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• W2022983114 abstract "Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24–72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an ∼30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention. Cryptosporidium sp. cause human and animal diarrheal disease worldwide. The molecular mechanisms underlying Cryptosporidium attachment to, and invasion of, host cells are poorly understood. Previously, we described a surface-associated Gal/GalNAc-specific lectin activity in sporozoites of Cryptosporidium parvum. Here we describe p30, a 30-kDa Gal/GalNAc-specific lectin isolated from C. parvum and Cryptosporidium hominis sporozoites by Gal-affinity chromatography. p30 is encoded by a single copy gene containing a 906-bp open reading frame, the deduced amino acid sequence of which predicts a 302-amino acid, 31.8-kDa protein with a 22-amino acid N-terminal signal sequence. The p30 gene is expressed at 24–72 h after infection of intestinal epithelial cells. Antisera to recombinant p30 expressed in Escherichia coli react with an ∼30-kDa protein in C. parvum and C. hominis. p30 is localized to the apical region of sporozoites and is predominantly intracellular in both sporozoites and intracellular stages of the parasite. p30 associates with gp900 and gp40, Gal/GalNAc-containing mucin-like glycoproteins that are also implicated in mediating infection. Native and recombinant p30 bind to Caco-2A cells in a dose-dependent, saturable, and Gal-inhibitable manner. Recombinant p30 inhibits C. parvum attachment to and infection of Caco-2A cells, whereas antisera to the recombinant protein also inhibit infection. Taken together, these findings suggest that p30 mediates C. parvum infection in vitro and raise the possibility that this protein may serve as a target for intervention. The intestinal apicomplexan parasite Cryptosporidium causes human and animal diarrheal disease worldwide (1Leav B.A. Mackay M. Ward H.D. Clin. Infect. Dis. 2003; 36: 903-908Crossref PubMed Scopus (77) Google Scholar, 2Tzipori S. Ward H. Microbes Infect. 2002; 4: 1047-1058Crossref PubMed Scopus (214) Google Scholar) The two major species that cause human disease are Cryptosporidium parvum, which infects humans as well as other animals, and Cryptosporidium hominis, which primarily infects humans (3Xiao L. Fayer R. Ryan U. Upton S.J. Clin. Microbial. Rev. 2004; 17: 72-97Crossref PubMed Scopus (708) Google Scholar). Cryptosporidial infection in immunocompetent hosts is asymptomatic or self-limiting. However, in immunocompromised hosts, such as patients with AIDS, Cryptosporidium may cause severe, chronic, and possibly fatal disease. Cryptosporidium is responsible for numerous outbreaks of waterborne diarrheal disease worldwide. Because of the potential for intentional contamination of water supplies with this organism, the Centers for Disease Control have listed Cryptosporidium as a category B pathogen for biodefense (4Rotz L.D. Khan A.S. Lillibridge S.R. Ostroff S.M. Hughes J.M. Emerg. Infect. Dis. 2002; 8: 225-230Crossref PubMed Scopus (696) Google Scholar). Treatment options for cryptosporidiosis are limited. Although nitazoxanide is approved by the Food and Drug Administration for use in immunocompetent individuals with the infection, this drug is not effective against cryptosporidiosis in immunocompromised patients (5Zardi E.M. Picardi A. Afeltra A. Chemotherapy. 2005; 51: 193-196Crossref PubMed Scopus (56) Google Scholar). Infection with Cryptosporidium occurs when oocysts are ingested with contaminated water or food or by direct person-to-person contact. Oocysts excyst in the small intestine, releasing sporozoites that attach to and invade intestinal epithelial cells. Intracellular replication occurs within a parasitophorus vacuole via asexual as well as sexual cycles. Although the pathogenic mechanisms by which Cryptosporidium cause disease are poorly understood, it is apparent that attachment of invasive stages (sporozoites and merozoites) of the parasite to epithelial cells and subsequent invasion of these cells are crucial events in the establishment of an infection. The ultrastructural characteristics of attachment and invasion and various factors influencing attachment have been described (6Fayer R. Guidry A. Blagburn B.L. Infect. Immun. 