All pathogenic trypanosomatids have a complex life cycle during which they alternate among insects and vertebrate hosts. In the case of Trypanosoma cruzi, the etiological agent of Chagas disease, this parasite navigates through different environments within the infected organisms, such as different regions of the triatomine midgut, the mammalian blood and host-cells cytoplasm. Indeed, this parasite goes through different adaptive stages depending on the regions it colonizes. The insect midgut is colonized by replicative non-infective forms termed epimastigotes (Epi). In the terminal portion of the insect digestive tube, Epi must differentiate into non-dividing infective forms called metacyclic trypomastigotes (MT) in a process called metacyclogenesis. Inside the mammalian host, MT invade nucleated cells and reach the cytoplasm where they differentiate into dividing amastigotes (Ama). After a number of cell divisions, Ama differentiate into infective, non-dividing tissue-derived trypomastigotes (Trypo), going through an intermediate stage called intracellular epimastigote (IE). Trypo lyse the infected cells and reach the bloodstream to initiate a new round of infection by invading other cells, or by infecting a new insect during its bloodmeal (Almeida-de-Faria et al., 1999; Tyler and Engman, 2001; Alves and Colli, 2007).

Metacyclogenesis, one of the critical events in the life cycle of the parasite, is a well investigated process. It is known that epimastigotes initiate differentiation when subjected to nutritional stress (Figueiredo et al., 2000) by adhering to the intestinal epithelium (Kollien et al., 1998). Several factors have been proposed as crucial for this process (for a comprehensive review, see (Melo et al., 2020)). In the mid-1980s, Contreras et al. described a simple and reliable method to mimic in vitro the metacyclogenesis by using a defined medium. In this method, stationary-phase Epi are subjected to metabolic stress for 2 hours in the physicochemical conditions provided by triatomine urine. This is achieved by incubating them in a medium known as TAU (an acronym for Triatomine Artificial Urine). After this initial metabolic stress, parasites are transferred to TAU supplemented with a combination of Proline, Glutamate, Aspartate, and Glucose (Pro, Glu, Asp and Glc respectively), referred to as TAU-3AAG (Contreras et al., 1985). These findings were complemented by other studies shedding light on the involvement of several other metabolites in this complex process of cell differentiation (Homsy et al., 1989; Damasceno et al., 2018). It is currently known that Pro alone is enough to promote metacyclogenesis, while Leu and Ile were identified as amino acids that do not induce metacyclogenesis. Furthermore, an intriguing observation was reported that Pro-dependent metacyclogenesis is abolished in the presence of Leu or Ile (Homsy et al., 1989).

The differentiation among intracellular stages that occur after Ama replication are much more challenging to study, as they involve a complex system of factors and events within the parasite, which is located in the cytoplasm of the infected cell. Therefore, the knowledge of the factors influencing the differentiation occurring during the intracellular infection is limited, and the process itself is not well understood. However, by utilizing a system that allows for synchronous intracellular infections, it has been demonstrated that, as occurs in metacyclogenesis, Pro is involved in the differentiation from IE to Trypo inside the infected mammalian host cells (Tonelli et al., 2004). To the best of our knowledge, Pro is the only factor identified thus far that promotes differentiation between these two intracellular stages of T. cruzi.

Interestingly, despite these data, and their relevance in metabolism and signaling, BCAA (Leu, Ile, and Val) have so far not been systematically assayed as possible players in the differentiation and proliferation of T. cruzi (Santos Souza et al., 2023). Based on the reported effect of Leu and Ile on the in vitro differentiation from the proliferative non-infective insect form Epi to the non-proliferative infective insect form MT, we were prompted to investigate the potential role of BCAAs as modulators of parasite differentiation during the mammalian host-cell infection. In this study, we revisit the inhibitory action of BCAAs on Pro-dependent metacyclogenesis, as previously reported by Homsy et al., and now also include Val (the BCAA which was not assessed for metacyclogenesis in previous studies). Furthermore, we assessed the ability of all three BCAAs to influence the differentiation of intracellular stages and, consequently, the release of trypomastigotes from infected host cells.