Abstract

Pathological processes such as bacterial, viral and parasitic infections can generate a plethora of responses such as, but not restricted to, oxidative stress that can be harmful to the host and the pathogen. This stress occurs when there is an imbalance between reactive oxygen species produced and antioxidant factors produced in response to the infection. This imbalance can lead to DNA lesions in both infected cells as well as in the pathogen. The effects of the host response on the parasite lead to several kinds of DNA damage, causing alterations in the parasite’s metabolism; the reaction and sensitivity of the parasite to these responses are related to the DNA metabolism and life cycle of each parasite. The present review will discuss the survival strategies developed by host cells and Trypanosoma cruzi, focusing on the DNA repair mechanisms of these organisms throughout infection including the relationship between DNA damage, stress response features, and the unique characteristics of these diseases.

Keywords:
Trypanosoma cruzi; reactive oxygen species; DNA repair

Introduction

The Kinetoplastida order comprises several Protozoa that can be parasitic or not. They share some unique characteristics, most remarkably the presence of a single and unique organelle called a kinetoplast. In this order, three of the major parasitic pathogens of medical importance are part of the Trypanosomatid family: Leishmania spp. (the etiological agent of the various forms of leishmaniasis), Trypanosoma brucei (the causative agent of African sleep sickness) and Trypanosoma cruzi (the etiological agent of Chagas disease, also named American trypanosomiasis). All these organisms have a heteroxenous life cycle, needing two hosts one invertebrate and another vertebrate; therefore, they present more than one parasitic form (Chagas, 1909Chagas C (1909) Nova tripanozomiaze humana: estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzi n. gen., n. sp., ajente etiolojico de nova entidade morbida do homem. Mem Inst Oswaldo Cruz 1:159–218.; Rassi et al., 2010Rassi A, Rassi A and Marin-Neto JA (2010) Chagas disease. Lancet 375:1388–1402.; Cantey et al., 2012Cantey PT, Stramer SL, Townsend RL, Kamel H, Ofafa K, Todd CW, Currier M, Hand S, Varnado W, Dotson E et al. (2012) The United States Trypanosoma cruzi Infection Study: Evidence for vector-borne transmission of the parasite that causes Chagas disease among United States blood donors. Transfusion 52:1922–1930.; Centers for Disease and Control Prevention, 2018Centers for Disease and Control Prevention (2018) Parasites – Leishmaniasis, https://www.cdc.gov/parasites/leishmaniasis/biology.html (accessed 14 December 2018).
https://www.cdc.gov/parasites/leishmania... ). Generally, diseases caused by trypanosomatids are zoonotic, i.e., they have animals as a natural reservoir and infection in humans is dependent on the maintenance of these reservoirs to sustain endemicity. Although T. cruzi and T. brucei are in the same genus, they have a distinct life cycle. Information about the T. cruzi life cycle is summarized in ^{Box 1} Box 1 Trypanosoma cruzi life cycle Trypanosoma cruzi is a heteroxenous parasite with a digenetic life cycle that involves two hosts: an invertebrate one, usually an insect of the order Triatomina and a vertebrate host. In each of these hosts and stages of development, the parasite assumes a different morphology. T. cruzi infection can occur by transfusion using contaminated blood, oral infection via contaminated food, or by direct infection by the insect vector. In a typical T. cruzi life cycle, the trypomastigote form of the parasite is deposited in the feces of the insect vector. The infection occurs when the feces come into contact with any mucosal tissue or the bloodstream. After that, the parasite is able to infect several types of cells, including macrophages, cardiomiocytes, adipocytes, epithelial cells, and neurons. During infection, T. cruzi trypomastigotes form a parasitophorous vacuole inside the host cell, and then escape from this vacuole into the cytoplasm. The parasite then differentiates into the replicative amastigote cell, further replicates, and transforms again into the trypomastigote form. Finally, the host cell bursts and releases new parasites to continue the infection cycle. When another insect vector feeds on blood containing trypomastigotes parasites, T. cruzi then reaches the intestinal tract of this insect. In the insect gut, the parasite differentiates into the replicative epimastigote form, replicates again, then differentiates into the trypomastigote, which then undergoes another infection cycle within the mammalian host. .

Several reviews have already addressed the particularities of how the trypanosomatids escape from the immune system (Oladiran and Belosevic, 2012Oladiran A and Belosevic M (2012) Immune evasion strategies of trypanosomes: A review. J Parasitol 98:284–292.; Geiger et al., 2016Geiger A, Bossard G, Sereno D, Pissarra J, Lemesre JL, Vincendeau P and Holzmuller P (2016) Escaping deleterious immune response in their hosts: Lessons from trypanosomatids. Front Immunol 7:1–21.; Weatherly et al., 2016Weatherly DB, Peng D and Tarleton RL (2016) Recombination-driven generation of the largest pathogen repository of antigen variants in the protozoan Trypanosoma cruzi. BMC Genomics 17:729.). Although T. brucei has recently been reported in the adipocytes of mice (Trindade et al., 2016Trindade S, Rijo-Ferreira F, Carvalho T, Pinto-Neves D, Guegan F, Aresta-Branco F, Bento F, Young SA, Pinto A, Van Den Abbeele J et al. (2016) Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice. Cell Host Microbe 19:837-848.), this parasite is present in a bloodstream-specific form in mammals and, therefore, has to deal with the attack of the host’s immune system. To be able to escape from the immune system, T. brucei has an extensive repertoire of variant surface glycoproteins (VSGs) that must be constantly exchanged (Taylor and Rudenko, 2006Taylor JE and Rudenko G (2006) Switching trypanosome coats: What’s in the wardrobe? Trends Genet 22:614-620.); this indicates a role for homologous recombination in the DNA metabolism of this parasite (McCulloch and Barry, 1999McCulloch R and Barry JD (1999) A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation. Genes Dev 13:2875–2888.; Glover et al., 2008Glover L, McCulloch R and Horn D (2008) Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes. Nucleic Acids Res 36:2608–2618.; Horn and McCulloch, 2010Horn D and McCulloch R (2010) Molecular mechanisms underlying the control of antigenic variation in African trypanosomes. Curr Opin Microbiol 13:700–705.).

For T. cruzi, the first barrier is immune system recognition in the bloodstream. In the mammalian host, bloodstream T. cruzi has to combat the host’s complement system, and therefore, the parasite expresses several proteins that interfere with the complement system. One of those proteins is T-DAF (trypomastigote decay-accelerating-factor), a protein capable of blocking the assembly of the C3 convertases from the complement system. The parasite also expresses several complement regulatory proteins (CRPs) and calreticulin (CRT) that also interfere with the complement system (Norris et al., 1991Norris KA, Bradt B, Cooper NR and So M (1991) Characterization of a Trypanosoma cruzi C3 binding protein with functional and genetic similarities to the human complement regulatory protein, decay-accelerating factor. J Immunol 147:2270-2277.; Tambourgi et al., 1993Tambourgi DV, Kipnis TL, Silva WD, Joiner KA, Sher A, Heath S, Hall BF and Ogden GB (1993) A partial cDNA clone of trypomastigote decay-accelerating factor (T-DAF), a developmentally regulated complement inhibitor of Trypanosoma cruzi, has genetic and functional similarities to the human complement inhibitor DAF. Infect Immun 61:3656-3663.; Cestari et al., 2009Cestari IS, Krarup A, Sim RB, Inal JM and Ramirez MI (2009) Role of early lectin pathway activation in the complement-mediated killing of Trypanosoma cruzi. Mol Immunol 47:426-37.; Ramírez et al., 2011Ramírez G, Valck C, Molina MC, Ribeiro CH, López N, Sánchez G, Ferreira VP, Billetta R, Aguilar L, Maldonado I et al. (2011) Trypanosoma cruzi calreticulin: A novel virulence factor that binds complement C1 on the parasite surface and promotes infectivity. Immunobiology 216:265-273.). The parasite also possesses trans-sialidases proteins that interfere with host lymphocytes as well as sialylated mucins and cruzipain that actively protect the parasite against antibodies from the host immune system (Berasain et al., 2003Berasain P, Carmona C, Frangione B, Cazzulo JJ and Goñi F (2003) Specific cleavage sites on human IgG subclasses by cruzipain, the major cysteine proteinase from Trypanosoma cruzi. Mol Biochem Parasitol 130:23-9.; Giorgi and De Lederkremer, 2011Giorgi EM and De Lederkremer RM (2011) Trans-sialidase and mucins of Trypanosoma cruzi: An important interplay for the parasite. Carbohydr Res 346:1389-1393.; Alvarez et al., 2012Alvarez VE, Niemirowicz GT and Cazzulo JJ (2012) The peptidases of Trypanosoma cruzi: Digestive enzymes, virulence factors, and mediators of autophagy and programmed cell death. Biochim Biophys Acta 1824:195-206.). The intracellular amastigote form requires an extensive repertoire of oxidative response proteins to cope with the new intracellular environment. During the intracellular phase, the host cell activates several oxidases in the parasitophorous vacuole, creating an oxidative burst via the generation of several reactive oxygen species (ROS) that can attack the parasite. The parasite’s response repertoire must also include DNA damage response proteins to oxidative stress (Passos-Silva et al., 2010Passos-Silva DG, Rajão MA, Nascimento De Aguiar PH, Vieira-Da-Rocha JP, Machado CR and Furtado C (2010) Overview of DNA repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major. J Nucleic Acids 2010:840768.; van Loon et al., 2010van Loon B, Markkanen E and Hübscher U (2010) Oxygen as a friend and enemy: How to combat the mutational potential of 8-oxo-guanine. DNA Repair (Amst) 9:604-616.; Alvarez et al., 2011Alvarez MN, Peluffo G, Piacenza L and Radi R (2011) Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: Consequences for oxidative killing and role of microbial peroxiredoxins in infectivity. J Biol Chem 286:6627-40.; Genois et al., 2014Genois MM, Paquet ER, Laffitte MCN, Maity R, Rodrigue A, Ouellette M and Masson JY (2014) DNA repair pathways in trypanosomatids: From DNA repair to drug resistance. Microbiol Mol Biol Rev 78:40–73.; Machado-Silva et al., 2016Machado-Silva A, Cerqueira PG, Grazielle-Silva V, Gadelha FR, Peloso EF, Teixeira SMR and Machado CR (2016) How Trypanosoma cruzi deals with oxidative stress: Antioxidant defence and DNA repair pathways. Mutat Res 767:8–22.).

