatment with 1% OsO4 for 1 h, dehydration in ethanol series, the parasites were embedded in Epon 812 (EMS). Thin sections were collected on Formvar/carbon-coated copper grids, stained with 2% uranyl acetate for 8 min and 1% lead citrate for 4 min, V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860852 washed, dried and observed in transmission electron microscope (Jeol 1200 EX II). 2.5. Detection of proteinase and trans-sialidase activities T. cruzi proteinase activity was determined as follows: 3 £ 107 metacyclic trypomastigotes were lysed in 0.4% Triton X-100, solubilised in sample buffer without 2- mercaptoethanol, and then loaded onto 10% SDS-poly- acrylamide gel containing 0.1% copolymerised gelatin as substrate. After electrophoresis, the gels were subjected to two 30 min washes with 2.5% Triton X-100 in 0.1 M acetate buffer, pH 6.0, to remove SDS, and incubated overnight in the same buffer without detergent. The gel was stained with Coomassie blue R250 and destained for visualisation of white bands against a blue background. Trans-sialidase activity was determined as previously described (Schenk- man et al., 1992). Briefly, 108 metacyclic forms were pelleted, detergent lysed and processed for measurement of trans-sialidase activity, by the transfer of sialic acid from sialyllactose to D-glucose-1-[14C]lactose and detection of sialylated products by chromatography in QAE-Sephadex A-25 (Pharmacia). 2.6. Treatment of parasites with neuraminidase and drugs that interfere with cell signaling Metacyclic trypomastigotes were treated at 37 8C for 1 h with 0.2 U/ml of neuraminidase (type III from Vibrio cholerae or type X from Clostridium perfringens, Sigma) in PBS, pH 6.0, containing 1 mM CaCl2. After washings in PBS, the parasites were used for reactivity towards lectin and for cell invasion assays. Treatment of metacyclic forms with different drugs included incubation with 250 mM genistein at 37 8C for 30 min, 1 mM U73122 at 37 8C for 4 min; 1 mM ionomycin plus 20 mM NH4Cl for 10 min at 37 8C. 2.7. Southern blot analysis T. cruzi DNA was digested with different restriction enzymes, separated by electrophoresis on 0.8% agarose gel and blotted onto nylon membranes. Hybridisation with the probe, which consisted of a DNA fragment corresponding to ORF of gp82 gene (whole insert of gp82 cDNA clone) labeled with [32P], and washings were performed as detailed (Araya et al., 1994). 2.8. Pulsed field gel electrophoresis Agarose blocks containing genomic DNA from 108 parasites were prepared, incubated at 50 8C for 16 h in lysis solution containing 10 mM Tris–HCl, pH 8.0, 500 mM EDTA, 1% sarkosyl, 1 mg/ml proteinase K, equilibrated in TE, washed and stored in 0.5 M EDTA at 4 8C. Small portions (equivalent to 107 parasites) were electrophoresed (1.2% agarose gel in 0.5 £ TBE) at 80 V for 132 h in Gene Navigatore System (Pharmacia), from pulse times varying from 90 to 800 s. DNA from Hansenula wingei was used as reference. After transfer to nylon membranes, chromosomal DNA bands were hybridised with the [32P]-labeled insert of gp82 cDNA clone and revealed by exposure to X-ray film (Hyperfilm-MP, Amersham). 2.9. Statistics Student’s t-test was used to determine significance in T. cruzi cell invasion assays, in which the infectivity between CL isolate and clone CL-14 was compared or the effect of treatment of parasites with neuraminidase or drugs that inhibit signal transduction was evaluated. 3. Results 3.1. In vivo and in vitro infection by metacyclic forms of T. cruzi isolate CL and clone CL-14 We compared the infectivity of CL isolate and clone CL-14 by oral administration, a highly efficient route leading to systemic T. cruzi infection (Hoft, 1996; Neira et al., 2003). Balb/c mice were injected orally with 2 £ 106 metacyclic forms of clone CL-14 or 4 £ 105 parasites of CL isolate. Starting on day 13 p.i., blood samples were examined twice a week. Parasitemia was not detectable in mice inoculated with clone CL-14 whereas the animals inoculated with five-fold fewer metacyclic forms of CL isolate developed high para- sitemias (Fig. 1A). The parasites used in these exper- iments were .95% pure typical metacyclic trypomastigotes, with the kinetoplast posterior to the nucleus, as visualised in Giemsa-stained preparations (Fig. 2). We have noted that CL isolate metacyclic forms were long and slender, with the kinetoplast localised closer to the nucleus than to the posterior end, whereas clone CL-14 parasites were shorter, with the kinetoplast very close to the posterior end. To examine whether the low infectivity of clone CL- 14 was associated with the impaired ability to invade host cells, we performed invasion assays by incubating metacyclic forms with HeLa cells for 1 h at 37 8C, at parasite:cell ratio of 10:1. The number of internalised parasites of clone CL-14 was approximately four-fold lower as compared to that of the CL isolate (Fig. 1B), a difference extremely significant ðP , 0:0001Þ: Exper- iments were also performed to determine the ability of clone CL-14 to replicate intracellularly. Parasites were incubated with HeLa cells for 1 h and, after washings in PBS, one set of duplicate coverslips were immediately stained with Giemsa whereas other sets were reconsti- tuted with DMEM containing 2% fetal calf serum V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 853 and maintained for up to 72 h. No apparent intracellular replication of clone CL-14 was observed by 72 h p.i., as opposed to the CL isolate (Fig. 1C) that multiplied actively as amastigotes. 3.2. Differential expression of gp82 on the surface of CL isolate and clone CL-14 In CL isolate, gp82 is the main surface molecule of metacyclic forms associated with the parasite infectivity (Ramirez et al., 1993; Neira et al., 2003). We examined whether clone CL-14 differed from CL isolate as regards the expression of gp82. Analysis of metacyclic forms by flow cytometry, upon reaction of live parasites with MAb 3F6, consistently revealed in CL isolate a major population expressing high gp82 levels and a minor population with lower fluorescence intensity, whereas in clone CL-14 the parasites reacting poorly with MAb 3F6 predominated over those with higher gp82 expression (Fig. 3A). We confirmed the reduced expression of gp82 Fig. 1. Differential infectivity of metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14. (A) The course of infection in mice inoculated with parasites by oral route was monitored by counting the number of parasites in 5 ml blood samples. Each data point corresponds to the mean parasitemia of five animals in each group of mice. Note the high parasitemias produced by CL isolate in contrast to the lack of patent infection by clone CL-14. (B) Host cell invasion was assayed by incubating parasites with HeLa cells at 37 8C for 1 h. After washings in PBS, the number of intracellular parasites was counted in a total of at least 500 Giemsa-stained cells. The difference in infectivity between CL isolate and clone CL-14 was extremely significant ðP , 0:0001Þ by Student’s t-test. (C) Intracellular T. cruzi replication was examined by incubating parasites with HeLa cells for 1 h. After washings in PBS, one set of coverslips were stained with Giemsa whereas another set was maintained for 72 h in Dulbecco’s minimum essential medium contain- ing 2% FCS. Values are the means ^ SD of eight experiments (B) and three experiments (C), performed in duplicate for each experiment. Fig. 2. Metacyclic trypomastigotes of Trypanosoma cruzi CL isolate and clone CL-14. Purified parasites were stained with Giemsa. Note the position of kinetoplast very close to the posterior end in clone CL-14 and more proximal to the nucleus in CL isolate. Fig. 3. Expression of surface glycoproteins gp82 in metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14. (A) Live parasites were reacted with MAb 3F6 directed to gp82, fixed and then processed as described in Section 2 for analysis by flow cytometry. Representative results of at least seven experiments are shown. Note the lower fluorescence of clone CL-14, reflex of reduced gp82 expression. (B) Detergent extracts, equivalent to 3 £ 107 