Mining the Giardia lamblia Genome for New Cyst Wall Proteins
2003; Elsevier BV; Volume: 278; Issue: 24 Linguagem: Inglês
10.1074/jbc.m302023200
ISSN1083-351X
AutoresChin‐Hung Sun, J. Michael McCaffery, David S. Reiner, Frances D. Gillin,
Tópico(s)Parasitic infections in humans and animals
ResumoThe Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by ∼50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation. The Giardia lamblia cyst wall (CW), which is required for survival outside the host and infection, is a primitive extracellular matrix. Because of the importance of the CW, we queried the Giardia Genome Project Database with the coding sequences of the only two known CW proteins, which are cysteine-rich and contain leucine-rich repeats (LRRs). We identified five new LRR-containing proteins, of which only one (CWP3) is up-regulated during encystation and incorporated into the cyst wall. Sequence comparison with CWP1 and -2 revealed conservation within the LRRs and the 44-amino-acid N-flanking region, although CWP3 is more divergent. Interestingly, all 14 cysteine residues of CWP3 are positionally conserved with CWP1 and -2. During encystation, C-terminal epitope-tagged CWP3 was transported to the wall of water-resistant cysts via the novel regulated secretory pathway in encystation-secretory vesicles (ESVs). Deletion analysis revealed that the four LRRs are each essential to target CWP3 to the ESVs and cyst wall. In a deletion of the most C-terminal region, fewer ESVs were stained in encysting cells, and there was no staining in cysts. In contrast, deletion of the 44 amino acids between the signal sequence and the LRRs or the region just C-terminal to the LRRs only decreased the number of cells with CWP3 targeting to ESVs and cyst wall by ∼50%. Our studies indicate that virtually every portion of the CWP3 protein is needed for efficient targeting to the regulated secretory pathway and incorporation into the cyst wall. Further, these data demonstrate the power of genomics in combination with rigorous functional analyses to verify annotation. Giardiasis is a major cause of waterborne diarrheal disease (1Marshall M.M. Naumovitz D. Ortega Y. Sterling C.R. Clin. Microbiol. Rev. 1997; 10: 67-85Crossref PubMed Google Scholar, 2Adam R.D. Microbiol. Rev. 1991; 55: 706-732Crossref PubMed Google Scholar, 3Farthing M.J.G. Blaser M.J. Smith P.D. Ravdin J.I. Infections of the Gastrointestinal Tract. Raven Press, New York1995: 1081-1105Google Scholar), contributing greatly to the global burden of malnutrition. Despite its importance, neither the basic biology of Giardia lamblia nor the pathophysiology of infection is well understood. Giardia infection initiates with ingestion of infectious cysts that can persist for months in cold fresh water (4Bingham A.K. Meyer E.A. Nature. 1979; 227: 301-302Crossref Scopus (130) Google Scholar). Exposure of cysts to gastric acid during passage through the stomach triggers excystation (4Bingham A.K. Meyer E.A. Nature. 1979; 227: 301-302Crossref Scopus (130) Google Scholar, 5Rice E.W. Schaefer F.W. III. J. Clin. Microbiol. 1981; 14: 709-710Crossref PubMed Google Scholar), and after entry into the small intestine, the parasite emerges and divides into two equivalent binucleate trophozoites (5Rice E.W. Schaefer F.W. III. J. Clin. Microbiol. 1981; 14: 709-710Crossref PubMed Google Scholar, 6Boucher S.-E. Gillin F.D. Infect. Immun. 1990; 58: 3516-3522Crossref PubMed Google Scholar). Trophozoites attach to and colonize the human small intestinal epithelium, where they can cause disease. However, if they detach and are carried downstream, they must encyst to survive outside the host. Our laboratory has reproduced the giardial life cycle in vitro (7Gillin F.D. Reiner D.S. Gault M.J. Douglas H. Das S. Wunderlich A. Sauch J.F. Science. 