During Chagas disease, T. cruzi has an intracellular form that must also resist the oxidative insult caused by the host cell. The primary response against the parasite is the oxidative burst caused during the infection, which consists mainly of superoxide anions and ROS (Müller et al., 2003Müller S, Liebau E, Walter RD and Krauth-Siegel RL (2003) Thiol-based redox metabolism of protozoan parasites. Trends Parasitol 19:320-328.; Alvarez et al., 2004Alvarez MN, Piacenza L, Irigoín F, Peluffo G and Radi R (2004) Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi. Arch Biochem Biophys 432:222-32.; Piacenza et al., 2009aPiacenza L, Alvarez MN, Peluffo G and Radi R (2009a) Fighting the oxidative assault: the Trypanosoma cruzi journey to infection. Curr Opin Microbiol 12:415-421.,bPiacenza L, Zago MP, Peluffo G, Alvarez MN, Basombrio MA and Radi R (2009b) Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39:1455-1464.). This insult is the most deleterious for the parasite and, therefore, the most studied. These pathological processes that generate oxidative stress can be very harmful to the individual and pathogen (Nathan and Shiloh, 2002Nathan C and Shiloh MU (2002) Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97:8841-8848.). Stress occurs when there is an unbalance between reactive oxygen species (ROS: hydroxyl radical, nitric oxide, superoxide, hydrogen peroxide, hypochlorous acid, and singlet oxygen) and antioxidant species (superoxide dismutase, glutathione reductase, glutathione, α-tocopherol, and ascorbic acid). The main reactive oxygen species (ROS) targets include DNA, RNA, lipids, proteins, and carbohydrates. However, DNA lesions result in cell cycle arrest and death, as it is the molecule responsible for genetic information of all cells from a single organism (Berra et al., 2006Berra CM, Menck CFM and Di Mascio P (2006) Estresse oxidativo, lesões no genoma e processos de sinalização no controle do ciclo celular. Quim Nova 29:1340-44.). Beyond oxidative stress, the importance of preserving genomic integrity is evident by the fact that DNA repair mechanisms are present in all organisms, as we have the description of DNA repair proteins and enzymes from bacteria to higher eukaryotes (Hoeijmakers, 2001Hoeijmakers JHJ (2001) Genome maintenance mechanism for preventing cancer. Nature 411:366–374.; DiRuggiero and Robb, 2004DiRuggiero J and Robb FT (2004) Early evolution of DNA repair mechanisms. In: Pouplana LR (ed) The Genetic Code and the Origin of Life. Kluwer Academic / Plenum Publishers, New York, pp 169–182.; Cagney et al., 2006Cagney G, Alvaro D, Reid RJD, Thorpe PH, Rothstein R and Krogan NJ (2006) Functional genomics of the yeast DNA-damage response. Genome Biol 7:7–10.).

During the cell cycle, DNA repair pathways ensure the fidelity of the DNA information transferred (Friedberg, 2008Friedberg EC (2008) A brief history of the DNA repair field. Cell Res 18:3–7.). The complexity of these processes becomes clear when we consider that, for any eukaryotic cell, there are numerous sources of DNA damage, which need to be detected and repaired (Hoeijmakers, 2001Hoeijmakers JHJ (2001) Genome maintenance mechanism for preventing cancer. Nature 411:366–374.; Hakem, 2008Hakem R (2008) DNA-damage repair; the good, the bad, and the ugly. EMBO J 27:589-605.; Tubbs and Nussenzweig, 2017Tubbs A and Nussenzweig A (2017) Endogenous DNA damage as a source of genomic instability in cancer. Cell 168:644-656.). DNA damage response leads to different cellular effects, such as cell cycle arrest, activation of distinct signaling pathways, and modulation of the DNA metabolism pathways (Hoeijmakers, 2001Hoeijmakers JHJ (2001) Genome maintenance mechanism for preventing cancer. Nature 411:366–374.; Marnett and Plastaras, 2001Marnett LJ and Plastaras JP (2001) Endogenous DNA damage and mutation. Trends Genet 17:214–221.; Harper and Elledge, 2007Harper JW and Elledge SJ (2007) The DNA damage response: Ten years after. Mol Cell 28:739–745.; Lord and Ashworth, 2012Lord CJ and Ashworth A (2012) The DNA damage response and cancer therapy. Nature 481:287–294.; Imlay, 2013Imlay JA (2013) The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium. Nat Rev Microbiol 11:443–454.). Therefore, although the DNA damage response is divided into pathways, the alterations in the cell comprise other metabolic pathways that are not so logical at first glance.

In this review, we will discuss what is already known about DNA metabolism and repair during T. cruzi infection and how these processes affect host cell metabolism. Thus, we will explore how DNA metabolism may be related to alterations in cellular metabolism and how these changes impact the pathogenesis of Chagas disease.

Metabolic effects of oxidative stress in T. cruzi

As an obligate intracellular parasite, T. cruzi has to combat oxidative stress generated by the host cell. The primary host response against the parasite is the oxidative burst caused by the infection, which consists mainly of superoxide anions and ROS (Müller et al., 2003Müller S, Liebau E, Walter RD and Krauth-Siegel RL (2003) Thiol-based redox metabolism of protozoan parasites. Trends Parasitol 19:320-328.; Alvarez et al., 2004Alvarez MN, Piacenza L, Irigoín F, Peluffo G and Radi R (2004) Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi. Arch Biochem Biophys 432:222-32.; Piacenza et al., 2009aPiacenza L, Alvarez MN, Peluffo G and Radi R (2009a) Fighting the oxidative assault: the Trypanosoma cruzi journey to infection. Curr Opin Microbiol 12:415-421.,bPiacenza L, Zago MP, Peluffo G, Alvarez MN, Basombrio MA and Radi R (2009b) Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39:1455-1464.).

With such specific environmental insults during the parasite life cycle, T. cruzi reacts with several metabolic responses to attempt to prevent the damage caused by ROS. A significant protein in this response is a parasite-specific superoxide dismutase (SOD), a protein already reported to protect the parasite from intracellular-generated ROS by macrophages (Mateo et al., 2008Mateo H, Marín C, Pérez-Cordón G and Sánchez-Moreno M (2008) Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi. Mem Inst Oswaldo Cruz 103:271-276.; Martinez et al., 2018Martinez A, Prolo C, Estrada D, Rios N, Piñeyro D, Robello C, Radi R and Piacenza L (2018) Cytosolic Fe-superoxide dismutase protects Trypanosoma cruzi from macrophage-derived superoxide radical increasing pathogen virulence in vivo. Free Radic Biol Med 20:S93.). SOD is an enzyme that catalyzes the dismutation of superoxide (O₂^-) into hydrogen peroxide (H₂O₂). In T. cruzi the SOD enzyme does not possess a copper/zinc or manganese mechanism; T. cruzi SODs are present in four iron-dependent versions of SOD (FeSOD) A, B1, B2, and C. It has been demonstrated that the overexpression of FeSOD C within the mitochondria was able to improve the resistance of the pathogen to ROS generated by the presence of bovine serum (Boveris and Stoppani, 1977Boveris A and Stoppani AOM (1977) Hydrogen peroxide generation in Trypanosoma cruzi. Experientia 33:1306-1308.; Piacenza et al., 2007Piacenza L, Irigoín F, Alvarez MN, Peluffo G, Taylor MC, Kelly JM, Wilkinson SR and Radi R (2007) Mitochondrial superoxide radicals mediate programmed cell death in Trypanosoma cruzi?: Cytoprotective action of mitochondrial iron superoxide dismutase overexpression. Biochem J 403:323-334.).

Contrary to most organisms, trypanosomatid glutathione is a tiny fraction of the pool of enzymes. Most eukaryotes rely on a system composed of glutathione and glutathione reductase in a system that uses glutathione as reductive power to neutralize oxidative molecules, in a reaction catalyzed by glutathione reductase (Krauth-Siegel et al., 2003Krauth-Siegel RL, Meiering SK and Schmidt H (2003) The parasite-specific trypanothione metabolism of trypanosoma and leishmania. Biol Chem 384:539-49.; Müller et al., 2003Müller S, Liebau E, Walter RD and Krauth-Siegel RL (2003) Thiol-based redox metabolism of protozoan parasites. Trends Parasitol 19:320-328.). In these parasites, the trypanothione system is responsible for protecting those organisms from oxidative insult (Fairlamb et al., 1985Fairlamb AH, Blackburn P, Ulrich P, Chait BT and Cerami A (1985) Trypanothione: A novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 227:1485-1487.; Fairlamb and Cerami, 1992Fairlamb AH and Cerami A (1992) Metabolism and functions of trypanothione in the kinetoplastida. Annu Rev Microbiol 46:695-729.; Meziane-Cherif et al., 1994Meziane-Cherif D, Aumercier M, Kora I, Sergheraert C, Tartar A, Dubremetz JF and Ouaissi MA (1994) Trypanosoma cruzi: Immunolocalization of trypanothione reductase. Exp Parasitol 79:536-41.). This protein is homologous to the human glutathione reductase, an enzyme capable of performing the reduction of glutathione disulfide into the sulfhydryl form glutathione (Fairlamb et al., 1985Fairlamb AH, Blackburn P, Ulrich P, Chait BT and Cerami A (1985) Trypanothione: A novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 227:1485-1487.; Piacenza et al., 2009bPiacenza L, Zago MP, Peluffo G, Alvarez MN, Basombrio MA and Radi R (2009b) Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39:1455-1464.). Although this protein is essential to the response of T. cruzi to oxidative assault, there is still some debate regarding its subcellular localization in the parasite, as the presence of trypanothione reductase was reported in the parasite’s kinetoplast (Meziane-Cherif et al., 1994Meziane-Cherif D, Aumercier M, Kora I, Sergheraert C, Tartar A, Dubremetz JF and Ouaissi MA (1994) Trypanosoma cruzi: Immunolocalization of trypanothione reductase. Exp Parasitol 79:536-41.), yet fractionation studies showed that this localization might be more diffuse than previously thought (Wilkinson et al., 2002Wilkinson SR, Meyer DJ, Taylor MC, Bromley EV, Miles MA and Kelly JM (2002) The Trypanosoma cruzi enzyme TcGPXI is a glycosomal peroxidase and can be linked to trypanothione reduction by glutathione or tryparedoxin. J Biol Chem 277:17062-17071.).

