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 5Ј 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 3Ј 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. INTRODUCTIONGiardia duodenalis (syn. G. intestinalis, G. lamblia) is a flagellated protozoan parasite of medical significance because it is a major cause of waterborne enteric disease worldwide (Adam, 1991). The biology of this parasite is of particular interest because it represents one of the earliest branching lineages of eukaryotic descent, based on phylogenetic analysis of ribosomal and protein coding sequences (Sogin et al., 1989;Leipe et al., 1993;Gupta et al., 1994;Drouin et al., 1995;Roger et al., 1999). Giardia also lacks organelles typical for higher eukaryotes such as mitochondria and peroxisomes. Asexually dividing, motile trophozoites colonize the upper intestine of humans and other mammals, where they attach to the intestinal epithelium (Adam, 1991). Encysting Giardia trophozoites undergo a series of developmental changes in the course of which they synthesize, export, and assemble an extensive fibrillar extracellular matrix composed of proteins but also a large proportion of carbohydrate, mainly in the form of N-acetylgalactosamine (Manning et al., 1992). Formation of environmentally resistant cysts is a key step in Giardia development as in other eukaryotic pathogens (Eichinger, 1997;Taratuto and Venturiello, 1997;Dubey et al., 1998) that has not been well characterized on a molecular level. Therefore, a better understanding of the mechanisms that govern stage-specific regulation of genes and the synthesis, transport, and assembly of the cyst wall is needed.Several genes that are up-regulated during Giardia encystation have been identified recently by biochemical approaches, molecular genetics, or metab...

Glucosylceramide synthesis inhibition affects cell cycle progression, membrane trafficking, and stage differentiation in Giardia lamblia

“…To investigate the nature of the fi rst N-terminal hydrophobic sequence, HA tags were introduced at the N-terminal end either of the full-length GlGCS or of a variant lacking the fi rst hydrophobic stretch (aa [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22]. Both reporters showed an equivalent signal and distribution, indicating that, like the mammalian homolog, the parasite protein is not proteolytically processed to remove the fi rst hydrophobic sequence (data not shown).…”

“…New subcultures were obtained by inoculating 5 × 10 4 trophozoites from confl uent cultures into new 11 ml culture tubes. Two-step encystation was induced as described previously ( 19,20 ) by cultivating the cells for ‫ف‬ 44 h in medium without bile (pre-encysting medium) and subsequently in medium with higher pH and porcine bile (encysting medium).…”

“…All constructs of giardial glucosylceramide synthase (GCS) (GL50803_11642) were based on the expression cassette C1-CWP for inducible expression under the control of the CWP1 promoter ( 19 ). For N-terminal tagging with the hemagglutinin (HA) epitope, full length GCS (aa 2-537) and variant without putative signal peptide (aa 23-537) coding regions were amplifi ed by PCR and cloned in a vector containing the HA epitope tag upstream of the NsiI restriction site.…”

An Ancestral Secretory Apparatus in the Protozoan Parasite Giardia intestinalis

“…The Giardia endomembrane system undergoes significant morphological changes during stage conversion, culminating in the emergence of large membrane-bound compartments containing cyst wall material (ESVs) and subsequent cyst wall assembly (15)(16)(17). Previous reports have described a Golgi apparatus structure (i.e.…”

Sterol and fatty acid regulatory pathways in a Giardia lamblia-derived promoter

“…It has been shown that 466 bp of the upstream regulatory region of the GL cwp-2 gene is sufficient to control regulated transcription (26). We chose to investigate the transcription of the cwp-2 promoter in a mammalian system because regulation of SREBP is well defined in mammalian cells.…”