Trypanosoma cruzi redox network as a virulence factor: role of tryparedoxin peroxidase in the pathogenesis of Chagas disease.

ZAGO MP; MESIAS, AC; PIÑEYRO MD; ROBELLO C; GARG NJ

Newport, Rhode Island

Conferencia; Gordon Research Conference on Host-Parasite Interactions, Biology of held; 2016

Gordon Research Conference

The aim of our study is to gain insight the involvement of T. cruzi redox system in the pathogenic mechanism of Chagas disease. Many groups have attributed to this pathway a role in the evasion of the host innate immune response. Previously, we described the up regulation of enzymes of this network, particularly tryparedoxine peroxidase (TXNPx), in trypomastigotes of DTUI isolates. In order to characterize its participation in the cellular response to infection, we employed a site-directed mutagenesis approach to construct the active site mutants of cytosolic (CPXC52S, CPXC173S) and mitochondrial (MPXC81S, MPXC204S) isoforms of TXNPx. The pRibotex plasmid encoding the cDNAs for mutant TXNPx isoforms was electroporated into Sylvio X10/4 and transfectants were selected under drug pressure (250-400µg/ml G418). Overexpression of the mutant proteins and the wild type protein in the transfectants was confirmed by western blotting with specific antibodies against CPX and MPX isoforms. Also, the differential peroxidase activity of the recombinant parasites was detected with the Peroxidase Assay Kit (Invitrogen). Evaluation of viability by Alamar Blue, revealed that T. cruzi expressing mutant isoforms of CPX and MPX were significantly more sensitive (p0.05) to oxidizing species, e.g. H2O2 and ONOO?−, when compared to the wild type parasites or T. cruzi transfected with CPX wild type isoform. In vitro infection studies in RAW macrophages showed that intracellular replication of Syl.MPXC81S and Syl.CPXC173S determined by qPCR was decreased, compared to that noted for SylWT and Syl.CPX (p