Active and Passive Mechanisms Drive Secretory Granule Biogenesis during Differentiation of the Intestinal Parasite Giardia lamblia
2006; Elsevier BV; Volume: 281; Issue: 26 Linguagem: Inglês
10.1074/jbc.m602081200
ISSN1083-351X
AutoresNatalia Gottig, E. Elias, Rodrigo Quiroga, María Jimena Nores, Alberto J. Solari, Marı́a C. Touz, Hugo D. Luján,
Tópico(s)Amoebic Infections and Treatments
ResumoThe parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors. The parasitic protozoan Giardia lamblia undergoes important changes to survive outside the intestine of its host by differentiating into infective cysts. During encystation, three cyst wall proteins (CWPs) are specifically expressed and concentrated within encystation-specific secretory vesicles (ESVs). ESVs are electron-dense secretory granules that transport CWPs before exocytosis and extracellular polymerization into a rigid cyst wall. Because secretory granules form at the trans-Golgi in higher eukaryotes and because Giardia lacks an identifiable Golgi apparatus, the aim of this work was to investigate the molecular basis of secretory granule formation in Giardia by examining the role of CWPs in this process. Although CWP1, CWP2, and CWP3 are structurally similar in their 26-kDa leucine-rich overlapping region, CWP2 is distinguished by the presence of a 13-kDa C-terminal basic extension. In non-encysting trophozoites, expression of different CWP chimeras showed that the CWP2 basic extension is necessary for biogenesis of ESVs, which occurs in a compartment derived from the endoplasmic reticulum. Nevertheless, the CWP2 basic extension per se is insufficient to trigger ESV formation, indicating that other domains in CWPs are also required. We found that CWP2 is a key regulator of ESV formation by acting as an aggregation factor for CWP1 and CWP3 through interactions mediated by its conserved region. CWP2 also acts as a ligand for sorting via its C-terminal basic extension. These findings show that granule biogenesis requires complex interactions among granule components and membrane receptors. Giardia lamblia, a parasitic protozoan of humans and other vertebrates, is a major source of waterborne disease worldwide. Clinical signs of giardiasis vary from asymptomatic infection to acute or chronic disease associated with diarrhea and malabsorption. Giardia is also of biological interest because it derives from one of the earliest branches of the eukaryotic line of descent (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar). Giardia undergoes important biological changes to survive in hostile environments, alternating between the motile trophozoite and the environmentally resistant cyst (see Fig. 1) (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar, 2Luján H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar). Trophozoites inhabit the upper small intestine and are responsible for symptoms of the disease, whereas cysts develop in the lower intestine and are excreted with the feces. This allows Giardia survival outside the intestine and transmission among susceptible hosts (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar). The encystation process includes cyst wall component synthesis and secretory organelle biogenesis. Encystation-specific secretory vesicles (ESVs) 2The abbreviations used are: ESVs, encystation-specific secretory vesicles; CWPs, cyst wall proteins; ER, endoplasmic reticulum; TGN, trans-Golgi network; VSP, variant-specific surface protein; HA, hemagglutinin; TM, transmembrane domain; mAbs, monoclonal antibodies; FITC, fluorescein isothiocyanate; DAPI, 4′,6-diamidino-2-phenylindole. (3Luján H.D. Marotta A. Mowatt M.R. Sciaky N. Nash Lippincott-Schwartz J.T.E. J. Biol. Chem. 1995; 270: 4612-4618Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 4Faubert G. Reiner D.S. Gillin F.D. Exp. Parasitol. 