Geração e caracterização de linhagens de Trypanosoma cruzi expressando proteínas fluorescentes como ferramentas para pesquisa em Doença de Chagas

Abstract

Stable transfection protocols have been described for a number of protozoan parasites such as Leishmania spp, Trypanosoma brucei and Trypanosoma cruzi. The transfection can be achieved by the integration of the foreign gene into the genome through homologous recombination, or by episomal maintenance of the transfected plasmid. In order to produce parasites expressing fluorescent proteins, epimastigotes forms of T.cruzi were transfected with the plasmid pROCKGFP/RFPNeo. This construction allowed the integration of green (GFP) and red (RFP) fluorescent protein genes by homologous recombination into -tubulin locus of several strains of the parasite. Parasites transfected clones were isolated by serial dilution and/or agar-blood plates. To evaluate the stability of the GFP and RFP markers in the transfected populations andclones, parasites were cultivated for 1 and 2 months in the absence or presence of G418 (200 g mL and 400 g mL) and were analyzed by FACS. The selected clones were able to maintain high expression levels of the fluorescent protein markers even in absence of G418, whereas in transfected population, expression of GFP and RFP markers wassignificantly reduced when cultured in the absence of the drug. The integration of the GFP and RFP markers in the tubulin locus was confirmed by chromoblot analyses. GFP and RFP expressing parasites were inoculated into Vero cells culture to evaluate the infectivity of the transfected population. When compared with the wild type population, no significant differences in the infectivity of Vero cells were found. Wedetected the fluorescent proteins in the tree forms of parasite life cicle by confocal and fluorescence microscopy. Furthermore, GFP expressing parasites were able produce significant levels of parasitemia 12 days after being inoculated into interferon-gamma knockout (GKO) mice. We observed fluorescent parasites in heart and diaphragm of infected animals. Cell cultures infected simultaneously with two cloned cell lines, each one expressing a distinct fluorescent marker, showed that two different parasites were able to infect the same cell. Double infected cells were also detected when GFP and RFP expressing parasites are derived from strains belonging to two distinct T. cruzi lineages Col1.7G2GFP (T. cruzi I) and TulahuénRFP (T. cruzi II). The parasites expressing GFP and RFP constitute a new tool for the study of various aspects of the T.cruzi biology. We can study the cellular invasion mechanism, genetic exchange, drug susceptibility and several aspects of interaction with invertebrate and vertebrate hosts, emphasizing the histotropic-clonal model of Chagas disease.