1990; 58: 2962-2965Crossref PubMed Google Scholar, 7Hamer D.H. Ward H. Tzipori S. Pereira M.E. Alroy J.P. Keusch G.T. Infect. Immun. 1994; 62: 2208-2213Crossref PubMed Google Scholar, 8Lumb R. Smith K. O'Donoghue P.J. Lanser J.A. Parasitol. Res. 1988; 74: 531-536Crossref PubMed Scopus (87) Google Scholar). However, little is known about specific parasite and host molecules involved in these processes. Knowledge of such molecules is crucial for understanding the pathogenic mechanisms involved in these interactions and for designing novel strategies to combat cryptosporidiosis. However, progress in identifying these molecules and their functional role has been severely hampered by the inability to propagate Cryptosporidium in vitro and to genetically manipulate the parasite (9Wanyiri J. Ward H. Future Microbiol. 2006; 1: 201-208Crossref PubMed Scopus (45) Google Scholar). In the human or animal host, Cryptosporidium preferentially infects small intestinal epithelial cells (10Yu J.R. Choi S.D. Kim Y.W. Korean J. Parasitol. 2000; 38: 59-64Crossref PubMed Scopus (16) Google Scholar, 11Upton S.J. Tilley M. Brillhart D.B. FEMS Microbiol. Lett. 1994; 118: 233-236Crossref PubMed Scopus (121) Google Scholar). This suggests that specific parasite and/or host determinants are involved in recognition and adhesion of the parasite to host cells. A number of such intercellular recognition and adhesion functions are mediated by lectins, or carbohydrate-binding proteins, via their interaction with specific carbohydrate residues (12Houzelstein D. Goncalves I.R. Fadden A.J. Sidhu S.S. Cooper D.N. Drickamer K. Leffler H. Poirier F. Mol. Biol. Evol. 2004; 21: 1177-1187Crossref PubMed Scopus (201) Google Scholar, 13Sharon N. Protein Sci. 1998; 7: 2042-2048Crossref PubMed Scopus (32) Google Scholar). Lectins or carbohydrate-binding proteins have been implicated as mediators of attachment and/or invasion of host cells in a number of protozoan parasites (14Bhat N. Ward H. Caron M. Seve A.P. Lectins and Pathology. Harwood Academic Publishers, Amsterdam, Netherlands2000: 173-200Google Scholar). Previous studies from our group reported the presence of surface-associated lectin activity in C. parvum sporozoites (15Thea D.M. Pereira M.E. Kotler D. Sterling C.R. Keusch G.T. J. Parasitol. 1992; 78: 886-893Crossref PubMed Scopus (27) Google Scholar, 16Joe A. Hamer D.H. Kelley M.A. Pereira M.E. Keusch G.T. Tzipori S. Ward H.D. J. Eukaryot. Microbiol. 1994; 41: S44PubMed Google Scholar). This activity was identified using a hemagglutination (HA) 3The abbreviations used are: HA, hemagglutination; Tg, Toxoplasma gondii; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DMEM, Dulbecco's modified Eagle medium; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; PIC, protease inhibitor mixture; mAb, monoclonal antibody; IFA, immunofluorescence assay; HRP, horseradish peroxidase; ORF, open reading frame; OGS, octyl glucoside; GPI, glycosylphosphatidylinositol; MALDI-TOF-MS, matrix-assisted laser desorption time-of-flight mass spectrometry. 3The abbreviations used are: HA, hemagglutination; Tg, Toxoplasma gondii; BSA, bovine serum albumin; PBS, phosphate-buffered saline; TBS, Tris-buffered saline; DMEM, Dulbecco's modified Eagle medium; RT, reverse transcription; ELISA, enzyme-linked immunosorbent assay; PIC, protease inhibitor mixture; mAb, monoclonal antibody; IFA, immunofluorescence assay; HRP, horseradish peroxidase; ORF, open reading frame; OGS, octyl glucoside; GPI, glycosylphosphatidylinositol; MALDI-TOF-MS, matrix-assisted laser desorption time-of-flight mass spectrometry. assay, is optimal at a pH of 7.5 and in the presence of divalent cations Ca2+ and Mn2+, and is most specific for the monosaccharides Gal and GalNAc (16Joe A. Hamer D.H. Kelley M.A. Pereira M.E. Keusch G.T. Tzipori S. Ward H.D. J. Eukaryot. Microbiol. 