parasites, were electrophoresed in 10% SDS-PAGE gels and analysed by immunoblotting using MAb 3F6. Gp82 bands of comparable intensity were revealed in CL isolate and clone CL-14. (C) Parasites were permeabilised or not by treatment with saponin before reaction with MAb 3F6 and processing for analysis by flow cytometry. Representative results of three experiments are shown. Note the increased gp82 levels in permeabilised clone CL-14 parasites. V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860854 on the surface of clone CL-14 metacyclic forms by immunocytochemical reactions visualised at the electron microscope (Fig. 4). In CL isolate, the presence of gp82 was revealed by MAb 3F6-colloidal gold complex throughout the parasite membrane, including the flagel- lum, whereas in clone CL-14 metacyclic forms the labeling was sparse. However, metacyclic forms of CL isolate and clone CL-14 displayed gp82 bands of comparable intensity when analysed by immunoblotting (Fig. 3B), a method that uses extracts of detergent lysed parasites and therefore can also detect internally localised gp82, not accessible to MAb 3F6 in live CL-14 Fig. 4. Distribution of surface molecule gp82 in metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14, revealed by immunogold. Parasites were incubated sequentially with MAb 3F6 directed to gp82 and anti-mouse IgG coupled to colloidal gold particles and then processed for electron microscopy. Note the labeling of gp82 in CL isolate metacyclic forms (arrows). K, kinetoplast. Bar, 0.5 mm. Insets show the position of kinetoplast relative to the nucleus (N) and to the posterior end of the parasite. Bar, 1 mm. V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 855 metacyclic forms. Accordingly, permeabilisation of CL- 14 parasites with saponin before reaction with MAb 3F6 increased the gp82 levels detectable by flow cytometry, whereas only a minor alteration in the profile of gp82 expression was observed in permeabilised CL isolate metacyclic forms as compared to the controls (Fig. 3C). We also compared the genomic organisation of gp82 gene family in CL isolate and clone CL-14. When hybridised with the insert of gp82 cDNA clone, Southern blots of genomic DNA digested with restriction enzyme Bam HI, Eco RI, Hind III or Hae III, displayed similar profiles in CL isolate and clone CL-14 (Fig. 5A). Chromosomal mapping of gp82 genes, carried out by hybridising the same probe with chromosomal size fragments separated by pulsed field gel electrophoresis, revealed some differences. A weak band of ,3.3 Mb and a ,2.1 Mb fragment of high intensity were detected only in CL isolate, whereas clone CL-14 displayed an exclusive fragment of ,2.9 Mb and a ,1.7 Mb band of much higher intensity than that of CL isolate (Fig. 5B). 3.3. Expression of mucin-like surface molecule gp35/50 and of gp90 in CL isolate and clone CL-14 We also examined the profile of other metacyclic trypomastigote surface molecules that are known to interact with host cells. By flow cytometry, CL isolate and clone CL- 14 parasites were found to express on the surface comparable levels of the mucin-like glycoprotein gp35/50 identified by MAb 2B10 (Fig. 6A) and immunoblots revealed gp35/50 bands of similar intensity but displaying some difference in size (Fig. 6B). The variant form of gp35/ 50 as well as gp90, the stage-specific glycoprotein that negatively modulates cell invasion (Málaga and Yoshida, 2001), recognised, respectively, by MAb 10D8 and MAb 1G7 in a number of poorly infective T. cruzi isolates (Ruiz et al., 1998), were not detected in CL isolate or clone CL-14, but both expressed a variant gp90 detectable by MAb 5E7 Fig. 5. Genomic organisation of gp82 genes in Trypanosoma cruzi CL isolate and clone CL-14. (A) Southern blot of genomic DNA digested with the indicated restriction enzyme was hybridised with the whole insert of gp82 cDNA clone. Samples 1, 2, 3 and 4 correspond to CL isolate and l0, 20, 30 and 40 to clone CL-14. (B) Chromosomal bands of parasites were separated by pulsed field gel electrophoresis, transferred to nylon membrane