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Feb 14, 2006 - wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process ...
Parasitol Res (2006) 100:59–68 DOI 10.1007/s00436-006-0236-6

ORIGINAL PAPER

Cell invasion by Trypanosoma cruzi amastigotes of distinct infectivities: studies on signaling pathways Adriana B. Fernandes & Ivan Neira & Alice T. Ferreira & Renato A. Mortara

Received: 14 February 2006 / Accepted: 9 May 2006 / Published online: 22 June 2006 # Springer-Verlag 2006

Abstract Trypanosoma cruzi metacyclic trypomastigotes of the major phylogenetic lineages use specific signaling pathways to invade host cells. Using a panel of drugs, we studied if the differences in the ability of extracellular amastigotes (EA) from G (T. cruzi I) and CL (T. cruzi II) strains to invade host cells could be associated to activation of specific signaling routes. Sonicated extracts from G or CL strain EA induced transient raises in HeLa cell intracellular Ca2+ levels in a dose-dependent manner. Treatment of EA with drugs that affect Ca2+ release from inositol-1,4,5-triphosphate-sensitive stores did not significantly affect the infectivity of either strain, whereas EA of both strains treated with ionomycin plus NH4Cl or nigericin that release Ca2+ from acidocalcisomes had their infectivity reduced. Treatment of parasites with adenylate cyclase activator forskolin increased the infectivity of both strains towards HeLa cells. These data, taken together, suggest that, for host cell invasion, G and CL strain EA engage

A. B. Fernandes : R. A. Mortara (*) Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de São Paulo-UNIFESP, Escola Paulista de Medicina, Rua Botucatu, 862 6th Floor, 04023-062 São Paulo, Brazil e-mail: [email protected] A. T. Ferreira Departamento de Biofísica, Universidade Federal de São Paulo-UNIFESP, Escola Paulista de Medicina, Rua Botucatu, 862 6th Floor, 04023-062 São Paulo, Brazil I. Neira Unidad de Parasitología, Facultad Ciencias de la Salud, Universidad de Antofagasta, Antofagasta, Chile

signaling pathways that lead to an increase of cyclic adenosine monophosphate and Ca2+ mobilization from acidocalcisomes. Moreover, treatment of EA with genistein reduced by ~45% the invasion of HeLa cells by G but not by CL strain, implicating a protein tyrosine kinase in the process. In line with this, HeLa cell extracts contained a protein tyrosine kinase activity that mediated the phosphorylation of 87- and 175-kDa polypeptides of EA from G but not from CL strain. Regarding the target cell response, the activation of host PI3 kinase appears to be required for invasion by either strain as treatment of HeLa cells with wortmannin reduced EA infectivity. These data overall reinforce the concept that cell invasion by T. cruzi EA markedly differs from the process involving metacyclic trypomastigotes.

Introduction The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas’ disease in humans that represents a significant public health problem in Latin America. During its life cycle that takes place in vertebrate and invertebrate hosts, proliferative forms, respectively known as amastigotes and epimastigotes, transform into the non-proliferative and highly infective trypomastigotes. In posterior portions of the digestive tract of the invertebrate host, epimastigotes transform into metacyclic trypomastigotes that may then be liberated with the insect’s feces and urine. In the vertebrate host, amastigotes multiplying within the host cell transform into trypomastigotes that are released after host cell rupture, reaching the extracellular space and the bloodstream where they may infect other cells (Brener 1973; De Souza 1984, 2002). The premature lysis of infected cells may release amastigotes to the extracellular

