Cells and parasites - Vero and HeLa cells obtained from Instituto Adolpho Lutz (São Paulo, SP, Brazil) were cultivated in RPMI 1640 medium (GIBCO BRL, Grand Island, NY, US) supplemented with 10% fetal calf serum (FCS/ CULTILAB, Campinas, SP, Brazil), penicillin (200 U/ml; Sigma Chemical, St. Louis, MO, US); streptomycin (2.5 µg/ml, Sigma); gentamycin (44 µg/ml; Schering-Plough, São Paulo, SP, Brazil), NaHCO3 (2 g/l), and N-(2-hidroxyetil)-piperazine-N'-(2-ethanosulfonic acid - HEPES (2.38 g/l) in a 5% CO2 humid atmosphere. Wild type CHO and sialic acid deficient Lec-2 cells (Deutscher et al. 1984) kindly provided by Sergio Schenkman from the Cell Biology division of our Department, and originally obtained from ATCC, American Type Culture Collection, were cultivated in a-MEM with ribonucleosides and ribonucleotides (GIBCO) supplemented with 5% FCS in a humid atmosphere with 5% CO2 at 36.5ºC.
Extracellular amastigotes [of the G strain (Yoshida 1983), sylvatic phylogenetic type 1 strain (Souto et al. 1996)] were obtained from the differentiation of cell-derived trypomastigotes isolated from infected Vero cells in LIT medium (Mortara 1991). Briefly cell-culture derived trypomastigotes are harvested from culture medium and resuspended in LIT medium at 37ºC for 48 h (Mortara 1991). Henceforth these forms will be referred as amastigotes. Metacyclic trypomastigotes were obtained from the axenic differentiation of epimastigotes grown in LIT (liver infusion tryptose) medium containing 10% FCS and 0.2% glucose at 28ºC and purified by ionic exchange chromatography (Yoshida 1983).
Cell invasion assays - Semi-confluent cells were infected with metacyclic trypomastigotes or extracellular amastigotes in a 10:1 (parasite:cell) ratio. Excess medium was aspirated and metacyclic trypomastigotes or extracellular amastigotes suspended in complete medium, with or without drugs, were added. Cells were immediately centrifuged at 2300 g for 20 min at 37ºC. After centrifugation, unattached parasites were removed by washing the coverslips three times with RPMI without serum and seven times with sodium phosphate-buffered saline pH 7.2 (PBS), both at 37ºC. Coverslips were fixed in 2% glutaraldehyde (Sigma, EM grade) in 0.1 M sodium phosphate buffer pH 7.0 for 1.5 h at room temperature. After three washings in PBS, coverslips were incubated with 0.138 M ethanolamine, pH 8.3 for 30 min at room temperature to quench excess aldehyde groups, washed with PBS, and soaked in PBS containing 0.25% gelatin and 0.05 M NaN3 (PGN) (Procópio et al. 1998).
After immunofluorescence incubations (described below) the invasion index was calculated according to the formula: number of parasites inside cells/ number cells infected x % of infected cells, counting 100 cells in triplicate coverslips (Procópio et al. 1998).
Antibodies and immunofluorescence labeling - For invasion assay quantitations, cells fixed in 2% glutaraldehyde were incubated with monoclonal antibodies (ascitic fluids diluted 1:40 in PGN): mAb 1G7 against the 90 kDa metacyclic trypomastigote glycoprotein (Teixeira & Yoshida 1986) or mAb 1D9, reactive against the Ssp-4 glycoprotein of amastigotes (Barros et al. 1997). After 1 h the coverlisps were washed 3 times with PBS and incubated 1 h with anti-mouse IgG-FITC conjugated (Sigma) diluted 1:50 in PGN and 10 µM DAPI (4',6-diamidino-2-phenylindole dihydrochloride, Molecular Probes, Eugene, Oregon, US). Using this methodology only parasites outside cells are labeled with antibodies and all parasites are visible after DAPI staining. Invasion index was calculated as described above. For conventional immunofluorescence infected cells were fixed with 3.5% formaldehyde, washed and then permeabilized with PGN solution containing 0.1% saponin (Barros et al. 1997). All statistical calculations describe here were done with SigmaStat (Version 1.0, Jandel Scientific), using the T-test for significance and standard deviations for paired data sets.
Determination of parasites within parasitophorous vacuoles - Coverslips fixed and processed for conventional immunofluorescence were incubated with anti LAMP-1 antibodies (clone H4A4 anti-human LAMP-1, and clone UH3, anti-hamster LAMP-1, both developed in mouse, supernatants from DSHB, Development Studies Hybridoma Bank, Iowa, US) for 1 h and finally incubated 1 h with anti-mouse IgG conjugated to Cy3 (Sigma) diluted 1:50 in PGN and 10 µM DAPI. Parasites within parasitophorous vacuoles appeared labeled with anti-LAMP-1 antibodies (Hall et al. 1992). The kinetic parameters analyzed here were the half-life times for formation and escape as well as the mean time of residence within the parasitophorous vacuole, described in Fig. 1. Invasion and kinetic experiments using triplicate coverslips were performed at least three times.
Hemolysin and transialidase assays - Hemolysin (TcTOX) activity was determined as described by Andrews and Whitlow (1989), with minor modifications. Briefly, freshly collected female albino mouse erythrocytes were washed three times with isotonic PBS/ 0.1% gelatin, 0.15% CaCl2 and 1mM MgCl2. Isolated amastigotes, Y strain tissue-culture derived (positive controls) and G strain metacyclic trypomastigotes (108/ml) were transferred to 10 mM sodium acetate buffer, pH 5.5, 0.15 M NaCl, containing 1% glucose and the erythrocytes (107/ml). Maximum hemolytic values were obtained by treating the erythrocytes with 0.1% saponin. Released hemoglobin was measured at 540 nm in a Labsystems Multiskan MS (Finland) ELISA reader, in duplicate samples collected at 3, 6, and 10 h. Transialidase activity with purified parasites was assayed as described (Schenkman et al. 1994). Briefly, the enzymatic transference of sialic acid to 14[C]-lactose generated 14[C]-sialyl-lactose that was captured in QAE-Sephadex A-25 columns, and we used Y strain tissue culture derived trypomastigotes as positive controls (Schenkman et al. 1994). These independent determinations were carried out at least three times.
Interference with cytosolic pH and calcium concentrations - In order to raise cytoplasmic pH, cells were exposed for 1 h at 36.5°C, to culture medium containing 100 µM chloroquine. Alternatively, cells were treated for 30 min with calcium ionophore A23187 or endoplasmic reticulum Ca2+ ATPase inhibitor thapsigargin (Sigma) that were used at final concentrations of 10 µM and 1 µM (in complete culture medium), respectively. Cells were then washed 3 times with PBS and complete medium containing the parasites was added to the flasks.
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