1991; 72: 345-354Crossref PubMed Scopus (48) Google Scholar, 5Gillin F.D. Reiner D.S. McCaffery J.M. Parasitol. Today. 1991; 7: 113-116Abstract Full Text PDF PubMed Scopus (19) Google Scholar), absent in non-encysting trophozoites, are necessary to transport cyst wall secretion components, leading to assembly of the extracellular cyst wall (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar). We previously characterized two Giardia cyst wall proteins (CWPs): CWP1 and CWP2 (6Mowatt M.R. Luján H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). A recent Giardia Genome Database search identified a new cyst wall protein, CWP3 (8Sun C.H. McCaffery J.M. Reiner D.S. Gillin F.D. J. Biol. Chem. 2003; 278: 21701-21708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). CWP1, CWP2, and CWP3 expression increases after trophozoites are exposed to the encystation stimulus (6Mowatt M.R. Luján H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Sun C.H. McCaffery J.M. Reiner D.S. Gillin F.D. J. Biol. Chem. 2003; 278: 21701-21708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). CWP1, CWP2, and CWP3 are acidic proteins of 26, 39, and 27 kDa, respectively. A hydrophobic N-terminal signal peptide targets them to the secretory pathway (6Mowatt M.R. Luján H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 8Sun C.H. McCaffery J.M. Reiner D.S. Gillin F.D. J. Biol. Chem. 2003; 278: 21701-21708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 9Hehl A.B. Marti M. Kohler P. Mol. Biol. Cell. 2000; 11: 1789-1800Crossref PubMed Scopus (85) Google Scholar). The central region of CWP1 and CWP2 consists of five tandem leucine-rich repeats, whereas CWP3 has four complete and one incomplete leucine-rich repeat (8Sun C.H. McCaffery J.M. Reiner D.S. Gillin F.D. J. Biol. Chem. 2003; 278: 21701-21708Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Leucine-rich repeat motifs in both prokaryotic and eukaryotic proteins have diverse functions and cellular localizations, but are always implicated in protein/protein interactions (10Kobe B. Kajava A.V. Curr. Opin. Struct. Biol. 2001; 11: 725-732Crossref PubMed Scopus (1289) Google Scholar). The C terminus of CWPs has a cysteine-rich domain involved in the formation of disulfide-bonded oligomers (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Although CWPs are closely related to each other, CWP2 is distinguished from CWP1 and CWP3 by the presence of a basic 121-residue C-terminal extension. In CWP2, this C-terminal region is present within ESVs, but is proteolytically cleaved before cyst wall assembly. The C-terminal processing role of CWP2 in early encystation is unknown (2Luján H.D. Mowatt M.R. Nash T.E. Microbiol. Mol. Biol. Rev. 1997; 61: 294-304Crossref PubMed Scopus (129) Google Scholar, 3Luján H.D. Marotta A. Mowatt M.R. Sciaky N. Nash Lippincott-Schwartz J.T.E. J. Biol. Chem. 1995; 270: 4612-4618Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). After synthesis in the endoplasmic reticulum (ER), CWPs are shuttled to the cell exterior within ESVs. ESVs are large, morphologically irregular, electron-dense granules that form de novo, and their presence is the earliest morphological change observed during Giardia encystation (Fig. 1) (4Faubert G. Reiner D.S. Gillin F.D. Exp. Parasitol. 1991; 72: 345-354Crossref PubMed Scopus (48) Google Scholar, 11McCaffery J.M. Faubert G.M. Gillin F.D. Exp. Parasitol. 