Estudo do papel diferencial de cada uma das isoformas da proteína lisossomal LAMP (LAMP-1E LAMP-2) no processo de infecção celular pelo Trypanosoma cruzi

Abstract

Trypanosoma cruzi, an obligatory intracellular parasite, is capable of triggering a series of signaling events in host cells, many of them leading to calcium mobilization from intracellular stocks. Increase in cytosolic calcium leads to lysosomal exocytosis, which in turn is followed by a compensatory endocytosis event that drives T. cruzi into the host cell. Parasite initially resides in a vacuole containing both lysosome and plasma membrane. To this initial vacuole more lysosomes fuse until parasite is all covered with lysosomal markers. Membranes from lysosome derived vacuole and T. cruzi are found tightly apposed and this interaction is thought to be important for parasite retention inside host cells. Besides its role during invasion, lysosomes also play a role in T. cruzi escape from its parasitophorous vacuole into the host cell cytosol. The presence of both sialic acid in vacuolar membrane and transsialidases on parasite surface have been shown to interfere with this process. Once in the cytosol, parasites transform into the amastigote replicative form and start colonizing the host cell. LAMP-1 and 2 are the major components of lysosomal membranes and are also highly sialilated. Previous studies from our group have demonstrated that the absence of these two proteins in cells decrease parasite invasion. On the other hand it also increases parasite intracellular multiplication. The mechanism by which LAMP absence leads to these phenotypes is still not clear. Both sialic acid that modify these proteins and the polipetide chain itself might be playing a role in both parasite invasion and intracellular multiplication. The main goal of this work is to better understand the role and mechanism by which both LAMP isoforms, LAMP-1 and 2 interfere with T. cruzi infection in host cells. For this we performed cell infection experiments using LAMP-2 and LAMP-1/2 knock out cells, as well as wild type cells. We have shown that both LAMP2 and LAMP-1/2 knock out cells present similar parasite invasion pattern, indicating that LAMP-2 plays a major role in this process. We have also shown that LAMP-2 absence compromises parasite adhesion without changing vacuole morphology. These data reinforce a role for the polypeptide chain itself and not its sialic acid residues in the phenotype observed. Parasite intracellular multiplication was also similar in LAMP-2 and LAMP-1/2 knock out cells. However in this case our data suggest that both polipetide chain and its sialic acid modifications might be playing a role. Although not statistically significant different from wild type infections in LAMP-2 knock out cells have shown a tendency for a faster rate of parasite escape from its parasitophorous vacuole. Based on our data we believe LAMP-2 plays an important role for T. cruzi infection in host cells, participating in both parasite invasion and intracellular multiplication.