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milieu (Behbehani 1973; Nogueira and Cohn 1976; Hudson et al. 1984; Ulisses de Carvalho and De Souza 1986) and extracellular differentiation of trypomastigotes may generate amastigote-like forms (Pan 1978; Andrews et al. 1987). These extracellular amastigote forms are capable of invading both professional and non-professional phagocytes, where they not only survive but can sustain the parasite’s life cycle (Behbehani 1973; Nogueira and Cohn 1976; Pan 1978; Hudson et al. 1984; Ley et al. 1988; Mortara 1991). Procópio et al. (1998) investigated the entry of extracellular amastigotes (EA) into HeLa and Vero cells as compared to the infectivity of metacyclic trypomastigote forms and confirmed that the mechanisms of cell invasion by the two forms is distinct, in line with the previous results of Schenkman et al. (1991) who observed no competition towards cell binding between them. When HeLa and Vero cells were treated with cytochalasin D before parasite invasion, amastigote invasion was inhibited indicating that, unlike what was observed with trypomastigotes, amastigotes had a more passive role in the entry process (Procópio et al. 1998). A number of studies have shown that invasion of mammalian cells by T. cruzi requires the activation of signal transduction pathways that lead to an increase in cytosolic Ca2+ concentration, both in the parasite and the host cell (Moreno et al. 1994; Tardieux et al. 1994; Yakubu et al. 1994; Ruiz et al. 1998). The efficiency in entering non-phagocytic mammalian cells may vary widely between T. cruzi strains and strains belonging to different phylogenetic lineages, and it has been well established that metacyclic trypomastigotes from CL strain (T. cruzi II) are highly invasive whereas parasites from the G strain (belonging to T. cruzi I) are poorly infective (Neira et al. 2002). However, EA of highly infective strains such as Y and CL are poorly infective when compared to type I parasites, particularly from the G strain, showing the opposite behavior of the corresponding metacyclic trypomastigote forms (Mortara et al. 1999; Fernandes and Mortara 2004). For metacyclic trypomastigotes, the developmental forms that initiate infection in mammalian hosts, the variability between these strains has been associated to the differential expression of surface glycoproteins that bind to target cells in a receptormediated manner and exhibit differential Ca2+ signaling activities (Ruiz et al. 1998; Yoshida et al. 2000). By treating parasites with different drugs, Neira et al. (2002) established that the metacyclic trypomastigote forms of G strain (T. cruzi I) engage in different signaling mechanisms to invade HeLa cells when compared to CL strain (T. cruzi II) parasites. They demonstrated that G strain engages adenylate cyclase activation for cellular invasion whereas CL strain depends on tyrosine phosphorylation to accom-

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plish this process, and Ca2+ appears to be mobilized from acidocalcisomes and 1,4,5-inositol-triphosphate-sensitive stocks, respectively. In this study, by using drugs that interfere with specific signaling processes, we investigated whether the differences in the ability of EA of G and CL strains to invade HeLa cells could be associated to the activation of specific signaling routes. Although substantial differences between the two strains were not disclosed, our results suggest, that for host cell invasion, G and CL EA engage signaling pathways that lead to an increase in cyclic adenosine monophosphate (cAMP) and parasite intracellular Ca2+ would be mobilized from acidocalcisomes. A protein tyrosine kinase activity also appears to be associated with G but not with CL strain EA invasion.

Materials and methods Cell lines and cultures Vero cells (African green monkey kidney fibroblasts, obtained from Institute Adolfo Lutz, São Paulo, Brazil) were grown at 37°C in a 5% CO2 humid atmosphere, in RPMI-1640 medium supplemented with 10% fetal bovine serum, streptomycin (100 ìg/ml) and penicillin (100 U/ml). These cells were used to maintain the in vitro T. cruzi cycle. HeLa cells, the human-carcinoma-derived epithelial cells (obtained from American Type Culture Collection), which were grown at 37°C in Dulbeccos’s minimum essential medium supplemented with 10% fetal bovine serum and antibiotics, were used for all cell invasion assays. Parasites In this study, two T. cruzi strains were used: G strain isolated in the Amazon from a marsupial of the Didephidae family (Yoshida 1983) and CL strain isolated in southern Brazil from Triatoma infestans in human dwellings where people were infected with T. cruzi (Brener and Chiari 1963). Extracellular amastigotes were derived from the axenic differentiation of cell-derived trypomastigotes. Vero-cell-derived trypomastigotes were isolated from culture supernatants of infected cells by centrifugation at 2,500×g for 5 min. The pellet was resuspended in liver infusion tryptose (LIT) medium and incubated for 24–48 h at 37°C, when at least 95% pure extracellular amastigotes were obtained (Ley et al. 1988; Mortara 1991). Control experiments, carried out to check for the predicted effect of each drug used, were performed with metacyclic trypomastigotes obtained from stationary-phase hemocultures of infected mice, in LIT medium (Camargo 1964). Grace’s medium was also used to obtain cultures enriched

