Rac Regulates Giardia lamblia Encystation by Coordinating Cyst Wall Protein Trafficking and Secretion

Abstract

Encystation of the common intestinal parasite Giardia lamblia involves the production, trafficking, and secretion of cyst wall material (CWM). However, the molecular mechanism responsible for the regulation of these sequential processes remains elusive. Here, we examined the role of GlRac, Giardia’s sole Rho family GTPase, in the regulation of endomembrane organization and cyst wall protein (CWP) trafficking. Localization studies indicated that GlRac is associated with the endoplasmic reticulum (ER) and the Golgi apparatus-like encystation-specific vesicles (ESVs). Constitutive GlRac signaling increased levels of the ER marker PDI2, induced ER swelling, reduced overall CWP1 production, and promoted the early maturation of ESVs. Quantitative analysis of cells expressing constitutively active hemagglutinin (HA)-tagged GlRac (HA-RacCA) revealed fewer but larger ESVs than control cells. Consistent with the phenotype of premature maturation of ESVs in HA-RacCA-expressing cells, constitutive GlRac signaling resulted in increased CWP1 secretion and, conversely, morpholino depletion of GlRac blocked CWP1 secretion. Wild-type cells unexpectedly secreted large quantities of CWP1 into the medium, and free CWP1 was used cooperatively during cyst formation. These results, in part, could account for the previously reported observation that G. lamblia encysts more efficiently at high cell densities. These studies of GlRac show that it regulates encystation at several levels, and our findings support its coordinating role as a regulator of CWP trafficking and secretion. The central role of GlRac in regulating membrane trafficking and the cytoskeleton, both of which are essential to Giardia parasitism, further suggests its potential as a novel target for drug development to treat giardiasis.

Myosin-independent cytokinesis in Giardia utilizes flagella to coordinate force generation and direct membrane trafficking

“…However, we propose that actin's most critical role is in positioning the tubulin cytoskeleton so that the flagella can direct trafficking and force generation along the furrow. The mechanism used to activate actin polymerization for organelle positioning and differential cortical organization remain to be determined, although it is likely that Giardia's sole Rho family GTPase, GlRac, has some role in coordinating actin polymerization and membrane remodeling during cytokinesis (31,79). In otherwise normal-looking cells, we observed many actindepleted and Rab11-depleted cells that were stuck at the tail-totail stage of cytokinesis.…”

“…Dataset S2 provides sequences and workflow. Note that N-terminal fusions result in morpholino-resistant constructs (62), and therefore we included the first 27 bp of the coding sequence to restore morpholino sensitivity (79) to the 3HA-Rab11 MS construct.…”

Succinylated Wheat Germ Agglutinin Colocalizes with the Toxoplasma gondii Cyst Wall Glycoprotein CST1

“…The actin cytoskeleton and Rab11 traffic vesicles to the cyst walls of Giardia and Entamoeba, supporting a model whereby cyst wall proteins are transported through the cytoskeleton (54,55). In addition, the Giardia Rho GTPase Rac regulates endomembrane organization and cyst wall protein trafficking in Giardia (56). How Toxoplasma cyst wall cargo for cyst wall building is delivered to the cyst periphery is currently unknown.…”

Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia

“…Although tEM showed no evidence for glycan fibrils on pseudocysts, we used a CWP1 capture assay to address the question whether some of the CW glycan was exported to the surface of differentiated Δcwp1 cells. Soluble CWP1 is released in significant amounts by encysting WB-C6 cells, presumably due to incomplete cross-linking on the surface of newly formed cysts and can be harvested from cell culture supernatants27. Soluble CWP1 binds efficiently to deproteinated ‘glycan cage' preparations of WB-C6 cysts26 but also to some extent to newly formed CWs27.…”

“…Soluble CWP1 is released in significant amounts by encysting WB-C6 cells, presumably due to incomplete cross-linking on the surface of newly formed cysts and can be harvested from cell culture supernatants27. Soluble CWP1 binds efficiently to deproteinated ‘glycan cage' preparations of WB-C6 cysts26 but also to some extent to newly formed CWs27. Using the anti-CWP1 mAb we could detect captured CWP1 on unfixed Δcwp1 pseudocysts incubated for 1 h in culture supernatant of encysting WB-C6 cells (Fig.…”