Abstract
American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, hence the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal (Trypano-soma cruzi). T. cruzi is an RNAi-negative parasite, therefore the silencing genes strategies by RNAi is not possible; for that reason, antibodies may be taken as a tool for studying the parasite proteins function by blocking these molecules with specific antibodies. The aim of this work was to establish a methodology for antibody delivery (antibody transfection) into viable parasites. We used anti-cyclin-A antibody (human origin) in western blot assay with epimastigote of T. cruzi proteins and this recognized a ~55 kDa polypeptide. Several methods for antibody transfection (electroporation, saponin permeabilization and a lipid-based formulation) were tested. The first two methods were unsuccessful. In electroporation was impossible to visualize the antibody inside parasites and with saponin permeabilization, antibodies were successfully introduced, but with loss of parasites viability. The lipid-based formulation method forms noncovalent complexes with antibodies. These complexes are internalized by cells and antibodies are released into the cytoplasm. With this method, a successful antibody delivery was achieved. Anti-cyclin antibodies were visualized in the cytoplasm from fixed transfected parasites (immunofluorescence assays). At 24 h post-transfection, parasites maintained their viability (90%) and were able to arrest the cell cycle in G0/G1-phase of cultured epimastigotes (cell population increased in G0/G1-phase from 50.5% to 66.2% and decreased in S-phase from 47.2% to 26%). It was also observed that anti-cyclin-A antibodies inhibit the parasite population doubling (p T. cruzi, with a simple and cheap technique, which will allows carrying out further studies of this protozoan.
Highlights
Chagas disease or American tripanosomiasis, is a zoonosis ranked as one the seventeen neglected tropical diseases in the world [1]
American trypanosomiasis is a zoonosis of worldwide medical importance and currently there is no effective treatment in chronic patients, the importance of the study of protein function of the parasite with the objective of finding new drug targets and to know better the biology of the agent causal (Trypanosoma cruzi)
The main objective of this study was to find a good method and optimal conditions for delivery antibodies into viable epimastigotes of T. cruzi, in order to have a simple tool for studying the parasite biology, for protein function tests or the study and validation of potential therapeutic targets
Summary
Chagas disease or American tripanosomiasis, is a zoonosis ranked as one the seventeen neglected tropical diseases in the world [1]. It is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) whose principal route of transmission to human is by feces of infected triatomine bugs. After more than 100 years of the discovery of Chagas disease, there is not any effective treatment available and the drugs used are lightly effective only during the early infection [3,4] This scenario requires the continuous search for strategies to eradicate this disease, including treatment, control and prevention. It is relevant the searching for new alternative treatments, identify new pharmacological targets and a better knowledge of the parasite biology
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