The overexpression of TcAP1 endonuclease confers resistance to infective Trypanosoma cruzi trypomastigotes against oxidative DNA damage

Abstract

Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.

Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival

“…9 The purified recombinant TcAP1DN-GFP as well as the human hAPE1DN-GFP fusion proteins did not show any AP endonuclease activity and substantially inhibited TcAP1 enzymatic activity in in vitro assays. 7,9 However, TcAP1DN-GFP expressed in T. cruzi epimastigotes did not decrease their viability when submitted to oxidative species. 9 Here, we show that a hAPE1DN-GFP expressed in epimastigotes significantly decrease their viability when exposed to H 2 O 2 as compared with control parasites (Figure 2).…”

“…7,9 However, TcAP1DN-GFP expressed in T. cruzi epimastigotes did not decrease their viability when submitted to oxidative species. 9 Here, we show that a hAPE1DN-GFP expressed in epimastigotes significantly decrease their viability when exposed to H 2 O 2 as compared with control parasites (Figure 2). This different behaviour may be explained by the low level of amino acid identity between TcAP1 and hAPE1 endonucleases (34% identity), which could result in a lower affinity of the parasite TcAP1 negative form for its AP DNA substrate than its human APE1 negative dominant counterpart.…”

“…2.1 | Epimastigote culture T. cruzi epimastigotes (Y strain) were cultivated at 28°C in LIT medium. 9 2.2 | pTREX-hape1dn-gfp expression vector construction…”

“…2.6 | hAPE1DN-GFP parasite immunolocalization Smears of epimastigotes, metacyclic trypomastigotes, and cell-derived trypomastigotes expressing the hAPE1DN-GFP protein were fixed, processed, and the recombinant enzyme recognized as indicated elsewhere. 9 For nuclear and kinetoplast DNA staining 4′,6-diamidino-2-phenylindole (DAPI) was used. Cells were observed and photographed under a Nikon Eclipse E400 fluorescence microscopy using a 430 ± 20 nm filter and a 520 ± 20 nm for blue and green fluorescence, respectively.…”

The Parasite Load of Trypanosoma cruzi Modulates Feeding and Defecation Patterns of the Chagas Disease Vector Triatoma infestans

“…Three Mus musculus (Linnaeus, 1758) (Rodentia: Muridae) of the BALB/c strain, from the Bioterio Central, Facultad de Medicina, Universidad de Chile, were used as acutely infected hosts under laboratory conditions to generate the rest of the infected T. infestans group, which was authorized by the Bioethics Committee of the Facultad de Medicina, Universidad de Chile (protocol N° 19262-MED-UCH). The infection process of the mice consisted of an intraperitoneal inoculation of the Dm28c strain (DTU TcI) of T. cruzi obtained from cell cultures (Vero cells, 60–70% confluence) as previously described [ 43 ], at a concentration of 1000 parasites in 100 microliters of RPMI 1640 medium (Biological Industries™, Beit Haemek, Israel). Infection of the mice was confirmed by the inspection of blood samples by light microscopy using a Neubauer chamber, measuring the concentration and viability of parasites from day 7 post-infection until reaching ranges between 80,000 and 500,000 parasites/mL.…”