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Human factors required for mycobacterium tuberculosis macrophage infection

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Resumo:Tuberculosis is a pathological manifestation of the infection by Mycobacterium tuberculosis in humans. Facultative intracellular parasites, like M. tuberculosis, evolved mechanisms to subvert the proper functioning of their host cells to their benefit. In this thesis we token advantage of the characteristics of the fluorescent proteins to develop methodologies based on fluorimetry to quantify the macrophage internalization and the number of intracellular mycobacteria. The application of these techniques allowed: 1) to demonstrate that the GFP expressing mycobacteria may be used to test the effects of new drugs namely pyrazinoic acid esters against M. tuberculosis growth in liquid cultures. However GFP is not a good fluorophore to quantify intracellular mycobacteria. Instead we found tdTomato the election fluorophore to this purpose 2) To determine the effect of 120 genes in the host vesicular traffic during macrophage infection with M. tuberculosis. We identified 10 genes, 8 lead to a reduction and 2 an increase of macrophage M. tuberculosis burden. 3) To demonstrate that a reduction in macrophage internalization was the cause for the reduction in intracellular M. tuberculosis observed in some of the genes identified in 2) and the modulation of internalization by the microRNA miR-142-3p. Additionally, we studied the effect of Rab GTPases silencing in exosome secretion. MicroRNAs (miR) are small non-coding RNA molecules that regulate gene expression. Previous studies in our laboratory shown that miR-142-3p was up-regulated during early stages of M. tuberculosis macrophage infection. MiR142-3p binds to Wasl gene controlling the expression of N-wasp protein involved in signal transduction of membrane receptors and actin cytoskeleton. N-wasp silencing reduces M. tuberculosis internalization by macrophage. These studies demonstrate that M. tuberculosis regulates its internalization through N-Wasp. Taken together our results demonstrate the effect of specific host factors in the survival and/or internalization of M. tuberculosis by macrophages. Tuberculosis bacilli apparently modulate some of them. The development of new molecules in formulations that inhibit resistant M. tuberculosis strains is another strategy for the eradication of this pathogen from humans.
Autores principais:Carmo, Nuno Baltazar do, 1976-
Assunto:Teses de doutoramento - 2014
Ano:2014
País:Portugal
Tipo de documento:tese de doutoramento
Tipo de acesso:acesso aberto
Instituição associada:Universidade de Lisboa
Idioma:inglês
Origem:Repositório da Universidade de Lisboa

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