Análise proteômica de células não fagocíticas na fase inicial da infecção por trypanosoma cruzi

Chagas disease is regarded as a neglected illness endemic from Latin Americ and, furthermore, available drugs have limited efficacy and high toxicity. It is estimated that 6 to 7 milion people worldwide are infected by Trypanosoma cruzi (the etiologic agent of the disease). Nonetheless, efforts toward more effective treatment have been made as well as a vaccine is being pursued. In the vectorial transmission of the parasite, the most common in endemic regions, triatominean bugs release trypomastigotes in the mammal host’s blood stream. The parasites through the contact with several host cell types initiate biochemical processes for cell interaction and infection. This early stage is crucial for the continuity of the parasite’s biological cycle and disease progression. Given its importance, and the limited information available regarding the modulation of the host cell, this study analyzed the T. cruzi infection effects in mammalian host cells at the early stages of the process through high-throughput proteomic approach. Therefore, a comparative quantitative proteome analysis by means of LCMS/MS was carried out between Hela cells which were incubated or not with trypomastigote forms (the main infective life form) The resulting list of regulated proteins showed known proteins, e.g calcium and cytoskeleton binding proteins. Regarding proteins with decreased expression, there were Major Histocompatibility Complex I proteins and subunits of the proteasome machinery important to its assembly and catalytic functions. This also corroborates with recent studies demonstrating its relevance on escaping the immune system’s surveillance by jeopardizing the host cell antigen presentation. Also there were proteins related to the ubiquitination process. And among those with no expression modulation reported so far, here we observed an increase in Rpn11, a proteasome subunit with deubiquitinase function, and decrease in cullin associated to NEDD8, a protein involved recognition and ubiquitination of proteins to be degraded. The interaction process between T. cruzi and HeLa cells also caused the modulation of proteins involved in metabolism, DNA binding and chaperones. This work underlines the need for further studies on processes of ubiquitination , degradation and targeting of T. cruzi proteins to MHC1 by host cells , which may be used by the immune system for the elimination of the parasite. Lista de Ilustrações Figura 1: Manifestação clínica no local de penetração do Tripanosoma cruzi. 8 Figura 2: Distribuição mundial de casos de transmissão da doença de chagas de forma vetorial. 9 Figura 3: Ciclo biológico do Trypanosoma cruzi no hospedeiro invertebrado (inseto vetor) e no hospedeiro vertebrado (homem e mamíferos). 12 Figura 4: Vias de sinalização para aumento de Ca2+ e invasão na célula hospedeira por T. cruzi.. 14 Figura 5: Vias de sinalização gerais utilizadas no processo de invasão da célula hospedeira por Trypanosoma cruzi 16 Figura 6 Fluxograma da sequência utilizada para marcação com iTRAQ. 20 Figura 7: Fluxograma geral do trabalho realizado 22 Figura 8 Determinação do tempo de interação.. 24 Figura 9 Perfil eletroforético de extrato total das duas condições.. 25 Figura 10 Vocano plot da análise estatística da expressão diferencial das proteínas.. ... 26 Figura 11 Agrupamento de proteínas diferencialmente expressas de acordo com a Função Molecular. . 27 Figura 12 Agrupamento de proteínas diferencialmente expressas relacionada a função de ligação. 28 Figura 13 Agrupamento de proteínas diferencialmente expressas relacionada a função de ligação a outras proteínas.. 29 Figura 14 Agrupamento de proteínas diferencialmente expressas relacionada a função de ligação a outras proteínas. 29 Figura 15 Agrupamento de proteínas diferencialmente expressas relacionadas a estrutura de moléculas da célula. 29 Figura 16 Agrupamento de proteínas diferencialmente expressas relacionadas a Processos Biológicos 30