Identification and Characterization of a Major Surface Antigen of Giardia Lamblia DAVID A

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INFECTION AND IMMUNITY, Nov. 1984, P. 377-383 Vol. 46, No. 2 0019-9567/84/110377-07$02.00/0 Copyright X 1984, American Society for Microbiology

Identification and Characterization of a Major Surface Antigen of Giardia lamblia DAVID A. EINFELDt* AND HENRY H. STIBBS Department of Pathobiology, University of Washington, Seattle, Washington 98195

Received 5 March 1984/Accepted 20 July 1984

The surface antigens of Giardia lamblia trophozoites were characterized by crossed immunoelectrophoresis, radioiodination, and immunoprecipitation. Crossed immunoelectrophoretic analysis of trophozoites with hyperimmune rabbit anti-trophozoite antiserum revealed a prominent precipitin peak that disappeared upon adsorption of the antiserum with live or formaldehyde-fixed trophozoites. This peak was intensely labeled when the antigen was derived from surface-radioiodinated trophozoites. An antiserum monospecific for the antigen contained in this precipitin peak was prepared. The precipitin peak was shown to contain an antigen with an apparent molecular weight of 82,000 by Western blotting. The antiserum also detected this 82,000-molecular- weight antigen on nitrocellulose blots of trophozoites analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On radioiodination of live trophozoites, an iodinated molecule of 82,000 apparent molecular weight was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was immunoprecipitated by the monospecific antiserum. Preliminary characterization of this antigen with the monospecific antiserum in crossed immunoelectrophoresis revealed that the surface antigen is hydrophobic and thus may be anchored in the plasma membrane, and that it is heat sensitive, but only partially sensitive to pronase or periodate. This antigen was shared by the four G. lamblia strains examined.

Giardia lamblia is the most frequently identified intestinal characterize cell surface antigens of cultured G. lamblia parasite in stool specimens submitted to U.S. public health trophozoites and to determine whether differences exist in laboratories (7). This parasite has been implicated in a the surface antigens of four different strains. number of epidemics of gastrointestinal illness (9), and its endemicity in the U.S. is well recognized, with prevalence in MATERIALS AND METHODS adults estimated at 4 to 7% (17). Higher prevalence rates Parasite cultures. Four strains of G. lamblia were cultured have been reported in children (49) and in homosexual males at 37°C in filter-sterilized TYI-S-33 medium (10; no bile (22). Giardiasis is highly variable in its presentation, ranging added) supplemented with 10% calf serum. Biosate (BBL from asymptomatic infection to acute disease with symp- Microbiology Systems, Cockeysville, Md.) was routinely toms including diarrhea, abdominal cramps, malabsorption, used in place of Trypticase (BBL) and yeast extract. Dia- and flatulence (40). The disease may also be chronic with mond TPS-1 vitamin solution (North American Biologicals, recurrent symptoms. Inc., Miami, Fla.) was used without additional vitamins, and The development of immunity to G. lamblia is suggested penicillin and streptomycin were each added at 50 ,ug or U by the self-limiting nature of acute infections and by the per ml. The designations and geographic origins of the appearance of G. lamblia-specific immunoglobulin G (IgG) strains are as follows: P-1, Oregon (29); WB, Afghanistan in the serum (35, 41). Acquired immunity to Giardia muris (43); LT, Puerto Rico (42); and RS, Ecuador (42). The P-1 has been shown to occur in some strains of mice (36). An strain was obtained from Govinda Visvesvara, Centers for understanding of the antigenic structure of G. lamblia would Disease Control, Atlanta, Ga., and the WB strain was from facilitate analysis of the host immune mechanisms responsi- Louis S. Diamond, National Institute of Allergy and Infec- ble for controlling the infection and might reveal antigenic tious Diseases, Bethesda, Md. Murray Wittner, Department differences among strains of G. lamblia. At this time little is of Pathology, Albert Einstein College of Medicine, provided known about the antigenic structure of G. lamblia. Smith et the RS and LT strains. An isolate of Trichomonas vaginalis al. (42) used crossed immunoelectrophoresis, sodium dode- (provided by Roberta Connelly and Bruce Torian of this cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), department) was cultured in the same medium under the and enzyme-linked immunoassay in comparing the antigens same conditions. The cultures were routinely tested for of four G. lamblia strains. Some minor differences were bacterial contamination by culturing in antibiotic-free medi- found in the crossed immunoelectrophoretic profiles of these um under aerobic conditions as well as the microaerophilic isolates, but the antigens were not characterized further. conditions of routine culture. Parasite cultures were also More recently, Nash et al. (31) reported the existence of cultured for mycoplasmata (25) and found to be negative. trophozoite surface components that were readily iodinated Trophozoites were cloned by limiting dilution in 96-well on live cells and then were gradually released into the microtiter plates that had been cut in half to allow incubation medium over a 24-h period. The surface antigens were in anaerobic jars (Gas-Pak, BBL). The cloning medium polydisperse in their molecular weights in SDS-PAGE, rang- consisted of TYI-S-33 plus 0.2% agarose (Seakem ME; FMC ing from 94,000 to 225,000. Corp., Rockland, Maine). After 10 days the medium from The purpose of the present study was to identify and wells containing a colony radiating from a single focus was transferred to sterile 1-dram (ca. 3.7-ml) vials, which were * Corresponding author. then filled with medium. When these cultures became con- t Present address: Genetic Systems Corp., Seattle, WA 98121. fluent they were transferred to 16- by 125-mm tubes. 377 378 EINFELD AND STIBBS INFECT. IMMUN.

