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

Abstract

In preparation for being shed into the environment as infectious cysts, trophozoites of Giardia spp. synthesize and deposit large amounts of extracellular matrix into a resistant extracellular cyst wall. Functional aspects of this developmentally regulated process were investigated by expressing a series of chimeric cyst wall protein 1 (CWP1)–green fluorescent protein (GFP) reporter proteins. It was demonstrated that a short 110 bp 5′ flanking region of the CWP1 gene harbors all necessary cis-DNA elements for strictly encystation-specific expression of a reporter during in vitro encystation, whereas sequences in the 3′ flanking region are involved in modulation of steady-state levels of its mRNA during encystation. Encysting Giardia expressing CWP1–GFP chimeras showed formation and maturation of labeled dense granule-like vesicles and subsequent incorporation of GFP-tagged protein into the cyst wall, dependent on which domains of CWP1 were included. The N-terminal domain of CWP1 was required for targeting GFP to regulated compartments of the secretory apparatus, whereas a central domain containing leucine-rich 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 investigating the organization of primitive eukaryotic vesicular transport.