Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum
2003; Elsevier BV; Volume: 278; Issue: 52 Linguagem: Inglês
10.1074/jbc.m306835200
ISSN1083-351X
AutoresJeffrey W. Priest, Angela Mehlert, Michael J. Arrowood, Michael W. Riggs, Michael A. J. Ferguson,
Tópico(s)Enterobacteriaceae and Cronobacter Research
ResumoCryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose3-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1–3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection. Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose3-glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1–3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection. Cryptosporidium parvum is an Apicomplexan protozoan parasite that has been recognized as a major cause of diarrheal illness in humans and in livestock around the world (1Dillingham R.A. Lima A.A. Guerrant R.L. Microbes Infect. 2002; 4: 1059-1066Crossref PubMed Scopus (172) Google Scholar, 2Chen X.-M. Kiethly J.S. Paya C.V. LaRusso N.F. N. Engl. J. Med. 2002; 346: 1723-1731Crossref PubMed Scopus (401) Google Scholar, 3Juranek D.D. Clin. Infect. Dis. 1995; 21: 57-61Crossref PubMed Scopus (139) Google Scholar). Cryptosporidiosis is spread by fecal contamination of food and water. Not surprisingly, outbreaks have also been linked to accidental ingestion of contaminated recreational water (4MacKenzie W.R. Kazmierczak J.J. Davis J.P. Epidemiol. Infect. 1995; 115: 545-553Crossref PubMed Scopus (46) Google Scholar, 5Centers for Disease Control Morb. Mortal. Wkly. Rep. 1998; 47: 856-860PubMed Google Scholar, 6Lee S.H. Levy D.A. Craun G.F. Beach M.J. Calderon R.L. Morb. Mortal. Wkly. Rep. 2002; 51: 1-48Google Scholar). Oocysts are ubiquitous in the environment and are commonly found in raw surface sources of drinking water. Because of the small size of the C. parvum oocyst and the resistance of the oocyst to standard chlorination treatments, they are particularly difficult to eliminate during treatment (7Korich D.G. Mead J.R. Madore M.S. Sinclair M.A. Sterling C.R. Appl. Environ. Microbiol. 1990; 56: 1423-1428Crossref PubMed Google Scholar). Numerous waterborne outbreaks have been linked to contaminated municipal water supplies (8Dworkin M.S. Goldman D.P. Wells T.G. 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Peterson D.E. Kazmierczak J.J. Addiss D.G. Fox K.R. Rose J.B. Davis J.P. N. Engl. J. Med. 1994; 331: 161-167Crossref PubMed Scopus (1593) Google Scholar, 13McDonald A.C. MacKenzie W.R. Addiss D.G. Gradus M.S. Linke G. Zembrowski E. Hurd M.R. Arrowood M.J. Lammie P.J. Priest J.W. J. Infect. Dis. 2001; 183: 1373-1379Crossref PubMed Scopus (52) Google Scholar). Infection in the immunocompetent host may be asymptomatic or may lead to a self-limiting diarrheal illness (14Chappell C.L. Okhuysen P.C. Sterling C.R. DuPont H.L. J. Infect. Dis. 1996; 173: 232-236Crossref PubMed Scopus (160) Google Scholar). However, in immunocompromised and immunosuppressed individuals, the disease is often severe and chronic and may contribute significantly to mortality (15Pozio E. Rezza G. Boschini A. Pezzotti P. Tamburrini A. Rossi P. Di Fine M. Smacchia C. Schiesari A. Gattei E. Zucconi R. Ballarini P. J. Infect. Dis. 1997; 176: 969-975Crossref PubMed Scopus (78) Google Scholar).Infection with C. parvum has been shown to elicit transient antibody responses that are directed mainly against two sporozoite antigens having apparent molecular masses of 27 and 17 kDa (16Reperant J.-M. Naciri M. Iochmann S. Tilley M. Bout D.T. Vet. Parasitol. 1994; 55: 1-13Crossref PubMed Scopus (53) Google Scholar, 17Mead J.R. Arrowood M.J. Sterling C.R. J. Parasitol. 