Trophozoites were harvested in the late log phase (48 to 72 adsorbed versus unadsorbed antisera. Polyspecific and h). Before harvesting, trophozoites that had settled to the monospecific antisera were adsorbed by incubation with live bottom of the culture tubes were aspirated with a Pasteur or formaldehyde-fixed cells (19). Washed trophozoites, pre- pipette to reduce the number of unattached cells in the pared as described above, were used immediately for live harvested population. These cells did not appear qualitative- cell adsorptions or were fixed overnight at 4°C in glucose- ly different from the attached cells, but attached cells have saline buffer containing 0.5% paraformaldehyde. Pellets of been reported to be more viable in terms of colony-forming live or fixed cells with a volume of approximately 0.1 ml efficiency (14). The culture tubes were chilled on ice for 10 were suspended in 400 ,ul of antiserum and incubated on ice min and then repeatedly inverted (10 to 15 times) to dislodge for 45 min. Agglutination of cells occurred with both poly- the trophozoites. Washed trophozoites were obtained by specific and monospecific antisera, but the cellular morphol- centrifugation at 400 x g for 10 min followed by four washes ogy did not change. Cells were removed by centrifugation at in ice-cold 25 mM P04 (pH 7.2) containing 100 mM NaCl, 6 400 x g for 7 min, and the supernatant was centrifuged at mM KCl, 2 mM MgSO4, and 10 mM glucose (glucose-saline 20,000 x g for 30 min. buffer). Preparation of antigens for electrophoretic analysis. Tro- Radioiodination. For radioiodination by the lactoperoxi- phozoites, harvested as described above, were employed dase procedure, 108 washed parasites (prepared as described immediately or after storage at -70°C in the preparation of above, but substituting 10 mM phosphate-buffered saline antigens for analysis. Antigens employed in crossed or [PBS], pH 7.2, for the glucose-saline buffer) were suspended rocket immunoelectrophoresis were prepared by sonication in 2 ml of PBS containing 10 mM D-glucose in disposable 15- of trophozoites on ice in 1% Triton X-100 in distilled water ml centrifuge tubes. After the addition of 10 pg of lactoper- with eight 10-s bursts (20 KC, Biosonik III; Bronwill Scien- oxidase (E.C. 1.11.1.7; Sigma Chemical Co., St. Louis, Mo.) tific Inc., Rochester, N.Y.), followed by centrifugation at 1 pug of glucose oxidase (E.C. 1.1.3.4; Sigmna; type VII) and 20,000 x g for 30 min at 4°C. The supernatant was collected, 300 RCi of Na125I (Amersham Corp., Arlington Heights, and the protein content was estimated by a modified Lowry Ill.), the cells were incubated at room temperature or on ice assay (11). For analysis of antigens by SDS-PAGE and for 20 min, after which the tubes were filled with ice-cold immunoblotting (see below), washed trophozoites were sus- PBS. The parasites were pelleted by centrifugation at 400 x pended in 2% SDS containing 1 mM N-alpha-p-tosyl-L-lysine g for 7 min and washed four times with ice-cold glucose- chloromethylketone (Sigma) and 1 mM phenylmethylsul- saline buffer. fonyl fluoride (Sigma) and incubated at room temperature for lodination with lodogen (1,3,4,6-tetrachloro-3a,6a-diphen- 20 min with periodic vortexing. The supernatant was collect- ylglycoluril; Pierce Chemical Co., Rockford, Ill.) was per- ed after centrifugation at 1,000 x g for 10 min and quantified formed in 3-dram (ca. 11.1-ml) capped vials, precoated by by the standard Lowry et al. assay (28). Before SDS-PAGE, evaporating 600 [L of chloroform containing 20 ,ug of lodo- the antigen was diluted with SDS sample buffer. Antigens gen. Suspensions (500 RI) of 108 washed trophozoites (pre- were obtained from radioiodinated trophozoites by extrac- pared