Host Cell Poly(ADP-Ribose) Glycohydrolase Is Crucial for Trypanosoma cruzi Infection Cycle

Abstract

Trypanosoma cruzi, etiological agent of Chagas’ disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas’ disease.

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei

“…Moreover, it is always located in the nucleus in procyclic cultures, independently of the occurrence of genomic harm (Additional file 4 A). This is similar to what we observed earlier for T. cruzi PARG ( Tc PARG) [ 33 ].…”

“…Immunolocalization experiments were carried out as detailed in our previous work [ 33 ] with 1:500 polyclonal anti- PAR antibody (BD) made in rabbit to detect PAR and 1:100 polyclonal anti- Tb PARP antibody (GenScript) made in rabbit to detect Tb PARP. Nuclear and kinetoplast DNA were stained with DAPI (2 μg/mL) (Sigma).…”

VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt

“…As alterations in PARP or poly-ADP-ribosylation (PARylation) levels are detected in several pathological conditions ( Cerboni et al, 2010 ; Masutani, Nakagama & Sugimura, 2005 ; Strosznajder et al, 2012 ; Virag & Szabo, 2002 ), and PARP or PARG inhibition interferes with T. cruzi infection and proliferation of the parasite ( Vilchez Larrea et al, 2012 ; Vilchez Larrea et al, 2013 ), PAR biology studies may have far reaching biomedical implications.…”

Poly(ADP-Ribose) Polymerases in Host-Pathogen Interactions, Inflammation, and Immunity

“…PARG was shown to be integral to the growth of Trypanosoma cruzi epimastigotes, and PARG inhibition caused changes to the cell cycle in the parasite. Knockdown of host cell PARG also abrogated T. cruzi infection (485). In Trypanosoma brucei, PARG knockdown led to increased sensitivity to oxidative stress (486).…”