1994; 41: S44PubMed Google Scholar). Gal and GalNAc-containing disaccharides and glycoconjugates, such as mucins, are potent inhibitors of lectin-induced hemagglutination. In addition, mucins block attachment to and invasion of host cells, implicating either host mucins and/or a C. parvum Gal/GalNAc-binding lectin in host cell recognition and adherence by C. parvum (17Joe A. Verdon R. Tzipori S. Keusch G.T. Ward H.D. Infect. Immun. 1998; 66: 3429-3432Crossref PubMed Google Scholar, 18Chen X.M. LaRusso N.F. Gastroenterology. 2000; 118: 368-379Abstract Full Text Full Text PDF PubMed Google Scholar, 19Hashim A. Mulcahy G. Bourke B. Clyne M. Infect. Immun. 2006; 74: 99-107Crossref PubMed Scopus (46) Google Scholar). Here we describe the identification and characterization of a 30-kDa Gal/GalNAc-specific lectin named C. parvum or C. hominis protein 30 (p30), which binds to intestinal epithelial cells and mediates attachment to and subsequent infection of these cells in vitro. Parasites—C. parvum (GCH1) and C. hominis (TU502) oocysts were obtained from Dr. Saul Tzipori, Tufts University Cummings School of Veterinary Medicine (Grafton, MA), and C. parvum (Iowa) was obtained from Pleasant Farms, Troy, ID, or Bunch Grass Farms, Deary, ID. Oocysts were stored at 4 °C. Prior to use oocysts were treated with 1.75% (v/v) sodium hypochlorite for 10 min on ice and then washed twice with 20 mm phosphate buffer, pH 7.2, containing 150 mm sodium chloride (PBS) by centrifugation at 5000 × g for 2 min at 4 °C. Hypochlorite-treated, washed oocysts were excysted for 1 h at 37 °C in the presence of 0.75% taurocholic acid in PBS. Excysted sporozoites were separated from oocysts by filtration through a 3.0-μm pore-size Nucleopore polycarbonate filter (Costar Scientific Corp., Cambridge, MA). Exocytosed or “shed proteins” were obtained as described previously (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar). Soluble phase proteins were obtained by Triton X-114 extraction and phase separation. Briefly, hypochlorite-treated oocysts (1.5 × 108/ml) were excysted for 1 h at 37 °C in the presence of 0.75% taurocholic acid, and the mixture of excysted oocysts and sporozoites was incubated with ice-cold pre-condensed 2% (v/v) Triton X-114 in 10 mm Tris-HCl, pH 8.0, containing 150 mm sodium chloride (TBS-1) for 1 h on ice followed by centrifugation at 10,000 × g for 10 min. The supernatant was incubated at 37 °C for 15 min to induce phase separation. The aqueous phase containing soluble proteins (soluble phase proteins) and the detergent phase enriched in membrane proteins (detergent phase proteins) were separated by centrifugation at 1000 × g for 10 min. Proteins in both phases were precipitated with 8 volumes of chilled (-20 °C) acetone overnight at 4 °C. After centrifugation at 10,000 × g for 10 min, the pellet was dried at room temperature, dissolved in SDS-PAGE reducing sample buffer, heated at 95 °C for 5 min, and resolved by SDS-PAGE. Cell Culture—Intestinal epithelial Caco-2A and HCT-8 cells were grown in 75-cm2 flasks in Dulbecco's modified Eagle's medium (DMEM; Invitrogen) containing 10% fetal calf sera, 25 mm HEPES, 100 units of penicillin, and 100 μg of streptomycin per ml at 37 °C in 5% CO2 as described earlier (17Joe A. Verdon R. Tzipori S. Keusch G.T. Ward H.D. Infect. Immun. 