1994; 79: 236-249Crossref PubMed Scopus (64) Google Scholar, 12McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar). In cells from higher eukaryotes, regulated secretory proteins concentrate into a dense core that buds off, forming an immature secretory granule in the last portion of the Golgi apparatus (13Bauerfeind R. Huttner W.B. Curr. Opin. Cell Biol. 1993; 5: 628-635Crossref PubMed Scopus (137) Google Scholar, 14Burgess T.L. Kelly R.B. Annu. Rev. Cell Biol. 1987; 3: 243-293Crossref PubMed Scopus (751) Google Scholar, 15Tooze S.A. FEBS Lett. 1991; 285: 220-224Crossref PubMed Scopus (65) Google Scholar). The Golgi apparatus consists of flattened cisternal membranes forming a stack and is remarkably conserved throughout eukaryotic evolution (16Griffiths G. Simons K. Science. 1986; 234: 438-443Crossref PubMed Scopus (764) Google Scholar); however, a typical Golgi complex is not apparent in vegetative Giardia trophozoites (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar). Evidence suggests that Giardia may possess organelle(s) in which typical Golgi functions take place, even though they do not have a Golgi-like appearance (1Adam R.D. Clin. Microbiol. Rev. 2001; 14: 447-475Crossref PubMed Scopus (912) Google Scholar). Constitutive and regulated mechanisms for protein transport exist in Giardia, suggesting Golgi functions, because the sorting and selection processes generally occur in the trans-Golgi network (TGN) (17Rothman J.E. Orci L. FASEB J. 1990; 4: 1460-1468Crossref PubMed Scopus (109) Google Scholar). The Giardia constitutive secretory pathway occurs by variant-specific surface protein (VSP) continuous transport to the plasma membrane and extracellular release. Additionally, hydrolytic enzyme sorting to lysosome-like peripheral vesicles is also a component of the constitutive pathway (3Luján H.D. Marotta A. Mowatt M.R. Sciaky N. Nash Lippincott-Schwartz J.T.E. J. Biol. Chem. 1995; 270: 4612-4618Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 11McCaffery J.M. Faubert G.M. Gillin F.D. Exp. Parasitol. 1994; 79: 236-249Crossref PubMed Scopus (64) Google Scholar, 18Touz M.C. Luján H.D. Hayes S.F. Nash T.E. J. Biol. Chem. 2003; 278: 6420-6426Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Nonetheless, knowledge of the regulated secretory pathway induced during Giardia encystation is limited and controversial (19Hehl A.B. Marti M. Mol. Microbiol. 2004; 53: 19-28Crossref PubMed Scopus (62) Google Scholar, 20Luján H.D. Touz M.C. Cell. Microbiol. 2003; 5: 427-434Crossref PubMed Scopus (43) Google Scholar). Immunoelectron microscopic studies indicate that synthesized CWPs are concentrated within flattened cisternae. These cisternae increase in size, forming large (>1-μm diameter) membrane-bound ESVs (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 12McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar, 21Reiner D.S. McCaffery J.M. Gillin F.D. Eur. J. Cell Biol. 1990; 53: 142-153PubMed Google Scholar). Detailed structural analyses of encysting cells (22Lanfredi-Rangel A. Attias M. Reiner D.S. Gillin F.D. De Souza W. J. Struct. Biol. 2003; 143: 153-163Crossref PubMed Scopus (39) Google Scholar) and the presence of BiP, an ER-resident chaperone, in these organelles (23Luján H.D. Mowatt M.R. Conrad Nash J. T.T.E. Biol. Cell. 1996; 86: 11-18PubMed Google Scholar, 24Stefanic S. Palm D. Svärd S.G. Hehl A.B. J. Biol. Chem. 2006; 281: 7595-7604Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) suggest that ESVs arise from modified ER cisternae. Whether these specific secretory granules form from an uncharacterized trans-Golgi or through condensation within the ER is unclear (20Luján H.D. Touz M.C. Cell. Microbiol. 