Full text
| |
| Author(s): |
Romario Lopes Boy
Total Authors: 1
|
| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) |
| Defense date: | 2022-04-28 |
| Examining board members: |
Maria Fernanda Laranjeira da Silva;
Daniella Castanheira Bartholomeu;
João Luiz Mendes Wanderley
|
| Advisor: | Maria Fernanda Laranjeira da Silva |
| Abstract | |
Protozoa of the genus Leishmania are the etiological agents responsible for the diseases known as leishmaniasis that affect millions of people worldwide. These organisms are parasites that alternate between the invertebrate and vertebrate host where they are able to survive and replicate in macrophages. One of the critical conditions found by Leishmania in the macrophage is the lack of nutrients, including iron, which is fundamental as a cofactor for several enzymes essential to the parasite. The identification and study of genes essential for iron uptake by these parasites revealed that iron availability plays a central role in infectivity. These studies also demonstrated that iron deprivation induces the expression of a series of genes whose function is still unknown, providing an excellent opportunity for the identification of additional components of the molecular machinery involved in the iron metabolism of these parasites. Trypanosomatids have unique organelles called glycosomes, which represent one of the main differences between parasite and host. Glycosomes compartmentalize enzymes involved in the glycolytic pathway, in the -oxidation of fatty acids, and also participate in cellular antioxidant processes. Iron-dependent superoxide dismutases (FeSOD) are particularly important enzymes due to their role in protecting the parasite against free radicals resulting from NADPH oxidase activation, and Leishmania glycosomes compartmentalize the B isoforms of superoxide dismutases (SODB1/2) that use exclusively iron as a cofactor. Several failed attempts to generate FeSODB knockouts showed that this isoform is essential for the survival of Leishmania, indicating that the transport of iron to the glycosome, not explored so far, is also an interesting target for identification and study. Therefore, the objective of this work is to identify and begin the characterization of genes potentially involved in iron transport to the Leishmania glycosome. From the in silico analysis of 576 L. amazonensis transcripts significantly modulated by iron deprivation, we searched for genes that contained predicted transmembrane domains and glycosomal targeting signals (PTS1 and/or PTS2). Among 11 genes found, we started the characterization of the 5 that have 3 or more predicted transmembrane domains, they are: LmxM.04.1030, LmxM.14.0540, LmxM.22.0580, LmxM.24.1090 and LmxM.30.0020. For this, we cloned the ORFs of these 5 genes in the Leishmania expression vectors pXG-GFP+ and pXG-GFP2+, for fusion of the target genes to the coding sequence of the fluorescent protein EGFP. The obtained constructs were transfected into L. amazonensis promastigotes. We selected several clones and confirmed the overexpression of target genes by qPCR. We evaluated the localization of the target proteins fused to EGFP in immunofluorescence assays and with that we showed the colocalization of the proteins encoded by LmxM.24.1090 and LmxM.30.0020 fused to EGFP with the glycosomal arginase protein. We performed assays to evaluate the in vitro replication of selected overexpressing promastigotes, and our results indicate that the lines overexpressing LmxM.04.1030, LmxM.14.0540, LmxM.22.0580, LmxM.24.1090 and LmxM.30.0020 grow slower than the wild type. Furthermore, we investigated whether the overexpression of target genes differentially modulates the expression of genes previously related to iron transport and metabolism, and to other glycosomal metabolic pathways. Our results revealed the gene expression induction of arginase and arginine transporter AAP3 5.1 in the overexpressing strains of LmxM.14.0540, LmxM.22.0580, LmxM.24.1090 and LmxM.30.0020. Thus, our results indicate that the genes LmxM.24.1090 and LmxM.30.0020 are potentially involved in glycosomal iron metabolism. The construction and characterization of knockout parasites of these genes will allow the characterization of their molecular functions, contributing to the elucidation of the mechanisms involved in the transport and metabolism of iron in the Leishmania glycosome. (AU) | |
| FAPESP's process: | 19/21185-5 - Identification and characterization of iron transporters in the glycossomes of Leishmania |
| Grantee: | Romário Lopes Boy |
| Support Opportunities: | Scholarships in Brazil - Master |