1998; 66: 3429-3432Crossref PubMed Google Scholar). Gal-affinity Chromatography—Hypochlorite-treated C. parvum oocysts (2 × 109) were resuspended in PBS containing a protease inhibitor mixture (PIC) consisting of 2 mm phenylmethylsulfonyl fluoride, 20 μm leupeptin (Sigma), 20 μm pepstatin (Sigma), and 20 μm E-64 (Sigma) and excysted at 37 °C for 1 h. The resultant mixture of sporozoites and unexcysted oocysts was washed three times with PBS by centrifugation at 4500 × g for 15 min, resuspended in PBS containing PIC, and lysed by sonication on ice using a Branson Sonifier 450 (Branson Ultrasonics Corporation, Danbury, CT) (five times for 1 min each with an interval of 2 min, 50% duty cycle, output 3) followed by detergent extraction in 1% octyl glucoside (1% OGS) (Sigma) in PBS overnight at 4 °C, and centrifugation at 10,000 rpm for 25 min. C. parvum lysate or C. hominis soluble phase proteins were applied to Gal-agarose (EY Laboratories Inc. San Mateo, CA) columns equilibrated with PBS containing 1% OGS overnight at 4 °C. After extensive washing with PBS containing 0.1% OGS, bound proteins were eluted with 1 m Gal in 0.01% OGS/PBS. Proteins present in the eluted fractions were detected by silver staining following SDS-PAGE. Eluted fractions were pooled and concentrated using Centricon-10 centrifugal filters (Millipore Corp., Bedford, MA). Protein concentration was determined using a BCA protein assay kit (Pierce). Gel Filtration—Gal affinity-purified proteins were separated by gel filtration on a Sephacryl S300-HR column (0.75 × 26.5 cm) equilibrated with TBS-1 containing 1 m Gal and 0.01% Triton X-100. 0.3-ml fractions were collected at a flow rate of 20 ml/h. The void volume (Vo) of the column was determined using blue dextran (2,000,000–4,000,000 kDa), and the column was calibrated using cytochrome c (14,700), carbonic anhydrase (29,000), albumin (68,000), amylase (200,000), and thyroglobulin (669,000) (all obtained from Sigma) as standards. Protein content of the fractions was monitored using a micro BCA assay kit (Pierce). Each fraction was concentrated 6-fold using Vivaspin 500 concentrators (Vivascience, Stonehouse, UK) and analyzed by Western blotting. The blots were probed with α-rp30 sera and mAb 4E9. p30, gp40, and gp900 present in the fractions were quantified by scanning densitometry of the Western blots using Quantity One software (Bio-Rad). The molecular mass of the proteins eluted in the two major included peaks was determined by the least squares line equation (21Andrews P. Biochem. J. 1965; 96: 595-606Crossref PubMed Scopus (2427) Google Scholar). N-terminal and Internal Peptide Amino Acid Sequence Determination—Gal affinity-purified proteins from C. parvum were resolved by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore Corp.), and stained with Coomassie Blue. The 30-kDa band was excised and processed for N-terminal amino acid microsequencing by automated Edman degradation using a PerkinElmer Life Sciences ABI 477A sequencer at the Tufts University Core Facility. The N-terminal sequence of the first 11 residues was determined. To obtain amino acid sequence data from internal peptides, the 30-kDa band was excised from Coomassie Blue-stained SDS gels and submitted to the Harvard Microchemistry Facility, Cambridge, MA. After in-gel tryptic digestion of the protein, sequence analysis of peptides was performed by microcapillary reverse-phase high pressure liquid chromatography nanoelectrospray tandem mass spectrometry on a Finnigan LCQ quadrupole ion trap mass spectrometer. Four peptides were selected and analyzed by matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS). These four peptides were then sequenced by Edman degradation. High confidence amino acid sequences (supported by the MALDI-TOF-MS results) were obtained for four peptides (Table 1).TABLE 1Sequence of p30-derived peptides and synthetic oligonucleotides used for PCR and RT-PCRPeptidesOligonucleotidesNGVC1LKKGaThis is an N-terminal peptide; P1 and P2 are degenerate primersP1, 5′-AATGGAGUCTGCATTCUUAAAAAAGG-3′ (Forward)FIAGSKVAYSLYKP2, 5′-TTTATAUUGGGUATAAGC-3′ (Reverse)QEWLATSSGSFVGR–FIYQSVSQGTMNVSR–P3, 5′-ATGAGCGGCGCGCGCAGCAGCATTTTTATT-3′ (Forward)P4, 5′-AACGGCGTGTGCATTCTGAAAAAAGGCGC-3′ (Forward)P5, 5′-ATAGCTGTTCATGGTGCCCTGGCTC-3′ (Reverse)a This is an N-terminal peptide; P1 and P2 are degenerate primers Open table in a new tab Hemagglutination (HA) and HA Inhibition Assays—The presence of lectin activity was determined by using an HA assay (22Goldhar J. Methods Enzymol. 