Differential Antioxidant Enzyme Profiles Reveal Early Molecular Signatures of Virulence in Trypanosoma cruzi DTU-TcI and DTU-TcVI Strains

Edward Valencia Ayala^1,2Joao Reis Cunha³Maritza Calderon Sanchez⁴Angela Vidal Riva²Daniella C. Bartholomeu³Alexandre F Marques^5*

• ¹Universidad Nacional Mayor de San Marcos Facultad de Ciencias Biologicas, Lima District, Peru
• ²Universidad de San Martin de Porres Facultad de Medicina Humana, La Molina, Peru
• ³Universidade Federal de Minas Gerais Instituto de Ciencias Biologicas, Belo Horizonte, Brazil
• ⁴Universidad Peruana Cayetano Heredia Facultad de Ciencias y Filosofia, Lima District, Peru
• ⁵University of Southern Mississippi, Hattiesburg, United States

Background: Trypanosoma cruzi, the causative agent of Chagas disease, exhibits significant genetic and phenotypic diversity that influences clinical outcomes. Antioxidant enzymes are crucial for parasite survival under host-induced oxidative stress, yet their role as determinants of virulence remains underexplored. Objective: This study aimed to characterize and compare early antioxidant responses between the virulent T. cruzi CL Brener clone and a non-virulent strain isolated from Arequipa, Peru, to test the hypothesis that elevated antioxidant enzyme expression is associated with parasite virulence. Methods: A reactivation protocol using Triatoma infestans restored infectivity in the nonvirulent Arequipa strain. Comparative analyses were performed between the non-virulent Arequipa strain (AQP300), the reactivated strain (AQP-RE), and the CL Brener clone using in vitro macrophage infection models, flow cytometry, gene expression profiling, and in vivo infection assays in C57BL/6 mice. Results: Both AQP-RE strain and CL Brener clone established infections in mice, whereas the AQP300 remained non-infective. Genome analysis confirmed the conservation of seven key antioxidant genes across strains. Despite similar phagocytosis rates, macrophages infected with CL Brener exhibited significantly lower nitric oxide and reactive oxygen species production. In contrast, CL Brener triggered robust upregulation of antioxidant genes (TcAPX, TcCPX, TcMPX, TcTR, and TcSODs) at 3-and 24-hours post-infection. In vivo, CL Brener induced significantly higher parasitemia, cardiac parasite burden, and sustained proinflammatory cytokine expression (IFN-γ, TNF-α) compared to AQP-RE. Elevated IL-10 expression in AQP-RE-infected mice during early infection suggests enhanced immune regulation in response to lower virulence. Conclusions: Enhanced early expression of antioxidant enzymes appears to correlate with T. cruzi virulence and persistence, suggesting a potential role for these molecules as biomarkers or therapeutic targets. These observations may help clarify strain-specific mechanisms contributing to Chagas disease pathogenesis.