1987; 235: 1040-1043Crossref PubMed Scopus (109) Google Scholar, 8Gillin F.D. Reiner D.S. Annu. Rev. Microbiol. 1996; 50: 679-705Crossref PubMed Scopus (188) Google Scholar, 9Eichinger D. Curr. Opin. Microbiol. 2001; 4: 421-426Crossref PubMed Scopus (87) Google Scholar). The "biological goal" of encystation is differentiation into a form that can survive in the environment and infect a new host. The construction of the extracellular cyst wall is therefore essential as it is the single structure that allows the parasite to persist in fresh water, resist disinfectants, pass through the stomach of the new host, and infect in the small intestine. This 300-nm-thick fibrous structure (10Erlandsen S.L. Bemrick W.J. Pawley J. J. Parasitol. 1989; 75: 787-797Crossref PubMed Scopus (33) Google Scholar, 11Erlandsen S.L. Bemrick W.J. Schupp D.E. Shields J.M. Jarroll E.L. Sauch J.F. Pawley J.B. J. Histochem. Cytochem. 1990; 38: 625-632Crossref PubMed Scopus (40) Google Scholar, 12Erlandsen S.L. Macechko P.T. van Keulen H. Jarroll E.L. J. Eukaryot. Microbiol. 1996; 43: 416-429Crossref PubMed Scopus (67) Google Scholar) is tight enough to exclude small molecules, such as water and gastric acid. On the other hand, it must be capable of transmitting the physiological stimuli that regulate activation and emergence (13Hetsko M.L. McCaffery J.M. Meng T-C. Que X. Gillin F.D. Exp. Parasitol. 1998; 88: 172-183Crossref PubMed Scopus (61) Google Scholar). Therefore, the encystation pathway may be thought of as a giardial virulence mechanism necessary for survival outside the host and infection. The cyst wall is at least 40% protein, and the remainder is carbohydrate (14Jarroll E.L. Macechko P.T. Steimle P.A. Bulik D. Karr C.D. van Keulen H. Paget T.A. Gerwig G. Kamerling J. Vliegenthart J. Erlandsen S. J. Eukaryot. Microbiol. 2001; 48: 22-26Crossref PubMed Scopus (42) Google Scholar, 15Gerwig G.J. van Kuik J.A. Leeflang B.R. Kamerling J.P. Vliegenthart J.F. Karr C.D. Jarroll E.L. Glycobiology. 2002; 12: 499-505Crossref PubMed Scopus (81) Google Scholar). The two known cyst wall proteins (CWPs) 1The abbreviations used are: CWP, CW protein; CW, cyst wall; LRR, leucine-rich repeat; ESV, encystation-secretory vesicle; RACE, rapid amplification of cDNA ends; PBS, phosphate-buffered saline; RT, reverse transcriptase; GFP, green fluorescent protein; nt, nucleotides; contig, group of overlapping clones. contain similar domains, including a putative N-terminal signal peptide and five tandem, leucine-rich repeats (LRR) (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar), and are reported to be phosphorylated (18Slavin I. Saura A. Carranza P.G. Touz M.C. Nores M.J. Lujan H.D. Mol. Biochem. Parasitol. 2002; 122: 95-98Crossref PubMed Scopus (40) Google Scholar). In the regions of similarity, both proteins are acidic, but CWP2 has a very basic 121-residue C-terminal extension. Expression of the two CWPs increases greatly soon after exposure to encystation signals (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 19Reiner D.S. Douglas D. Gillin F.D. Infect. Immun. 1989; 57: 963-968Crossref PubMed Google Scholar, 20McCaffery J.M. Faubert G.M. Gillin F.D. Exp. Parasitol. 1994; 79: 236-249Crossref PubMed Scopus (64) Google Scholar). LRR motifs of both prokaryotic and eukaryotic proteins have diverse functions and cellular localizations but are associated with protein-protein interactions (21Marino M. Braun L. Cossart P. Ghosh P. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8784-8788Crossref PubMed Scopus (47) Google Scholar, 22Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (546) Google Scholar, 23Kobe B. Kajava A.V. Curr. Opin. Struct. Biol. 2001; 11: 725-732Crossref PubMed Scopus (1289) Google Scholar). Earlier, we discovered a novel regulated secretory pathway that is induced during giardial encystation (19Reiner D.S. Douglas D. Gillin F.D. Infect. Immun. 1989; 57: 963-968Crossref PubMed Google Scholar, 24Reiner D.S. McCaffery M. Gillin F.D. Eur. J. Cell Biol. 1990; 53: 142-153PubMed Google Scholar). The appearance of large membrane-bounded encystation secretory vesicles (ESVs) at 6–24 h is the earliest morphological change in giardial encystation (17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 19Reiner D.S. Douglas D. Gillin F.D. Infect. Immun. 1989; 57: 963-968Crossref PubMed Google Scholar, 25McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar, 26Faubert G. Reiner D.S. Gillin F.D. Exp. Parasitol. 1991; 72: 345-354Crossref PubMed Scopus (48) Google Scholar). CWP1 and -2 are concentrated within the ESVs for transport to the nascent cyst wall (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 20McCaffery J.M. Faubert G.M. Gillin F.D. Exp. Parasitol. 1994; 79: 236-249Crossref PubMed Scopus (64) Google Scholar, 25McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar). CWP1 and -2 also contain 18 and 19 cysteine residues, respectively, and form disulfide-bonded heterodimers and oligomers soon after synthesis (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 27Reiner D.S. McCaffery J.M. Gillin F.D. Cell Microbiol. 2001; 3: 459-472Crossref PubMed Scopus (37) Google Scholar, 28Hehl A.B. Marti M. Kohler P. Mol. Biol. Cell. 2000; 11: 1789-1800Crossref PubMed Scopus (85) Google Scholar). However, the mechanism by which CWPs are targeted to the regulated secretory pathway is not well understood (28Hehl A.B. Marti M. Kohler P. Mol. Biol. Cell. 2000; 11: 1789-1800Crossref PubMed Scopus (85) Google Scholar). Giardia belongs to one of the earliest diverging eukaryotic lineages (29Sogin M.L. Gunderson J.H. Elwood H.J. Alonso R.A. Peattie D.A. Science. 1989; 243: 75-77Crossref PubMed Scopus (591) Google Scholar). There is little understanding of the biology of the origins of eukaryotes, particularly of the endomembrane-mediated system of protein modification, sorting, and transport (30Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. 2002; (Garland, NY): 551-651Google Scholar). The Giardia cyst wall is a relatively simple form of eukaryotic extracellular matrix with crucial structural and signaling functions whose assembly requires regulated protein expression and transport. Therefore, formation of the ESVs and cyst wall in this protist is an excellent model for the study of early forms of biosynthesis of extracellular structures in eukaryotes. Because of the evolutionary and functional importance of the cyst wall, we queried the Giardia Genome Project Database (www.mbl.edu/Giardia, Ref. 31McArthur A.G. Morrison H.G. Nixon J.E.J. Passamaneck N.Q.E. Kim U. Reich C.I. Holder M.E. Hinkle G. Crocker M.K. Farr R. Olsen G.E. Aley S.B. Adam R. Gillin F.D. Sogin M.L. FEMS Microbiol. Lett. 2000; 189: 271-273Crossref PubMed Google Scholar) with the coding sequences of CWP1 and CWP2 (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. Chem. 1995; 270: 29307-29313Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) to identify additional CWPs. We identified five putative leucine-rich proteins. Only one of these (CWP3) is up-regulated during encystation and is transported by the ESV pathway and incorporated into the cyst wall. In contrast, the genes encoding the other four proteins are actually down-regulated during encystation. Therefore, we characterized the expression and detailed the targeting