TcI isolates of Trypanosoma cruzi exploit the antioxidant network for enhanced intracellular survival in macrophages and virulence in mice

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/ matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P<0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO-]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi.Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Hosakote, Yashoda M.. University of Texas Medical Branch; Estados UnidosFil: Koo, Sue Jie. University of Texas Medical Branch; Estados UnidosFil: Dhiman, Monisha. University of Texas Medical Branch; Estados UnidosFil: Piñeyro, María Dolores. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Parodi­ Talice, Adriana. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Basombrío, Miguel Ángel Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Robello, Carlos. Instituto Pasteur de Montevideo. Unidad de Biología Molecular; Uruguay. Universidad de la Republica; UruguayFil: Garg, Nisha J.. University of Texas Medical Branch; Estados Unido

Early Trypanosoma cruzi Infection Triggers mTORC1-Mediated Respiration Increase and Mitochondrial Biogenesis in Human Primary Cardiomyocytes

Chagasic chronic cardiomyopathy is one of the most frequent and severe manifestations of Chagas disease, caused by the parasite Trypanosoma cruzi. The pathogenic and biochemical mechanisms responsible for cardiac lesions remain not completely understood, although it is clear that hypertrophy and subsequent heart dilatation is in part caused by the direct infection of cardiomyocytes. In this work, we evaluated the initial response of human cardiomyocytes to T. cruzi infection by transcriptomic profiling. Immediately after infection, cardiomyocytes dramatically change their gene expression patterns, up regulating most of the genes encoding for respiratory chain, oxidative phosphorylation and protein synthesis. We found that these changes correlate with an increase in basal and maximal respiration, as well as in spare respiratory capacity, which is accompanied by mitochondrial biogenesis pgc-1α independent. We also demonstrate that these changes are mediated by mTORC1 and reversed by rapamycin, resembling the molecular mechanisms described for the non-chagasic hypertrophic cardiomyopathy. The results of the present work identify that early during infection, the activation of mTORC1, mitochondrial biogenesis and improvement in oxidative phosphorylation are key biochemical changes that provide new insights into the host response to parasite infection and the pathogenesis of chronic chagasic cardiomyopathy. The finding that this phenotype can be reversed opens a new perspective in the treatment of Chagas disease, through the identification of host targets, and the use of combined parasite and host targeted therapies, in order to prevent chagasic cardiomyopathy

Trypanothione synthetase confers growth, survival advantage and resistance to anti-protozoal drugs in Trypanosoma cruzi

Background: Chagas cardiomyopathy, caused by Trypanosoma cruzi infection, continues to be a neglected illness, and has a major impact on global health. The parasite undergoes several stages of morphological and biochemical changes during its life cycle, and utilizes an elaborated antioxidant network to overcome the oxidants barrier and establish infection in vector and mammalian hosts. Trypanothione synthetase (TryS) catalyzes the biosynthesis of glutathione-spermidine adduct trypanothione (T(SH)2) that is the principal intracellular thiol-redox metabolite in trypanosomatids. Methods and Results: We utilized genetic overexpression (TryShi) and pharmacological inhibition approaches to examine the role of TryS in T. cruzi proliferation, tolerance to oxidative stress and resistance to anti-protozoal drugs. Our data showed the expression and activity of TryS was increased in all morphological stages of TryShi (vs. control) parasites. In comparison to controls, the TryShi epimastigotes (insect stage) recorded shorter doubling time, and both epimastigotes and infective trypomastigotes of TryShi exhibited 36-71% higher resistance to H2O2 (50-1000 M) and heavy metal (1-500 M) toxicity. Treatment with TryS inhibitors (5-30 M) abolished the proliferation and survival advantages against H2O2 pressure in a dose-dependent manner in both TryShi and control parasites. Further, epimastigote and trypomastigote forms of TryShi (vs. control) T. cruzi tolerated higher doses of benznidazole and nifurtimox, the drugs currently administered for acute Chagas disease treatment. Conclusions: TryS is essential for proliferation and survival of T. cruzi under normal and oxidant stress conditions, and provides an advantage to the parasite to develop resistance against currently used antitrypanosomal drugs. TryS indispensability has been chemically validated with inhibitors that may be useful for drug combination therapy against Chagas disease.Fil: Mesias, Andrea Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Sasoni, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Arias, Diego Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Pérez Brandan, Cecilia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Orban, Oliver C. F.. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; AlemaniaFil: Kunick, Conrad. Technische Universitat Carolo Wilhelmina Zu Braunschweig.; AlemaniaFil: Robello, Carlos. Instituto Pasteur de Montevideo; UruguayFil: Comini, Marcelo. Instituto Pasteur de Montevideo; UruguayFil: Garg, Nisha J.. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentin

Innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease.