as described above with glucose-saline buffer) were tion into PBS containing 1% Triton X-100, 1 mM N-alpha-p- placed in the vials, to which 200 pLCi of Na125I was then tosyl-L-lysine chloromethylketone, and 1 mM phenylmethyl- added. After a 5-min incubation at room temperature, the sulfonyl fluoride. After a 30-min incubation on ice with suspensions were transferred to 15-ml disposable centrifuge periodic vortex mixing, the supernatant from centrifugation tubes, which were then filled with ice-cold glucose-saline at 20,000 x g for 30 min (4°C) was obtained. This superna- buffer. The trophozoites were washed as described above for tant also provided the antigen for radioimmunoprecipitation the lactoperoxidase procedure. Microscopic examination of experiments (see below). In addition, other samples of the trophozoites at the end of the labeling period revealed radioiodinated parasites were solubilized in SDS as de- that nearly all were motile and retained normal morphology, scribed for the immunoblot antigen and analyzed by SDS- and that at least 90% excluded trypan blue. Labeled tropho- PAGE. zoites were solubilized (see below) immediately or after Electrophoresis. Crossed immunoelectrophoresis was em- storage at -70°C. ployed to obtain an overall profile of the precipitable anti- Antisera. Organisms utilized as immunogens for the pro- gens of G. lamblia trophozoites. This technique was then duction of hyperimmune polyspecific antisera were obtained used in identifying surface antigens, in isolating antigens for after at least three passages in TYI-S-33 medium prepared preparation of monospecific antiserum, in identifying radio- with a dialysate of Biosate. For each liter of complete labeled surface antigens, and in physicochemically charac- medium, 30 g of Biosate was suspended in 40 to 50 ml of terizing antigens. Crossed immunoelectrophoresis (26, 46) distilled water and dialyzed against 800 ml of distilled water was performed on 43- by 43-mm glass slides covered with 3 for 48 h at 4°C. The dialysis tubing was then removed, and ml of 0.5% agarose (Seakem ME; Mr of 0.16 to 0.19) in the other medium components were added to the dialysate. Veronal buffer, pH 8.6 (ionic strength, 0.05) containing This medium contained calf serum at 10% (vol/vol). Rabbits 0.25% Triton X-100. For first-dimension electrophoresis a were immunized by a previously described procedure (24) field strength of 4 V/cm (measured on the agarose) was with washed trophozoites (prepared as described above) that applied for 70 min. Bovine serum albumin (BSA; 2 pug; had been killed by freezing at -20°C. A total of 10 mg of Sigma) was added to the antigen for use as a migration protein (estimated by the Lowry et al. assay [28]) was used standard. Before electrophoresis in the second dimension, a per rabbit. Monospecific antiserum to precipitin arcs on gel containing 10% rabbit antiserum and 0.05% rabbit anti- crossed immunoelectrophoresis plates was produced by the BSA antiserum (to detect the BSA marker) was poured procedure of Alexander and Kenny (2). Briefly, the precipi- above the 1-cm strip containing the separated antigen prepa- tin peaks in agarose were excised from 40 washed plates, ration, and then electrophoresis was performed for 15 h at 1 emulsified in Freund adjuvant, and used to immunize two V/cm. An intermediate antiserum-free gel was sometimes rabbits. Antisera were stored at -70°C. employed to allow greater migration and separation of Adsorption of antisera. Cell surface antigens were first slowly migrating antigens. Antigens will precipitate in the identified through the comparison of antigens detected by intermediate gel because of downward migration of antibod- VOL. 46, 1984 SURFACE ANTIGEN OF G. LAMBLIA 379

