Papel das GTPases Cdc42, RhoA e Rac1 no citoesqueleto de actina durante a invasão celular pelas formas tripomastigotas metacíclicas e de cultura de tecido de Trypanosoma cruzi

• Ano de defesa: 2019
• Autor(a) principal: Bonifacio, Bruno Souza [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Trypanosoma Cruzi; Cell Invasion; Actin; Gtpases; Metacyclic Trypomastigotes; Tissue Culture Trypomastigotes.; Invasão Celular; Actina; Tripomastigotas Metacíclicos; Tripomastigotas De Cultura De Tecido.
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=7741216; https://repositorio.unifesp.br/handle/11600/59405

Cell invasion by Trypanosoma cruzi metacyclic trypomastigotes (TMs) and tissue culture trypomastigotes (TCTs) is a complex process involving parasite-host interactions, and the role of the host cell actin cytoskeleton is still controversial in the literature. The GTPases Cdc42, RhoA and Rac1 and their effectors are key regulators of the actin cytoskeleton promoting its polymerization according to cellular stimuli. In this work we aimed to evaluate actin dynamics and the participation of Cdc42, RhoA and Rac1 during cell invasion by T. cruzi trypomastigote forms. Invasion assays with GTPase-depleted (kd) HeLa cells showed reduced TM invasion in the Cdc42-kd and RhoA-kd groups while TCT presented reduced invasion in Cdc42-kd RhoA-kd and Rac1-kd groups. We also performed invasion assays with HeLa cells overexpressing the studied GTPases, in wild type, constitutively active, dominant negative constructs. Our results showed diverse invasion profiles for either TCTs and TMs, varying between increase or reduction of parasite invasion, depending on the GTPase and construct. Additionally, we investigated the distribution of lysosomes in cells depleted for GTPases. We identified augmented spreading of these organelles even in the absence of parasites. Regarding the GTPases, our results suggest the participation of Cdc42 in both rypomastigote forms whereas Rac1 plays a significant role only for TCTs. On the other hand, the RhoA protein is likely to play a negative role in the invasion of TMs and TCTs. We also performed invasion assays with cells pretreated with Cytocalasin D, Latrunculin B, Phaloidin and Jasplakinolide. Our results showed that all toxins inhibited TMs and TCTs invasion. Finally, actin recruitment assays showed in both TCTs and TMs an inconsistent recruitment profiles, in other words, some parasites do recruit while others do not recruit actin during the invasion process. Taken together, our results suggest that actin cytoskeleton plays an indirect role in the invasion of TCTs and TMs, but other mechanisms that might play a role still need to be elucidated.