Unraveling the Role of cAMP Signaling in Giardia: Insights into PKA-Mediated Regulation of Encystation and Subcellular Interactions

Abstract

cAMP plays a crucial role as a second messenger in the stage transition of various protozoan parasites. This signaling pathway relies on multiple effectors, such as protein kinase A (PKA), exchange protein activated by cAMP (EPAC), and cAMP-response element binding protein (CREB) transcription factors, to initiate signal transduction in humans. Even though Giardia has been observed to have only two adenylate cyclases (ACs) and a single cAMP effector, the specific functions of these components are not fully understood. In our previous research, we demonstrated the essential role of AC2-dependent cAMP signaling in the upregulation of the encystation program. Using NanoBit technology, we emphasized the significance of AC2-dependent cAMP biosynthesis in regulating the dissociation of PKAr and PKAc. In this study, our objectives are twofold: first, we used newly developed Split-Halo to examine subcellular interactions of GlPKAr and GlPKAc; second, we investigated whether PKAc regulates encystation markers. Our findings revealed distinct subcellular locations where GlPKAr and GlPKAc interacted during the trophozoite stage, including the flagella, basal bodies, and cytosol. Upon exposure to encystation stimuli, the interaction shifted from the flagella to the cytosol. Additionally, interaction occurred at an unidentified cell compartment between twelve to sixteen hours of encystation and persisted to the cyst stage. Knockdown of GlPKAc resulted in the downregulation of encystation-specific genes, leading to the production of fewer viable and water-resistant cysts indicating a role for PKA in transcriptional regulation. These discoveries contribute to a deeper understanding of the cAMP signaling pathway and its pivotal role in governing Giardias encystation process.