2003; 5: 427-434Crossref PubMed Scopus (43) Google Scholar, 22Lanfredi-Rangel A. Attias M. Reiner D.S. Gillin F.D. De Souza W. J. Struct. Biol. 2003; 143: 153-163Crossref PubMed Scopus (39) Google Scholar). Previously, we (6Mowatt M.R. Luján H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) and others (12McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar, 22Lanfredi-Rangel A. Attias M. Reiner D.S. Gillin F.D. De Souza W. J. Struct. Biol. 2003; 143: 153-163Crossref PubMed Scopus (39) Google Scholar) observed that CWPs aggregate within membrane-bound clefts. These aggregates appear to grow by direct addition of newly synthesized CWPs, forming large ESVs, suggesting that CWP accumulation is an important factor for granule formation. During encystation, inhibition of CWP synthesis abolishes ESV formation (25Touz M.C. Nores M.J. Slavin I. Piacenza L. Acosta D. Carmona C. Luján H.D. Biochem. J. 2002; 364: 703-710Crossref PubMed Scopus (21) Google Scholar). Furthermore, blocking CWP transport at low temperatures indicates that ESV formation depends on CWP export from the ER (26Marti M. Li Y. Schraner E.M. Wild P. Kohler P. Hehl A.B. Mol. Biol. Cell. 2003; 14: 1433-1447Crossref PubMed Scopus (66) Google Scholar). We hypothesized that ESV formation is a direct consequence of CWP synthesis (20Luján H.D. Touz M.C. Cell. Microbiol. 2003; 5: 427-434Crossref PubMed Scopus (43) Google Scholar). We investigated the molecular basis of secretory granule formation by examining the role of CWPs and CWP chimeras in ESV biogenesis in Giardia. Our results suggest that secretory granule formation requires complex interactions between granule components (aggregation/condensation) and granule membrane receptors (sorting), involving both passive and active mechanisms. Construction of Expression Vectors—For CWP expression in Giardia, the corresponding genes were amplified with sense primers containing NcoI or ApaI sites and antisense primers containing EcoRV or SmaI sites. The products were purified, digested, and cloned into the pTubH7HApac vector as reported previously (18Touz M.C. Luján H.D. Hayes S.F. Nash T.E. J. Biol. Chem. 2003; 278: 6420-6426Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Strategy and Oligonucleotides Used to Construct Different CWP Variants—To constitutively express cwp1, cwp2, cwp3, and cwp2 without the basic extension (cwp2(-TCWP2)), all fragments were PCR-amplified from genomic DNA with the following sense (s) and antisense (as) oligonucleotides and cloned into the pTubHApac vector: CWP1s, 5′-CCA CCA TGG TGA TGC TCG CTC TCC TT-3′; CWP1as, 5′-GTT GAT ATC AGG CGG GGT GAG GCA G-3′; CWP2s, 5′-CCA CCA TGG TCG CAG CCC TTG TTC-3′; CWP2as, 5′-AGC GAT ATC CCT TCT GCG GAC AAT AGG-3′; CWP3s, 5′-CCA CCA TGG TTT CTC TGC TTC TTC TCC-3′; CWP3as, GTT GAT ATC TCT GTA GTA GGG CGG CTG-3′; CWP2-Ts, CCA CCA TGG TCG CAG CCC TTG TTC-3′; and CWP2-Tas, 5′-GTT GAT ATC GAC TAC TGT CTG CCT GTA GTA-3′. To constitutively express the CWP2 basic extension (TCWP2), the signal peptide of CWP2 was added in front of TCWP2. For this purpose, the fragment corresponding to the basic extension was amplified with a sense primer containing an XhoI site (TAILs, 5′-ACT CTC GAG AGA GAT GGA TGC ACG-3′) and antisense primer CWP2as. The fragment corresponding to the signal peptide was amplified with sense primer CWP2s and an antisense oligonucleotide containing an XhoI site (Spas, 5′-ATT CTC GAG AGC GGC GCG AGC A-3′). The two fragments were purified, digested with XhoI, and then ligated. The ligated product was re-amplified using primers CWP2s and CWP2as and cloned into the pTubHApac vector. The chimera cwp1(+TCWP2)-hemagglutinin (HA) was generated by PCR. cwp1 was amplified using primer CWP1s and an antisense oligonucleotide with a 5′-region complementary to the beginning of the cwp2 basic extension (MIX1, 5′-CTT TCG TCC CGA CGC ATT GCG AGG CGG GGT GAG GCA GTA C-3′). The basic tail (T-HA) was amplified using a sense oligonucleotide with a 5′-region complementary to the end of cwp1 (MIX2, 5′-GTA CTG CCT CAC CCC GCC TCG CA ATG CGT CGG GAC GAA AG-3′) and antisense primer CWP2as. The two