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in metacyclic trypomastigotes, which were purified by passage through diethylaminoethyl cellulose columns (Yoshida 1983). Cell invasion assays Host cell invasion assays were carried out by seeding parasites onto each well of 24-well plates containing 13-mmdiameter round glass coverslip coated with 1.5×105 HeLa cells, at a parasite-to-cell ratio of 20:1 for G strain and 40:1 for CL strain. After 1 h of incubation, the triplicate coverslips were washed in phosphate-buffered saline (PBS) and stained with Giemsa. Intracellular parasites were scored in a total of 100 cells to calculate the percentage of cell invasion (Fernandes and Mortara 2004). Determination of intracellular Ca2+ fluctuations in HeLa cells after addition of extracellular amastigote extracts To measure the fluctuations in cytosolic free Ca2+ ([Ca2+]i), the following procedure, based on previously described techniques (Dorta et al. 1995), was used. In brief, HeLa cells were washed in buffer A, pH 7.2, containing 116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, and 5.5 mM N-(2-hydroxyethyl)-piperazine-N′-2-ethanesulfonic acid (HEPES). After adjusting the concentration to 108 cells/ ml, the cells were loaded with 5 μM Fura2/AM (Sigma) for 3 h at room temperature and the non-incorporated Fura2 was washed out. Fluorescence was measured in a fluorophotometer SPEX AR-CM system (Spex) with dual wavelength excitation (340 and 380 nm) and emission set at 510 nm. The variations in HeLa cell [Ca2+]i, resulting from the addition of parasite extracts (50 μg protein/100 μl extract) to 2.5 ml of cell suspension, were calculated as previously described (Grynkiewicz et al. 1985). Rmax and Rmin that correspond to the fluorescence ratios at 340 and 380 nm in the presence of saturating Ca2+ after treatment with 50 μM digitonin and in the absence of Ca2+ upon addition of 10mM EGTA, respectively, were determined for each preparation. The use of cellular extracts instead of live parasites was necessary due to the potential hazard of contamination. Treatment of parasites and HeLa cells with different drugs Treatment of parasites or HeLa cells with 250 μM genistein or 10 μM forskolin was performed by incubation of the parasites or HeLa cells at 37°C for 30 min in the presence of the drug, followed by washing in PBS. Other treatments included incubation with 1 μM thapsigargin for 30 min at 37°C, 1 μM U73122 for 4 min at 37°C, 100 μM neomycin for 4 h at 37°C, 10 μM caffeine for 30 min at 37°C, ionomycin (1μM) plus NH4Cl (20mM) or nigericin (1 μM)

