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.
Highlights
While some level of synchronization could be achieved from this method, cells remained in various stages within the process of cyst development, as judged by localizing CWP1 and visualizing Encystation Specific Vesicles (ESVs) morphology (Figure 1)
Based on our current understanding of the sequence of events involved in encystation, we propose the following key for staging encysting cells (Figure 1): Stage I – CWP1 localizes to the endoplasmic reticulum (ER); Stage II – CWP1 localizes in the ER and in ER-associated punctate structures thought to be ER-exit sites (Faso et al, 2013); Stage III – CWP1 localizes homogenously in small ESVs; Stage IV – CWP1 localizes to doughnut-shaped structures as a result of CWP2 being proteolytically processed to drive core condensation which pushes fluid phase CWP1 to the vesicle periphery
We have developed a staging system for encysting cells and have identified novel molecular markers for ESV stages that correlate with the progression of encystation
Summary
Giardia intestinalis and Giardia duodenalis) is a major intestinal parasite which infects more than 280 million people every year (Lane and Lloyd, 2002). The lifecycle of this diplomonad protozoan is simple, featuring only two stages – the binucleate, double-diploid, proliferative trophozoites which non-invasively colonize host intestines and the environmentally resistant, infectious, non-motile cysts that are shed in host’s feces. Regulation of encystation ensures the production of viable cysts and promotes transmission of this ubiquitous parasite. Being a popular life-cycle strategy adopted by other protozoan parasites, studying this differentiation process is important and Giardia is the best-developed model available (Eichinger, 2001).
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