Overexpression of cyclophilins in the unicellular parasite Trypanosoma cruzi: a tool for protein function analysis.

ALINA PERRONE, PATRICIA BUSTOS, MARIA DE LOS MILAGROS CÁMARA, GABRIELA A. GARCÍA AND JACQUELINE BÚA.

Boston

Congreso; Congreso Mundial de Biotecnología 2013 (World Biotechnology Congress 2013); 2013

Cyclophilins (CyPs) are enzymes involved in protein folding and are target of Cyclosporin A. We have previously described and biochemically characterized the cyclophilin gene family in Trypanosoma cruzi, the causative agent of Chagas disease, of health importance in Latin America. T. cruzi expresses two cyclophilins TcCyP19 and TcCyP21, which are homologues to the mammal cytosolic CyPA, involved in many interesting pathways and the mitochondrial CyPD, involved in programmed cell death events. To further elucidate the functions of TcCyP19 and TcCyP21 parasite cyclophilins, their genes were cloned in the vector pTEXOmni-GFP, and T. cruzi parasites were transfected with each construction. A significant decrease of infected cells were observed in vitro with transfected parasites overexpressing the cytosolic and secreted TcCyP19 cyclophilin. Overexpression of the mitochondrial TcCyP21 allowed us to assess its role in oxidative stress stimulation with 5mM H2O2, and observed significant differences in programmed cell death features, as mitochondrial membrane potential decrease and an increment in TUNNEL positive parasites, among others. A pre-incubation of parasites with 1mM Cyclosporin A inhibited the T. cruzi cyclophilins, and reverted the TcCyP19 action in parasite cell invasion and TcCyP21 role in the programmed cell death features observed.