One of the main classes of proteins involved in the cytosolic response against ROS in T. cruzi is the family of peroxiredoxins. Two of these well-characterized proteins in T. cruzi are the mitochondrial and cytosolic tryparedoxin perioxidase proteins (TcMPx and TcCPx, respectively) (Piacenza et al., 2009bPiacenza L, Zago MP, Peluffo G, Alvarez MN, Basombrio MA and Radi R (2009b) Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39:1455-1464.). Overexpression of these proteins can confer resistance to peroxynitrite. Expression of both proteins differs among the T. cruzi forms, as it is higher in metacyclic trypomastigotes than in epimastigotes (Piacenza et al., 2009bPiacenza L, Zago MP, Peluffo G, Alvarez MN, Basombrio MA and Radi R (2009b) Enzymes of the antioxidant network as novel determiners of Trypanosoma cruzi virulence. Int J Parasitol 39:1455-1464.). The existence of several proteins to deal with oxidative stress indicates how important the oxidative response is for the parasite.

DNA repair and response to oxidative stress in T. cruzi

The oxidative stress that surpasses the first line of defense of the parasite, composed by the aforementioned antioxidant proteins, constitutes a potential source of damage to the intracellular components of the organism. One primary target of ROS in living organisms is DNA. Among many effects, oxidative stress can generate apurinic/apyrimidinic (AP) sites and base modifications, one of the most common of which is the modified base 7,8-dihydro-8-oxoguanine also known as 8-oxoguanine (8-oxodG) (Kanvah et al., 2010Kanvah S, Joseph J, Schuster GB, Barnett RN, Cleveland CL and Landman UZI (2010) Oxidation of DNA: Damage to nucleobases. Acc Chem Res 43:280-87.). Further studies of the response of T. cruzi to the damage caused by 8-oxoguanine in each forms of the parasite are necessary since one of the two crucial drugs used against this parasite, benznidazole (BZN), is able to induce oxidative stress primarily by targeting guanine in the nucleotide pool of the parasite (Rajão et al., 2014Rajão MA, Furtado C, Alves CL, Passos-Silva DG, De Moura MB, Schamber-Reis BL, Kunrath-Lima M, Zuma AA, Vieira-da-Rocha JP, Garcia JBF et al. (2014) Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55:309–321.).

8-oxoguanine can be generated directly on double-stranded DNA, but also by the oxidation of the guanine present on the cell nucleotide pool (Mahon et al., 2007Mahon KP, Potocky TB, Blair D, Roy MD, Stewart KM, Chiles TC and Kelley SO (2007) Deconvolution of the cellular oxidative stress response with organelle-specific peptide conjugates. Chem Biol 14:923–930.; Aguiar et al., 2013Aguiar PHN, Furtado C, Repolês BM, Ribeiro GA, Mendes IC, Peloso EF, Gadelha FR, Macedo AM, Franco GR, Pena SDJ et al. (2013) Oxidative stress and DNA lesions: The role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability. PLoS Negl Trop Dis 7:e2279.). This modified base mutagenicity is due to its ability to cause transversions when not corrected by the DNA repair system since replicative polymerases can incorporate cytosine or adenine in opposition to 8-oxodG. Therefore, this modified base can be incorporated in opposition to an adenine, causing the transversion AT to CG (Cadet et al., 2003Cadet J, Douki T, Gasparutto D and Ravanat JL (2003) Oxidative damage to DNA: Formation, measurement and biochemical features. Mutat Res 531:5-23.; Barzilai and Yamamoto, 2004Barzilai A and Yamamoto KI (2004) DNA damage responses to oxidative stress. DNA Repair (Amst) 3:1109-15.; van Loon et al., 2010van Loon B, Markkanen E and Hübscher U (2010) Oxygen as a friend and enemy: How to combat the mutational potential of 8-oxo-guanine. DNA Repair (Amst) 9:604-616.).

There are several pieces of evidence indicating that DNA repair factors are not restricted to one pathway, but instead can be involved with the regulation and control of multiple DNA repair pathways (Nemzow et al., 2015Nemzow L, Lubin A, Zhang L and Gong F (2015) XPC: Going where no DNA damage sensor has gone before. DNA Repair (Amst) 36:19–27.). Although the response against oxidative insult is very complex and involves multiple metabolic alterations (Pascucci et al., 2011Pascucci B, D’Errico M, Dogliotti E, Giovannini S and Parlanti E (2011) Role of nucleotide excision repair proteins in oxidative DNA damage repair: An updating. Biochem 76:4-15.; Melis et al., 2013Melis JPM, van Steeg H and Luijten M (2013) Oxidative DNA damage and nucleotide excision repair. Antioxid Redox Signal 18:2409–2419.), the primary pathway involved in DNA repair is base excision repair (BER) (Hoeijmakers, 2001Hoeijmakers JHJ (2001) Genome maintenance mechanism for preventing cancer. Nature 411:366–374.; Barzilai and Yamamoto, 2004Barzilai A and Yamamoto KI (2004) DNA damage responses to oxidative stress. DNA Repair (Amst) 3:1109-15.). In general terms, the BER pathway consists of several DNA glycosylases, each one specifically recognizing one type of modified base. These proteins can flip the modified base out of the DNA strand and cleave it from the sugar-phosphate backbone. This cleavage will generate an AP site, cleaned by some endonucleases; sometimes this hydrolysis occurs spontaneously (Meadows et al., 2003Meadows KL, Song B and Doetsch PW (2003) Characterization of AP lyase activities of Saccharomyces cerevisiae Ntg1p and Ntg2p: Implications for biological function. Nucleic Acids Res 31:5560-5567.). From this point forward the pathway can take two directions: the short-patch BER, in which a polymerase, such as polymerase β (Polβ), performs a single nucleotide gap fill on the damage site, or the long-patch BER, in which, in coordination with proliferating cell nuclear antigen (PCNA), polymerization of a more substantial portion of the DNA and displacement of the damaged single-strand occurs, generating a DNA flap. Next, the DNA flap is removed by flap endonuclease 1 (FEN1), and the DNA nick is closed by a specific DNA ligase (Hoeijmakers, 2001Hoeijmakers JHJ (2001) Genome maintenance mechanism for preventing cancer. Nature 411:366–374.; David et al., 2010David SS, Shea VLO and Kundu S (2010) Base excision repair of oxidative DNA damage. Nature 447:941–950.; Wallace 2014Wallace SS (2014) Base excision repair: A critical player in many games. DNA Repair (Amst) 19:14–26.).

The oxidative response is one of the main fields of study for T. cruzi and proteins involved in all steps of both BER pathways have already been described (Passos-Silva et al., 2010Passos-Silva DG, Rajão MA, Nascimento De Aguiar PH, Vieira-Da-Rocha JP, Machado CR and Furtado C (2010) Overview of DNA repair in Trypanosoma cruzi, Trypanosoma brucei, and Leishmania major. J Nucleic Acids 2010:840768.; Genois et al., 2014Genois MM, Paquet ER, Laffitte MCN, Maity R, Rodrigue A, Ouellette M and Masson JY (2014) DNA repair pathways in trypanosomatids: From DNA repair to drug resistance. Microbiol Mol Biol Rev 78:40–73.). One significant protein already reported in T. cruzi is the homolog for AP endonuclease, the enzyme responsible for cleaning AP sites from the genome, thereby preventing cytotoxic effects during replication. Two AP endonuclease (APE) homologs were reported: TcAPE1 (orthologous to Homo sapiens APE1) and TcAP2 (orthologous to Homo sapiens APE 2 and Schizosaccharomyces pombe Apn2p) (Sepúlveda et al., 2014Sepúlveda S, Valenzuela L, Ponce I, Sierra S, Bahamondes P, Ramirez S, Rojas V, Kemmerling U, Galanti N and Cabrera G (2014) Expression, functionality, and localization of apurinic/apyrimidinic endonucleases in replicative and non-replicative forms of Trypanosoma cruzi. J Cell Biochem 115:397–409.). These proteins were identified in replicative forms of the parasite, demonstrating the importance of their expression for accurate DNA replication. The overexpression of these proteins confers higher resistance to oxidative stress, especially in a continuous and persistent oxidative environment, when compared with wildtype (WT) strains, thus demonstrating how essential DNA repair is to the survival of the parasite in the face of oxidative attacks (Sepúlveda et al., 2014Sepúlveda S, Valenzuela L, Ponce I, Sierra S, Bahamondes P, Ramirez S, Rojas V, Kemmerling U, Galanti N and Cabrera G (2014) Expression, functionality, and localization of apurinic/apyrimidinic endonucleases in replicative and non-replicative forms of Trypanosoma cruzi. J Cell Biochem 115:397–409.). Although there is much investigation into the epimastigote form, there is lack information regarding the influence of these genes in the amastigote form of the parasite.

