Stage-specific expression and targeting of cyst wall protein-green fluorescent protein chimeras in Giardia

In preparation for being shed into the environment as infectious cysts, tro phozoites of Giardia spp. synthesize and deposit large amounts of extracell ular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a s eries of chimeric cyst wall protein 1 (CWP1)-green fluorescent protein (GFP ) reporter proteins. It was demonstrated that a short 110 by 5' flanking re gion of the CWP1 gene harbors all necessary cis-DNA elements for strictly e ncystation-specific expression of a reporter during in vitro encystation, w hereas sequences in the 3' flanking region are involved in modulation of st eady-state levels of its mRNA during encystation. Encysting Giardia express ing CWP1-GFP chimeras showed formation and maturation of labeled dense gran ule-like vesicles and subsequent incorporation of GFP-tagged protein into t he cyst wall, dependent on which domains of CWP1 were included. The N-termi nal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-ri ch repeats mediated association of the chimera with the extracellular cyst wall. We show that analysis of protein transport using GFP-tagged molecules is feasible in an anaerobic organism and provides a useful tool for invest igating the organization of primitive eukaryotic vesicular transport.