and hybridised with the labeled insert of gp82 cDNA clone. Numbers correspond to molecular sizes. Arrows point to fragments detectable only in CL isolate and arrowheads to bands exclusive for or that appear in higher intensity in clone CL-14. Fig. 6. Molecular profile of metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14. (A) Live parasites were reacted with MAb 2B10 directed to gp35/50, fixed and processed for analysis by flow cytometry. Representative results of seven experiments are shown. The comparable fluorescence intensity indicates that there is no difference in gp35/50 expression. (B) Detergent extracts, equivalent to 3 £ 107 parasites, were electrophoresed in 10% SDS-PAGE gels and analysed by immunoblotting with the indicated monoclonal antibodies. (C) For detection of proteases, Triton X-100 extracts, equivalent to 3 £ 107 parasites, pre-incubated or not with 100 mM E-64, were electrophoresed in 10% SDS-PAGE gel containing gelatin. Afterwards, the gel was washed, incubated overnight and stained with Coomassie blue. Note the high expression of cysteine protease activity in clone CL-14. V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860856 (Fig. 6B) directed to an epitope that is cryptic in live parasites (Teixeira and Yoshida, 1986). 3.4. Cruzipain and trans-sialidase activities in CL isolate and clone CL-14 Cruzipain, the major T. cruzi cysteine proteinase (gp57/51) has been implicated in host cell invasion of tissue culture trypomastigotes as well as in the replication of amastigotes (Meirelles et al., 1992). We determined the cruzipain activity of CL isolate and clone CL-14 metacyclic forms in gelatin gels, by loading parasite preparations in absence or in the presence of 20 mM E-64, an inhibitor of cysteine proteinase. Expression of functional cruzipain, inhibitable by E-64, was higher in clone CL-14 (Fig. 6C). In addition to the gp57/51 bands, a minor band migrating slower than BSA and resistant to E-64 was detected in clone CL-14. As regards trans-sialidase, also implicated in cell invasion of tissue culture trypomastigotes (Schenkman et al., 1991), the activity in clone CL-14 was ,1.6-fold higher than in CL isolate. 3.5. Differential effect of neuraminidase treatment of CL isolate and clone CL-14 metacyclic forms on host cell invasion A previous study had shown that, depending on the T. cruzi isolate, treatment of metacyclic trypomastigotes with neuraminidase, which removes sialic acid from mucin-like molecule gp35/50, increases the ability to invade target cells (Yoshida et al., 1997). To determine whether removal of sialic acid affected the infectivity of clone CL-14 metacyclic forms, the parasites were treated with neuraminidase as described in Section 2. Enzyme-treated parasites had augmented their reactivity with Bauhinia purpurea lectin, which has affinity for D-galactose, an indication that sialic acid was removed, exposing galactosyl residues. As shown in Fig. 7A, the infectivity of clone CL-14 metacyclic forms Fig. 7. Differential effect of treatment of Trypanosoma cruzi metacyclic forms with different drugs on target cell invasion of CL isolate and clone CL-14. (A) Parasites were either untreated or treated with neuraminidase (0.2 U/ml) for 1 h, washed and then seeded onto HeLa cells. After 1 h incubation at 37 8C, followed by washings in PBS, the number of intracellular parasites was counted in a total of at least 500 Giemsa-stained cells. Values are the means ^ SD of four experiments performed in duplicate. The difference between untreated controls and neuraminidase-treated CL-14 parasites was significant, whether the neuraminidase used was from Vibrio cholerae ðP , 0:05Þ or from Clostridium perfringens ðP , 0:005Þ; by Student’s t-test. (B) Parasites, pre-treated or not with the indicated drug, were incubated with HeLa cells, at parasite:cell ratio of 50:1 for clone CL-14 and 10:1 for CL isolate. After 30 min at 37 8C, the number of intracellular parasites was counted in at least 500 Giemsa-stained cells. Values are the means ^ SD of three experiments performed in