Inhibition of the poly (ADP-ribose) polymer metabolism affects infection levels of Trypanosoma cruzi in HL-1cardiomyocytes.

MARÍA LAURA KEVORKIAN; SALOMÉ VILCHEZ LARREA; SILVIA H. FERNÁNDEZ VILLAMIL

Buenos Aires

Congreso; Drug Discovery for Neglected Diseases International Congress 2018 - 4th Scientific Meeting of ResNet NPND; 2018

ResNet NPND

Poly(ADP-ribosyl)ation is a reversible posttranslational modification of proteins involved in many cellular processessuch as DNA repair, cell signaling, gene regulation, inflammation and cell death. Poly(ADP-ribose)polymerase (PARP)catalyzes the addition of poly(ADP-ribose)polymers (PAR) under different stimulus, including DNA strand breaks,increased calcium concentration or ERK mediated phosphorylation. This covalent modification is then catabolized byPoly(ADP-ribose)glycohydrolase (PARG), generating free PAR. We have studied the importance of PAR in the host cellduring Trypanosoma cruzi infection, demonstrating that the presence and activity of PARP and PARG are important forthe persistence of the infection in VERO and A549 epithelial cell line. Inhibition of PARP-1 by nanomolar concentrationsof Olaparib (third generation inhibitor), as well as inhibition of PARG using DEA in a micromolar range, leads to asignificant decrease in infection levels. Given these results, we proposed to characterize the role of PAR metabolismduring infection in HL-1 cardiomyocytes, a cell type for which Trypanosoma cruzi presents tropism. Progression of PARsynthesis during the course of infection was analyzed by Western Blot (WB) and Indirect Immunofluorescence (IFI)assays. PAR levels increased alongside the infection after 6, 24 and 72 hours post infection (PI), decreased in infectedcells treated with Olaparib 50 nM and increased after DEA 1 µM treatment. Following PARP and PARG inhibition,no changes were observed in their expression in infected cells, via WB and IFI. However, PARG relocalized from thecytoplasm to the nucleus 72 hs PI. In order to study the importance of PAR metabolism on host cell infection levels,HL-1 monolayers where infected with β-galactosidase-expressing trypomastigotes and treated with PARP or PARGinhibitors. Absorbance of β-galactosidase derived metabolite showed a significant decrease in PARG inhibited cells72 hours PI. Furthermore, the number of trypomastigotes released to the supernatant was diminished upon DEAtreatment, five days PI. In contrast, infection levels or trypomastigote number were not altered by PARP inhibition. Thenumber of cells with more than 30 amastigotes, when PARG was inhibited, was 10 times fewer than that of untreatedcells, and 5 times fewer than Olaparib-treated cells, even though the percentage of infected cells remained the same.In conclusion, PAR metabolism proved to play an important role in cardiomyocyte infection by affecting the levels ofintracellular and extracellular stages of the parasite when inhibiting PARG. The mechanism of action by which PARGaccomplishes to disturb Trypanosoma cruzi infection in the host cell remains to be elucidated.