One piece of evidence for the presence of the short-patch BER in T. cruzi is the observation that the parasite can cope with uracil by using the protein Tc-uracil DNA glycosylase (TcUNG) that removes misincorporated uracil in single-strand DNA in front of cytosine and adenine substrates (Peña-Diaz et al., 2004Peña-Diaz J, Akbari M, Sundheim O, Esther Farez-Vidal M, Andersen S, Sneve R, Gonzalez-Pacanowska D, Krokan HE and Slupphaug G (2004) Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair. J Mol Biol 342:787–799.). Evidence of a canonical BER pathway was provided by a study that reported that the function of TcUNG is increased in vitro in the presence of AP endonuclease (Farez-Vidal, 2001Farez-Vidal ME (2001) Characterization of uracil-DNA glycosylase activity from Trypanosoma cruzi and its stimulation by AP endonuclease. Nucleic Acids Res 29:1549–55.), suggesting that these two proteins act on the same pathway. The long-patch repair pathway also seems to be present in all forms of T. cruzi since a recent paper described the presence of Tc-Flap endonuclease 1 (TcFEN1), a homolog of human FEN1. In this work, TcFEN1 increased parasite resistance to oxidative stress when overexpressed; this protein deals with DNA intermediates containing a 5’ flap, showing a protein specific to LP-BER in this parasite (Ponce et al., 2017Ponce I, Aldunate C, Valenzuela L, Sepúlveda S, Garrido G, Kemmerling U, Cabrera G and Galanti N (2017) A flap endonuclease (TcFEN1) is involved in Trypanosoma cruzi cell proliferation, DNA repair, and parasite survival. J Cell Biochem 118:1722–1732.).

Among DNA modifications caused by oxidative stress, the most prevalent is the generation of 8-oxodG. Nevertheless, several groups of organisms have a BER subpathway, the GO system, which can handle damage caused by 8-oxodG (Michaels and Miller, 1992Michaels ML and Miller JH (1992) The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J Bacteriol 174:6321-6325.; Nash et al., 1996Nash HM, Bruner SD, Schärer OD, Kawate T, Addona TA, Spooner E, Lane WS and Verdine GL (1996) Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily. Curr Biol 6:968-980.; Slupska et al., 1996Slupska MM, Baikalov C, Luther WM, Chiang JH, Wei YF and Miller JH (1996) Cloning and sequencing a human homolog (hMYH) of the Escherichia coli mutY gene whose function is required for the repair of oxidative DNA damage. J Bacteriol 178:3885-3892.; Radicella et al., 1997Radicella JP, Dherin C, Chantal D, Fox MS and Boiteux S (1997) Cloning and characterization of hOGG1, a human homolog of the OGG1 gene of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 94:8010-8015.; Kuipers et al., 2000Kuipers GK, Slotman BJ, Poldervaart HA, Reitsma-Wijker CA and Lafleur MVM (2000) The influence of combined Fpg- and MutY-deficiency on the spontaneous and γ-radiation-induced mutation spectrum in the lacZα gene of M13mp10. Mutat Res 461:189-195.; Barzilai and Yamamoto, 2004Barzilai A and Yamamoto KI (2004) DNA damage responses to oxidative stress. DNA Repair (Amst) 3:1109-15.; Hwang et al., 2014Hwang BJ, Shi G and Lu AL (2014) Mammalian MutY homolog (MYH or MUTYH) protects cells from oxidative DNA damage. DNA Repair (Amst) 13:10-21.; Boiteux et al., 2017Boiteux S, Coste F and Castaing B (2017) Repair of 8-oxo-7,8-dihydroguanine in prokaryotic and eukaryotic cells: Properties and biological roles of the Fpg and OGG1 DNA N-glycosylases. Free Radic Biol Med 107:179-201.). Three proteins from the GO system are significant in prokaryotic cells: MutM (also named FPG), repairs 8-oxodG already misincorporated to cytosine. MutY, removes an adenine misincorporated in front of 8-oxodG in double-stranded DNA, and MutT hydrolyzes 8-oxod-GTP into its monophosphate form, preventing its misincorporation into the DNA molecule by DNA polymerase (Michaels and Miller, 1992Michaels ML and Miller JH (1992) The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J Bacteriol 174:6321-6325.).

For T. cruzi, all three proteins of the GO system have been characterized (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.; Aguiar et al., 2013Aguiar PHN, Furtado C, Repolês BM, Ribeiro GA, Mendes IC, Peloso EF, Gadelha FR, Macedo AM, Franco GR, Pena SDJ et al. (2013) Oxidative stress and DNA lesions: The role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability. PLoS Negl Trop Dis 7:e2279.; Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.). Initially, only TcOGG1 (FPG homolog for T. cruzi) and TcMYH (MutY homolog for T. cruzi) were identified as part of the T. cruzi CL Brener strain genome deposited in TritrypDB (El-Sayed et al., 2005El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, Ghedin E, Worthey EA, Delcher AL, Blandin G et al. (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309:409–415.). Heterogeneous expression of TcOGG1 in Ogg1 mutants of Saccharomyces cerevisiae could complement the deficient phenotype of the yeast. In T. cruzi, OGG1 is located on nuclear and kinetoplast DNA. The protein was also functional in both organelles as overexpression sensitized the parasite to oxidative stress and led to lower rates of 8-oxodG (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.). A similar result was observed for TcMYH, which is found in nuclear and kinetoplast DNA; overexpression of this protein led to increased sensitivity of the modified strain (Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.). In addition, the parasite possesses a functional MutT homolog, which can complement MutT-deficient E. coli; overexpression of this protein increased resistance, decreasing parasite DNA damage (Aguiar et al., 2013Aguiar PHN, Furtado C, Repolês BM, Ribeiro GA, Mendes IC, Peloso EF, Gadelha FR, Macedo AM, Franco GR, Pena SDJ et al. (2013) Oxidative stress and DNA lesions: The role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability. PLoS Negl Trop Dis 7:e2279.). The difference observed after overexpression of each protein can be explained by their function. MutY and OGG1 are proteins that act directly on the lesion already incorporated into the DNA strand, generating AP sites for the BER pathway to repair; however, MutT acts before incorporation, by removing the modified base as a substrate for the DNA polymerase (Michaels and Miller, 1992Michaels ML and Miller JH (1992) The GO system protects organisms from the mutagenic effect of the spontaneous lesion 8-hydroxyguanine (7,8-dihydro-8-oxoguanine). J Bacteriol 174:6321-6325.) and their homologs may act in the same way in T. cruzi. Thus, TcMYH and TcOGG1 overexpression may cause an imbalance in the DNA repair system of these modified strains, generating more AP sites than the parasite has the ability to efficiently repair. In fact, strains overexpressing TcMYH accumulate more AP sites after treatment with H₂O₂ and have a higher and earlier peak of AP sites in the genome as compared to the WT strain (Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.). By using these mutants it was demonstrated that overexpression of TcMTH, but not TcMYH or TcOGG1, leads to higher resistance to BZN (Rajão et al., 2014Rajão MA, Furtado C, Alves CL, Passos-Silva DG, De Moura MB, Schamber-Reis BL, Kunrath-Lima M, Zuma AA, Vieira-da-Rocha JP, Garcia JBF et al. (2014) Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55:309–321.). This result indicates that BZN has a preference for action on the nucleotide pool and also targets parasite DNA; thus this is a direct link between DNA metabolism and Chagas disease treatment.

Regarding infection capability, overexpression of TcMTH increased both parasitemia and the number of amastigotes per cell in vitro as compared to the WT strain (Aguiar et al., 2013Aguiar PHN, Furtado C, Repolês BM, Ribeiro GA, Mendes IC, Peloso EF, Gadelha FR, Macedo AM, Franco GR, Pena SDJ et al. (2013) Oxidative stress and DNA lesions: The role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability. PLoS Negl Trop Dis 7:e2279.; Goes et al., 2016Goes GR, Rocha PS, Diniz ARS, Aguiar PHN, Machado CR and Vieira LQ (2016) Trypanosoma cruzi needs a signal provided by reactive oxygen species to infect macrophages. PLoS Negl Trop Dis 10:1–25.). An intriguing finding was that, after pre-treatment with a sublethal dose of H₂O₂, the number of intracellular parasites per cell after infection was higher when the authors compared the non-treated overexpressing cells with the pre-treated overexpressing cells (Aguiar et al., 2013Aguiar PHN, Furtado C, Repolês BM, Ribeiro GA, Mendes IC, Peloso EF, Gadelha FR, Macedo AM, Franco GR, Pena SDJ et al. (2013) Oxidative stress and DNA lesions: The role of 8-oxoguanine lesions in Trypanosoma cruzi cell viability. PLoS Negl Trop Dis 7:e2279.). Also, when an experiment was performed using Phox KO macrophage (i.e., mice deficient in the gp91^phox subunit of the NADPH oxidase), which impairs ROS production post-infection, the authors found that the parasitemia in modified macrophages was lower than in control cells (Goes et al., 2016Goes GR, Rocha PS, Diniz ARS, Aguiar PHN, Machado CR and Vieira LQ (2016) Trypanosoma cruzi needs a signal provided by reactive oxygen species to infect macrophages. PLoS Negl Trop Dis 10:1–25.). These results are in agreement with previous works that had described oxidative stress as a factor that enhances the parasite infection (Paiva et al., 2012Paiva CN, Feijó DF, Dutra FF, Carneiro VC, Freitas GB, Alves LS, Mesquita J, Fortes GB, Figueiredo RT, Souza HSP et al. (2012) Oxidative stress fuels Trypanosoma cruzi infection in mice. J Clin Invest 122:2531–2542.; Paiva and Bozza, 2014Paiva CN and Bozza MT (2014) Are reactive oxygen species always detrimental to pathogens? Antioxid Redox Signal 20:1000–1037.), but the exact signal that these ROS give remains unclear.