1994; 236: 211-231Crossref PubMed Scopus (29) Google Scholar). Serial doubling dilutions (in PBS containing 1 mg/ml bovine serum albumin (BSA) and 2 mm CaCl2) of starting material or eluate were incubated with equal volumes of a 1% (v/v) suspension of rabbit erythrocytes in PBS in the wells of a U-bottom microtiter plate (Falcon Micro Test III™ BD Biosciences) for 1 h at 4 °C. Lectin activity was expressed (in HA units) as the reciprocal of the highest dilution of the test solution showing visible hemagglutination. Carbohydrate specificity was determined using an HA inhibition assay. Serial 2-fold dilutions of mono- and disaccharides (Sigma) in PBS were preincubated with equal volumes of test solution for 1 h at room temperature, and an equal volume of 1% rabbit erythrocytes in PBS was added. The plate was incubated for 2 h at 4 °C, and HA titer was determined as described above. DNA Isolation, PCR, and DNA Sequencing—DNA was isolated from hypochlorite-treated, freeze-thawed GCH1 oocysts using a Fast DNA kit (Bio 101, Carlsbad, CA). Degenerate primers (P1 and P2, see Table 1) were constructed based on the amino acid sequence of the N-terminal and internal peptides shown in Table 1 (Invitrogen). Nondegenerate primers P3, P4, and P5 (Table 1) were synthesized by the Tufts University School of Medicine Core Facility. Conditions for PCR using degenerate primers were as follows: 95 °C for 2 min; 95 °C for 40 s, 37 °C for 60 s, and 72 °C for 50 s (5 cycles); 94 °C for 40 s, 55 °C for 50 s, and 72 °C for 50 s (35 cycles); and 72 °C for 5 min. The first five cycles were excluded for PCR using nondegenerate primers. Reagents were used at the following final concentrations: deoxynucleoside triphosphates, 0.4 mm; degenerate primers, 1 μm; nondegenerate primers, 0.5 μm; MgCl2, 2.5 mm; and TaqDNA polymerase (Invitrogen). The 909-bp fragment generated by PCR using primers P3 and P5 was cloned into the pCR 2.1 vector (Invitrogen) for sequencing. Plasmids were purified using a Qiagen miniprep kit (Qiagen, Inc., Valencia, CA). DNA sequencing was performed by the dye-terminator method at the Tufts University School of Medicine Core Facility using a PerkinElmer Life Sciences ABI 377 sequencer. The BLAST algorithm was used to compare DNA and protein sequences to sequences in data bases. Analysis of nucleotide and amino acid sequences was performed using Vector NTI software (Informax, North Bethesda, MD) and the ExPASy Molecular Biology Server. The entire coding sequences of p30 were obtained from the C. parvum (23Abrahamsen M.S. Templeton T.J. Enomoto S. Abrahante J.E. Zhu G. Lancto C.A. Deng M. Liu C. Widmer G. Tzipori S. Buck G.A. Xu P. Bankier A.T. Dear P.H. Konfortov B.A. Spriggs H.F. Iyer L. Anantharaman V. Aravind L. Kapur V. Science. 2004; 304: 441-445Crossref PubMed Scopus (746) Google Scholar) and C. hominis (24Xu P. Widmer G. Wang Y. Ozaki L.S. Alves J.M. Serrano M.G. Puiu D. Manque P. Akiyoshi D. Mackey A.J. Pearson W.R. Dear P.H. Bankier A.T. Peterson D.L. Abrahamsen M.S. Kapur V. Tzipori S. Buck G.A. Nature. 2004; 431: 1107-1112Crossref PubMed Scopus (418) Google Scholar) genome sequences using CryptoDB (25Heiges M. Wang H. Robinson E. Aurrecoechea C. Gao X. Kaluskar N. Rhodes P. Wang S. He C.Z. Su Y. Miller J. Kraemer E. Kissinger J.C. Nucleic Acids Res. 2006; 34: 419-422Crossref PubMed Scopus (117) Google Scholar, 26Puiu D. Enomoto S. Buck G.A. Abrahamsen M.S. Kissinger J.C. Nucleic Acids Res. 2004; 32: D329-D331Crossref PubMed Google Scholar). The nucleotide sequences of p30 were deposited in GenBank™ under accession numbers AY308041 (GCH1, C. parvum) and AY308040 (TU502, C. hominis). RT-PCR and Northern Blot Analysis—Caco-2A cells were grown to confluence in T25 tissue culture flasks and infected with C. parvum GCH1 oocysts (3 × 106 per flask) for 24 h at 37 °C as described previously (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar). RNA was extracted from uninfected and infected Caco-2A monolayers at different time points (24, 48, and 72 h) using an RNeasy kit (Qiagen). Contaminating DNA was removed by RNase-free DNase treatment using a DNA-free™ kit (Ambion). 