Characterization of cAMP signaling microdomains in the human pathogen Trypanosoma cruzi

Abstract Chagas disease (CD) is a leading cause of disability and premature death in the Americas. This vector-borne infectious disease is caused by the protozoan parasite Trypanosoma cruzi. There is no vaccine to prevent CD or satisfactory treatment for chronic patients. If untreated, the infection persists for a lifetime, causing serious irreversible heart and gastrointestinal damage in one-third of the infected population, several years or even decades after the initial contagion. Unfortunately, most affected individuals remain undiagnosed and untreated. Understanding T. cruzi biology is crucial to develop alternative strategies to control Chagas disease. T. cruzi life cycle involves a triatomine vector and a mammalian host. To survive microenvironmental stress, the parasite differentiates into four main developmental stages. The molecular mechanisms driving these transformations are highly unexplored. Cyclic AMP (cAMP) is a universal second messenger that regulates diverse cellular functions, including cell differentiation. In mammalian cells cAMP signals are spatiotemporal regulated by compartmentalization in subcellular microdomains to ensure specificity, duration, and intensity of the signal, where phospodiesterases (PDEs) precisely regulate cAMP levels. Our laboratory recently discovered two putative cAMP microdomains in T. cruzi: the flagellar distal domain (flagellar tip) and the contractile vacuole complex (CVC). However, the specific role of cAMP in each one of these compartments remains unknown. Three PDEs have been reported in these two structures: TcPDEB1, TcPDEB2 in the flagellum and TcPDEC2 in the CVC. This study aims to characterize the flagellar tip and the CVC as signaling domains in T. cruzi. We hypothesize that these two compartments are cAMP signaling microdomains involved in specific cellular processes: cellular adhesion and metacyclogenesis (flagellar tip), and response to osmotic (CVC), where the specificity of the signals is regulated by PDEs. To address this hypothesis, we propose to perform the functional and structural characterization of cAMP microdomains in T. cruzi. To elucidate the specific function of each microdomain, we will modulate the expression of flagellar and CVC PDEs by generation of overexpression and knockout cell lines of the genes encoding these proteins. We will then evaluate cAMP content, cell adhesion, metacyclogenesis, response to osmotic stress, host cell invasion, intracellular replication, and gene expression profiles. For the structural characterization, we will use lipid raft markers for co-localization with cAMP signaling proteins. This result will confirm the membrane assembly of cAMP signaling proteins in lipid rafts. We will also use the fluorescent dye NR12S to monitor microdomain stability (membrane fluidity) in response to different extracellular conditions. In addition, we will evaluate the interactome of specific proteins in each microdomain to identify new signaling players present in each compartment. Results from this study will provide new insights into the role of compartmentalized cAMP signals in T. cruzi, specifically during developmental transformations, significantly contributing to understand environmental sensing in unicellular eukaryotes.