The investigation into the importance of the response to ROS for T. cruzi was based on the finding that the parasite lacks, in its annotated genome, the sequence for the catalase gene (Boveris et al., 1980Boveris A, Sies H, Martino EE, Docampo R, Turrens JF and Stoppani AO (1980) Deficient metabolic utilization of hydrogen peroxide in Trypanosoma cruzi. Biochem J 188:643-648.; El-Sayed et al., 2005El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, Ghedin E, Worthey EA, Delcher AL, Blandin G et al. (2005) The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science 309:409–415.; Freire et al., 2017Freire ACG, Alves CL, Goes GR, Resende BC, Moretti NS, Nunes VS, Aguiar PHN, Tahara EB, Franco GR, Macedo AM et al. (2017) Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi. Parasitology 144:1498–1510.). Catalase is an antioxidant enzyme found in nearly all aerobic organisms. It performs the decomposition of hydrogen peroxide into water and oxygen (Chelikani et al., 2004Chelikani P, Fita I and Loewen PC (2004) Diversity of structures and properties among catalases. Cell Mol Life Sci 61:192-208.), two molecules that are not harmful to most organisms. The lack of such an essential gene was unexpected given the importance for T. cruzi to fight oxidative stress, and given the central role catalase plays in oxidative defense in several organisms (Chelikani et al., 2004Chelikani P, Fita I and Loewen PC (2004) Diversity of structures and properties among catalases. Cell Mol Life Sci 61:192-208.). The most exciting finding was that the heterologous expression of the catalase gene from E. coli (KatE) in T. cruzi increased parasite resistance to hydrogen peroxide. However, when the cells were pre-treated with a sub-lethal dose of H₂O₂, there was no difference in survival as compared with KatE in WT strains (Freire et al., 2017Freire ACG, Alves CL, Goes GR, Resende BC, Moretti NS, Nunes VS, Aguiar PHN, Tahara EB, Franco GR, Macedo AM et al. (2017) Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi. Parasitology 144:1498–1510.). These results suggest that the pre-treatment can induce a cellular adaptation in WT strains of T. cruzi, a condition that is abrogated with the expression of the heterologous KatE gene. T. cruzi modified with KatE also exhibited higher parasitemia, infection index, and midgut proliferation in the invertebrate host when compared to WT cells (Freire et al., 2017Freire ACG, Alves CL, Goes GR, Resende BC, Moretti NS, Nunes VS, Aguiar PHN, Tahara EB, Franco GR, Macedo AM et al. (2017) Catalase expression impairs oxidative stress-mediated signalling in Trypanosoma cruzi. Parasitology 144:1498–1510.). Taken together, these results help illustrate a scenario in which ROS provides a signal for T. cruzi proliferation in cells, as observed in other works (Paiva et al., 2012Paiva CN, Feijó DF, Dutra FF, Carneiro VC, Freitas GB, Alves LS, Mesquita J, Fortes GB, Figueiredo RT, Souza HSP et al. (2012) Oxidative stress fuels Trypanosoma cruzi infection in mice. J Clin Invest 122:2531–2542.) and that, the abolishment of this signal caused by KatE, which degrades H₂O₂ into two non-signaling molecules, can alter the parasitic response to this stress and allow T. cruzi to benefit from oxidative stress to replicate in their hosts.

The parasite response to the insults caused by the host cell is, therefore, very complex and is summarized in Figure 1. Oxidative stress is related to several kinds of damage (Imlay, 2013Imlay JA (2013) The molecular mechanisms and physiological consequences of oxidative stress: Lessons from a model bacterium. Nat Rev Microbiol 11:443–454.), but the investigation into the relevant parasitic machinery to combat the resulting DNA damage demonstrates that repair and signaling machinery is essential for the parasite to sustain infection both in vitro and in vivo. However, the exact role of this signal in amastigotes is not yet clear. The majority of experiments were performed on the epimastigote and trypomastigote forms of the parasite, and investigation of the particularities of the amastigote form remains an interesting field of study.

Mitochondrial DNA damage response

Although the response to oxidative stress is a significant area of research in T. cruzi, since the oxidative burst is the major insult the parasite faces during infection, little is known regarding parasitic mitochondrial ROS generation and mitochondrial DNA repair. It has long been reported that some DNA repair pathways are absent from the mitochondria. For over thirty years, there have been reports of BER proteins in the mitochondria (Anderson and Friedberg, 1980Anderson CT and Friedberg EC (1980) The presence of nuclear and mitochondrial uracil-DNA glycosylase in extracts of human KB cells. Nucleic Acids Res 8:875-88.; Pettepher et al., 1991Pettepher CC, LeDoux SP, Bohr VA and Wilson GL (1991) Repair of alkali-labile sites within the mitochondrial DNA of RINr 38 cells after exposure to the nitrosourea streptozotocin. J Biol Chem 266:3113-3117.). Repair of double-strand breaks has already been described in some organisms such as fungi and plants (Manchekar et al., 2006Manchekar M, Scissum-Gunn K, Song D, Khazi F, McLean SL and Nielsen BL (2006) DNA recombination activity in soybean mitochondria. J Mol Biol 356:288-99.; Morel et al., 2008Morel F, Renoux M, Lachaume P and Alziari S (2008) Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired. Mutat Res 637:111-117.; Mileshina et al., 2011Mileshina D, Koulintchenko M, Konstantinov Y and Dietrich A (2011) Transfection of plant mitochondria and in organello gene integration. Nucleic Acids Res 39:e115.), but mammalian cells possess only a basic recombination machinery in the mitochondria (Thyagarajan et al., 1996Thyagarajan B, Padua RA and Campbell C (1996) Mammalian mitochondria possess homologous DNA recombination activity. J Biol Chem 271:27536-27543.). Mitochondrial recombination events are rare, and some of the necessary components are involved in other DNA metabolic functions (Minczuk et al., 2011Minczuk M, He J, Duch AM, Ettema TJ, Chlebowski A, Dzionek K, Nijtmans LGJ, Huynen MA and Holt IJ (2011) TEFM (c17orf42) is necessary for transcription of human mtDNA. Nucleic Acids Res 39:4284-99.). Mismatch resolution in mitochondrial DNA has only been recently described in some human cell lines and some other mammals (Mason et al., 2003Mason PA, Matheson EC, Hall AG and Lightowlers RN (2003) Mismatch repair activity in mammalian mitochondria. Nucleic Acids Res 31:1052-1058.; Souza-Pinto et al., 2009Souza-Pinto NC, Mason PA, Hashiguchi K, Weissman L, Tian J, Guay D, Lebel M, Stevnsner TV, Rasmussen LJ and Bohr VA (2009) Novel DNA mismatch-repair activity involving YB-1 in human mitochondria. DNA Repair (Amst) 8:704–719.). Interestingly, general MMR nuclear proteins are not located in mitochondria, suggesting that different pathways occur in this organelle (Kazak et al., 2012Kazak L, Reyes A and Holt IJ (2012) Minimizing the damage: Repair pathways keep mitochondrial DNA intact. Nat Rev Mol Cell Biol 13:659–671.). Further details about specific pathways present in mitochondria of higher eukaryotes have already been reviewed in other works (Shokolenko et al., 2011Shokolenko I, LeDoux S, Wilson G and Alexeyev M (2011) Mitochondrial DNA damage, repair, degradation and experimental approaches to studying these phenomena. In: Storici F (ed) DNA Repair - Pathways to Fixing DNA Damage Errors. IntechOpen, London, pp 339-355.; Kazak et al., 2012Kazak L, Reyes A and Holt IJ (2012) Minimizing the damage: Repair pathways keep mitochondrial DNA intact. Nat Rev Mol Cell Biol 13:659–671.; Alexeyev et al., 2013Alexeyev M, Shokolenko I, Wilson G and LeDoux S (2013) The maintenance of mitochondrial DNA integrity - critical analysis and update. Cold Spring Harb Perspect Biol 5:a012641-a012641.). T. cruzi and other trypanosomatids possess a single and modified mitochondria, allowing their use as great model organisms to study mitochondrial DNA repair. T. cruzi has been reported to be able to repair both the nuclear and mitochondrial DNA damaged by oxidative stress, although the level of kDNA damage is lower and more consistent than in the nuclear DNA. In addition, DNA repair in the kinetoplast does not eliminate all damage, as detected by a qPCR assay (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.).

The main type of DNA damage generated by oxidative stress is the 8-oxodG base modification, which can be repaired by the GO system. In the T. cruzi GO system, two proteins colocalize with kDNA. Although TcOGG1 is generally thought to be localized within the nucleus, it was also identified in the kDNA T. cruzi. In addition, mitochondrial 8-oxodG levels were reduced in TcOGG1 overexpressing strains when compared to the control strain (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.). TcMYH was also co-localized in the kinetoplast of T. cruzi, although the localization demonstrated that the protein was also found in the parasite’s nucleus. Overexpression of this protein caused a small elevation in the number mtDNA damage sites, as measured in mitochondria 30 minutes after the damage was induced and sensitized the cells to treatment with a specific mitochondrial stress agent (Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.).

During the resolution of oxidative damage by BER, one pathway involves the insertion of the correct nucleotide into the 3’-OH of the AP site previously generated, followed by the excision of the remaining sugar-phosphate bone (DRP-lyase activity), a process that can be catalyzed by DNA polymerase β (Pol β) in some organisms (Allinson et al., 2001Allinson SL, Dianova II and Dianov GL (2001) DNA polymerase β is the major dRP lyase involved in repair of oxidative base lesions in DNA by mammalian cell extracts. EMBO J 20:6919-26.). In T. cruzi, Pol β is present and is also able to perform these activities (Lopes et al., 2008Lopes DO, Schamber-Reis BLF, Regis-da-Silva CG, Rajão MA, DaRocha WD, Macedo AM, Franco GR, Nardelli SC, Schenkman S, Hoffmann JS et al. (2008) Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance. DNA Repair (Amst) 7:1882–1892.; Schamber-Reis et al., 2012Schamber-Reis BLF, Nardelli S, Régis-Silva CG, Campos PC, Cerqueira PG, Lima SA, Franco GR, Macedo AM, Pena SDJ, Cazaux C et al. (2012) DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions. Mol Biochem Parasitol 183:122–131.). It was demonstrated that this protein can deal with oxidative lesions in the kinetoplast and that its localization is dependent on the cell cycle. During oxidative stress, the protein translocates into the antipodal sites of the kinetoplast (Schamber-Reis et al., 2012Schamber-Reis BLF, Nardelli S, Régis-Silva CG, Campos PC, Cerqueira PG, Lima SA, Franco GR, Macedo AM, Pena SDJ, Cazaux C et al. (2012) DNA polymerase beta from Trypanosoma cruzi is involved in kinetoplast DNA replication and repair of oxidative lesions. Mol Biochem Parasitol 183:122–131.). Pol β PAK, a homolog of Pol β that contains a domain rich in proline, alanine, and lysine, has also been characterized in T. cruzi (Lopes et al., 2008Lopes DO, Schamber-Reis BLF, Regis-da-Silva CG, Rajão MA, DaRocha WD, Macedo AM, Franco GR, Nardelli SC, Schenkman S, Hoffmann JS et al. (2008) Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance. DNA Repair (Amst) 7:1882–1892.). Pol β PAK has a strict localization in the kDNA of T. cruzi and also possesses DRP-lyase activity. However, unlike Pol β, only Pol β PAK is capable of performing a bypass of 8-oxodG (Lopes et al., 2008Lopes DO, Schamber-Reis BLF, Regis-da-Silva CG, Rajão MA, DaRocha WD, Macedo AM, Franco GR, Nardelli SC, Schenkman S, Hoffmann JS et al. (2008) Biochemical studies with DNA polymerase β and DNA polymerase β-PAK of Trypanosoma cruzi suggest the involvement of these proteins in mitochondrial DNA maintenance. DNA Repair (Amst) 7:1882–1892.).

