Trypanosoma redox network as a virulent factor. Role of TXNPs in macrophage infection

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

Conferencia; Gordon Research Conferences- Host Parasite interaction; 2016

The aim of our study was to understand he role of T. cruzi redox system in the pathogenic mechanism of Chagas disease. 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 SylCPX (p