Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed in the infective and intracellular stages of Trypanosoma cruzi and are recognized as antigenic targets by both the humoral and cellular arms of the immune system. Previously, we demonstrated the efficacy of genes encoding GPI-anchored proteins in eliciting partially protective immunity to T. cruzi infection and disease, suggesting their utility as vaccine candidates. For the identification of additional vaccine targets, in this study we screened the T. cruzi expressed sequence tag (EST) and genomic sequence survey (GSS) databases. By applying a variety of web-based genomemining tools to the analysis of ϳ2,500 sequences, we identified 348 (37.6%) EST and 260 (17.4%) GSS sequences encoding novel parasite-specific proteins. Of these, 19 sequences exhibited the characteristics of secreted and/or membrane-associated GPI proteins. Eight of the selected sequences were amplified to obtain genes TcG1, TcG2, TcG3, TcG4, TcG5, TcG6, TcG7, and TcG8 (TcG1-TcG8) which are expressed in different developmental stages of the parasite and conserved in the genome of a variety of T. cruzi strains. Flow cytometry confirmed the expression of the antigens encoded by the cloned genes as surface proteins in trypomastigote and/or amastigote stages of T. cruzi. When delivered as a DNA vaccine, genes TcG1-TcG6 elicited a parasitespecific antibody response in mice. Except for TcG5, antisera to genes TcG1-TcG6 exhibited trypanolytic activity against the trypomastigote forms of T. cruzi, a property known to correlate with the immune control of T. cruzi. Taken together, our results validate the applicability of bioinformatics in genome mining, resulting in the identification of T. cruzi membrane-associated proteins that are potential vaccine candidates.

In this study, we investigated the role of Trypanosoma cruzi invasion and inflammatory processes in reactive oxygen species (ROS) production in mouse atrial cardiomyocyte line (HL-1) and primary adult rat ventricular cardiomyocytes. Cardiomyocytes were incubated with T. cruzi (Tc) trypomastigotes, Tc lysate (TcTL) or Tc secreted proteins (TcSP) for 0-72 h, and ROS measured by amplex red assay. Cardiomyocytes infected by T. cruzi (but not those incubated with TcTL or TcSP) exhibited a linear increase in ROS production during 2-48 h post-infection (max.18-fold increase) which was further enhanced by recombinant cytokines (IL-1β, TNF-α and IFN-γ). We observed no increase in NADPH oxidase, xanthine oxidase, and myeloperoxidase activities, and specific inhibitor of these enzymes did not block the increased rate of ROS production in infected cardiomyocytes. Instead, the mitochondrial membrane potential was perturbed, and resulted in inefficient electron transport chain (ETC) activity, and enhanced electron leakage and ROS formation in infected cardiomyocytes. HL-1 rho (ρ) cardiomyocytes lacked a functional ETC, and exhibited no increase in ROS formation in response to T. cruzi. Together, these results demonstrate that invasion by T. cruzi and inflammatory milieu affect mitochondrial integrity and contribute to electron transport chain inefficiency and ROS production in cardiomyocytes.

Seroanalysis of parasite circulation in dogs can help identify T. cruzi infection in humans.

In this study, we evaluated the activities of respiratory chain complexes and oxidative phosphorylation (OXPHOS) capacity of the heart to gain insights into the pathological significance of mitochondrial dysfunction in chagasic cardiomyopathy (CCM). In a murine model of Trypanosoma cruzi infection, biochemical and histochemical analysis of the cardiac mitochondria revealed deficiency of the respiratory chain complexes (CI-CV) in infected mice; the inhibition of CI activity was more pronounced in the acute infection phase, CIII was constitutively repressed throughout the infection and disease phase, and the CV defects appeared in chronic phase only. A substantial decline in cardiac mtDNA content (54-60%) and mitochondria-encoded transcripts (50-65%) with disease development indicated that the alterations in mtDNA contribute to the quantitative deficiencies in respiratory chain activity in chagasic hearts. The observations of a selective inhibition of redox-sensitive CI and CIII complexes that are also the site of free radical generation in mitochondria, and the decline in cardiac mtDNA content in infected mice, all support the free radical hypothesis of mitochondria dysfunction in CCM. Consequently, OXPHOS-mediated ATP synthesis capacity of the cardiac mitochondria in infected mice was substantially reduced (37-50%), suggesting an energy homeostasis in the affected tissue.

In this study, we investigated the mechanism(s) of mitochondrial functional decline in acute Chagas' disease. Our data show a substantial decline in respiratory complex activities (39 to 58%) and ATP (38%) content in Trypanosoma cruzi-infected murine hearts compared with normal controls. These metabolic alterations were associated with an approximately fivefold increase in mitochondrial reactive oxygen species production rate, substantial oxidative insult of mitochondrial membranes and respiratory complex subunits, and >60% inhibition of mtDNA-encoded transcripts for respiratory complex subunits in infected myocardium. The antioxidant phenyl-␣-tert-butyl nitrone (PBN) arrested the oxidative damage-mediated loss in mitochondrial membrane integrity, preserved redox potential-coupled mitochondrial gene expression, and improved respiratory complex activities (47 to 95% increase) and cardiac ATP level (>40% increase) in infected myocardium. Importantly, PBN resulted twofold decline in mitochondrial reactive oxygen species production rate in infected myocardium. Taken together, our data demonstrate the pathological significance of oxidative stress in metabolic decay and energy homeostasis in acute chagasic myocarditis and further suggest that oxidative injuries affecting mitochondrial integrity-dependent expression and activity of the respiratory complexes initiate a feedback cycle of electron transport chain inefficiency, increased reactive oxygen species production, and energy homeostasis in acute chagasic hearts. PBN and other mitochondria-targeted antioxidants may be useful in altering mitochondrial decay and oxidative pathology in Chagas' disease. Chagas' disease is a pathological process induced by infections with the hemoflagellate protozoan Trypanosoma cruzi and is a major human health problem in the southern parts of the American continent.1 In Ͼ95% of acutely infected individuals, parasitemia is controlled by the immune system. After several years of a clinically silent but ongoing process of organelle and myocardial degeneration, Ͼ40% of seropositive patients develop chronic cardiomyopathy.Since early findings of abnormal mitochondria in cardiac biopsies obtained from seropositive patients, 2 mitochondrial impairment has been associated with cardiac dysfunction in Chagas' disease. Quantitative light and electron microscopic analysis of the myocardial biopsy samples from human chagasic patients and experimental models has revealed that mitochondrial degenerative changes occur early in the course of T. cruzi infection and are exacerbated with progressive disease severity. 2-5The functional decline of cardiac mitochondria in experimental models of Chagas' disease was shown by impaired activities of the respiratory complexes that contain subunits encoded by mtDNA and nDNA.6 -8 The demonstration of inefficient ATP production in infected mice 8 provided the first indication of the physiological effects of mitochondrial dysfunction in chagasic hearts.Toward understanding the mechanism(s) of mitochondrial decay, it...