1988; 74: 135-143Crossref PubMed Scopus (84) Google Scholar, 18Moss D.M. Bennett S.N. Arrowood M.J. Wahlquist S.P. Lammie P.J. Am. J. Trop. Med. Hyg. 1998; 58: 110-118Crossref PubMed Scopus (49) Google Scholar, 19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar). In a study of C. parvum infections in human volunteers, antibody responses to these antigens were associated with protection from diarrheal symptoms (20Moss D.M. Chappell C.L. Okhuysen P.C. DuPont H.L. Arrowood M.J. Hightower A.W. Lammie P.J. J. Infect. Dis. 1998; 178: 827-833Crossref PubMed Scopus (93) Google Scholar). Both of these antigens are associated with the sporozoite surface, and subsets of the antigens can be partially purified from sonicated oocysts by phase partitioning into Triton X-114 detergent (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar). In earlier work we used this technique to purify the 17-kDa antigen for peptide sequence analysis and to generate a native antigen fraction suitable for use in an enzyme-linked immunoassay for the detection and quantitation of serum IgG antibodies (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar, 21Priest J.W. Kwon J.P. Arrowood M.J. Lammie P.J. Mol. Biochem. Parasitol. 2000; 106: 261-271Crossref PubMed Scopus (57) Google Scholar). While analyzing the antibody response to the Triton X-114 detergent extract by Western blot, we noted that a number of cryptosporidiosis patients also reacted with a novel low molecular weight antigen. In this work we report that this antigen is actually a family of glycosylinositol phospholipids (GIPLs). 1The abbreviations used are: GIPLglycosylinositol phospholipidGPIglycosylphosphatidylinositolOSoctyl-SepharoseHPTLChigh performance thin layer chromatographyPVDFpolyvinylidene difluoridePBSphosphate-buffered salinePIphosphatidylinositolGC-MSgas chromatography-mass spectrometryJBAMjack bean α-mannosidaseASAMAspergillus satoi α-mannosidaseES-MSelectrospray-mass spectrometryCIDcollision-induced dissociationAHM2,5-anhydromannitolmAbmonoclonal antibody.1The abbreviations used are: GIPLglycosylinositol phospholipidGPIglycosylphosphatidylinositolOSoctyl-SepharoseHPTLChigh performance thin layer chromatographyPVDFpolyvinylidene difluoridePBSphosphate-buffered salinePIphosphatidylinositolGC-MSgas chromatography-mass spectrometryJBAMjack bean α-mannosidaseASAMAspergillus satoi α-mannosidaseES-MSelectrospray-mass spectrometryCIDcollision-induced dissociationAHM2,5-anhydromannitolmAbmonoclonal antibody.GIPLs and related structures that anchor some surface proteins into the membrane, glycosylphosphatidylinositol (GPI) anchors, are present in large quantities in the surface membranes of many protozoan parasites and have been recognized recently as important effectors of the host immune response during infection (reviewed in Refs. 22Ferguson M.A.J. J. Cell Sci. 1999; 112: 2799-2809Crossref PubMed Google Scholar and 23Ropert C. Gazzinelli R.T. Curr. Opin. Microbiol. 2000; 3: 395-403Crossref PubMed Scopus (84) Google Scholar). GPI anchors and/or GIPLs from medically important parasites such as Trypanosoma cruzi, Leishmania mexicana, Trypanosoma brucei, Plasmodium falciparum, and Toxoplasma gondii have been shown to modulate immune system function as both suppressors and activators (23Ropert C. Gazzinelli R.T. Curr. Opin. Microbiol. 2000; 3: 395-403Crossref PubMed Scopus (84) Google Scholar, 24Tachado S.D. Gerold P. Schwarz R. Novakovic S. McConville M. Schofield L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4022-4027Crossref PubMed Scopus (185) Google Scholar). A GPI-derived toxin has been implicated in pathogenesis in cases of severe malaria (25de Souza J.B. Todd J. Krishegowda G. Gowda D.C. Kwiatkowski D. Riley E.M. Infect. Immun. 