duplicated. For CL isolate, a significant inhibition of cell invasion was observed upon treatment of parasites with genistein ðP , 0:0001Þ or U73122 ðP , 0:0001Þ; whereas for clone CL-14 the difference between untreated controls and parasites treated with the combination ionomycin þ NH4Cl was significant ðP , 0:05Þ; by Student’s t-test. This suggests phospholipase C-mediated Ca2þ release in CL isolate and phospholipase C-independent Ca2þ mobilisation from acidocalcisomes in clone CL-14. V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 857 towards HeLa cells increased significantly upon treatment with neuraminidase from Vibrio cholerae ðP , 0:05Þ or Clostridium perfringens ðP , 0:005Þ; whereas that of CL isolate remained unaltered. Neuraminidase-treated clone CL-14 parasites failed to replicate intracellularly. 3.6. Activation of distinct signaling pathways in metacyclic forms of CL isolate and clone CL-14 during host cell invasion In this set of short time (30 min) cell invasion assays, in which parasites were pre-treated with drugs that act on signal transduction pathways leading to Ca2þ mobilisation, the parasite:cell ratio used for clone CL-14 was 50:1. Genistein, a specific inhibitor of protein tyrosine kinase (Akiyama et al., 1987), that inhibits Ca2þ response and the infectivity of CL isolate metacyclic forms (Yoshida et al., 2000), did not affect HeLa cell entry of clone CL-14 parasites (Fig. 6B). Treatment with U73122, a specific inhibitor of phospholipase C (Bleasdale et al., 1990), which mediates inositol 1,4,5-triphosphate production (Berridge, 1993), inhibited HeLa cell invasion of CL isolate but not of clone CL-14 metacyclic forms, whereas Ca2þ ionophore ionomycin plus NH4Cl, a combination that potentiates Ca 2þ release from acidocalcisomes (Docampo et al., 1995), had an opposite effect, by affecting only clone CL-14 internal- isation (Fig. 7B). 4. Discussion Our study supports the hypothesis that the metacyclic stage-specific surface glycoprotein gp82 is a key molecule in promoting efficient T. cruzi infection. Of the various parasite molecules implicated in host cell invasion analysed in this study, gp82 was found to be expressed at much lower levels on thesurfaceofcloneCL-14,ascompared to thehighly infective parentalCLisolate, butotherwise the molecularprofiles of the isolate and the clone were similar. In CL isolate, gp82 plays a central role in target cell penetration in vitro (Ramirez et al., 1993; Ruiz et al., 1998) as well as in promoting mucosal infection, leading to high parasitemias, upon oral challenge (Neira et al., 2003). The expression of reduced levels of surface gp82 in metacyclic froms of clone CL-14 could explain the poor cell invasive capacity of these parasites in vitro and why they do not produce patent infection in mice whenadministeredorally.Gp82,which binds togastricmucin in a dose-dependent manner (Neira et al., 2003), may first adhere to mucin that lines the mucosal surfaces, in order to initiate mucosal infection. If engagement of gp82 is required to adhere to and traverse the mucin coat, before parasite penetration into underlying epithelial cells, the reduced expression of gp82 in CL-14 metacyclic forms would be an impediment for this initial interaction with the host. Based on several pieces of evidence, we had proposed that the most efficient target cell penetration of metacyclic trypomastigotes is mediated by gp82, which triggers in both cells the activation of signaling cascades leading to Ca2þ mobilisation from intracellular reservoirs (Ruiz et al., 1998; Neira et al., 2003). Binding of gp82 to its receptor on the host cell induces in CL isolate metacyclic forms the activation of protein tyrosine kinase and Ca2þ release, probably from endoplasmic reticulum, in a manner dependent on inositol 1,4,5-triphosphate (Yoshida et al., 2000). In accord with this, cell invasion of clone CL-14 that is deficient in the expression of surface gp82 is not dependent on protein tyrosine kinase. An alternative signal transduction pathway must be induced in metacyclic forms of clone CL-14 