requirements of cwp3. Other important parasites, e.g. Cryptosporidium, Entamoeba, Toxoplasma, several tapeworms, and nematodes, are transmitted as cysts or oocysts with a resistant extracellular wall. Many of these parasites also emerge in the host small intestine after exposure to gastric and intestinal signals (32Despommier D.D. Karapelou J.W. Parasite Life Cycles. Springer-Verlag New York Inc., New York1987Google Scholar). Study of these organisms, however, is limited by the inability to generate viable cysts in vitro (9Eichinger D. Curr. Opin. Microbiol. 2001; 4: 421-426Crossref PubMed Scopus (87) Google Scholar). Our completion of the life cycle of G. lamblia in vitro and the availability of the Genome Project Database make it a valuable model for other parasites whose differentiation has not been achieved. Giardia Culture and Encystation—Trophozoites of G. lamblia isolate WB (ATCC 50803), clone C6, were cultured in modified TYI-S33 medium (33Keister D.B. Trans. R. Soc. Trop. Med. Hyg. 1983; 77: 487-488Abstract Full Text PDF PubMed Scopus (792) Google Scholar). Encystation was induced by a modification (34Sun C.H. Palm D. McArthur A.G. Svard S.G. Gillin F.D. Mol. Microbiol. 2002; 46: 971-984Crossref PubMed Scopus (81) Google Scholar) of our earlier procedure (35Que X. Svard S.G. Meng T.C. Hetsko M.L. Aley S.B. Gillin F.D. Mol. Biochem. Parasitol. 1996; 81: 101-110Crossref PubMed Scopus (38) Google Scholar). Briefly, trophozoites that were grown to late log phase in growth medium were harvested and encysted for the times indicated in TYI-S-33 medium containing 12.5 mg/ml bovine bile at pH 7.8 at a beginning density of 5 × 105 cells/ml. Isolation and Analysis of the cwp3 Gene—We searched the G. lamblia Genome Project Database (www.mbl.edu/Giardia, Ref. 31McArthur A.G. Morrison H.G. Nixon J.E.J. Passamaneck N.Q.E. Kim U. Reich C.I. Holder M.E. Hinkle G. Crocker M.K. Farr R. Olsen G.E. Aley S.B. Adam R. Gillin F.D. Sogin M.L. FEMS Microbiol. Lett. 2000; 189: 271-273Crossref PubMed Google Scholar) with amino acid sequences of the cwp1 and cwp2 genes (GenBank™ accession numbers S61925 and S61924, respectively), using the BLAST program (36Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59923) Google Scholar). We amplified a 1.2-kb sequence containing the cwp3 gene from genomic DNA by PCR using primers cwp35F (5′-CCAGTATCACGGAATTAATTTCTT-3′) and cwp33R (5′-TGTGACACGGGGAAAAGCCATAGG-3′). For RT-PCR, 5 μg of total RNA from vegetative and 24-h encysting cells was mixed with oligo(dT)12–18 and Superscript II RNase H– reverse transcriptase (Invitrogen). PCR on the first-strand cDNA used primers cwp3F (5′-ATGTTTTCTCTGCTTCTTCTCCTT-3′) and cwp3R (5′-TTATCTGTAGTAGGGCGGCTGTAT-3′). Genomic and RT-PCR products were cloned into pGEM-T easy vector (Promega) and sequenced (Applied Biosystems). For semiquantitative RT-PCR of cwp3, total RNA from 24-h encysting cells was mixed with primer AU1 (5′-GATGTATCGATACGTATC-3′), and reverse transcriptase reaction was performed. PCR on the first-strand cDNA used primers cwp3F and cwp3R. For semiquantitative RT-PCR of neomycin, primer NEOR (5′-TCAGAAGAACTCGTCAAGAAGGCG-3′) was used for reverse transcriptase reaction. Primers NEOF (5′-ATGATTGAACAAGATGGATTGCAC-3′) and NEOR were used for PCR. Primer Extension and 3′-RACE Analysis—Primer extension was performed as described. Briefly, the labeled cwp5R primer (5′-TCCGACTGTAGCCAATTGTTTGGC-3′) (2 pmol) was added to RNA (50 μg), and hybridization was carried out by incubating the mixture at 80 °C for 3 min and then at 50 °C for 90 min. After addition of 100 units of Superscript II reverse transcriptase (Invitrogen), the reaction mixtures were incubated at 42 °C for 1 h. 3′-RACE was performed