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for 10 min at 37°C, 10μM dibutyryl cAMP at room temperature for 15 min, and 10 nM wortmannin for 60 min at 37°C. All drugs were obtained from Sigma and the stock solutions were prepared in dimethylsulphoxide or PBS. To verify that the different treatments were effective, parallel experiments using appropriate G or CL strain metacyclic trypomastigotes as controls (Neira et al. 2002) were carried out in each experiment. Transmission electron microscopy The parasites (107/ml) were washed three times with buffer A (119 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 50 mM HEPES, and 5.5 mM glucose, pH 7.2) and resuspended in the same buffer (1:4). Drops (5–10 μl) of whole parasites, unfixed and unstained, were applied to Formvar/carboncoated copper grids, and the cells were allowed to adhere for 30 min and then carefully blotted dry (Lu et al. 1998). The specimens were observed directly by transmission electron microscopy (TEM) using a JEOL 1200 EXII microscope operated at 80 kV. Preparation of HeLa cell extracts HeLa cells washed in PBS were detached by scraping, suspended in PBS containing protease inhibitors (1 mM phenylmethylsulphonyl fluoride, 1 mM iodoacetamide, 25 μg/ml leupeptin, and 25 μg/ml antipain), and then sonicated on ice in an ultrasonic processor XL (two pulses of 30 s each). Once checked under phase contrast microscopy that the cells were disrupted, the sonicated preparation was centrifuged at 12,000×g for 5 min, and the supernatant was collected and stored at −80°C until it was used. Detection of T. cruzi tyrosine-phosphorylated proteins In standard experiments, 5×107 parasites were incubated for 20 min at 37°C in the absence or presence of HeLa cell extract (equivalent to 160 μg/ml protein) in a total volume of 200 μl. After washing in PBS, the parasites were disrupted at 4°C in a lysis solution containing phosphatase and protease inhibitors (50mM Tris–HCl, pH 7.4; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 1 mM NaVO4; 1 mM NaF; 1mM phenylmethylsulphonyl fluoride; 1 mM iodoacetamide; 25 μg/ml leupeptin and 25 μg/ml antipain). The lysates were dissolved in loading buffer and subjected to electrophoresis in a 10% sodium dodecyl sulfate– polyacrylamide gel, under reducing conditions, and the proteins were transferred to nitrocellulose membrane. After blockage with PBS containing 5% defatted milk, the nitrocellulose membrane was incubated for 1 h at room temperature with anti-phosphotyrosine antibodies (mouse

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monoclonal antibody from Sigma) diluted in PBS–milk. After several washes in PBS containing 0.05% Tween 20, the membrane was incubated with anti mouse IgG conjugated to peroxidase. The final reaction was revealed by chemiluminescence using the ELC Western blotting detection reagent and Hyperfilm-MP from Amersham (Neira et al. 2002). Quantitation of experiments and statistical calculations All experiments were performed in triplicate using three coverslips. On average, 100 cells per coverslip were analyzed. The statistical calculations were done with SigmaStat (version 1.0, Jandel Scientific, USA), using the t test for significance and standard deviations.

Results Increase of HeLa cell intracellular Ca2+ concentration is induced by extracts of G and CL strain EA The experiments of intracellular Ca2+ mobilization were carried out by adding parasite extracts to HeLa cells preloaded with Fura-2. As shown in Fig. 1, significant transient increases in [Ca2+]i were detected after addition of EA extracts from either strain, an effect comparable to that were that of metacyclic trypomastigotes used as positive control (Neira et al. 2002). The relatively small [Ca2+]i transients induced by epimastigote form extracts used as negative controls could possibly be due to either a basal Fig. 1 EA extracts from G and CL strains induce increases in HeLa cell intracellular Ca2+ concentration in a dose-dependent manner. Intracellular calcium transients induced by different T. cruzi infective forms. Sonicated extracts of EA, containing an equivalent of 5×107 parasites/50 μl were added separately to live HeLa cells preloaded with Fura2. Extracts of epimastigotes and metacyclic trypomastigotes (G strain, 5×107 parasites/50 μl) were used as negative and positive controls, respectively. These traces are representative of at least four independent experiments

