Dissecting the role and pathogen benefits of trypanothione synthetase overexpression in Trypanosoma cruzi

GARG NJ; SASONI N; ARIAS DG; ZAGO MP; MESIAS AC

Galveston, Texas

Congreso; IHII/McLaughlin Colloquium on Infection and Immunity; 2018

University of Texas

Chagas disease is a tropical neglected illness caused by Trypanosoma cruzi that remains to be endemic in Latin America. This pathogen has to deal with different oxidant challenges such as the respiratory burst triggered inside the macrophage. Thus, it has an effective antioxidant system capable of overcoming the host barriers and maintaining the redox balance. The parasite antioxidant network utilizes trypanothione (TSH), a low MW dithiol, as substrate. The trypanothione synthetase (TryS) enzyme produces TSH metabolite and is uniquely present in kinetoplastids, so it is a good candidate for drug design. In order to characterize its role in the host-parasite interaction, we have overexpressed pTREX encoding TryS in T. cruzi SylvioX10 isolate by electroporation. Recombinant parasites (TrySHI) exhibited stable overexpression (>2-fold increase) of the TryS protein and a significant increase in TryS enzymatic activity as compared to controls. The TrySHI showed a higher rate of metacyclogenesis, and ~20% more of infective forms were obtained in TrySHI cultures compared to the cultures of parasites transfected with empty pTREX. Furthermore, transfectant parasites tolerated higher doses of benznidazole (IC50 value: 21.3 µM and 11 µM, TrySHI vs. controls, respectively, p0.05). To get insight into TryS role in host-parasite interaction, Raw 264.7 cells were infected. After 48 h incubation, cells infected with TrySHI (vs. control) T. cruzi exhibited lower levels of intracellular ROS, determined by H2DCFDA fluorescence (p0.05). Our results suggest that T. cruzi utilizes TryS to promote differentiation from insect stage to infective forms and to maintain the redox balance in the host cells that support intracellular survival of the parasite.