using the 3′-RACE system (Invitrogen). For 3′-RACE, oligonucleotide cwp33RA (5′-GCACGATGCAACAGACTGCAATAC-3′) was used as amplification primer. RNA and DNA Extraction and Northern Blot Analysis—Total RNA was extracted from G. lamblia at the indicated differentiation stages using TRIzol reagent (Invitrogen). Genomic DNA was isolated from trophozoites using DNA STAT-60 reagent (Tel-Test). Standard procedures were used in electrophoresis, blotting, and hybridization of genomic DNA and total RNA (37Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual,2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 10 μg of total RNA was fractionated and transferred to Zeta-Probe blotting membrane (Bio-Rad). Strand-specific probes were labeled using the Prime-It II kit (Stratagene). A full-length coding region probe of cwp3 was made with cwp3F and cwp3R and primed with cwp3R. The membranes were hybridized and washed as described (38Knodler L.A. Svard S.G. Silberman J.D. Davids B.J. Gillin F.D. Mol. Microbiol. 1999; 34: 327-340Crossref PubMed Scopus (74) Google Scholar). Hybridization signals were imaged and quantified using a Storm system (Molecular Dynamics). Plasmid Construction—All cwp3 fragments were amplified from genomic DNA by PCR. The cwp3 gene and its 262-bp 5′-flanking region was amplified from genomic DNA with oligonucleotides cwp35NF (5′-GGCGGCTAGCCAGTATCACGGAATTAATTTCTTTGG-3′) and cwp3AUER (5′-GGCGGAATTCTTAGATGTATCGATACGTATCTCTGTAGTAGGGCGGCTGTATCTT-3′), digested with NheI/EcoRI, and ligated in place of the NheI/EcoRI-excised luciferase gene, 32-bp ran promoter, and two copies of a 19-bp tet operator sequence in pNLop2-1 (34Sun C.H. Palm D. McArthur A.G. Svard S.G. Gillin F.D. Mol. Microbiol. 2002; 46: 971-984Crossref PubMed Scopus (81) Google Scholar). The resulting plasmid, pNC3, contained the cwp3 gene controlled by its own promoter with an AU1 tag fused at its C terminus. For constructing ND, a PCR with oligonucleotide cwp3NDF (5′-GGCGCCATGGATCAATATGATGCACTCGTTCAG-3′) and cwp3AUER generated a 0.7-kb product that was digested with NcoI and EcoRI. Another PCR with primers cwp3NDR (5′-GGCGCCATGGATCAGTAGTAACTTATTTTTTGGG-3′) and cwp35NF generated a 0.3-kb PCR product that was digested with NcoI and NheI and cloned into NheI/EcoRI-digested pNLop2-1 with the 0.7-kb NcoI/EcoRI fragment. The resulting ND contains a cwp3 gene lacking the predicted signal peptide sequence (residues 1–16). For constructing ND2, a PCR with oligonucleotide cwp3ND2F (5′-GGCGGGATCCTCACTAAATGTGCAGCACATGGGG-3′) and cwp3AUER generated a 0.6-kb product that was digested with BamHI and EcoRI. Another PCR with primers cwp3ND2R (5′-GGCGGGATCCGTCTACTAAACCATATCCAACCTC-3′) and cwp35NF generated a 0.4-kb PCR product that was digested with BamHI and NheI and cloned into NheI/EcoRI-digested pNLop2-1 with the 0.6-kb BamHI/EcoRI fragment. The resulting ND2 contains a cwp3 gene lacking the 44 amino acids N-terminal to LRRs (residues 17–60). A similar strategy was used to construct R1D, R2D, R3D, R4D, R1–4D, and CD1, which contain cwp3 gene with deletion of first LRR (residues 61–83), second LRR (residues 84–107), third LRR (residues 108–131), fourth LRR (residues 132–155), first to fourth (residues 61–155) LRRs, and the region C-terminal to LRRs (residues 156–201), respectively. The product obtained with primers cwp35NF and cwp3CD2R was digested with NheI and EcoRI and cloned into NheI/EcoRI-digested pNLop2-1. The resulting CD2 contains a cwp3 gene with a C-terminal (residues 202–247) deletion. The cwp2 gene and its 356-bp 5′-flanking region was amplified from genomic DNA with oligonucleotides cwp25XF (5′-GGCGTCTAGAAAACATGGGATGTGATCGATGTGACAC-3′) and cwp2AUER (5′-GGCGGAATTCTCAGATGTATCGATACGTATCCCTTCTGCGGACAATAGGCTT-3′), digested with XbaI/EcoRI, and ligated in place of the NheI/EcoRI-excised cwp3 gene in pNC3. The resulting plasmid, pNC2, contained the cwp2 gene controlled by its own promoter with an AU1 tag fused at its C terminus. The cwp1 gene and its 402-bp 5′-flanking region was amplified from genomic DNA with oligonucleotides cwp15NF (5′-GGCGGCTAGCTTCGCTTGCTTTTGCCGTTAGAAGAG-3′) and cwp1AU5ER (5′-GGCGGAATTCTCACTTGAGGTAGAAATCGGTAGGCGGGGTGAGGCAGTACTCTCCG-3′), digested with NheI/EcoRI, and ligated in place of the NheI/EcoRI-excised cwp1 gene in pNC1. The resulting plasmid, pNC1, contained the cwp1 gene controlled by its own promoter with an AU5 tag fused at its C terminus. A NheI/ClaI fragment containing the luciferase gene, 32-bp ran promoter, and two copies of a 19-bp tet operator sequence from pPop2 (34Sun C.H. Palm D. McArthur A.G. Svard S.G. Gillin F.D. Mol. Microbiol. 2002; 46: 971-984Crossref PubMed Scopus (81) Google Scholar) was replaced by the NheI/ClaI-excised cwp1 gene and its 5′-flanking region from pNC1, resulting in pPC1. The 262-bp 5′-flanking region of the genomic cwp3 gene was amplified with oligonucleotides cwp35NF and cwp35NR (5′-GGCGCCATGGATCAGTAGTAACTTATTTTTTGGG-3′), digested with NheI/NcoI, and ligated in place of the NheI/NcoI-excised 32-bp ran promoter and two copies of a 19-bp tet operator sequence in pNLop2-1. The resulting plasmid, pNC35, contained the luciferase gene under the control of the cwp3 promoter. The same strategy was used to clone pNC35+, which contains the 262-bp 5′-flanking region of the genomic cwp3 gene and includes 47 nt of the coding sequence upstream of the luciferase gene, using primers cwp35NF and cwp7NlucR (5′-GGCGCCATGGTCTACTAAACCATATCCAACCTCAA-3′). Transfection and Luciferase Assay—Cells were transfected with pNC3, ND, ND2, R1D, R2D, R3D, R4D, R1–4D, CD1, CD2, pNC2, pNC35, or pNC35+ and selected with G418 as described (39Sun C.H. Chou C.F. Tai J.H. Mol. Biochem. Parasitol. 1998; 92: 123-132Crossref PubMed Scopus (54) Google Scholar). Stable transfectants were maintained at 150 μg/ml G418. Cells transfected with pPC1 were selected and maintained at 54 μg/ml puromycin (40Singer S.M. Yee J. Nash T.E. Mol. Biochem. Parasitol. 1998; 92: 59-69Crossref PubMed Scopus (111) Google Scholar). Western blots were probed with anti-AU1 or AU5 monoclonal antibody (Berkeley Antibody, 1/5000 in blocking buffer) and detected with peroxidase-conjugated goat anti-mouse IgG (Pierce, 1/5000). The control membrane was incubated with anti-gPDI-1 serum (38Knodler L.A. Svard S.G. Silberman J.D. Davids B.J. Gillin F.D. Mol. Microbiol. 1999; 34: 327-340Crossref PubMed Scopus (74) Google Scholar) and detected with goat anti-rabbit antibody (Pierce, 1/5000) and enhanced chemiluminescence (Amersham Biosciences). Immunofluorescence Assay—Stably transfected cells were harvested after 24 h in growth or encystation medium under G418 selection, washed in PBS, and attached to glass coverslips (2 × 106 cells/coverslip) and then fixed and stained (41Abel E.S. Davids B.J. Robles L.D. Loflin C.E. Gillin F.D. Chakrabarti R. J. Biol. Chem. 2001; 276: 10320-10329Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Water-resistant cysts were harvested after 48 h by centrifugation and five treatments with double distilled water for 5 min and then kept in cold distilled water overnight. Cells were reacted with anti-AU1 or AU5 monoclonal antibody (Covance, Princeton, NJ, 1/300 in blocking buffer) and anti-mouse ALEXA 568 (Molecular Probes, 1/500 in blocking buffer) as the detector. The images were evaluated on a Zeiss LSM 510 laser scanning confocal microscope. Immunoelectron Microscopy—Encysting cells were processed as described (25McCaffery J.M. Gillin F.D. Exp. Parasitol. 