Active and Passive Mechanisms Drive Secretory Granule Biogenesis during Differentiation of the Intestinal Parasite Giardia lamblia

Abstract

The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors.

Encystation commitment inGiardia duodenalis: a long and winding road

“…Likewise the TCWP2 moiety may confer on CWP2 a role as sorting factor for CWP complexes through a likely ligand activity with Giardia-specific ER membrane receptors. This is in turn consistent with the observation that nonencysting trophozoites transfected with haemagglutinin-tagged CWP1-TCWP2 chimera or the whole CWP2 display granules morphologically similar to ESV (Gottig et al, 2006) but these granules are not formed in similarly transfected mammalian cells (Elias et al, 2007).…”

“…Encystation-specific vesicles (ESV) are specialized secretory granules that mature during their traffic from ER to the cell surface that act as the equivalents of late Golgi structures and are also a hallmark of encystation induction. ESV arise from: a) specialized regions of the ER, a trans-Golgi equivalent (Gottig et al, 2006); b) enlarged ER cisternae (Lanfredi-Rangel et al, 2003); or c) homotypic fusion of ER-derived COPII-coated transport vesicles into "pre-Golgi" vesicles (Marti et al, 2003). The modestly increased mRNA expression levels of structural and cargo processing molecules of ESV during encystation induction (Marti et al, 2003) suggest that these structures are derived from structural templates in the ER that differentially recruit components required for sorting and cargo maturation.…”

Characterization of SNAREs Determines the Absence of a Typical Golgi Apparatus in the Ancient Eukaryote Giardia lamblia

“…Additional evidence, however, plays against the possibility that vesicles containing gQb1, gQb2, or gQa3 are Golgi equivalents. For example, (a) when we expressed an HA-tagged version of a heterologous glycosyltransferase or p115 in trophozoites, they localized in the perinuclear region of Giardia and not in a punctate pattern dispersed throughout the cytoplasm (55); (b) a similar perinuclear pattern was observed by constitutive expression of the Giardia KDEL receptor (55); and (c) brefeldin A treatment of trophozoites expressing gQb1, gQb2, or gQa3 failed to disrupt these vesicular structures, whereas the secretory pathway was certainly affected, as determined by relocalization of gARF3 (the only gARF showing similar localization pattern) (not shown). Although those experiments do not provide a definitive answer whether the vesicles containing SNAREs Qb1 and Qb2 are Golgi structures, our results strongly suggest that if Giardia has a Golgi, it is completely atypical.…”

“…In addition, all SNAREs were analyzed under two developmental conditions, non-encysting as well as encysting cells. The latter displays a unique secretory compartment, secretory granules called ESVs, which seem to develop directly from the ER, as shown recently (55).…”

Novel Role of Sphingolipid Synthesis Genes in Regulating Giardial Encystation

“…During encystation, various molecular and cellular changes take place that allow this protozoan to transport cyst wall proteins (CWPs) through regulatory secretory pathways (35). In encysting cells, three encystation-specific CWPs (CWP-1, -2, and -3 encoded by cwp-1, -2, and -3, respectively) are synthesized and concentrated within encystation-specific vesicles (ESVs) before targeting into the cyst wall (17,36,44).Recent studies suggest that all three CWPs are essential for forming ESVs and that CWP-2 functions as an aggregation factor to regulate ESV formation by interacting with CWP-1 and CWP-3 via conserved regions (17). It has been proposed that transient Golgi body-like membranes synthesized during encystation are involved in modifying the CWPs and other membrane proteins (27-29).…”