Trypanosoma cruzi extracellular amastigotes (EA) from G strain (T. cruzi I) show high infectivity towards host cells in vitro when compared to the traditionally more infectious CL strain (T. cruzi VI). We have performed a DNA microarray from EA from G and CL strain, in order to identify components that would modulate infectivity of these strains. It was identified a 21 kDa protein that upregulates cell invasion by EA from G strain. On the other hand, we have not investigated the mechanisms beneath the low infection rate of EA from CL strain. The possibility of negative modulators was considered once previous studies showed that poorly infectious metacyclic trypomastigotes express high levels of gp90, a stage-specific surface glycoprotein that modulates negatively cell invasion. We observed in a microarray data that amastin, a stage specific surface protein, was 21 times more expressed in EA from CL strain. In order to study the putative involvement of amastin in cell invasion, probably as negative modulator, we cloned, expressed and purified the less hydrophobic region of amastin in fusion with GST. We then developed a polyclonal antibody in rabbit. Immunolocalization of amastin in amastigotes of T. cruzi confirmed its location on the parasite surface. The recombinant protein adhered to HeLa cells in a dose-dependent and saturatable manner. HeLa cells pre-incubated with 5 ¼g/mL of recombinant protein showed a decreased in cell invasion by EA. To confirm the role of amastin in T. cruzi cell invasion, parasites from G strain were transfected with vector pTREX-Amastin-GFP and pTREX-GFP: a significant decrease in cell invasion of EA that over-expressed amastin was observed when compared to control. However, the number of trypomastigotes released into the supernatant of infected HeLa cells 96h and 120h after the invasion was higher in cells infected with the parasites that over-expressed amastin. As a result, amastin can compensate the lower invasiveness and provide a more propitious environment for parasite multiplication and differentiation providing a patent infection.
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