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

Abstract

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.

mSphere of Influence: Compartmentalized cAMP signals in American trypanosomes

“…Understanding the role of cAMP in environmental sensing could lead to the development of strategies to interrupt the parasite’s life cycle and control this silent but deadly disease. Our recent work revealed the presence of two putative cAMP microdomains in T. cruzi : the flagellar distal domain and the contractile vacuole complex, compartments specialized in cell adhesion and osmoregulation, respectively ( 4 ). Studies involving cAMP signaling in three different eukaryotic models have strongly influenced the current direction of my research ( 1 , 7 , 8 ).…”

“…Altogether, these findings led me to hypothesize that microenvironmental cues could activate T. cruzi ACs in at least two putative cAMP signaling microdomains: the flagellar distal domain and the contractile vacuole complex. Three cAMP signaling components share dual localization in these microdomains: TcAC1, TcAC2, and TcCARP3, and are involved in cell adhesion, metacyclogenesis, and osmoregulation in T. cruzi ( 4 ). But there are still key unanswered questions.…”

A sticky situation: When trypanosomatids attach to insect tissues

“…A PDE localized to the CVC could terminate the signal [118]. Recent work on T. cruzi AC1 and AC2 found that they are present at both the flagellar tip and the CVC [119]. When TcAC1 is overexpressed, it increases both attachment and metacyclogenesis, an effect that can be rescued by expression of truncated mutants with altered localization.…”

“…When Tc AC1 is overexpressed, it increases both attachment and metacyclogenesis, an effect that can be rescued by expression of truncated mutants with altered localization. These truncations also reduced the parasites’ ability to recover from hyperosmotic stress through regulatory volume decrease [ 119 ].…”

Gene editing of putative cAMP and Ca2+‐regulated proteins using an efficient cloning‐free CRISPR/Cas9 system in Trypanosoma cruzi

“…In mammalian cells, the basic components of this signaling pathway are well defined, and the expression of these proteins in different microdomains determines spatiotemporal regulation of cAMP signals (Musheshe et al, 2018;Wang et al, 2022). We have recently identified two putative cAMP signaling microdomains in T. cruzi: the flagellar distal domain (flagellar tip) and the contractile vacuole complex (CVC), structures involved in cell adhesion and osmoregulation, respectively (Chiurillo et al, 2023). However, just a few components of this pathway have been characterized in T. cruzi (Lander et al, 2021;Schoijet et al, 2019).…”