From Trypomastigotes to Trypomastigotes: Analyzing the One-Way Intracellular Journey of by Ultrastructure Expansion Microscopy.

The protozoan parasite is the causative agent of Chagas disease, also called American trypanosomiasis. This neglected tropical disease affects millions of individuals across the Americas. To complete its life cycle, parasitizes both vertebrate hosts and its vector, commonly known as the 'kissing bug'. The parasite's survival and proliferation strategies are driven by the diverse environments it encounters. Despite being described by Carlos Chagas in 1909, significant knowledge gaps persist regarding the parasite's various life forms and adaptive capabilities in response to environmental cues. In this study, we employed Ultrastructure Expansion Microscopy to explore the intricate journey of within the host cell. Upon entry into the host cell, trypomastigotes undergo folding, resulting in intermediate forms characterized by a rounded cell body, anterior positioning of basal bodies, and a shortened flagellum. The repositioning of basal bodies and the kinetoplast and the shortening of the flagella mark the culmination of intracellular amastigogenesis. Furthermore, we analyzed intracellular trypomastigogenesis, identifying discrete intermediate forms, including leaf-shaped stages and epimastigote-like forms, which suggests a complex differentiation process. Notably, we did not observe any dividing intracellular epimastigotes, indicating that these may be non-replicative forms within the host cell. Our detailed examination of amastigote cell division revealed semi-closed nuclear mitosis, with mitotic spindle formation independent of basal bodies. This study provides new insights into the morphological and cytoskeletal changes during the intracellular stages of , providing a model for understanding the dynamics of intracellular amastigogenesis and trypomastigogenesis.