during target cell entry in vitro. One possibility is that the mucin-like surface glycoprotein gp35/ 50 is involved. Gp35/50, which also induce target cell Ca2þ response, although to a lesser degree than gp82 (Dorta et al., 1995), has been implicated in host cell invasion of metacyclic forms of poorly infective T. cruzi isolates (Ruiz et al., 1998). This molecule is the main acceptor of sialic acid in a reaction mediated by trans-sialidase (Schenkman et al., 1993), but sialic acid appears to impair, rather than promote, the interaction of gp35/50 with target cells. Treatment of metacyclic forms with neuraminidase removes sialic acid from gp35/50, increases the Ca2þ signal-inducing activity and, depending on the T. cruzi isolate, augments the parasite infectivity (Yoshida et al., 1997). Neuraminidase treatment of CL-14 metacyclic forms increased the reactivity with lectin recognising D-galactose as well as the invasive capacity of clone CL-14 metacyclic forms towards HeLa cells, suggesting that the removal of sialic acid from gp35/ 50 facilitates parasite–target cell interaction by exposing D- galactose residues, which putatively are required for recognition of target cell receptors. Neuraminidase treat- ment did not affect CL isolate infectivity, which is not dependent on gp35/50. Our results indicate that distinct signaling cascades are activated in CL isolate and clone CL-14 parasites during host cell invasion, leading to Ca2þ mobilisation from different intracellular reservoirs. Ca2þ release in CL isolate, possibly from endoplasmic reticulum (Neira et al., 2002) is mediated by inositol 1,4,5-triphosphate gener- ated by phospholipase C, an enzyme whose inhibition results in decreased parasite infectivity. In clone CL-14, the Ca2þ necessary for parasite internalisation appears, at least in part, to be mobilised from acidocalcisomes in a manner independent of inositol 1,4,5-triphosphate (Table 1). In addition to a reduced ability to enter host cells, clone CL-14 also appears to have impaired capacity for intra- cellular development (Fig. 1C). As T. cruzi cysteine proteinase cruzipain plays a role in intracellular parasite replication (Meirelles et al., 1992), one possibility could be a deficient replication due to the reduced cruzipain activity. However, this is apparently not the case. Expression of functional cruzipain was higher in clone CL-14 as compared V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860858 to CL isolate. In addition, Paiva et al. (1998) have found similar expression of cruzipain on flagellar and cellular membranes of trypomastigotes of CL isolate and clone CL- 14. Another possibility could be an impaired capacity to escape from the parasitophorous vacuole to the cytoplasm, due to a deficient expression of trans-sialidase, an enzyme that also displays neuraminidase activity (Schenkman et al., 1992). According to Hall et al. (1992), T. cruzi neurami- nidase facilitates the parasite’s escape into the cytoplasm by desialylating the vacuole membrane and rendering it more susceptible to lysis by a parasite hemolysin. That possibility is not compatible with trans-sialidase activity of clone CL- 14, which was ,1.6-fold higher than that of the CL isolate. An intriguing question is whether the deficient expression in clone CL-14 of gp82, which is a member of T. cruzi gp85/ sialidase family (Araya et al., 1994) but is apparently devoid of enzymatic activity, impairs functions essential for parasite development other than the ability to bind to gastric mucin and to enter epithelial cells. Taken together, our data suggest that the non-virulence of clone CL-14 metacyclic forms is associated with the reduced expression on the surface of gp82, the molecule involved in mucosal infection in vivo and activation of parasite signaling pathway leading to efficient host cell invasion. Acknowledgements This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). We thank Evania B. Azevedo for help in the detection of parasite trans-sialidase activity, and Dr Tania A.T. Gomes for reading the manuscript. References Akiyama, T., Ishida, J., Nakagawa, S., Ogawara, H., Watanabe, S., Itoh, N., Shibuya, M., Fukami, Y., 1987. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J. Biol. Chem. 262, 5592–5595. Araya, J.E., Cano, M.I., Yoshida, N., Franco da Silveira, J., 1994. Cloning and characterization of a gene for the stage-specific 82-kDa surface antigen of metacyclic trypomastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 65, 161–169. Barbiéri, C.L., Giorgio, S., Merjan, A.J., Figueiredo, E.N., 1993. Glyco- sphingolipid antigens of Leishmania (Leishmania) amazonensis amastigotes identified by use of a monoclonal antibody. Infect. Immun. 61, 2131–2137. Berridge, M.J., 1993. Inositol triphosphate and calcium signalling. Nature 361, 315–325. Bleasdale, J.E., Thakur, R., Gremban, R.S., Bundy, G.L., Fitzpatrick, F.A., Smith, R.J., Bunting, S., 1990. Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and poly- morphonuclear neutrophils. Exp. Ther. 255, 756–768. Brener, Z., Chiari, E., 1963. Variações morfológicas observadas em diferentes amostras de Trypanosoma cruzi. Rev. Inst. Med. Trop. São Paulo 5, 220–224. Chiari, E., 1981. Diferenciação do Trypanosoma cruzi em cultura. PhD Thesis, Universidade Federal de Minas Gerais, M.G., Brasil. Coura, J.R., Junqueira, A.C.V., Fernandes, O., Valente, S.A.S., Miles, M.A., 2002. Emerging Chagas disease in Amazonian Brazil. Trends Parasitol. 18, 171–176. Docampo, R., Scott, D.A., Vercesi, A.E., Moreno, S.N.J., 1995. Intracellular Ca2þ storage in acidocalcisomes of Trypanosoma cruzi. Biochem. J. 310, 1005–1012. Dorta, M.L., Ferreira, A.T., Oshiro, M.E.M., Yoshida, N., 1995. Ca2þ signal induced by Trypanosoma cruzi metacyclic trypomastigote surface molecules implicated in mammalian cell invasion. Mol. Biochem. Parasitol. 73, 285–289. Favoreto, S. Jr., Dorta, M.L., Yoshida, N., 1998. Trypanosoma cruzi 175- kDa protein tyrosine phosphorylation is associated with host cell invasion. Exp. Parasitol. 89, 188–194. Hall, B.F., Webster, P., Ma, A.K., Joiner, K.A., Andrews, N.W., 1992. Desialylation of lysosomal glycoprotein by Trypanosoma cruzi: a role for the surface neuraminidase in facilitating parasite entry into the host cell cytoplasm. J. Exp. Med. 176, 313–325. Hoft, D.F., 1996. Differential mucosal infectivity of different life stages of Trypanosoma cruzi. Am. J. Trop. Med. Hyg. 55, 360–364. Hoft, D.F., Farrar, P.L., Kratz-Owens, K., Shaffer, D., 1996. Gastric invasion by Trypanosoma cruzi and induction of protective mucosal immune responses. Infect. Immun. 64, 3800–3810. Lima, M.T., Jansen, A.M., Rondinelli, E., Gattass, C.R., 1990. Trypano- soma cruzi: properties of a clone isolated from CL strain. Parasitol. Res. 77, 77–81. Lima, M.T., Lenzi, H.L., Gattass, C.R., 1995. Negative tissue parasitism with a noninfective clone of Trypanosoma cruzi. Parasitol. Res. 81, 6–12. Málaga, S., Yoshida, N., 2001. Targeted reduction in expression of Trypanosoma cruzi surface glycoprotein gp90 increases parasite infectivity. Infect. Immun. 69, 353–359. Meirelles, M.N., Juliano, L., Carmona, E., Silva, S.G., Costa, E.M., Murta, A.C., Scharfstein, J., 1992. Inhibitors of the major cysteinyl proteinase (gp57/51) impair host cell invasion and arrest the intracellular development of Trypanosoma cruzi in vitro. Mol. Biochem. Parasitol. 52, 175–184. Moreno, S.N.J., Silva, J., Vercesi, A.E., Docampo, R., 1994. Cytosolic-free calcium elevation in Trypanosoma cruzi is required for cell invasion. J. Exp. Med. 180, 1535–1540. Neira, I., Ferreira, A.T., Yoshida, N., 2002. Activation of distinct signal transduction pathways in Trypanosoma cruzi isolates with differential capacity to invade host cells. Int. J. Parasitol. 32, 405–414. Neira, I., Silva, F.A., Cortez, M., Yoshida, N., 2003. Involvement of Trypanosoma cruzi metacyclic trypomastigote surface molecule gp82 in adhesion to gastric mucin and invasion of epithelial cells. Infect. Immun. 