fragments cwp1 (726 bp) and TCWP2 (363 bp) were purified and hybridized by a denaturation-hybridization round (95 °C for 2 min, 65 °C for 1 min, and 73 °C for 10 min). The product was re-amplified by PCR using primers CWP1s and CWP2as and cloned into the pTubH7HApac vector. Strategy and Oligonucleotide Primers Used to Construct Different VSPH7 Chimeras—vspH7 without its transmembrane domain (vspH7(-TM)HA) was PCR-amplified from genomic DNA with the following sense (s) and antisense (as) oligonucleotides and then cloned into pTubH7HApac: VSPH7-TMs, 5′-ATC GGG CCC ATG TTT CTA TTA ATT AAT TG-3′; and VSPH7-TMas, 5′-AGC GAT ATC GGA GAG GTT GGG GCC AC-3′. The chimera of vspH7 to which the basic extension of cwp2 was added, vspH7(-TM+TCWP2)-HA, was generated by PCR using the QuikChange site-directed mutagenesis kit (Stratagene) following the protocol described by Geiser et al. (27Geiser M. Cebe R. Drewello D. Schmitz R. BioTechniques. 2001; 31: 88-90Crossref PubMed Scopus (212) Google Scholar). Briefly, vspH7 cloned in pTubH7HApac was modified to construct the chimera using primers that have sequences complementary to vspH7, to the basic extension of cwp2, and to the vector. The antisense oligonucleotide used had a 5′-region complementary to the vector and a 3′-region complementary to the end of cwp2 (TAILas, 5′-CAG GCA CAT TCA TAT GGA TAG ATA TCC CTT CTG CGG ACA ATA GGC TTG TTC-3′). The sense oligonucleotide had a 5′-region complementary to the region of interest of vspH7 and a 3′-region complementary to the beginning of the cwp2 basic extension (VSPH7-TM+Ts, 5′-CGG CGA TAG TGG CCC CAA CCT CTC CCT CGA GAG ATG GAT GCA CGT AC-3′). All constructs were verified by sequencing. Giardia Culture and Transfection—Clone WB/1267 trophozoites (28Nash T.E. Aggarwal A. Adam R.D. Conrad J.T. Merritt J.W. J. Immunol. 1988; 141: 636-641PubMed Google Scholar) were cultured and induced to encyst as described previously (18Touz M.C. Luján H.D. Hayes S.F. Nash T.E. J. Biol. Chem. 2003; 278: 6420-6426Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Trophozoites were transfected with the constructs by electroporation and selected with puromycin as described (18Touz M.C. Luján H.D. Hayes S.F. Nash T.E. J. Biol. Chem. 2003; 278: 6420-6426Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). After the transfection, clones with low expression were selected by immunoblot and immunofluorescence analyses. For transient coexpression, trophozoites were transfected with both plasmids at the same concentration (10 μg) and selected with puromycin, and clones were analyzed with the corresponding anti-CWP1 and anti-CWP2 monoclonal antibodies (mAbs). Giardia Trophozoite Immunofluorescence Analysis—Cells cultured in growth, pre-encystation, or encystation medium were harvested and processed as described previously (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). The cells were fixed with 4% paraformaldehyde and permeabilized for 1 h at room temperature in phosphate-buffered saline, 0.1% Triton X-100, and 10% normal goat serum. The cells were then incubated with the antibodies diluted in phosphate-buffered saline, 0.1% Triton X-100, and 3% goat serum. For indirect staining, slides were incubated with the specific mAbs (final dilution of 1:200) or anti-HA mAb (final dilution of 1:1000; Sigma) for 1 h at 37 °C, followed by anti-mouse secondary antibody labeled with fluorescein isothiocyanate (FITC) or rhodamine (final dilution of 1:250; ICN Biomedicals) for1hat37 °C. For direct double staining, FITC-conjugated anti-HA mAb (final dilution of 1:500) was used to detect the transgenic proteins; mAb 9C9 was directly labeled with Texas Red (Zenon One, Molecular Probes) for GRP78/BiP detection; and mAbs 5-3C and 7D2 were directly labeled with Texas