2002; 70: 5045-5051Crossref PubMed Scopus (47) Google Scholar, 26Naik R.S. Branch O.H. Woods A.S. Vijaykumar M. Perkins D.J. Nahlen B.L. Lal A.A. Cotter R.J. Costello C.E. Ockenhouse C.F. Davidson E.A. Gowda D.C. J. Exp. Med. 2000; 192: 1563-1575Crossref PubMed Scopus (200) Google Scholar, 27Schofield L. Hewitt M.C. Evans K. Siomos M.-A. Seeberger P.H. Nature. 2002; 418: 785-789Crossref PubMed Scopus (410) Google Scholar). In this work, we report that many cryptosporidiosis patients have a serum antibody response to sporozoite-derived GIPLs, and we report the purification and structural analysis of the sporozoite GIPL antigens.MATERIALS AND METHODSPurification of Antigen—C. parvum oocysts (Maine isolate) were purified from the feces of experimentally infected Holstein calves as described by Arrowood and Sterling (28Arrowood M.J. Sterling C.R. J. Parasitol. 1989; 73: 314-319Crossref Scopus (345) Google Scholar). A crude antigen preparation was obtained by sonication and freeze/thaw of the oocysts as described previously (29Moss D.M. Lammie P.J. Am. J. Trop. Med. Hyg. 1993; 49: 393-401Crossref PubMed Scopus (31) Google Scholar). The crude antigen preparation was extracted with Triton X-114 using a modification of the method of Ko and Thompson (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar, 30Ko Y.-G. Thompson Jr., G.A. Anal. Biochem. 1995; 224: 166-172Crossref PubMed Scopus (37) Google Scholar). Antigens were acetone-precipitated from the detergent fraction (4 volumes of cold acetone with overnight incubation at -20 °C) and dissolved in buffer containing 0.1% SDS, 25 mm Tris, pH 8.0, and 1 mm EDTA for a final protein concentration of ∼1 mg/ml (BCA protein microassay; Pierce). Glycolipids were extracted from the antigen fraction with water-saturated 1-butanol (2 times, 1 volume) and then dried under vacuum. Butanol-extracted glycolipids were further purified by octyl-Sepharose (OS) chromatography (1 cm inner diameter × 10 cm length) as described by McConville et al. (31McConville M.J. Collidge T.A.C. Ferguson M.A.J. Schneider P. J. Biol. Chem. 1993; 268: 15595-15604Abstract Full Text PDF PubMed Google Scholar). The fractions having the strongest orcinol/H2SO4 reaction (1 μl of each 1-ml fraction was spotted on a silica gel HPTLC plate (EM Science, Gibbstown, NJ)) (32Schneider P. Ralton J.E. McConville M.J. Ferguson M.A.J. Anal. Biochem. 1993; 210: 106-112Crossref PubMed Scopus (27) Google Scholar) were pooled (fractions 86–92), dried under vacuum, and dissolved in 40% 1-propanol. An aliquot of OS-purified glycolipid was further fractionated by preparative silica gel HPTLC using a solvent system of 10:10:3 (v/v/v) chloroform, methanol, 1.0 m NH4OH (31McConville M.J. Collidge T.A.C. Ferguson M.A.J. Schneider P. J. Biol. Chem. 1993; 268: 15595-15604Abstract Full Text PDF PubMed Google Scholar). A strip was cut from the side of the preparative lane and stained with orcinol/H2SO4 reagent to locate the positions of the component glycolipid bands. The regions corresponding to each glycolipid band were scraped into a tube and extracted once with water-saturated butanol and once with chloroform/methanol/water, 1:2:0.8. The combined eluates were dried under vacuum, dissolved in water-saturated butanol, and spotted onto polyvinylidene difluoride (PVDF) membranes (Immobilon P; Millipore Corp., Bedford, MA) for Western blot analysis.Western Blots and Treatment of Blot Strips with NaIO4and Proteinase K—Triton X-114-extracted oocyst/sporozoite antigens were resolved on 10–22.5% SDS-PAGE by the method of Laemmli (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206057) Google Scholar) and then electrotransferred to PVDF membrane for further analysis. For Western blots, membrane strips were incubated overnight at 4 °C with sera diluted 1:100 in phosphate-buffered saline (0.85% NaCl and 10 mm Na2HPO4, pH 7.2) (PBS) with 0.3% Tween 20. Bound antibodies were visualized using biotinylated mouse anti-human monoclonal antibodies (IgG, clone HP6017; IgG1, clone HP6069; IgG2, clone HP6002; IgG3, clone HP6047; IgG4, clone HP6025; IgA, clone GA112; and IgM, clone HP6083; Zymed Laboratories Inc., South San Francisco) and alkaline phosphatase-labeled streptavidin as described previously (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar). A mouse monoclonal antibody (antibody 18.44) and a biotinylated rat anti-mouse IgG monoclonal secondary antibody (Zymed Laboratories Inc.) were used to detect the C. parvum CPS-500 glycolipid (34Riggs M.W. McGuire T.C. Mason P.H. Perryman L.E. J. Immunol. 1989; 143: 1340-1345PubMed Google Scholar).Strips of PVDF membrane containing Triton X-114-soluble antigens were incubated 1–48 h at room temperature with 0–25 mm NaIO4 in 50 mm sodium acetate buffer, pH 4.5, in the dark, washed extensively, blocked, then incubated overnight at 4 °C with diluted human patient serum, and developed for bound IgG antibodies as described above (35Woodward M.P. Young Jr., W.W. Bloodgood R.A. J. Immunol. Methods. 1985; 78: 143-153Crossref PubMed Scopus (595) Google Scholar). A strip of PVDF membrane containing Triton X-114-soluble antigens was incubated overnight at 37 °C in PBS containing 100 μg/ml proteinase K. The strip was then washed twice for 1 h with PBS, 0.3% Tween 20 containing 1 mm phenylmethylsulfonyl fluoride, incubated overnight with diluted patient serum, and developed for bound IgG antibodies as described above.Analysis of myo-Inositol and Phosphatidylinositol (PI) from C. parvum Antigens—Triton X-114-extracted antigens (120 μg of protein) were resolved by gradient SDS-PAGE and transferred to PVDF as described previously (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar). After staining the proteins with Amido Black, strips of membrane of approximately equal sizes were cut from the following: 1) above the 17-kDa antigen; 2) the stained 17-kDa antigen protein band near the 14.3-kDa marker; 3) immediately below the 17-kDa antigen band; and 4) a region between the 6- and 3-kDa markers. About 15% (20 μg of protein) of each strip was subjected to 6 m HCl acid hydrolysis (16 h at 110 °C) with 50 pmol of d6-myo-inositol added as an internal standard. The hydrolysis products were dried, derivatized with trimethylsilane, and analyzed by gas chromatography and selected ion gas chromatography-mass spectrometry (GC-MS) as described previously (36Priest J.W. Xie L.-T. Arrowood M.J. Lammie P.J. Mol. Biochem. Parasitol. 2001; 113: 117-126Crossref PubMed Scopus (33) Google Scholar, 37Ferguson M.A.J. Fukada M. Kobata A. Glycobiology: A Practical Approach. IRL Press at Oxford University Press, Oxford1994: 349-383Google Scholar).The remainder of each strip (about 100 μg of total protein loaded) was used for PI analysis (38Fontaine T. Magnin T. Mehlert A. Lamont D. Latge J.P. Ferguson M.A. Glycobiology. 