8 μg of total RNA was used in each RT reaction, which was performed using primers P3 and P5 (Table 1) and the Access RT-PCR System according to the manufacturer's instructions (Promega, Madison, WI). RNA from uninfected Caco-2A cells was used as a negative control. RT-PCRs without added reverse transcriptase were performed in parallel to confirm that the PCR products were not because of the amplification of contaminating DNA. The product obtained by RT-PCR was cloned into the TOPO pCR2.1 vector (Invitrogen), and the nucleotide sequence was determined as described above. As a loading control for the RT-PCRs, an 800-bp fragment of the C. parvum small subunit ribosomal RNA gene (SSU rRNA) was amplified using previously described primers and conditions (27Tosini F. Agnoli A. Mele R. Gomez Morales M.A. Pozio E. Mol. Biochem. Parasitol. 2004; 134: 137-147Crossref PubMed Scopus (42) Google Scholar). Northern blot analysis was performed as described previously (28O'Connor R.M. Thorpe C.M. Cevallos A.M. Ward H.D. Mol. Biochem. Parasitol. 2002; 119: 203-215Crossref PubMed Scopus (17) Google Scholar). Briefly, mRNA was isolated from uninfected and infected Caco-2A cells using an Oligotex QIA kit (Qiagen). 3.5 μg of mRNA was separated by 1% glyoxyl-agarose electrophoresis, transferred to a BrightStar Plus nylon membrane (Ambion) using a Northern-Gly blotting kit (Ambion) and fixed by UV radiation using a UV Stratalinker 1800 (Stratagene). The 909-bp product generated by PCR using genomic DNA (Table 1) was extracted from an agarose gel using a GEL QIAquick extraction kit (Qiagen), labeled with [32P]dCTP by random priming, and the membrane hybridized with this probe. Hybridization was carried out in prehybridization/hybridization solution at 65 °C for 16 h in a PersonalHyb™ chamber (Stratagene, La Jolla, CA). The blot was washed three times with 40 mm phosphate buffer, pH 7.4, containing 0.1% SDS and exposed to film, and reactive bands were detected by autoradiography. Expression, Purification, and Gal Binding of Recombinant p30—The p30 sequence (excluding the signal sequence) was cloned from genomic DNA and primers P4 and P5 into the pET-32 Xa/LIC vector (Novagen, Madison WI), which contains an internal factor Xa cleavage site (8 amino acids), a thrombin site (8 amino acids), an S tag (15 amino acids), a His tag (6 amino acids), and an N-terminal thioredoxin tag (109 amino acids), and the plasmid was designated pNBp30. The recombinant fusion protein designated rp30 was overexpressed in Escherichia coli AD494 (DE3) cells following induction with 1 mm isopropyl β-d-thiogalactopyranoside. E. coli cells expressing rp30 were lysed in BugBuster reagent (Novagen), and the mixture was centrifuged at 10,000 × g for 30 min in the presence of Benzonase 1 mg/ml (Novagen). The supernatant and pellet (containing inclusion bodies) were resuspended in TBS-1, resolved by 10% SDS-PAGE under reducing conditions, and transferred to a nitrocellulose membrane, and the S tag present in the fusion protein was detected by probing with HRP-conjugated S-protein according to the manufacturer's instructions (Novagen). The majority of rp30 was detected in the pellet that contains inclusion bodies. rp30 present in inclusion bodies was solubilized and refolded using a protein refolding kit (Novagen). The refolded protein was suspended in binding buffer (50 mm phosphate buffer, pH 7.0, containing 300 mm NaCl, 50 mm imidazole, and 5 mm CaCl2), and rp30 was purified by metal-affinity chromatography using Talon metal-affinity resin (Clontech). Purity of rp30, which migrated at the expected relative molecular mass of 48 kDa, was verified by SDS-PAGE and Western blotting with HRP-conjugated S protein. Equal amounts of purified rp30 in binding buffer