Red or FITC (Zenon One) for endogenous CWP1 and CWP2 detection. The nuclei were visualized with 4′,6-diamidino-2-phenylindole (DAPI). Controls included no primary antibodies and untransfected cells. Confocal images were collected using a Zeiss LSM5 Pascal laser-scanning confocal microscope equipped with an argon/helium/neon laser and a ×100 (numerical aperture = 1.4) oil immersion objective (Zeiss Plan-Apochromat). Single confocal sections of 0.3 μm were taken parallel to the coverslip (z sections). Images were acquired using a Zeiss charge-coupled device camera and processed with LSM and Adobe Photoshop software. Immunoblot and Secretion Assays—SDS-PAGE of total G. lamblia proteins was performed under reducing or nonreducing conditions as reported previously (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). For Western blot comparative analysis between different CWPs and CWP chimeras, five times more protein from transgenic trophozoites (100 μg instead of 20 μg) was loaded onto the gel because of the lower level of expression of the constructs expressed by the pTubHApac vector compared with CWP expression during encystation. For secretion, transfected cells were cultured in growth medium for 24 h. The cultured medium was collected and centrifuged at 800 × g for 10 min to eliminate Giardia trophozoites. The collected medium was incubated with trichloroacetic acid (10% final concentration) for 1 h at 4 °C. Trichloroacetic acid precipitates were centrifuged, and the resulting pellets were washed with iced-cold ethanol, dried, resuspended in 30 μl of sample buffer with 2-mercaptoethanol, and boiled for 5 min. The samples were analyzed by Western blotting using anti-HA mAb. Electron Microscopy—Cultures were fixed in situ to preserve the cell organization when attached to the culture flask wall. After 30 min in fixative (2.5% glutaraldehyde in 0.1 m sodium cacodylate buffer, pH 7.0), the flask wall was lightly scraped to detach the trophozoites. The resulting suspension was centrifuged at 500 × g, and the pellet was placed in fresh fixative. Fixation was performed for 2 h, followed by post-fixation in 1% OsO4 with the addition of 1.25% potassium ferrocyanide for 1-2 h. During dehydration, the cells were prestained with 1% uranyl acetate in 70% ethanol for 2 h. The pellets were embedded in Araldite, and sections were cut in a Poter-Blum ultramicrotome. Thin, silver interference color (∼60 nm in thickness), serial sections were used. Single whole grids (oval, 2 × 1 mm; Pelco International) were used for collecting segments of 20 sections from series of 100-120 thin sections. The sections were stained first with saturated uranyl acetate in water and then with lead citrate. Sections were examined in a Siemens Elmiskop at magnifications standardized with diffraction grids. The CWP2 Basic Tail Is Necessary for Secretory Granule Biogenesis—To analyze CWP involvement in ESV biogenesis, different tagged versions of CWPs (Fig. 2A) were constitutively expressed in non-encysting trophozoites (Fig. 2B). We used a C-terminal HA epitope tag because CWPs have an N-terminal signal peptide that is processed during their trafficking through the secretory pathway (6Mowatt M.R. Luján H.D. Cotten D.B. Bowers B. Yee J. Nash T.E. Stibbs H.H. Mol. Microbiol. 