2003; 13: 169-177Crossref PubMed Scopus (69) Google Scholar). The strips were treated with 0.5 m NaNO2 in 0.15 m sodium acetate, pH 4.0, for 2 h at 37 °C. The strips were then washed twice in water and extracted three times for 15 min with water-saturated butanol at 37 °C. Samples were sonicated to improve the extraction efficiency. The combined butanol extracts were dried under vacuum, dissolved in 4:1 chloroform/methanol, and loaded onto 200-μl silica gel mini columns (70–230 mesh). After extensive washing with 4:1 chloroform/methanol, bound PI was eluted with 1:4 chloroform/methanol for analysis.Methylation of GIPL Glycan—Approximately 200 pmol of OS-purified GIPLs (based on GC-MS myo-inositol analysis) were deacylated in 1:1 (v/v) 40% methanol/ammonium hydroxide at 37 °C for 1 h and then deaminated with NaNO2 as described above. At the end of the deamination reaction, boric acid was added to a final concentration of 73 mm, and the pH was adjusted to 10.7 with NaOH. NaB2H4 was added, and the deutero-reduction proceeded overnight at room temperature. The reaction was acidified by the addition of 0.5 volumes of 1 m acetic acid, and the reaction products were passed over a 0.5-ml Dowex AG50-X12(H+) column. The eluate was collected, dried by rotary evaporation, and sequentially dried from methanol, 1% acetic acid, toluene (2 times), and methanol (2 times). The deuterated products were permethylated with methyl iodide in NaOH/Me2SO as described previously (37Ferguson M.A.J. Fukada M. Kobata A. Glycobiology: A Practical Approach. IRL Press at Oxford University Press, Oxford1994: 349-383Google Scholar).C. parvum GIPL Neutral Glycan Labeling and Analysis—A membrane strip from region 4 (between 3 and 6 kDa) of a blot containing 140 μg of Triton X-114-extracted antigen was minced into 2-mm squares and washed sequentially with excess methanol and water. The membrane fragments were pre-reduced with 200 mm NaBH4 for 1 h at room temperature and then washed sequentially with excess water, 50 mm acetic acid, and water again. Antigens were deaminated for 2 h at 37 °C with 0.5 m NaNO2 in 0.15 m sodium acetate, pH 4.0, then washed with excess water (2 times) and with 0.1 mm NaOH. Those antigens still bound to the membrane were labeled for 2 h at room temperature with 2.5 mCi of NaB3H4 in 10 mm NaOH (70-μl volume) (38Fontaine T. Magnin T. Mehlert A. Lamont D. Latge J.P. Ferguson M.A. Glycobiology. 2003; 13: 169-177Crossref PubMed Scopus (69) Google Scholar). Unlabeled NaBH4 (5 μl of a 1 m solution) was added to the sample, and incubation was continued for an additional 1 h at room temperature. The membrane fragments were removed and washed sequentially with excess water, 50 mm acetic acid, PBS (2 times), and water again (2 times). HF treatment (50% aqueous solution at 4 °C overnight) was used to release labeled neutral glycans from the membrane fragments. The HF solution was collected and lyophilized. The resulting labeled neutral glycans were re-N-acetylated with acetic anhydride and treated with neuraminidase (50 milliunits for 1 h at 37 °C) prior to desalting on a column containing 0.2 ml of Chelex 100 (Na+), 0.2 ml of Dowex AG50-X12(H+), 0.4 ml of Dowex AG3-X4 (OH), and 0.2 ml of QAE-Sephadex-A25(OH-) as described previously (37Ferguson M.A.J. Fukada M. Kobata A. Glycobiology: A Practical Approach. IRL Press at Oxford University Press, Oxford1994: 349-383Google Scholar). Approximately 100,000 cpm were recovered in the final neutral glycan fraction.Exoglycosidase reactions with jack bean α-mannosidase (JBAM), Aspergillus satoi α-mannosidase (ASAM), green coffee bean α-galactosidase, Helix pomatia β-mannosidase, bovine testes β-galactosidase, and jack bean β-galactosidase (all enzymes from Glyko, Novato, CA) were conducted (6400 cpm/reaction) as described in Treumann et al. (39Treumann A. Guther M.L.S. Schneider P. Ferguson M.A.J. Methods Mol. Biol. 1998; 76: 213-235PubMed Google Scholar) or as directed by the manufacturer. Reaction products were resolved by HPTLC by using the solvent system described above and were visualized