DNA polymerase kappa was also implicated in kDNA metabolism in T. cruzi (Rajão et al., 2009Rajão MA, Passos-Silva DG, DaRocha WD, Franco GR, Macedo AM, Pena SDJ, Teixeira SM and Machado CR (2009) DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate. Mol Microbiol 71:185–197.). Although the parasite possesses more than one sequence of DNA polymerase kappa, one of the copies possesses a mitochondrial localization signal and specific kinetoplast localization. Like polymerase β and β-PAK, DNA polymerase kappa can bypass 8-oxodG damage, but can also replicate an intermediate recombination structure in vitro that mimics the D-loop (Rajão et al., 2009Rajão MA, Passos-Silva DG, DaRocha WD, Franco GR, Macedo AM, Pena SDJ, Teixeira SM and Machado CR (2009) DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate. Mol Microbiol 71:185–197.). Overexpression of these three polymerases increases parasite survival against BZN (Rajão et al., 2014Rajão MA, Furtado C, Alves CL, Passos-Silva DG, De Moura MB, Schamber-Reis BL, Kunrath-Lima M, Zuma AA, Vieira-da-Rocha JP, Garcia JBF et al. (2014) Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55:309–321.), another direct link between DNA metabolism and Chagas disease treatment. It is worth noting that only DNA polymerase kappa (Rajão et al., 2009Rajão MA, Passos-Silva DG, DaRocha WD, Franco GR, Macedo AM, Pena SDJ, Teixeira SM and Machado CR (2009) DNA polymerase kappa from Trypanosoma cruzi localizes to the mitochondria, bypasses 8-oxoguanine lesions and performs DNA synthesis in a recombination intermediate. Mol Microbiol 71:185–197.), TcOGG1 (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.), and TcMYH (Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.) possess a predicted mitochondrial localization signal; also, some kinetoplast associated proteins have no mitochondrial localization prediction, even though they are located in the organelle (Souza et al., 2010Souza FSP, Rampazzo RCP, Manhaes L, Soares MJ, Cavalcanti DP, Krieger MA, Goldenberg S and Fragoso SP (2010) Knockout of the gene encoding the kinetoplast-associated protein 3 (KAP3) in Trypanosoma cruzi: Effect on kinetoplast organization, cell proliferation and differentiation. Mol Biochem Parasitol 172:90–98.). Some other proteins, such as Rad51 can also be localized to the mitochondria, as seen by by immunofluorescence experiments, although they are also not predicted to be present in this organelle (Cerqueira et al., 2017Cerqueira PG, Passos-Silva DG, Vieira-da-Rocha JP, Mendes IC, Oliveira KA, Oliveira CFB, Vilela LFF, Nagem RAP, Cardoso J, Nardelli SC et al. (2017) Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system. Mol Biochem Parasitol 212:55–67.), suggesting that the exportation of proteins to the mitochondria of T. cruzi still needs further study.

These data contribute to a scenario in which T. cruzi possesses, on the kinetoplast, proteins from all steps of the BER pathway, suggesting that the parasite can repair oxidative damages on the kDNA. Although the decrease in the number of lesions in the mitochondrial DNA observed in WT cells by qPCR is not significant (Furtado et al., 2012Furtado C, Kunrath-Lima M, Rajão MA, Mendes IC, Moura MB, Campos PC, Macedo AM, Franco GR, Pena SDJ, Teixeira SMR et al. (2012) Functional characterization of 8-oxoguanine DNA glycosylase of Trypanosoma cruzi. PLoS One 7:e42484.), the increase in the number of lesions on the kDNA caused by the overexpression of TcMYH (Kunrath-Lima et al., 2017Kunrath-Lima M, Repolês BM, Alves CL, Furtado C, Rajão MA, Macedo AM, Franco GR, Pena SDJ, Valenzuela L, Wisnovsky S et al. (2017) Characterization of Trypanosoma cruzi MutY DNA glycosylase ortholog and its role in oxidative stress response. Infect Genet Evol 55:332–342.) demonstrates that the kinetoplast does possess proteins for combating oxidative damage within this organelle. The quantification of DNA damage from other sources and repaired by other DNA repair pathways remain to be investigated.

Chronic Chagas disease and DNA metabolism

A remarkable characteristic of Chagas disease is the heterogeneity of its clinical manifestations. Immediately after the infection, patients initiate an acute phase of the disease that can last from 4-8 weeks and is, in most cases, asymptomatic (Prata, 2001Prata A (2001) Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis 1:92–100.; Pérez-Molina and Molina, 2018Pérez-Molina JA and Molina I (2018) Chagas disease. Lancet 391:82–94.). Symptomatic acute phase patients can present symptoms like fever, inflammation at the location of infection, and unilateral palpebral edema (a clinical condition called Romaña sign). In severe cases, the acute phase can lead to acute myocarditis, meningoencephalitis, and pericardial effusion (Laranja et al., 1956Laranja FS, Dias E, Nóbrega G and Miranda A (1956) Chagas’ Ddsease: A clinical, epidemiologic, and pathologic study. Circulation 14:1035-1060.; Teixeira et al., 2006Teixeira ARL, Nitz N, Guimaro MC, Gomes C and Santos-Buch CA (2006) Chagas disease. Postgrad Med J 82:788-798.; Pinto et al., 2008Pinto AYN, Valente SA, Valente VC, Ferreira Junior AG and Coura JR (2008) Acute phase of Chagas disease in the Brazilian Amazon region: Study of 233 cases from Pará, Amapá and Maranhão observed between 1988 and 2005. Rev Soc Bras Med Trop 41:602-614.; Pereira and Navarro, 2013Pereira PCM and Navarro EC (2013) Challenges and perspectives of Chagas disease: A review. J Venom Anim Toxins Incl Trop Dis 19:1–8.). However, the disease usually persists in an asymptomatic form and enters a chronic stage, which is characterized by low parasitemia (Pereira and Navarro, 2013Pereira PCM and Navarro EC (2013) Challenges and perspectives of Chagas disease: A review. J Venom Anim Toxins Incl Trop Dis 19:1–8.). Only 30% to 40% of these patients develop cardiomyopathy, megaesophagus, or megacolon (Prata, 2001Prata A (2001) Clinical and epidemiological aspects of Chagas disease. Lancet Infect Dis 1:92–100.; Rassi et al., 2010Rassi A, Rassi A and Marin-Neto JA (2010) Chagas disease. Lancet 375:1388–1402.; Pérez-Molina and Molina, 2018Pérez-Molina JA and Molina I (2018) Chagas disease. Lancet 391:82–94.). It has been long known that treatment with BZN and nifurtimox (NX) provides a high cure rate when given during the acute phase (Bahia-Oliveira et al., 2000Bahia-Oliveira LM, Gomes JA, Cançado JR, Ferrari TC, Lemos EM, Luz ZM, Moreira MC, Gazzinelli G and Correa-Oliveira R (2000) Immunological and clinical evaluation of chagasic patients subjected to chemotherapy during the acute phase of Trypanosoma cruzi infection 14-30 years ago. J Infect Dis 182:634-8.), but this treatment is 4 to 16 times less effective during the chronic phase (Cançado, 2002Cançado JR (2002) Long term evaluation of etiological treatment of Chagas disease with benznidazole. Rev Inst Med Trop Sao Paulo 44:29–37.). In this context, one of the biggest questions regarding Chagas disease is why the parasite can persist in such a long period of dormancy, in which the host exhibits low levels of parasitemia, lasting for years, and the parasite becomes resistant to treatment.

Recently, an interesting report revealed that during cellular infection the parasite develops a non-proliferating intracellular amastigote form (Sánchez-Valdéz et al., 2018Sánchez-Valdéz FJ, Padilla A, Wang W, Orr D and Tarleton RL (2018) Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure. eLife 7:34039). By using a cell division tracker and the 5-ethynyl-2’-deoxyuridine (EdU) thymidine analog, the authors were able to demonstrate that, although the BZN treatment is effective in reducing the number of trypomastigotes during the acute phase, there are still some amastigotes in dormancy and in a non-replicative state. Remarkably, the authors observed that, in vivo and in situ, even a single dormant amastigote cell can resume infection after this latency period, where the cells are dormant and in a non-replicative state (Sánchez-Valdéz et al., 2018Sánchez-Valdéz FJ, Padilla A, Wang W, Orr D and Tarleton RL (2018) Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure. eLife 7:34039). When those cells were challenged with BZN, those dormant amastigotes were resistant to treatment (Sánchez-Valdéz et al., 2018Sánchez-Valdéz FJ, Padilla A, Wang W, Orr D and Tarleton RL (2018) Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure. eLife 7:34039). Although it is not clear if BZN causes this dormancy, or if the parasite is already dormant and the treatment is unable to affect it since, as stated previously, BZN causes oxidation mainly on the parasite nucleotide pool (Rajão et al., 2014Rajão MA, Furtado C, Alves CL, Passos-Silva DG, De Moura MB, Schamber-Reis BL, Kunrath-Lima M, Zuma AA, Vieira-da-Rocha JP, Garcia JBF et al. (2014) Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55:309–321.).

