Role of intracellular cAMP in oxidative stress responses through Trypanosoma cruzi life cycle

ALEJANDRA C. SCHOIJET; GUILLERMO D. ALONSO; TAMARA STERNLIEB; SALOME VILCHEZ LARREA; PATRICIO GENTA

Santa Fe

Congreso; XXVIII Reunión Anual de la Sociedad Argentina de Protozoología y Enfermedades Parasitarias.; 2016

Cyclic adenosine monophosphate (cAMP) is a key second messenger in several metabolic pathways. In Trypanosoma cruzi it was found to participate in proliferation, differentiation and osmoregulation. Here we explore the role of cAMP in the response to oxidative stress in T. cruzi epimastigotes and trypomastigotes. To determine the role of cAMP in oxidative stress responses, we first set up the conditions for a proliferation measurement method using tritiated thymidine, based on the incorporation of the radioactive nucleotide during DNA replication. Through this technique, we established an optimal work concentration of hydrogen peroxide of 150 μM, which presents a moderate effect on proliferation, allowing the recovery of the parasites? normal growth after 24h. Our results suggest a possible protective role of cAMP over the hydrogen peroxide stressed cells, through the use of cAMP analogs (pCPT-AMPc and 8-Bromoadenosine 3′,5′-cyclic monophosphate). We also generated transgenic parasite lines that overexpress different phosphodiesterases and assessed their involvement in these responses. In addition, we evaluated these treatments on tripomastigotes through their viability and infectivity. Parasites expressing bacterial β-galactosidase allowed us to compare infection levels in Vero cells after 48 h, using trypomastigotes that were treated with H2O2, with or without pCPT-cAMP. Our results show that cAMP increases the number of amastigotes produced during infection for parasites under no stress or treated with low doses of H2O2. To establish if cAMP enhanced infectivity or survival under oxidative stress we used fluorescent vital staining with resazurine dye and the stable tetrazolium salt (WST-1) colorimetric indicator. Taken together, our results unveil an unknown role for cAMP as a protective regulator against oxidative stress in T. cruzi and point to identify potential components of these signaling pathways.