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Role of the ubiquitin proteasome system in proliferation, differentiation and infectivity of Leishmania infantum


The ubiquitin proteasome system (UPS) is primarily responsible for intracellular proteolysis in eukaryotes, consisting of enzymes E1 (ubiquitin-activating enzyme), E2 (ubiquitin-carrying enzyme) and E3 (ubiquitin-ligase) that promote the ubiquitination of protein substrates and the proteasome, a proteolytic macrostructure responsible for the degradation of ubiquitinated targets. In parasitic protozoa, intracellular proteolysis is essential for the alternation of hosts in their life cycles and, consequently, for the success of parasitism. No study to date has characterized the components of UPS in Leishmania infantum, the etiologic agent of visceral leishmaniasis (VL) in Brazil, the most severe form of leishmaniasis that can be fatal if untreated. Through bioinformatics analyses, searching for conserved domains of H. sapiens UPS in L. infantum, we identified 91 genes related to UPS in this parasite, being 3 E1 enzymes, 15 E2s and 47 genes of E3 ligases subdivided into: 15 components of CRLs, 5 Single RING ligases, 13 HECT-like, 4 U-box, 9 components of APCs -Anaphase Promoting Complex, 1 RING between RING, 3 COP9 signalosome and 25 components of the proteasome. Given the importance of UPS in cellular homeostasis and the lack of studies of this system in L. infantum, this proposal aims to understand the role of UPS in promastigotes and amastigotes, as well as in the infectivity in mammalian cells through CRISPR-Cas9 and Bar-seq strategies. We will produce null mutants of UPS-related genes identified and individually tagged with a unique nucleotide sequence (barcode). Viable strains will be pooled in a culture of promastigotes to be grow at different times (0h, 24h, 48h and 168h), differentiated into axenic amastigotes (24h and 72h) and used for in vitro infection (12h and 72h). We will identify and quantify, through sequencing and bioinformatics analysis, the genes required for the development of the parasites in each experimental condition. After validating the effects of gene knockout on parasite proliferation, differentiation and infectivity, the genes of interest will be functionally characterized by identifying protein partners in the parasite using mass spectrometry and intracellular localization by confocal microscopy. The results of this project will contribute to the knowledge of the physiology of this parasite and its relationship with the host, which may lead to the identification of new targets for pharmacological intervention aimed at the treatment of visceral leishmaniasis. (AU)

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