We investigated the pathological and diagnostic role of selected markers of inflammation, oxidant/antioxidant status, and cellular injury in human Chagas disease. METHODS: Seropositive/chagasic subjects characterized as clinically-symptomatic or clinically-asymptomatic (n = 116), seronegative/cardiac subjects (n = 102), and seronegative/healthy subjects (n = 45) were analyzed for peripheral blood biomarkers. RESULTS: Seropositive/chagasic subjects exhibited an increase in sera or plasma levels of myeloperoxidase (MPO, 2.8-fold), advanced oxidation protein products (AOPP, 56%), nitrite (5.7-fold), lipid peroxides (LPO, 12-17-fold) and malondialdehyde (MDA, 4-6-fold); and a decline in superoxide dismutase (SOD, 52%) and glutathione (GSH, 75%) contents. Correlation analysis identified a significant (p0.95). The MPO (r = 0.664) and LPO (r = 0.841) levels were also correlated with clinical disease state in chagasic subjects (p<0.001). Seronegative/cardiac subjects exhibited up to 77% decline in SOD, 3-5-fold increase in LPO and glutamate pyruvate transaminase (GPT) levels, and statistically insignificant change in MPO, AOPP, MDA, GPX, GSH, and creatine kinase (CK) levels. CONCLUSIONS: The interlinked effects of innate immune responses and antioxidant/oxidant imbalance are major determinants of human Chagas disease. The MPO, LPO and nitrite are excellent biomarkers for diagnosing seropositive/chagasic subjects, and MPO and LPO levels have potential utility in identifying clinical severity of Chagas diseaseFil: Dhiman, Monisha. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Coronado, Yun A.. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Vallejo, Cecilia K.. University Of Texas Medical Branch. Department Of Microbiology & Immunology And Pathology; United State of America;Fil: Petersen, John R.. University of Texas Medical Branch. Department of Pathology; United States of America;Fil: Ejilemele, Adetoum. University of Texas Medical Branch. Department of Pathology; United States of America;Fil: Nuñez, Sonia. Hospital Público de Gestión Descentralizada San Bernardo (HPGDSA); Argentina;Fil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico - CONICET - Salta. Instituto de Patologia Experimental; Argentina;Fil: Spratt, Heidi. Departments of Biochemistry and Molecular Biology and Preventive Medicine and Community Health. University of Texas Medical Branch; United States of America;Fil: Garg, Nisha Jain. University of Texas Medical Branch. Department of Pathology; United States of America

Immunity and vaccine development efforts against Trypanosoma cruzi

Artículo de revisión especializadoTrypanosoma cruzi (T. cruzi) is the causative agent for Chagas disease (CD). There is a critical lack of methods for prevention of infection or treatment of acute infection and chronic disease. Studies in experimental models have suggested that the protective immunity against T. cruzi infection requires the elicitation of Th1 cytokines, lytic antibodies and the concerted activities of macrophages, T helper cells, and cytotoxic T lymphocytes (CTLs). In this review, we summarize the research efforts in vaccine development to date and the challenges faced in achieving an efficient prophylactic or therapeutic vaccine against human CD.UTM

Cardiac-Oxidized Antigens Are Targets of Immune Recognition by Antibodies and Potential Molecular Determinants in Chagas Disease Pathogenesis

Trypanosoma cruzi elicits reactive oxygen species (ROS) of inflammatory and mitochondrial origin in infected hosts. In this study, we examined ROS-induced oxidative modifications in the heart and determined whether the resultant oxidized cardiac proteins are targets of immune response and of pathological significance in Chagas disease. Heart biopsies from chagasic mice, rats and human patients exhibited, when compared to those from normal controls, a substantial increase in protein 4-hydroxynonenal (4-HNE), malondialdehyde (MDA), carbonyl, and 3-nitrotyrosine (3-NT) adducts. To evaluate whether oxidized proteins gain antigenic properties, heart homogenates or isolated cardiomyocytes were oxidized in vitro and one- or two-dimensional gel electrophoresis (2D-GE)/Western blotting (WB) was performed to investigate the proteomic oxidative changes and recognition of oxidized proteins by sera antibodies in chagasic rodents (mice, rats) and human patients. Human cardiomyocytes exhibited LD50 sensitivity to 30 µM 4-HNE and 100 µM H2O2 at 6 h and 12 h, respectively. In vitro oxidation with 4-HNE or H2O2 resulted in a substantial increase in 4-HNE- and carbonyl-modified proteins that correlated with increased recognition of cardiac (cardiomyocytes) proteins by sera antibodies of chagasic rodents and human patients. 2D-GE/Western blotting followed by MALDI-TOF-MS/MS analysis to identify cardiac proteins that were oxidized and recognized by human chagasic sera yielded 82 unique proteins. We validated the 2D-GE results by enzyme-linked immunosorbent assay (ELISA) and WB and demonstrated that oxidation of recombinant titin enhanced its immunogenicity and recognition by sera antibodies from chagasic hosts (rats and humans). Treatment of infected rats with phenyl-α-tert-butyl nitrone (PBN, antioxidant) resulted in normalized immune detection of cardiac proteins associated with control of cardiac pathology and preservation of heart contractile function in chagasic rats. We conclude that ROS-induced, cardiac-oxidized antigens are targets of immune recognition by antibodies and molecular determinants for pathogenesis during Chagas disease

