Quantitative proteomics of intracellular and axenic amastigote-like forms of Trypanosoma cruzi under replicative and non-replicative conditions

Trypanosoma cruzi , the etiologic agent of Chagas disease, has a complex life cycle that involves both arthropods and mammals. Amastigotes are the T. cruzi life forms capable of replication in mammalian hosts. Here we report a quantitative LC–MS/MS–based proteomics of intracellular amastigotes (AI) and amastigote-like forms produced in axenic cultures. AI were purified from T. cruzi- infected HeLa cells using DEAE-cellulose, whereas three different axenic amastigote-like forms were produced as follows. Incubation of trypomastigotes in DMEM, pH 5.0, for 9 h led to AD5 forms. Further incubation of AD5 cells for 18 h at pH 7.2 in DMEM or LIT media produced AD7 and AL7 cells, respectively. AD5 cells were non-replicative, whereas AD7 and AL7 cells displayed active replication. Proteomic analysis confirmed the efficiency of the AI isolation method. Further comparison of AI, AD5, AD7 and AL7 proteomes suggested the relevance of ubiquitination, phosphorylation and kinetoplast-associated proteins in amastigote replication and highlighted similarities between amastigote-like forms and intracellular amastigotes. In contrast, substantial differences in protein translation and energy metabolism distinguished axenic amastigote-like forms from intracellular amastigotes. Together, these results demonstrate that axenic amastigote-like forms only partially reflect the proteomic landscape of intracellular amastigotes. In this study we have adapted a previously published method for intracellular Trypanosoma cruzi amastigote purification, highlighting the great enrichment of the protozoan proteins at the proteomic level. Comparing the intracellular amastigotes with axenic amastigote-like grown life forms, we showed differences in the replication process, mainly related to energy, amino acid metabolism and translation. This study also revealed important processes for amastigote cell division, such as protein phosphorylation, ubiquitination and kDNA associated proteins, that are likely conserved on both intracellular and amastigote-like forms. We expect that this study can provide a guideline of axenic amastigote-like form replication biology for future T. cruzi experiments. Created in BioRender. Magalhaes melo, R. (2026) https://BioRender.com/d7h6quy • Quantitative proteomics revealed proteome differences between intracellular amastigotes isolated from infected mammalian cells and axenic amastigote-like forms. • The abundance of proteins involved in energy and amino acid metabolism differed markedly between intracellular and axenic amastigotes. • Proteins involved in cell cycle regulation, ubiquitination, and kinetoplast DNA maintenance were associated with replicative and non-replicative states.