Biological material
C. abyssinica seeds were supplied by the Fundação MS para a Pesquisa e Difusão de Tecnologias Agropecuárias, Maracaju, Mato Grosso do Sul, Brazil. Epimastigotes of T. cruzi (DM28 strain) and the cell line LLC-MK2 were mantained by Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense Darcy Ribeiro, Campos dos Goytacazes, Rio de Janeiro, Brazil.
Protein extraction and characterization
Fractions were obtained as described [12] with minor modifications. The seeds were ground and immersed in 0.2 mol/L sodium phosphate pH 7.2 [1:5 [w/v]] for 3 h. The crude extract was centrifuged (15 000 × g at 4°C for 10 min), and the supernatant was recentrifuged under the same conditions. The resulting supernatant was frozen at -20°C until needed.
Gel-filtration chromatography
The crude extract [1 mL containing 10 mg of protein] was fractioned in a Sephadex G-50 [Sigma-Aldrich] gel-filtration column. The proteins were eluted with 0,1% trifluoroacetic acid (TFA; Sigma-Aldrich) at a flow rate of 1 mL/min. The absorbance of the eluted proteins was monitored at 280 nm and 220 nm. The eluted fraction with molecular weights of 5–20 kDa [FCrF5-20] was freeze-dried for biological assay.
SDS-PAGE and Western analysis
The FCrF5-20 was submitted to SDS-PAGE − 12.5%. The proteins resolved by SDS-PAGE 12.5% were transferred onto nitrocellulose or PVDF membrane using 25 mM Tris, 192 mM glycine, and 20% MeOH pH 8.3. Polyclonal mice antibody against 2S albumin from Ricinus communis was diluted at 1:500 for immunoblotting experiments. The immunoreactive band was developed using protein A-peroxidase (Sigma) and 3,3’-diaminobenzidine (DAB) from BioRad as a substrate.
N-terminal sequence
After separation by SDS-PAGE, the protein transferred onto PVDF membrane was subjected to Edman degradation using a PPSQ-33A Protein Sequencer Shimadzu Scientific Instruments, Inc. [Columbia, Maryland, USA] to determine N-terminal partial amino acid sequence. The polypeptide sequence obtained was submitted to automatic alignment using a BLAST [Basic Local Alignment Search Tool] for sequence similarity search.
Host-cell maintenance
LLC-MK2 [Macaca mulatta kidney epithelial cell] cells were maintained in RPMI 1640 medium supplemented with 5% FBS [fetal bovine serum, Gibco] in sterile plastic flasks. The bottles were incubated at 37°C in 5% CO2, and the culture medium was changed every 48 h. The monolayer cell cultures were washed three times with phosphate-buffered saline [PBS] and treated with trypsin. The suspended cells were transferred to new flasks or sterile 24-well plates [3 × 104 cells per well] with coverslips for 24 h to allow adhesion before infection with parasites [13].
Parasite maintenance
T. cruzi epimastigotes were cultivated in 5 mL of liver infusion tryptose (LIT) medium supplemented with 10% FBS and 4% hemin at 28°C. An aliquot of 1mL parasites in the exponential growth phase was added to 4 mL of new medium every 5 d. Trypomastigotes were obtained with the acclimatization of the epimastigotes in RPMI 1650 medium at 37°C for 72 h [13,14].
Determination of cytotoxic activity [2S albumin] in LLC-MK2 cells
The plated LLC-MK2 cells were incubated with various concentrations (50, 100, 500, 1000 µg/mL) of FCrF5-20, identified as 2S albumin by N-terminal partial sequence, and maintained at 37°C for 24 h in a humidified 5% CO2 atmosphere to determine its cytotoxic effect. The cells were then quantified by counting the number of cells per visual field by light microscopy using the 40x objective [14]. Controls without the protein fraction were treated under the same conditions. All assays were performed in triplicates.
Cytotoxicity assays of the 2S albumin fraction with T. cruzi epimastigotes
Epimastigotes in the exponential growth phase were counted in a Neubauer chamber using a light microscope (Zeiss Axioinvert 135, 20× objective), and the cell number was adjusted to 1.8 × 106/mL. Aliquots of the 2S albumin were diluted in dimethyl sulphoxide and added to the LIT medium [final concentration of dimethyl sulphoxide was 1.5%], sterilized by filtration [Millex-GV 0.22 µm, Millipore], added to epimastigotes, and incubated at 28°C in a humidified 5% CO2 atmosphere. After 24 and 48 h, samples were quantified in a Neubauer chamber. The assays were performed in triplicate [15].
The activity of the fractions on T. cruzi amastigotes
Host cells were plated over coverslips or culture flasks as described above for 24 h and were infected at a ratio of 10 parasites [trypomastigotes] per cell and incubated at 37°C for 48 h to allow the establishment of the infection. The 2S albumin (50, 100, 500, and 1000 µg/mL) was added to the cells and further incubated at 37°C and 5% CO2 for 24 h [13]
Morphological and amastigotes number analysis by light microscopy
Infected LLC-MK2 samples were washed three times with PBS, fixed in Bouin’s solution (19:1 picric acid: acetic acid, v/v) for 5 min, and washed four times with PBS. The cells were then stained with Giemsa stain (10%, v/v) at room temperature for six h. The coverslips containing the cells were dehydrated in decreasing concentrations of an acetone-xylene solution and mounted on histological slides using Entellan. After the incubation period, epimastigotes were centrifuged at 900 × g for 10 min and washed with PBS, pH 7.2, at room temperature. They were then fixed in a solution containing 4% paraformaldehyde in PBS and stained with Giemsa stain [10% v/v] at room temperature for two h. Aliquots of 100 µL were spread on microscope slides, dried at 37°C, and examined under a light microscope equipped with a 40× objective. Images were obtained using an Olympus DP72 camera and processed using CellˆF [13].
Ultrastructural analyses by transmission electron microscopy
Uninfected and infected LLC-MK2 cells in culture flasks and epimastigotes were treated with 2S albumin at 500 µM for 24 h and then processed for transmission electron microscopy. Epimastigotes were centrifuged at 900 × g for 10 minutes, washed in PBS for 10 minutes, and recentrifuged under the same conditions. Uninfected and infected LLC-MK2 cells were mechanically removed with a cell scraper and centrifuges. The Pellet of the cells was fixed with 4% formaldehyde, 1% glutaraldehyde, 0.2 M sodium cacodylate buffer, and 5% sucrose at room temperature for one h. The cells were washed and post-fixed with a solution containing 2% OsO4, 0.8% potassium ferrocyanide, and 5 mM CaCl2 in 0.1 M cacodylate buffer, pH 7.2. Samples were washed in sodium cacodylate buffer and dehydrated in an increasing concentration series of acetone. The dehydrated samples were incubated in a solution of 100% acetone-EPON resin in proportions of 2:1, 1:1, 1:2, and pure EPON resin for six h each step. The samples were then embedded in EPON resin and polymerized at 60 ºC for 48 h. Ultrathin sections were obtained using an ultramicrotome (Reichert Ultracuts Leica Instruments®). The cells were stained in the dark with 5% aqueous uranyl acetate for 20 min, lead citrate for 5 min, and subsequently observed and photographed with a Jeol 1400 plus transmission electron.