Inhibition of Poly(ADP-Ribose)Glycohydrolase activity affects lysosomal function and hampers T. cruzi infection in Vero cells

Abstract

Chagas disease is a potentially life-threatening protozoan infection but, despite its high incidence and large economic costs associated to it, effective pharmacological treatments are lacking. The search for new anti-chagasic drugs has focused on potential targets in the parasite itself but heterogeneity among different strains of Trypanosoma cruzi - the etiological agent- has hampered these efforts. Like other protozoa, T. cruzi invades the host cell and this complex interplay can determine the outcome of the infection: the parasite must manipulate host cell signaling pathways to achieve its purpose. Therefore, targeting the host signals that promote T. cruzi infection can be therapeutically valuable. Poly(ADP-ribose) (PAR) ?crucial for DNA damage response among other processes- participates in host cell response to the parasitic infection: Poly(ADP-ribose)polymerase-1 inhibitors decrease T. cruzi infection while Poly(ADP-ribose)glycohydrolase (PARG) inhibition or silencing almost completely abrogates it, raising interest in PAR signaling and the role of PARG in the host-parasite interaction.PAR levels raised early after infection (15 min) and remained elevated during the complete cell infection cycle, as determined by ELISA using a PAR-detecting reagent. PARG inhibition by DEA 1 μM or silencing by shRNA caused reduced T. cruzi cell invasion, indicating that PARG might be important during this initial step. T. cruzi can invade the host cell by lysosome-independent, lysosome-dependent and autophagic pathways but they must all culminate in the fusion of the trypomastigote-bearing parasitophorous vacuole (TcPV) to lysosomes. Absence of PARG activity did not hamper the formation of TcPV with early endosomal characteristics as shown by staining against EEA1 (early endosomal antigen) or the use of a FYVE-eGFP probe to detect PIP3-rich vacuoles, nor did it affect infection levels when cells were subjected to nutritional stress, suggesting PARG is not participating in the initial stages of the lysosome-independent and autophagic pathways. However, PARG activity seems crucial for lysosomal function: PARG-inhibited or silenced Vero cells showed reduced DQ-BSA Red and Lysotracker DND-99 staining, indicating proteolytic activity and pH are altered. A drastic reduction in LAMP-1 signal was also detected. PARG inhibition and silencing also appears to affect the reorganization of host cell cytoskeleton during T. cruzi invasion. These results indicate that PARG activity is important for the maintenance of lysosomal activity, which is crucial for the initial steps of T. cruzi infection.