Abel Sana^1,2, Sarah Beatriz de Fucio Barros^1,2, Daniella dos Santos Courrol³, Angela Silva Barbosa³, Marcel Ivan Ramirez^1,2

Introduction: Trypanosoma cruzi, the aetiological agent of Chagas disease, must convert bloodstream trypomastigotes (TCTs) into replicative epimastigotes, an event called epimastigogenesis, inside the triatomine midgut to complete its life-cycle. Methodology: We analysed three parasite populations, trypomastigotes, epimastigotes, and trypomastigotes differentiating into epimastigotes under the influence of extracellular vesicles (EVs) and, by means of complement-lysis assays, complement-factor deposition studies, and measurements of C3b and C5b cleavage, sought to define the fine association between differentiation, EVs, complement activation, and resistance, as well as to identify the complement pathway predominantly affected. Epimastigogenesis was induced in CL Brener and Dm28c strains at 28 °C or 37 °C. Results: Differentiation occurred exclusively at 28 °C, with higher conversion in Dm28c (56 %) than in CL Brener (35 %). Although both strains still invaded Vero cells after 72 h, infectivity declined sharply by 120 h and was abolished by 168 h. In parallel, trypomastigotes undergoing 72 h of differentiation cleaved C3b and generated C5b, disrupting the amplification loops shared by the classical, lectin and alternative complement pathways and preventing membrane-attack-complex formation. EVs isolated from 24 h or 72 h cultures, validated by nanoparticle-tracking analysis, increased the epimastigote yield in CL Brener by 10 % and 20 %, respectively, and intensified C3b/C5b cleavage. Conclusion: These findings indicate that EV-associated proteases and regulators modulate complement activation, linking immune evasion with morphological differentiation. Determining the specific pathway preferentially targeted by EVs will clarify how T. cruzi controls sensitivity to host immunity and may reveal novel strategies to block parasite development and transmission.

Agradecimentos: We would like to thank CNPq, the Postgraduate Program in Cellular and Molecular Biology of the Federal University of Paraná, the Carlos Chagas Institute/Fiocruz Paraná and the EVAHPI research group for all the support provided, which made this work possible.