1994; 79: 220-235Crossref PubMed Scopus (76) Google Scholar). Briefly, washed parasites were fixed in suspension for 15 min in freshly prepared 4% formaldehyde, in PBS, pH 7.4, and then pelleted and resuspended in 4% formaldehyde in PBS and fixed for an additional 18–24 h at 4 °C. The cells were washed briefly in PBS and resuspended in 1% low temperature gelling agarose. After cooling, the agarose blocks were trimmed into mm3 pieces; cryoprotected with 2.3 m sucrose/20% polyvinyl pyrrolidone (10 K), pH 7.4, for 2 h; and mounted onto cryopins and rapidly frozen in liquid nitrogen. Ultrathin cryosections were cut on a Leica UCT ultramicrotome equipped with an FC-S cryoattachment and collected onto formvar/carbon-coated nickel grids. Grids were washed through several drops of 1× PBS containing 5% fetal calf serum, 10 mm glycine, pH 7.4; blocked in 10% fetal calf serum for 30 min; and incubated overnight in 20 μg/ml anti-AU-1 antibody. After washing, the grids were incubated for 2 h in 5 nm AU-conjugated donkey anti-mouse (Jackson Immunoresearch Laboratories). The grids were then washed through several drops of PBS followed by several drops of ddH2O and embedded in an aqueous solution containing 3.2% polyvinyl alcohol (10 K), 0.2% methyl cellulose (400 centiposes), and 0.1% uranyl acetate. The grids were observed and photographed on a Philips 420 transmission electron microscope at 80 kV. Sequence Analysis and Alignment—The cwp3 and lrp1–4 genes were amplified from genomic DNA. An alignment of the LRRs of Cwp1, Cwp2, and Cwp3 was prepared with ClustalW (42Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55757) Google Scholar). The cwp3 sequence was previously deposited in GenBank™ under accession number AY061927. The lrp3 sequence was previously deposited in GenBank™ under accession number AF310726. We have deposited lrp1, lrp2, and lrp4 in GenBank™ under accession numbers AY225413, AY225414, and AY225415. Identification and Characterization of the cwp3 Gene—To identify possible new cyst wall proteins, we queried the G. lamblia Genome Project Database (www.mbl.edu/Giardia, Ref. 31McArthur A.G. Morrison H.G. Nixon J.E.J. Passamaneck N.Q.E. Kim U. Reich C.I. Holder M.E. Hinkle G. Crocker M.K. Farr R. Olsen G.E. Aley S.B. Adam R. Gillin F.D. Sogin M.L. FEMS Microbiol. Lett. 2000; 189: 271-273Crossref PubMed Google Scholar) with amino acid sequences of the cwp1 and cwp2 genes. This search detected five putative open reading frames with E values <10–4, lrp1, lrp2, lrp3, lrp4, and cwp3. During the course of this study, two of these proteins, LRP3 ("virus receptor protein") and CWP3 were deposited in GenBank™ by others but not published. We found that lrp1, lrp2, lrp3, lrp4 were expressed at significantly lower levels during encystation (Fig. 1A). Therefore, we focused on cwp3, which was up-regulated (Fig. 1B). The cwp3 coding region with 262 nt of 5′- and 120 nt of 3′-flanking regions was retrieved and amplified. Comparing genomic and cDNA sequences showed that the cwp3 gene contained no introns. Primer extension analysis revealed a major transcription start site, –13, and three minor transcription start sites, –16, –17, and –18, relative to the translation start site of cwp3 in 24-h encysting cells. No primer extension product was detected in vegetative cells. All transcription start sites are located within a 9-bp AT-rich sequence, –13/-21 (AAAAAATAA), as has frequently been observed for initiation of giardial transcripts (16Mowatt M.R. Lujan 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, 17Lujan H.D. Mowatt M.R. Conrad J.T. Bowers B. Nash T.E. J. Biol. 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