Dual localization of receptor-type adenylate cyclases and cAMP response protein 3 unveils the presence of two putative signaling microdomains in Trypanosoma cruzi

Trypanosoma cruzi is the etiologic agent of Chagas disease, a leading cause of disability and premature death in the Americas. This parasite spends its life between a triatomine insect and a mammalian host, transitioning between developmental stages in response to microenvironmental changes. Among the second messengers driving differentiation in T. cruzi, cAMP has been shown to mediate metacyclogenesis and response to osmotic stress, but this signaling pathway remains largely unexplored in this parasite. Adenylate cyclases (ACs) catalyze the conversion of ATP to cAMP. They comprise a multigene family encoding putative receptor-type ACs in T. cruzi. Using protein sequence alignment, we classified them into five groups and chose a representative member from each group to study their localization (TcAC1–TcAC5). We expressed an HA-tagged version of each protein in T. cruzi and performed immunofluorescence analysis. A peculiar dual localization of TcAC1 and TcAC2 was observed in the flagellar distal domain and in the contractile vacuole complex (CVC), and their enzymatic activity was confirmed by gene complementation in yeast. Furthermore, TcAC1 overexpressing parasites showed an increased metacyclogenesis, a defect in host cell invasion, and a reduced intracellular replication, highlighting the importance of this protein throughout T. cruzi life cycle. These mutants were more tolerant to hypoosmotic stress and showed a higher adhesion capacity during in vitro metacyclogenesis, whereas the wild-type phenotype was restored after disrupting TcAC1 localization. Finally, TcAC1 was found to interact with cAMP response protein 3 (TcCARP3), co-localizing with this protein in the flagellar tip and CVC. IMPORTANCE: We identified three components of the cAMP signaling pathway (TcAC1, TcAC2, and TcCARP3) with dual localization in Trypanosoma cruzi: the flagellar distal domain and the CVC, structures involved in cell adhesion and osmoregulation, respectively. We found evidence on the role of TcAC1 in both cellular processes, as well as in metacyclogenesis. Our data suggest that TcACs act as signal sensors and transducers through cAMP synthesis in membrane microdomains. We propose a model in which TcACs sense the harsh conditions in the triatomine hindgut (nutrient deprivation, acidic pH, osmotic stress, ionic composition, hydrophobic interactions) and become active. Synthesis of cAMP then triggers cell adhesion prior completion of metacyclogenesis, while mediating a response to osmotic stress in the parasite. These results shed light into the mechanisms driving cAMP-mediated cell differentiation in T. cruzi, while raising new questions on the activation of TcACs and the role of downstream components of this pathway.