1995; 15: 955-963Crossref PubMed Scopus (137) Google Scholar, 7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). After transfection, we analyzed protein expression by Western blotting and immunofluorescence microscopy using either anti-HA or anti-CWP mAb. The expression level of the CWP chimera was always lower than that of endogenous CWP during encystation (∼5-fold lower compared with CWP expression in wild-type encysting cells) (data not shown). Localization of HA-tagged CWP during encystation was similar to that of native CWPs (see below), indicating that the HA tag did not affect their sub-cellular localization. Immunofluorescence assays using anti-HA mAb with constitutively HA-tagged CWP1 and CWP3 showed that both proteins localized to the ER in non-encysting trophozoites (Fig. 2B), as determined by their co-localization with the ER-resident chaperone BiP (23Luján H.D. Mowatt M.R. Conrad Nash J. T.T.E. Biol. Cell. 1996; 86: 11-18PubMed Google Scholar). When the same assay was performed with HA-tagged CWP2, formation of large vesicles with characteristics similar to those of native ESVs was observed (Fig. 2B). Like ESVs in encysting trophozoites, these vesicles contained BiP (Fig. 2B). CWP2 differs from CWP1 and CWP3 by the presence of a 13-kDa basic extension at the C-terminal end (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). We deleted the CWP2 basic tail (TCWP2) to examine its role in ESV biogenesis. HA-tagged CWP2 without the basic extension (CWP2(-TCWP2)-HA) and CWP1 containing the CWP2 extension at its C terminus (CWP1(+TCWP2)-HA) were expressed in trophozoites (Fig. 2A). Immunofluorescence assays performed on non-encysting cells showed that CWP2 minus the basic extension localized in a cytoplasmic meshwork resembling the ER, a pattern similar to those of CWP1-HA, CWP3-HA, and BiP (Fig. 2B). The formation of ESV-like vesicles in cells expressing CWP1(+TCWP2)-HA was similar to that observed in non-encysting trophozoites with CWP2-HA (Fig. 2B)orin encysting trophozoites with wild-type CWP2 (7Luján H.D. Mowatt M.R. Conrad J.T. Nash T.E. BowersB. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). These vesicles also co-localized with BiP (Fig. 2B). We confirmed the aforementioned localization with additional mAbs that detect CWP conformational states. These data suggest that the exogenous proteins undergo normal folding (supplemental Fig. 1). Negative staining in non-encysting trophozoites with mAb against granule-specific protein, an ESV-specific calcium-binding protein induced during encystation (29Touz M.C. Gottig N. Nash T.E. Luján H.D. J. Biol. Chem. 2002; 277: 50557-50563Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), confirmed that these cells were not encysting (data not shown). In encysting cells, HA-tagged CWP1 and CWP3 constructs were sorted to secretory granules and co-localized with endogenous CWPs (Fig. 3A), indicating that native CWP2 reroutes CWP1 and CWP3 from the constitutive to the regulated pathway. In addition, immunofluorescence assays with anti-HA mAb were performed to confirm that these ESV-like granules behave like native ESVs. These experiments showed that CWP1-HA, CWP3-HA, and CWP2(-TCWP2)-HA were incorporated into the cyst wall similar to native CWPs (Fig. 3B). CWP2-HA and CWP1(+TCWP2)-HA were not detected in cyst walls using anti-HA mAb, in agreement with previous results showing that this basic extension is cleaved from CWP2 by an encystation-specific cysteine protease before cyst wall assembly (30Touz M.C. Nores M.J. Slavin I. Carmona C. Conrad J.T. Mowatt M.R. Nash T.E. Coronel C.E. Luján H.D. J. Biol. Chem. 2002; 277: 8474-8481Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In addition, the cysts generated in vitro from transfected cells were identical in shape to those obtained from untransfected organisms (Figs. 1 and 3B) and were resistant to hypo-osmotic shock (data not shown), suggesting that the HA tag in CWPs does not interfere with cyst wall formation. We also examined CWP expression by immunoblotting reduced and nonreduced protein extracts obtained from encysting and non-encysting Giardia trophozoites (Fig. 4). When extracts from cells expressing CWP1-HA (Fig. 4A), CWP3-HA (Fig. 4B), and CW
Referência(s)