A state that resembles dormancy has already been previously reported for T. cruzi in epimastigote cultures under laboratory conditions. After exposure to high doses of gamma irradiation (i.e. 500 Gy), epimastigotes enter a non-replicative state for a period of time that could be as long as 10 days (Regis-da-Silva et al., 2006Regis-da-Silva CG, Freitas JM, Passos-Silva DG, Furtado C, Augusto-Pinto L, Pereira MT, DaRocha WD, Franco GR, Macedo AM, Hoffmann JS et al. (2006) Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation. Mol Biochem Parasitol 149:191–200.; Alves et al., 2018Alves CL, Repolês BM, Silva MS, Mendes IC, Marin PA, Aguiar PHN, Santos SS, Franco GR, Macedo AM, Pena SDJ et al. (2018) The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. Sci Rep 8:13335.; Silva et al., 2018Silva DGP, Santos SS, Nardelli SC, Mendes IC, Freire ACG, Repolês BM, Resende BC, Costa-Silva HM, Silva VS, Oliveira KA et al. (2018) The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions. PLoS Negl Trop Dis 12:e0006875.), although parasite DNA is repaired during the first 48 hours after the damaging insult (Regis-da-Silva et al., 2006Regis-da-Silva CG, Freitas JM, Passos-Silva DG, Furtado C, Augusto-Pinto L, Pereira MT, DaRocha WD, Franco GR, Macedo AM, Hoffmann JS et al. (2006) Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation. Mol Biochem Parasitol 149:191–200.). This growth impairment was coincident with an arrest in S/G2 phase, which was diminished by the time of growth resumption (Garcia et al., 2016Garcia JBF, Rocha JPV, Costa-Silva HM, Alves CL, Machado CR and Cruz AK (2016) Leishmania major and Trypanosoma cruzi present distinct DNA damage responses. Mol Biochem Parasitol 207:23–32.), indicating that those cells were not replicating. It has also been shown that resumption of parasite DNA repair and growth are dependent on Rad51 levels (Regis-da-Silva et al., 2006Regis-da-Silva CG, Freitas JM, Passos-Silva DG, Furtado C, Augusto-Pinto L, Pereira MT, DaRocha WD, Franco GR, Macedo AM, Hoffmann JS et al. (2006) Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation. Mol Biochem Parasitol 149:191–200.; Silva et al., 2018Silva DGP, Santos SS, Nardelli SC, Mendes IC, Freire ACG, Repolês BM, Resende BC, Costa-Silva HM, Silva VS, Oliveira KA et al. (2018) The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions. PLoS Negl Trop Dis 12:e0006875.). Rad51 seems to be related to specific kinds of replicative stress, being necessary for the response to methyl methanesulfonate (MMS), an alkylating agent capable of causing stalled replication forks; however, RAD51 is not recruited in response to replicative stress caused by hydroxyurea (Silva et al., 2018Silva DGP, Santos SS, Nardelli SC, Mendes IC, Freire ACG, Repolês BM, Resende BC, Costa-Silva HM, Silva VS, Oliveira KA et al. (2018) The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions. PLoS Negl Trop Dis 12:e0006875.). The relationship between those two dormant states needs to be investigated as it is not yet clear if they represent correlated events. Therefore, the control of replication and dormancy could be essential for T. cruzi survival.

It has recently been reported that in Chagas disease, dormancy is a mechanism by which T. cruzi can resist treatment with BZN, as demonstrated in both during in vivo and in vitro systems (Sánchez-Valdéz et al., 2018Sánchez-Valdéz FJ, Padilla A, Wang W, Orr D and Tarleton RL (2018) Spontaneous dormancy protects Trypanosoma cruzi during extended drug exposure. eLife 7:34039). This dormancy can be replicated in the laboratory by causing DNA damage and replication stress (Regis-da-Silva et al., 2006Regis-da-Silva CG, Freitas JM, Passos-Silva DG, Furtado C, Augusto-Pinto L, Pereira MT, DaRocha WD, Franco GR, Macedo AM, Hoffmann JS et al. (2006) Characterization of the Trypanosoma cruzi Rad51 gene and its role in recombination events associated with the parasite resistance to ionizing radiation. Mol Biochem Parasitol 149:191–200.; Cerqueira et al., 2017Cerqueira PG, Passos-Silva DG, Vieira-da-Rocha JP, Mendes IC, Oliveira KA, Oliveira CFB, Vilela LFF, Nagem RAP, Cardoso J, Nardelli SC et al. (2017) Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system. Mol Biochem Parasitol 212:55–67.; Alves et al., 2018Alves CL, Repolês BM, Silva MS, Mendes IC, Marin PA, Aguiar PHN, Santos SS, Franco GR, Macedo AM, Pena SDJ et al. (2018) The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. Sci Rep 8:13335.; Silva et al., 2018Silva DGP, Santos SS, Nardelli SC, Mendes IC, Freire ACG, Repolês BM, Resende BC, Costa-Silva HM, Silva VS, Oliveira KA et al. (2018) The in vivo and in vitro roles of Trypanosoma cruzi Rad51 in the repair of DNA double strand breaks and oxidative lesions. PLoS Negl Trop Dis 12:e0006875.). Furthermore, it was recently demonstrated that different T. cruzi discrete typing units (DTUs), clusters of parasites based on biogeographical data, sequencing, and similarity among strains, exchange genetic information (Alves et al., 2018Alves CL, Repolês BM, Silva MS, Mendes IC, Marin PA, Aguiar PHN, Santos SS, Franco GR, Macedo AM, Pena SDJ et al. (2018) The recombinase Rad51 plays a key role in events of genetic exchange in Trypanosoma cruzi. Sci Rep 8:13335.), but the relationship between these phenomena and the differential virulence observed among these clusters has not yet been explored.

As with the discovery of the mechanism of BZN resistance (Rajão et al., 2014Rajão MA, Furtado C, Alves CL, Passos-Silva DG, De Moura MB, Schamber-Reis BL, Kunrath-Lima M, Zuma AA, Vieira-da-Rocha JP, Garcia JBF et al. (2014) Unveiling Benznidazole’s mechanism of action through overexpression of DNA repair proteins in Trypanosoma cruzi. Environ Mol Mutagen 55:309–321.), researchers have demonstrated a direct interplay between DNA metabolism and repair during Chagas disease development. It will be of interest to determine how the DNA damage response can alter the parasite metabolism in a broader sense. Although the kinetics of some DNA repair pathways were previously elucidated, much more investigation is needed on the sensing and signaling to the oxidative environment and their related responses.

Effects of oxidative stress induced by T. cruzi in the host cell

The intracellular component of the T. cruzi life cycle begins with metacyclic or bloodstream trypomastigotes actively invading a host cell by disrupting the host cell membrane, stimulating cytosolic Ca²⁺ influx (Moreno, 2004Moreno SN (2004) Cytosolic-free calcium elevation in Trypanosoma cruzi is required for cell invasion. J Exp Med 180:1535-1540.; Tardieux, 2004Tardieux I (2004) Role in host cell invasion of Trypanosoma cruzi-induced cytosolic-free Ca2+ transients. J Exp Med 179:1017-1022.). This event leads to several changes in the host cell, including mitochondrial membrane permeabilization and the induction of high levels of cytosolic ROS, which can signal apoptosis (Kroemer et al., 2007Kroemer G, Galluzzi L and Brenner C (2007) Mitochondrial membrane Permeabilization in cell death. Physiol Rev 87:99-163.; Giorgi et al., 2012Giorgi C, Baldassari F, Bononi A, Bonora M, De Marchi E, Marchi S, Missiroli S, Patergnani S, Rimessi A, Suski JM et al. (2012) Mitochondrial Ca2+ and apoptosis. Cell Calcium 52:36-43.; Webster, 2012Webster KA (2012) Mitochondrial membrane permeabilization and cell death during myocardial infarction: Roles of calcium and reactive oxygen species. Future Cardiol 8:863-884.). The imbalance in ROS and antioxidant barriers has been described in many pathogen infections (Piacenza et al., 2019Piacenza L, Trujillo M and Radi R (2019) Reactive species and pathogen antioxidant networks during phagocytosis. J Exp Med 216:501-516.). During T. cruzi infection, NADP oxidase (NOX) 2 was reported to be present in the plasma membrane of macrophages (Cardoni et al., 1997Cardoni RL, Antunez MI, Morales C and Rodriguez Nantes I (1997) Release of reactive oxygen species by phagocytic cells in response to live parasites in mice infected with Trypanosoma cruzi. Am J Trop Med Hyg 56:329-334.). In addition, high levels of superoxide (O₂^-) and nitric oxide were released approximately 60-90 min after infection (Muñoz-Fernández et al., 1992Muñoz-Fernández MA, Fernández MA and Fresno M (1992) Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-α and IFN-γ through a nitric oxide-dependent mechanism. Immunol Lett 33:35-40.; Alvarez et al., 2004Alvarez MN, Piacenza L, Irigoín F, Peluffo G and Radi R (2004) Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi. Arch Biochem Biophys 432:222-32.). ROS generated in the intraphagosomal space is likely to have microbicidal effects on T. cruzi, which were reversed when T. cruzi cytosolic tryparedoxin was overexpressed. Higher levels of parasite survival after infection were observed. In addition, membrane potential loss from host cell mitochondria was observed after parasite cardiomyocyte infection. At 48 h post-infection, increased levels of cytosolic O₂^- were observed, even when ROS producing enzymes from host cells were pharmacologically inhibited (Gupta et al., 2009Gupta S, Bhatia V, Wen JJ, Wu Y, Huang MH and Garg NJ (2009) Trypanosoma cruzi infection disturbs mitochondrial membrane potential and ROS production rate in cardiomyocytes. Free Radic Biol Med 47:1414–1421.). The relationships between infection and oxidative stress are reviewed elsewhere (Gupta et al., 2011Gupta S, Dhiman M, Wen JJ and Garg NJ (2011) ROS Signalling of inflammatory cytokines during Trypanosoma cruzi infection. Adv Parasitol 76:153-170.; Paiva et al., 2018Paiva CN, Medei E and Bozza MT (2018) ROS and Trypanosoma cruzi: Fuel to infection, poison to the heart. PLoS Pathog 14:e1006928.).