were applied to two 0.5-ml Gal-agarose columns overnight at 4 °C. After washing with 10 bed volumes of binding buffer, the bound proteins were eluted with 1 m Gal or 1 m Glc in the same buffer. Eluted fractions were concentrated by ultrafiltration and analyzed by SDS-PAGE and Western blotting. Antibodies—mAb 4E9 is an IgM mAb directed at an αGal-NAc-containing carbohydrate epitope present on C. parvum gp900 and gp40 (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar). Antisera to purified rp30 were produced as follows. Purified rp30 was resolved by SDS-PAGE and stained with Coomassie Blue. The 48-kDa band (which was shown to represent rp30 by S tag Western blotting of a parallel strip of gel transferred to nitrocellulose) was excised and emulsified with complete Freund's adjuvant for the initial immunization and incomplete complete Freund's adjuvant for subsequent boosts. 6-Week-old BALB/c mice were immunized by intraperitoneal injection at 6–10-week intervals for a total of three immunizations, and the presence of α-rp30 antibodies in sera was monitored by Western blotting of C. parvum oocysts and rp30. Antisera were depleted of antibodies to rp30 by incubation with rp30 immobilized via the His tag on Talon metal affinity resin for 2 h on ice. Western Blot Analysis—Proteins were separated by SDS-PAGE, transferred to nitrocellulose, and probed with mAb 4E9 and α-rp30 sera as described (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar). HRP-conjugated goat anti-mouse IgM and IgG (heavy and light chain) (Pierce) were used as secondary antibodies for mAb 4E9 and α-rp30, respectively. The S tag present in fusion proteins was identified in blots by reactivity with HRP-conjugated S protein according to the manufacturer's instructions (Novagen). Blots were developed with SuperSignal substrate (Pierce). Perfect Protein markers, 10–225 kDa (Novagen); were used as molecular weight standards and were detected by reactivity with HRP-conjugated S protein according to the manufacturer's protocol. Immunofluorescence Assay (IFA)—Hypochlorite-treated oocysts (2 × 107/ml in PBS) were placed on 0.1% poly-l-lysine-coated 8-well chamber slides (Falcon), allowed to excyst at 37°C for 1 h in a moist chamber, then air-dried, fixed, and permeabilized in methanol for 10 min at room temperature. IFAs were performed as described earlier (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar, 29Verdon R. Keusch G.T. Tzipori S. Grubman S.A. Jefferson D.M. Ward H.D. J. Infect. Dis. 1997; 175: 1268-1272Crossref PubMed Scopus (30) Google Scholar). Localization of p30 in intracellular stages in infected HCT-8 cells was performed by IFA as described previously (30O'Connor R.M. Kim K. Khan F. Ward H.D. Infect. Immun. 2003; 71: 6027-6034Crossref PubMed Scopus (39) Google Scholar). Glycosidase Digestion—Hypochlorite-treated oocysts (2 × 108/ml) were excysted in the presence of 0.75% taurocholic acid for 1 h at 37 °C. The mixture of excysted oocysts and sporozoites was resuspended in 50 mm Tris-HCl, pH 7.5, containing 10 mm sodium chloride (TBS-2) and PIC and lysed by freeze-thawing (20 cycles). The lysate was incubated with an equal volume of 2% Triton X-100 in TBS-2 overnight at 4 °C with gentle shaking. After centrifugation at 10,000 × g for 30 min, the supernatant was treated with recombinant peptide N-glycosidase F, 300 units/ml (New England Biolabs, Beverley, MA), or α-N-acetylgalactosaminidase, 500 units/ml (New England Biolabs), at 37 °C overnight according to the manufacturer's instructions. As a control for these glycosidases, aliquots of lysate were treated with buffer alone under identical conditions. p30 Binding Assay—Binding of p30 to Caco-2A cells was assessed by ELISA (20Cevallos A.M. Bhat N. Verdon R. Hamer D.H. Stein B. Tzipori S. Pereira M.E. Keusch G.T. Ward H.D. Infect. Immun. 2000; 68: 5167-5175Crossref PubMed Scopus (99) Google Scholar) and IFA. Soluble pha" @default.
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