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

MESIAS ANDREA; ARIAS DG; ROBELLO C; GARG NJ; ZAGO MP

Galveston, Texas

Jornada; 2018 McLaughlin Colloquium; 2018

University of Texas Medical Branch

Chagas disease is a tropicalneglected illness caused byTrypanosoma cruzi that remains tobe endemic in Latin America. Thispathogen has to deal with differentoxidant challenges such as therespiratory burst triggered inside themacrophage. Thus, it has an effectiveantioxidant system capable ofovercoming the host barriers andmaintaining the redox balance. Theparasite antioxidant network utilizestrypanothione (TSH), a low MWdithiol, as substrate. Thetrypanothione synthetase (TryS)enzyme produces TSH metaboliteand is uniquely present inkinetoplastids, so it is a goodcandidate for drug design. In order tocharacterize its role in the host parasiteinteraction, we haveoverexpressed pTREX encoding TrySin T. cruzi SylvioX10 isolate byelectroporation. Recombinantparasites (TrySHI) exhibited stableoverexpression (>2-fold increase) ofthe TryS protein and a significantincrease in TryS enzymatic activity ascompared to controls. The showed a higher rate ofmetacyclogenesis, and ~20% more ofinfective forms were obtained inTrySHI cultures compared to thecultures of parasites transfected withempty pTREX. Furthermore,transfectant parasites toleratedhigher doses of benznidazole (IC50value: 21.3 μM and 11 μM, TrySHI vs.controls, respectively, p􀀀0.05). To getinsight into TryS role in host-parasiteinteraction, Raw 264.7 cells wereinfected. After 48 h incubation, cellsinfected with TrySHI (vs. control) T.cruzi exhibited lower levels ofintracellular ROS, determined byH2DCFDA fluorescence (p􀀀0.05). Ourresults suggest that T. cruzi utilizesTryS to promote differentiation frominsect stage to infective forms and tomaintain the redox balance in thehost cells that support intracellularsurvival of the parasite.