Epidemiology and pathogenesis of maternal-fetal transmission of Trypanosoma cruzi and a case for vaccine development against congenital Chagas disease

Trypanos o ma cruzi (T. cruzi or Tc) is the causative agent of Chagas disease (CD). It is common for patients to suffer from non-specific symptoms or be clinically asymptomatic with acute and chronic conditions acquired through various routes of transmission. The expecting women and their fetuses are vulnerable to congenital transmission of Tc. Pregnant women face formidable health challenges because the frontline antiparasitic drugs, benznidazole and nifurtimox, are contraindicated during pregnancy. However, it is worthwhile to highlight that newborns can be cured if they are diagnosed and given treatment in a timely manner. In this review, we discuss the pathogenesis of maternal-fetal transmission of Tc and provide a justification for the investment in the development of vaccines against congenital CD.Fil: Rios, Lizette. University of Texas Medical Branch; Estados UnidosFil: Campos, Emiliano Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Menon, Ramkumar. University of Texas Medical Branch; Estados UnidosFil: Zago, María Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; ArgentinaFil: Garg, Nisha J.. University of Texas Medical Branch; Estados Unido

Manganese superoxide dismutase deficiency exacerbates the mitochondrial ROS production and oxidative damage in Chagas disease.

In this study, we have investigated the effects of manganese superoxide dismutase (SOD2 or MnSOD) deficiency on mitochondrial function and oxidative stress during Chagas disease. For this, C57BL/6 wild type (WT) and MnSOD+/- mice were infected with Trypanosoma cruzi (Tc), and evaluated at 150 days' post-infection that corresponded to chronic disease phase. Genetic deletion of SOD2 decreased the expression and activity of MnSOD, but it had no effect on the expression of other members of the SOD family. The myocardial expression and activity of MnSOD were significantly decreased in chronically infected WT mice, and it was further worsened in MnSOD+/- mice. Chronic T. cruzi infection led to a decline in mitochondrial complex I and complex II driven, ADP-coupled respiration and ATP synthesis in the myocardium of WT mice. The baseline oxidative phosphorylation (OXPHOS) capacity in MnSOD+/- mice was decreased, and it had an additive effect on mitochondrial dysregulation of ATP synthesis capacity in chagasic myocardium. Further, MnSOD deficiency exacerbated the mitochondrial rate of reactive oxygen species (ROS) production and myocardial oxidative stress (H2O2, protein carbonyls, malondialdehyde, and 4-hydroxynonenal) in Chagas disease. Peripheral and myocardial parasite burden and inflammatory response (myeloperoxidase, IL-6, lactate dehydrogenase, inflammatory infiltrate) were increased in all chagasic WT and MnSOD+/- mice. We conclude that MnSOD deficiency exacerbates the loss in mitochondrial function and OXPHOS capacity and enhances the myocardial oxidative damage in chagasic cardiomyopathy. Mitochondria targeted, small molecule mitigators of MnSOD deficiency will offer potential benefits in averting the mitochondrial dysfunction and chronic oxidative stress in Chagas disease

A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against <i>Trypanosoma cruzi</i>

<div><p>In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against <i>Trypanosoma cruzi</i> infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with <i>T</i>. <i>cruzi</i> at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting <i>T</i>. <i>cruzi</i>. Our data showed that D/P vaccine elicited CD4⁺ (30-38%) and CD8⁺ (22-42%) T cells maintained an effector phenotype up to 180 dpv, and were capable of responding to antigenic stimulus or challenge infection by a rapid expansion (CD8>CD4) with type 1 cytokine (IFNγ⁺ and TFNα⁺) production and cytolytic T lymphocyte (CTL) activity. Subsequently, challenge infection at 120 or 180 dpv, resulted in 2-3-fold lower parasite burden in vaccinated mice than was noted in unvaccinated/infected mice. Co-delivery of IL-12- and GMCSF-encoding expression plasmids provided no significant benefits in enhancing the anti-parasite efficacy of the vaccine-induced T cell immunity. Booster immunization (bi) with recombinant TcG2/TcG4 proteins 3-months after primary vaccine enhanced the protective efficacy, evidenced by an enhanced expansion (1.2-2.8-fold increase) of parasite-specific, type 1 CD4⁺ and CD8⁺ T cells and a potent CTL response capable of providing significantly improved (3-4.5-fold) control of infecting <i>T</i>. <i>cruzi</i>. Further, CD8⁺T cells in vaccinated/bi mice were predominantly of central memory phenotype, and capable of responding to challenge infection 4-6-months post bi by a rapid expansion to a poly-functional effector phenotype, and providing a 1.5-2.3-fold reduction in tissue parasite replication. We conclude that the TcG2/TcG4 D/P vaccine provided long-term anti-<i>T</i>. <i>cruzi</i> T cell immunity, and bi would be an effective strategy to maintain or enhance the vaccine-induced protective immunity against <i>T</i>. <i>cruzi</i> infection and Chagas disease.</p></div