by fluorography.Electrospray-Mass Spectrometry (ES-MS) Analysis—Mass spectra were collected on a Micromass Q-Tof2 mass spectrometer using nanoflow tips. For negative ion mode mass spectrometry, samples were dissolved in chloroform/methanol (2:3) at ∼1 pmol/μl. For positive ion mode mass spectrometry, OS-purified GIPLs were dissolved in 30% 1-propanol with 10% acetic acid, and permethylated glycans were dissolved in 80% acetonitrile with 1 mm sodium acetate. Capillary and cone voltages were 900 and 60 V, respectively. Collision-induced dissociation (CID) daughter ion spectra were collected using argon as collision gas at 2.5 × 10-3 torr and an accelerating voltage of 43–55 V.Serum Specimens—Patient sera were available from individuals who were naturally infected during several C. parvum outbreaks. Samples were chosen for further analysis based upon the presence of Cryptosporidium-specific IgG antibody responses (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar). Informed consent was obtained from patients prior to sample collection. This study was reviewed and approved by the Institutional Review Board at the Centers for Disease Control and Prevention.RESULTSSerum Antibodies from Cryptosporidiosis Patients Recognize a Family of Very Low Molecular Weight Antigens—In previous work, we demonstrated that human infection with C. parvum elicits an IgG antibody response to the 17- and 27-kDa surface antigens within 10–14 days of symptom onset (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar, 40Priest J.W. Li A. Khan M. Arrowood M.J. Lammie P.J. Ong C.S. Roberts J.M. Isaac-Renton J. Clin. Diagn. Lab. Immunol. 2001; 8: 415-423Crossref PubMed Scopus (50) Google Scholar). Although both of these antigens include post-translational carbohydrate and/or lipid modifications, we demonstrated that much of the antibody response was directed against the protein component (19Priest J.W. Kwon J.P. Moss D.M. Roberts J.M. Arrowood M.J. Dworkin M.S. Juranek D.D. Lammie P.J. J. Clin. Microbiol. 1999; 37: 1385-1392Crossref PubMed Google Scholar, 21Priest J.W. Kwon J.P. Arrowood M.J. Lammie P.J. Mol. Biochem. Parasitol. 2000; 106: 261-271Crossref PubMed Scopus (57) Google Scholar). While examining the IgG antibody reactivity to the subset of those antigens that were extracted into Triton X-114 detergent (the 27- and 17-kDa antigen families include both soluble and membrane-bound forms), we noted that an additional antigen having a molecular mass of <6-kDa was recognized by serum IgG antibodies from ∼50% of the patients (Fig. 1 and data not shown). Although the resolution in that area of the Western blot was insufficient to allow the visual identification of distinct bands, we did note that there were differences in the apparent molecular weights of the antigens recognized by the various patients; some patients had antibodies that recognized antigens spread between the 3- and 6-kDa markers, whereas others recognized a narrow band of antigen closer to the 3-kDa marker. A subclass analysis of the IgG antibody response to the 3–6-kDa antigen indicated that most of the antibodies were of the IgG1 subclass, although IgG3 and IgG4 responses were present (Fig. 2A). In contrast, most of the antibodies to the 27-kDa antigen were of the IgG2 subclass. In addition to the IgG response, serum IgA and IgM antibodies to the 3–6-kDa antigens were also present in some patients (Fig. 2A and data not shown). Recognition of the 3–6-kDa antigen by total IgG antibodies was abolished in a concentration- and time-dependent fashion by preincubation of the antigen in sodium periodate (Fig. 2B). As expected for a protein-directed response, antibody