Molecular events occurred during Trypanosoma cruzi invasion

ARBOIT, MA1; LAVANDERO, S2; COLOMBO, MI1 AND ROMANO, PS1.

Mendoza

Otro; Second Cell Biology Summer Course; 2010

Instituto Curie, IHEM-CONICET

Trypanosoma cruzi, the etiologic agent of Chagas` disease, exploits different mechanisms to infect the mammalian cells. In a previous work, we have demonstrated that the infective form of this parasite recruits autophagic compartments during the invasion and that the induction of autophagy increase parasite infection. To follow this work we next analyzed the molecular components involved in the fusion between autophagosomes and the plasma membrane, a process required during infection, as well as the capacity of T. cruzi to activate the autophagosome formation. Using CHO cells overexpressing GFP-NSF-wt and GFP-NSF-D1EQ (negative mutant: NM) we have observed a significantly increase of infection in wild type cells comparing with control cells; whereas there is a significant lower level of infection in NM cells. Furthermore, we found a high level of association between the v-SNARE protein GFP-VAMP 7-wt and the T. cruzi parasitophorous vacuole (TcPV) whereas the non-functional protein GFP-VAMP 7-NT decreases the number of parasites which invade the cell. VAMP 7-wt also presents an elevated colocalization with the autophagosomal marker LC3 in TcPV during starvation. The expression of both proteins produce a very significantly increase in the number of infected cells indicating that there is a synergism in both proteins during T. cruzi infection.  Finally, we have used two different methods to asses if this parasite can induce the autophagic pathway in the host cell. The analysis of GFP-LC3 degradation by flow cytometry showed that cells infected with T. cruzi during 6 h have the same behavior that the control, whereas a significantly increase in the fluorescence values was produced at 24 h after infection. Moreover, we have observed by western blot a high expression in the LC3-II protein at 3, 6 and 24 h after infection. Based on these results we are conducting a new set of experiments to confirm the probably different regulation of autophagy at different times after T. cruzi infection.