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| Author(s): |
Emerson Augusto Castilho Martins
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2011-04-06 |
| Examining board members: |
Lucile Maria Floeter Winter;
Marcello Andre Barcinski;
Regina Pekelmann Markus;
Nancy Amaral Reboucas;
Maria Carolina Quartim Barbosa Elias Sabbaga
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| Advisor: | Lucile Maria Floeter Winter |
| Abstract | |
Protozoan of genus Leishmania are digenetic parasites that present a stage in the life in insect gut (promastigotes) and an intracellular phase (amastigotes) inside vertebrate host macrophages. The study of L-arginine influx consists in an interesting matter, since the amino acid is used on NO production pathway (the main macrophage microbial pathway) but are also important for parasites survival. The aim of this work was to perform a genetic and physiological characterization of the arginine transport in Leishmania (Leishmania) amazonensis. To verify how does the arginine uptake occurs in the phagolisosomes, we used J774 macrophages infected with L. (L.) amazonensis to establish a flow cytometry sorting protocol to purify the organelle. Microscopic validation of organelle integrity was achieved by acidic pH marker treatment and detection of fluorescent parasites. The arginine transporter coding gene was characterized. We found two copies in tandem that produces two transcripts, named 5.1AAP3 and 4.7AAP3, with distinct 5\'UTR and 3\'UTR. By quantitative real time PCR we found that 5.1AAP3 mRNA expression varies along parasite development. This copy was, also, more abundant than 4.7AAP3 mRNA. This last mRNA showed a half-life of 32.6±5.0 min, while the 5.1AAP3 mRNA did not decay until 180min. As response to arginine starvation, wild type parasites increase the uptake of arginine, as well as the abundance of 5.1AAP3 mRNA. Arginase null parasites starvation responses showed lower arginine uptake rates compared to wild type responses. Unlike wild type, the null mutants also respond to starvation in stationary phase. This data set allow us to propose that arginine internal pool can downregulate its transporter expression in a feed-back mechanism. (AU) | |