As mentioned above, ROS produced by the host cell have a microbicidal effect on T. cruzi in the host cell environment (Muñoz-Fernandez et al., 1992Muñoz-Fernández MA, Fernández MA and Fresno M (1992) Activation of human macrophages for the killing of intracellular Trypanosoma cruzi by TNF-α and IFN-γ through a nitric oxide-dependent mechanism. Immunol Lett 33:35-40.; Alvarez et al., 2004Alvarez MN, Piacenza L, Irigoín F, Peluffo G and Radi R (2004) Macrophage-derived peroxynitrite diffusion and toxicity to Trypanosoma cruzi. Arch Biochem Biophys 432:222-32.). Recently, it was proposed that T. cruzi utilize their response to oxidative stress to survive inside the host cell cytosol. Briefly, macrophages and mice treated with cobalt protoporphyrin (CoPP), an antioxidant that induces heme oxygenase (HO-1) expression through nuclear erythroid factor-2 (NRF-2), reduced parasite growth. Paiva et al. (2012)Paiva CN, Feijó DF, Dutra FF, Carneiro VC, Freitas GB, Alves LS, Mesquita J, Fortes GB, Figueiredo RT, Souza HSP et al. (2012) Oxidative stress fuels Trypanosoma cruzi infection in mice. J Clin Invest 122:2531–2542. observed that the treatment of infected cells with several antioxidants (apocynin, superoxide dismutase, N-acetyl-L-cysteine, resveratrol) reduces parasite burden. Also, there is evidence that BZN, the drug used to treat Chagas disease, stimulates NRF2 (Rigalli et al., 2016Rigalli JP, Perdomo VG, Ciriaci N, Francés DEA, Ronco MT, Bataille AM, Ghanem CI, Ruiz ML, Manautou JE and Catania VA (2016) The antitripanocide benznidazole promotes adaptive response to oxidative injury: Involvement of the nuclear factor-erythroid 2-related factor-2 (Nrf2) and multidrug resistance associated protein 2 (MRP2). Toxicol Appl Pharmacol 304:90-98.). Conversely, when they treated infected cells with a respiratory burst inducer, phorbol 12-myristate 13-acetate (PMA) and H₂O₂, parasite burden increased significantly. These results raise an important question: could oxidative stress not only benefit the parasite but also prejudice host cell? Thus, how the oxidative stress induced by T. cruzi could harm the host cell is not yet fully understood.

There are some studies reporting that T. cruzi can interfere with the host cell cycle, although the mechanisms behind this process have not yet been fully described. The transcriptome revealed downregulation of cell cycle and cell division genes (Shigihara et al., 2008Shigihara T, Hashimoto M, Shindo N and Aoki T (2008) Transcriptome profile of Trypanosoma cruzi-infected cells: Simultaneous up- and down-regulation of proliferation inhibitors and promoters. Parasitol Res 102:715-722.; Costales et al., 2009Costales JA, Daily JP and Burleigh BA (2009) Cytokine-dependent and-independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling. BMC Genomics 10:252.), though there was no difference in S-phase between uninfected cells and infected cells after 48 h of infection (Costales et al., 2009Costales JA, Daily JP and Burleigh BA (2009) Cytokine-dependent and-independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling. BMC Genomics 10:252.). Our recent findings suggest that infected cells are less likely to be in S phase as compared to control.

Several genes related to DNA metabolism are up-regulated during early timepoints of infection (3 h and 6 h post-infection), such as the DNA polymerases POLK and POLB, DNA helicase RECQL, and DNA glycosylase SMUG1, that are involved in various types of DNA repair, including mismatch repair, base excision repair, and direct repair (Costales et al., 2009Costales JA, Daily JP and Burleigh BA (2009) Cytokine-dependent and-independent gene expression changes and cell cycle block revealed in Trypanosoma cruzi-infected host cells by comparative mRNA profiling. BMC Genomics 10:252.). It was also reported that T. cruzi induces DNA breaks in cardiomyocytes during late stages of infection; the breaks are repaired when cells are treated with a free radical ion trap (PBN). PAR levels were increased in T. cruzi infected cells (Ba et al., 2010Ba X, Gupta S, Davidson M and Garg NJ (2010) Trypanosoma cruzi induces the reactive oxygen species-PARP-1-RelA pathway for up-regulation of cytokine expression in cardiomyocytes. J Biol Chem 285:11596-606.). These results suggest that oxidative stress leads to BER pathway activation in the host cell during infection. The mechanisms behind genotoxicity in host cells are not yet fully understood and research efforts toward this end are underway.

There is some evidence that T. cruzi infection can induce not only host cell cycle arrest, but might also lead to senescence (Guimarães-Pinto et al., 2018Guimarães-Pinto K, Nascimento DO, Corrêa-Ferreira A, Morrot A, Freire-de-Lima CG, Lopes MF, DosReis GA and Filardy AA (2018) Trypanosoma cruzi infection induces cellular stress response and senescence-like phenotype in murine fibroblasts. Front Immunol 9:1569.). It was revealed that a senescence-associated secretory phenotype (SASP) characterized by the production of a specific set of cytokines and chemokines such as IL-6, TNF-α, IL-1β, and MCP-1 and also secretion of SA-β-galactose are higher in infected fibroblast cells when compared to non-infected cells. The authors observed that host cell nuclei presented a characteristic senescence-associated heterochromatin focus. In addition, they observed that antioxidants reduced SASP molecules in the host cell and inhibited parasite growth. Cellular senescence, by definition, impairs cell cycle progression, which could benefit amastigote multiplication in the host environment. These results suggest that parasite growth in fibroblasts leads to host cell senescence, which benefits T. cruzi reproduction in the host cell cytosol.

In cell culture after parasite growth, the host cell will eventually die, spreading parasites to the supernatant. How the parasite induces host cell death is controversial. It has been reported that T. cruzi can induce apoptosis in the host cell (Stahl et al., 2013Stahl P, Ruppert V, Meyer T, Schmidt J, Campos MA, Gazzinelli RT, Maisch B, Schwarz RT and Debierre-Grockiego F (2013) Trypomastigotes and amastigotes of Trypanosoma cruzi induce apoptosis and STAT3 activation in cardiomyocytes in vitro. Apoptosis 18:653-663.). The authors observed activation of caspases 3/7, 8, and 9 in cardiomyocytes infected with T. cruzi when compared to control cells. In this model, STAT3 activation was induced by infection. Therefore, the Janus Kinase (JAK) signal-STAT pathway could be responsible for apoptosis and cardiomyopathy. Conversely, it was reported that T. cruzi could prevent cardiac cells from undergoing apoptosis via activation of NF-kB and IL-1 (Petersen et al., 2006Petersen CA, Krumholz KA, Carmen J, Sinai AP and Burleigh BA (2006) Trypanosoma cruzi infection and nuclear factor kappa B activation prevent apoptosis in cardiac cells. Infect Immun 74:1580-1587.). How the parasite contributes to cell death and how the cell fate decision might spread neighboring cells is an important issue that needs to be further study.

Concluding remarks

As described above, DNA metabolism and responses to DNA damage in both the parasite and host vary depending on the parasite in question and its specific life form. In Chagas disease, success of the infection and associated pathological effects are influenced by both parasite and host factors. (Andrade et al., 1999Andrade LO, Machado CRS, Chiari E, Pena SDJ and Macedo AM (1999) Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice. Mol Biochem Parasitol 100:163–172.; Andrade and Andrews, 2005Andrade LO and Andrews NW (2005) The Trypanosoma cruzi - host-cell interplay: Location, invasion, retention. Nat Rev Microbiol 3:819–823.; Albertti et al., 2010Albertti LAG, Macedo AM, Chiari E, Andrews NW and Andrade LO (2010) Role of host lysosomal associated membrane protein (LAMP) in Trypanosoma cruzi invasion and intracellular development. Microbes Infect 12:784–789.; Kayama and Takeda 2010Kayama H and Takeda K (2010) The innate immune response to Trypanosoma cruzi infection. Microbes Infect 12:511–517.; Henrique et al., 2016Henrique PM, Marques T, Silva MV, Nascentes GAN, Oliveira CF, Rodrigues V, Gómez-Hernández C, Norris KA, Ramirez LE and Meira WSF (2016) Correlation between the virulence of T. cruzi strains, complement regulatory protein expression levels, and the ability to elicit lytic antibody production. Exp Parasitol 170:66–72.). Parasite-host interactions have been studied for a long time and seem to be important for both cell types (Caradonna and Burleigh, 2011Caradonna KL and Burleigh BA (2011) Mechanisms of host cell invasion by Trypanosoma cruzi. Adv Parasitol 76:33–61.; Caradonna et al., 2013Caradonna KL, Engel JC, Jacobi D, Lee CH and Burleigh BA (2013) Host metabolism regulates intracellular growth of Trypanosoma cruzi. Cell Host Microbe 13:108-17). The findings that T. cruzi can alter the mitochondrial membrane potential and the oxidative burst within the host cell to their benefit gives a new perspective on the infection process. Investigation into how the parasite regulates both its own and the host cell cycle is also essential, as this information could lead to new insights into the understanding of parasite/host interactions. Reports of how T. cruzi can regulate host cell cycle arrest and/or cell death in cardiomyocytes and further understanding of how the parasite enters a dormant state can lead to a new understanding of the disease mechanisms.

Acknowlegments

This work was supported by funds of CNPq, FAPEMIG, CAPES and FAPESP.

References

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