Chagas' disease is caused by the flagellated protozoan Trypanosoma cruzi. The extracellular amastigotes (EA) cellular invasion is highly dependent on the actin cytoskeleton of the host cell whose regulation during this specific interaction remains poorly characterized. The Cdc42 and Rac1 GTPases are involved in a variety of in various cellular processes, but mainly in the regulation of the actin cytoskeleton where its main effector molecules, N-WASP and Wave2 respectively, crucially act as nucleators of actin polymerization during the formation of filopodia and lamelipodia. Several studies have shown the involvement of both pathways - Cdc42/N-WASP and Rac1/WAVE2 - in the regulation of actin cytoskeleton during cellular invasion by parasites and bacteria but there is no information regarding mammalian cell invasion by T. cruzi. Thus, the present project aims to evaluate the role of these two signaling pathways in actin dynamics during cell invasion by EAs. To assess so the (i) recruitment and (ii) invasion by EAs and the (iii) interaction of these proteins will be evaluated in cells stably overexpressing their native and mutant constructs fused to fluorescent proteins and also expressing interference RNA sequences. These three aspects which properly characterize the role of both pathways in actin polymerization during cell invasion by AEs will be evaluated in living and fixed cells; the latter by means of confocal microscopy. Finally, this strategy will also be used in coinfections studies with cells colonized by the bacteria Coxiella burnetii, whose infection alters the organization of the host cell cytoskeleton and probably the signaling underlying its regulation.
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