Chagas disease is caused by the protozoan T.cruzi. Pathogenesis of Chagas heart disease (CHD) involves low-grade incessant systemic infection and triggered autoimmune reaction. Cruzipain (CZP) is a key virulence factor of T. cruzi.
Autophagy is an intracellular (IC) pathway that affects survival and/or virulence of parasites.
A recent study showed that Crv was effective in reducing oxidative damage in CHD.
K777 is a CZP inhibitor with antiproliferative effect on T. cruzi. On the other hand, a virtual screening of K777-similar structures selected Crv as a candidate (Stanford University, USA). Although Crv didnt inhibit CZP, we observed some effect of Crv on T. cruzi autophagy and continued studies in this regard.
The effect of Crv on T. cruzi epimastigotes was evaluated on Y-GFP strains maintained in BHT medium. Crv10 uM was added and compared with control. Replication was counted in Neubauer chamber (Fig 1).
To observe effect of Crv on amastigotes, rat cardiomyoblast H9C2 cell lines were infected with T. cruzi trypomastigotes Y-GFP for 24 h, then treated with Crv10 uM for 96 h. IC replication was evaluated counting amastigotes per cell by microscopy.
Parasite autophagy was studied staining epimastigotes with (monodansylcadaverine) MDC (Fig 3) and TcAtg8 (T. cruzi specific protein of autophagosomes). Acidic and degradative compartments of T. cruzi were studied by Lysotracker and DQ BSA staining.
Crv at doses of 10 uM has significant inhibitory effect on proliferation of epimastigotes (p<0.001) and IC amastigotes (p <0.001).Morphological studies showed multiple vacuoles and double membrane vesicles with membranes juxtaposed inside, similar to autophagic compartments (fig 2). The increased number of autophagic compartments was confirmed by detection of TcAtg8 and MDC staining. The percentage of acidic and degradative compartments detected with specific markerswas reduced in presence of Crv.
Autophagy is related to infectioncapacity of T. cruzi.
Crv acts on the autophagic pathway of T. cruzi leading to accumulation of autophagic non-degradative compartments affecting its growth and vitality.
This study shows that Crv has a direct effect on T. cruzi and should be explore as treatment for CHD.
C. V. Rivero,
J. Cueto, None
P. Romano, None