recognition of the 17- and 27-kDa antigens was largely unaffected by periodate treatment. In contrast, pretreatment of the antigens with proteinase K did not significantly affect the recognition of the 3–6-kDa antigens but completely eliminated antibody binding to both the 17- and 27-kDa antigens (Fig. 2C). Taken together, these results suggest that the antigens in the 3–6-kDa size range are composed mostly of carbohydrate or carbohydrate-dependent epitopes.Fig. 2Characterization of the human serum antibody response to the 3–6-kDa antigens.A, strips of PVDF membrane containing Triton X-114-soluble C. parvum antigens were incubated with the diluted serum (1:100 in PBS, 0.3% Tween 20) from a single cryptosporidiosis patient. The bound IgA, IgM, IgG, and IgG subclass antibodies were detected using biotinylated class- and subclass-specific mouse anti-human monoclonal antibodies as described under "Materials and Methods." B, strips of PVDF membrane containing Triton X-114-soluble antigens were incubated at room temperature at the indicated times with the indicated concentrations of NaIO4 in 50 mm sodium acetate buffer, pH 4.5, in the dark. The strips were then washed twice with buffer and incubated for 1 h at room temperature with 1% glycine in PBS. The strips were then incubated overnight at 4 °C with a human patient serum (diluted 1:100 in PBS, 0.3% Tween 20) and developed for bound IgG antibodies as described previously. C, a strip of PVDF membrane containing Triton X-114-soluble antigens was incubated overnight at 37 °C in PBS containing 100 μg/ml proteinase K. The strip was then washed twice for 1 h with PBS, 0.3% Tween 20 containing 1 mm phenylmethylsulfonyl fluoride, incubated overnight with diluted patient serum as in B, and developed for bound IgG antibodies as described previously.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Identification of the 3–6-kDa C. parvum Antigens as GIPLs— A selected ion GC-MS technique that was previously used for the detection of GPI-anchored proteins (36Priest J.W. Xie L.-T. Arrowood M.J. Lammie P.J. Mol. Biochem. Parasitol. 2001; 113: 117-126Crossref PubMed Scopus (33) Google Scholar) was used to demonstrate that the 3–6-kDa antigen region of blotted Triton X-114 extract contained myo-inositol. Analysis of a blot of Triton X-114-extracted antigens between 18 and 3 kDa demonstrated the presence of two distinct peaks of myo-inositol, one corresponding to the GPI-anchored 17-kDa antigen (apparent molecular mass of 12–14 kDa) and another corresponding to the newly identified 3–6-kDa antigen (Table I).Table IDetection of trimethylsilyl-derivatized inositols from acid-hydrolyzed PVDF membranes using gas chromatography and selected ion mass spectrometryRegionaRegion 1 was cut from the vicinity of the 18-kDa marker. Region 2 was cut from the stained 17-kDa antigen protein band near the 14-kDa marker. Region 3 was cut immediately below the 17-kDa antigen protein band. 3-6-kDa antigen was cut from region 4 between the 3- and 6-kDa markersSample identificationmyo-InositolbThe integrated intensities of the characteristic fragment ions at m/z of 307 and 321 (for D6-labeled internal standard) and at m/z of 305 and 318 (for the H6 species) were used to calculate the relative amounts of myo-inositol in each membrane sample (37)pmol1Above 17-kDa antigen7217-kDa antigen443Below 17-kDa antigen843-6-kDa antigen90a Region 1 was cut from the vicinity of the 18-kDa marker. Region 2 was cut from the stained 17-kDa antigen protein band near the 14-kDa marker. Region 3 was cut immediately below the 17-kDa antigen protein b
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