Formation of the Giardia Cyst Wall: Studies on Extracellular Assembly Using Immunogold Labeling and High Resolution Field Emission SEM

Abstract

Encystment of the intestinal protozoan, Giardia, is a key step in the life cycle that enables this parasite to be transmitted from host to host via either fecal oral, waterborne, or foodborne transmission. The process of encystment was studied by localizing cyst wall specific antigens with immunofluorescence for light microscopy and immunogold staining for field emission scanning electron microscopy. Chronological sampling of Giardia cultures stimulated with endogenous bile permitted identification of an intracellular and extracellular phase in cyst wall formation, a process which required a total of 14-16 h. The intracellular phase lasted for 8-10 h, while the extracellular phase, involved the appearance of cyst wall antigen on the trophozoite membrane, and the assembly of the filamentous layer, a process requiring an additional 4-6 h for completion of mature cysts. The extracellular phase was initiated with the appearance of cyst wall antigen on small protrusions of the trophozoite membrane (approximately 15 nm), which became enlarged with time to caplike structures ranging up to 100 nm in diameter. Caplike structures involved with filament growth were detected over the entire surface of the trophozoite including the adhesive disc and flagella. Encysting cells rounded up, lost attachment to the substratum, and became enclosed in a layer of filaments. Late stages in encystment included a "tailed" cyst in which flagella were not fully retracted into the cyst. Clusters of cysts were seen in which filaments at the surface of one cyst were connected with the surface of adjacent cysts or the "tailed" processes of adjacent cysts, suggesting that the growth of cyst wall filaments may be at the terminal end. In conclusion, the process of encystment has been shown to consist of two morphologically different stages (intracellular and extracellular) which requires 16 h for completion. Further investigation of the extracellular stage with regard to assembly of the filamentous layer of the cyst wall may lead to innovative methods for interfering with production of an intact functional cyst wall, and thereby, regulation of viable Giardia cyst release from the host.

The Most Abundant Glycoprotein of Amebic Cyst Walls (Jacob) Is a Lectin with Five Cys-Rich, Chitin-Binding Domains

“…In contrast, the Giardia cyst wall contains polymers of GalNAc rather than GlcNAc, and the abundant Leu-rich proteins of the Giardia cyst walls (CWP1 and CWP2) show no similarity in structure to Jacob or peritrophins (27,30). Like Giardia, the amebic cyst wall is synthesized simultaneously from numerous loci across the surface of parasites, while cyst walls of budding yeast or elongating fungal hyphae are secreted from particular loci (6,18,26). While chitin is a primary structural component of the amebic cyst wall and peritrophic matrices, it is often used to shape fungal walls, which have a complex architecture and composition (2,5,6).…”

Stage-Specific Expression and Targeting of Cyst Wall Protein–Green Fluorescent Protein Chimeras inGiardia

“…Previous work has shown that ESV membranes can become continuous with the plasma membrane, suggesting that ESV contents are secreted by exocytosis (Reiner et al, 1990). On the other hand, scanning electron microscopy has shown the presence of numerous knob-like structures on the plasma membrane and even on flagella of encysting cells that react with an antibody specific for cyst wall components and were postulated to be the attachment points for the fibrils observed on mature cyst walls (Erlandsen et al, 1996). Outgrowth of fibrils from many different locations on the plasma membrane, however, has not been demonstrated and would be difficult to reconcile with the localization of the membrane-bound ESVs that we see before deposition of the cyst wall.…”

Encystation commitment inGiardia duodenalis: a long and winding road

“…Near to the plasma membrane, ESV appears to carry out two processes: a) fusion with PV (Luján & Touz, 2003) that acidifies the cargo to limit CP2 activity and/or allow ESCP activity and favor other undefined processing mechanisms preceding the release of CW material on the cell surface; or b) fission (dispersal) into small secretory vesicles that eventually fuse with the plasma membrane to discharge its content (Hehl & Marti, 2004). Proposal 'a' is supported by the PV-to-ESV redistribution of CLH (Marti et al, 2003) while 'b' is consistent with microscope observations of the initial secretion of antigenic [(glyco)polypeptide] CW material as small protrusions on the surface of encysting cells (Erlandsen et al, 1990(Erlandsen et al, , 1996. CWP complexes are concentrated, stabilized, sorted and bud in ESV together with chaperons that allow their correct folding (by immunoglobulin heavy chain-binding protein (BiP)) and oligomerization (by protein disulfide isomerases (PDI) 1-3).…”

“…The release of ESV contents and CWF formation/assembly may occur stepwise in late encysting cells, i.e., CWP and HCNCp (glyco)polypeptide exposure on the cell surface precedes their co-polymerization with the [GalNAc(β1➝3)GalNAc(β1➝3)] n complex (Argüello-García et al, 2002). This proposal is supported by independent estimations of the time for ESV content release (up to 1 min) (Hehl & Marti, 2004) compared with that required for complete CW assembly (5-6 h.), a process occurring by fibril tip growth (Erlandsen et al, 1996;Argüello-García et al, 2002) and by the presence of CW (glyco) polypeptides in 15-to-100 nm-sized protrusions laid on the surface of encysting cells (Erlandsen et al, 1996) which do not display typical CWF architecture. The cytolocalization of cyst wall synthase (CWS) activity in encysting cells will provide further insights into the process of CW formation.…”