71, 557–561. Paiva, C.N., Souto-Padron, T., Costa, D.A., Gattass, C.R., 1998. High expression of a functional cruzipain by a non-infective and non- pathogenic Trypanosoma cruzi clone. Parasitology 117, 483–490. Ramirez, M.I., Ruiz, R.C., Araya, J.E., Franco da Silveira, J., Yoshida, N., 1993. Involvement of the stage-specific 82-kilodalton adhesion Table 1 Activation of distinct signal transduction pathways in metacyclic forms of Trypanosoma cruzi CL isolate and clone CL-14 during host cell invasion Parasite component involved Isolate CL Clone CL-14 Surface glycoprotein gp82 gp35/50 Protein tyrosine kinase Yes No Phospholipase C Yes No Intracellular Ca2þ store Inositol 1,4,5-triphosphate- sensitive compartment Acidocalcisome V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860 859 molecule of Trypanosoma cruzi metacyclic trypomastigotes in host cell invasion. Infect. Immun. 61, 3636–3641. Ruiz, R.C., Favoreto, S. Jr., Dorta, M.L., Oshiro, M.E.M., Ferreira, A.T., Manque, P.M., Yoshida, N., 1998. Infectivity of Trypanosoma cruzi isolates is associated with differential expression of surface glycoproteins with differential Ca2þ signalling activity. Biochem. J. 330, 505–511. Schenkman, S., Jiang, M., Hart, G.W., Nussenzweig, V., 1991. A novel cell surface trans-sialidase of Trypanosoma cruzi generates a stage- specific epitope required for invasion of mammalian cells. Cell 65, 1117–1125. Schenkman, S., Pontes de Carvalho, L., Nussenzweig, V., 1992. Trypanosoma cruzi trans-sialidase and neuraminidase activities can be mediated by the same enzymes. J. Exp. Med. 175, 567–575. Schenkman, S., Ferguson, M.A.J., Heise, N., Cardoso de Almeida, M.L., Mortara, R.A., Yoshida, N., 1993. Mucin-like glycoproteins linked to the membrane by glycosylphosphatidylinositol anchor are the major acceptors of sialic acid in a reaction catalysed by trans-sialidase in metacyclic forms of Trypanosoma cruzi. Mol. Biochem. Parasitol. 59, 293–304. Tardieux, I., Nathanson, M.H., Andrews, N.W., 1994. Role in host cell invasion of Trypanosoma cruzi-induced cytosolic free Ca2þ transients. J. Exp. Med. 179, 1017–1022. Teixeira, M.M.G., Yoshida, N., 1986. Stage-specific surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi identified by monoclonal antibodies. Mol. Biochem. Parasitol. 18, 271–282. Yoshida, N., 1983. Surface antigens of metacyclic trypomastigotes of Trypanosoma cruzi. Infect. Immun. 40, 836–839. Yoshida, N., Mortara, R.A., Araguth, M.F., Gonzalez, J., Russo, M., 1989. Metacyclic neutralizing effect of monoclonal antibody 10D8 directed to the 35- and 50-kilodalton surface glycoconjugates of Trypanosoma cruzi. Infect. Immun. 57, 1663–1667. Yoshida, N., Dorta, M.L., Ferreira, A.T., Oshiro, M.E.M., Mortara, R.A., Acosta-Serrano, A., Favoreto, S. Jr., 1997. Removal of sialic acid from mucin-like surface molecules of Trypanosoma cruzi metacyclic trypomastigotes enhances parasite–host cell interaction. Mol. Biochem. Parasitol. 84, 57–67. Yoshida, N., Favoreto, S. Jr., Ferreira, A.T., Manque, P.M., 2000. Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells. Braz. J. Med. Biol. Res. 33, 269–278. V.D. Atayde et al. / International Journal for Parasitology 34 (2004) 851–860860 Molecular basis of non-virulence of Trypanosoma cruzi clone CL-14 Introduction Materials and methods Trypanosoma cruzi, mammalian cells and cell invasion assay Oral infection of mice with T. cruzi Flow cytometry TEM and immunocytochemistry Detection of proteinase and trans-sialidase activities Treatment of parasites with neuraminidase and drugs that interfere with cell signaling Southern blot analysis Pulsed field gel electrophoresis Statistics Results In vivo and in vitro infection by metacyclic forms of T. cruzi isolate CL and clone CL-14 Differential expression of gp82 on the surface of CL isolate and clone CL-14 Expression of mucin-like surface molecule gp35/50 and of gp90 in CL isolate and clone CL-14 Cruzipain and trans-sialidase activities in CL isolate and clone CL-14 Differential effect of neuraminidase treatment of CL isolate and clone CL-14 metacyclic forms on host cell invasion Activation of distinct signaling pathways in metacyclic forms of CL isolate and clone CL-14 during host cell invasion Discussion Acknowledgements References