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contamination of epimastigote cultures with metacyclic trypomastigote forms or a non-specific basal effect. Parasites and HeLa cells are differentially affected by drugs that interfere with intracellular Ca2+ mobilization We tested several drugs that interfere with intracellular Ca2+ mobilization on EA infectivity towards HeLa cells. First, we confirmed that, in spite of using twice as much CL parasites (40:1, parasites to HeLa cells), G strain EA (used at 20:1 ratio) displayed much higher infectivity. Using the protocol for cell infection, we found in control samples (untreated parasites) around 320 G strain EA per 100 cells, compared to around 180 of the CL strain. To determinate the effect of U73122 (Bleasdale et al. 1990) and neomycin (Ma and Michel 1998), phospholipase C inhibitors, the parasites were incubated in the absence or presence of the drugs. As shown in Fig. 2, G and CL strains infectivity was unaltered by pretreatment with either compound. By contrast, the treatment of HeLa cells with U73122 or neomycin inhibited to some extent the penetration of parasites from both strains. To test whether cell invasion involved Ca2+ mobilization from intracellular stores, EA from G or CL strains were treated with thapsigargin or caffeine. Similarly to what was observed with phospholipase C inhibitors, EA infectivity was somewhat reduced only when HeLa cells were pretreated with these drugs (Fig. 2). Positive controls for drug treatment were run in parallel with CL strain metacyclic trypomastigotes, which display marked reduction in their infectivity upon treatment of parasites with these compounds (not shown; Neira et al. 2002).

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whole EA, mounted without any treatment such as fixation or staining, appear as electron-dense granules of different sizes (Fig. 4). The acidocalcisomes were equally abundant in EA from G and CL strains: 23.2±8 (G strain) and 22.5±6 (CL strain) electron-dense granules/parasite (n=23). Adenylyl cyclase activation increases infectivity of EA from G and CL strains

Fig. 2 Inhibition of HeLa cell intracellular Ca2+ mobilization reduced the infectivity of both G and CL strains EA. EA or HeLa cells were pretreated or not with the indicated inhibitors. The results of invasion assays are expressed as percentage of parasites in relation to controls, counted in 100 cells in triplicate coverslips (*p< 0.05). These results represent the means ± standard deviations of three independent experiments

Cyclic AMP, synthesized from ATP by adenylyl cyclase, is a ubiquitous messenger that has been shown to play a role in the control of T. cruzi growth and differentiation (Oliveira et al. 1984; Gonzales-Perdomo et al. 1988). The activation of parasite adenylyl cyclase with forskolin increased HeLa cell invasion by EA from both G and CL strains (Fig. 5). By contrast, the treatment of HeLa cells with this drug did not affect infection by EA of either strain. The involvement of cAMP in EA internalization was further examined in experiments in which the parasites were pre-incubated with dibutyryl cAMP, a phosphodiesterase inhibitor. Pretreatment of parasites with 10 μM dibutyryl cAMP also resulted in an increase (∼35%) of

Drugs that interfere with Ca2+ release from acidocalcisomes inhibit HeLa cell invasion by EA from both T. cruzi strains Acidocalcisomes, the acidic vacuoles containing Ca2+/H+ exchange system rich in Ca2+, have been detected in all developmental stages of T. cruzi (Docampo et al. 1995, 2005). To examine the participation of acidocalcisomes in the Ca2+ response required for host cell invasion by EA, the parasites or HeLa cells were treated with calcium ionophore ionomycin, combined with NH4Cl or nigericin, compounds that increase the pH of acidic compartments and release Ca2+ (Neira et al. 2002). As shown in Fig. 3, the treatment of parasites with these drugs caused a small but significant inhibition of HeLa cell invasion by EA from both G and CL strains but had no direct effect upon the target cells. G strain metacyclic trypomastigotes that mobilize Ca2+ from acidocalcisomes to invade HeLa cells were used in these experiments as positive controls for drug treatments (not shown; Neira et al. 2002). The detection and quantification of acidocalcisomes in the EA from G and CL strains was by transmission electron microscopy. As the drugs that interfere with calcium recruitment from acidocalcisomes inhibit EA invasion, we carried out morphological observations by TEM of EA from G and CL strain to verify possible differences in the relative abundance of these organelles. Acidocalcisomes in

Fig. 3 Release of Ca2+ from parasite acidocalcisomes reduced the infectivity of EA from G and CL strains towards HeLa cells. EA or HeLa cells were pretreated or not with the indicated drugs. Results of invasion assays are expressed as % of intracellular parasites in relation to controls, counted in 100 cells, in triplicate coverslips. *p