Staging Encystation Progression in Giardia lamblia Using Encystation-Specific Vesicle Morphology and Associating Molecular Markers

Abstract

Differentiation into environmentally resistant cysts is required for transmission of the ubiquitous intestinal parasite Giardia lamblia. Encystation in Giardia requires the production, processing and transport of Cyst Wall Proteins (CWPs) in developmentally induced, Golgi-like, Encystation Specific Vesicles (ESVs). Progress through this trafficking pathway can be followed by tracking CWP localization over time. However, there is no recognized system to distinguish the advancing stages of this process which can complete at variable rates depending on how encystation is induced. Here, we propose a staging system for encysting Giardia based on the morphology of CWP1-stained ESVs. We demonstrate the molecular distinctiveness of maturing ESVs at these stages by following GlRab GTPases through encystation. Previously, we established that Giardia’s sole Rho family GTPase, GlRac, associates with ESVs and has a role in regulating their maturation and the secretion of their cargo. As a proof of principle, we delineate the relationship between GlRac and ESV stages. Through proteomic studies, we identify putative interactors of GlRac that could be used as additional ESV stage markers. This staging system provides a common descriptor of ESV maturation regardless of the source of encysting cells. Furthermore, the identified set of molecular markers for ESV stages will be a powerful tool for characterizing trafficking mutants that impair ESV maturation and morphology.

Encystation stimuli sensing mediated by adenylate cyclase AC2-dependent cAMP signaling in Giardia

“…Our results provide clear evidence that encystation stimuli alter plasma membrane lipid ordering, activate AC2-dependent intracellular cAMP elevation accompanied with dissociation of PKAr and PKAc, and this increase of intracellular cAMP upregulates MYB2, CWPs, and four enzymes in the GalNAc pathway to enable successful cyst production. It is noteworthy that AC2 overexpression induces encystation but ESVs only progress to Stage III which is correlated with 6-8 h of encystation stimuli 27 . The lack of fully formed cysts reveals that additional signaling pathways that co-regulate differentiation remain to be discovered.…”

The Giardia ventrolateral flange is a lamellar membrane protrusion that supports attachment

“…P21 activated Kinase (PAK, GL50803_2796) contains the only CRIB/PBD domain in the Giardia genome. We developed a CRIB based Gl Rac biosensor by fusing the CRIB/PBD domain of Giardia’s PAK kinase with mNG to create CRIB-mNG [ 52 ]. We found that CRIB-mNG was robustly recruited to the base of the ventrolateral flange, suggesting a role for active GTP-loaded Gl Rac in regulating flange assembly from this position ( Fig 5B ).…”

Giardia intestinalis and its endomembrane system*

“…Recently, it was reported a staging system for encysting Giardia based on the morphology of CWP1‐stained ESVs (Thomas et al, 2021). The authors proposed a system using molecular distinctiveness of maturing ESVs at these stages by following Gl Rab GTPases through encystation.…”