A B C

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.05 t , .4,.dow,

.89 FIG. 1. Effect of adsorption of antiserum on the precipitin profile of P-1. The antigen in each case consisted of 60 ,ug of a Triton X-100 sonicate of P-1. The 1.8-ml antibody bed contained 10% anti-P-1 antiserum that was untreated (A) or adsorbed with formaldehyde-fixed (B) or live (C) P-1 trophozoites. The anode was to the left and top, respectively, for the two dimensions. The prominent peak running off the top of the plate is BSA added as a migration standard. The peaks designated by their mobilities (relative to that of BSA, assigned a value of 1.00) are those most affected by adsorption of the antiserum. ies into this gel by diffusion and by the process of electroen- buffered agarose containing 10% rabbit antiserum. Precipitin doosmosis. Precipitin peaks were stained with Coomassie peaks were excised from these plates and analyzed in SDS- blue. Charge-shift electrophoresis was employed to deter- PAGE and immunoblots by the procedure of Norrild et al. mine whether antigens were amphiphilic and thus likely to be (33). Briefly, agarose pieces containing precipitates were intrinsic membrane components. The procedure outlined by dried to a thin film by laying them on chromatography paper. Alexander and Kenny (3) was followed. Briefly, 0 to 0.25% These were collected and incubated in SDS sample buffer for sodium deoxycholate was incorporated in the first-dimen- 20 h at 37°C. After removing the agarose by centrifugation, sion gel, which also contained 0.25% Triton X-100. the supernatant was run in SDS-PAGE and immunoblots. Rocket immunoelectrophoresis was performed on 1-by 3- Discontinuous SDS-PAGE was performed under reducing in. (ca. 2.5- by 7.5-cm) microscope slides coated with 2.5 ml conditions as described by O'Farrell (34). Samples were of buffered agarose (described above). A section of the electrophoresed through a 10% acrylamide gel at 40 mA. agarose was removed and replaced with an equal volume of A B

2004 A *. 92.5+ 9944 ) 694 92.5+ 53+ 674 E; 9 + -. _w 'p 44 53+ 4 3+ I&J

30+

30+ 4

1 2 1 2 FIG. 3. SDS-PAGE analysis of surface-iodinated trophozoites. (A) Autoradiograph of a protein blot from SDS-PAGE of P-1 1 2 trophozoites iodinated with lodogen. Lanes: 1, 14C-labeled standards of the indicated molecular weights (x103; top to bottom: FIG. 2. SDS-PAGE immunoblot of material precipitated by the myosin, phosphorylase B, BSA, gamma globulin heavy chain, and monospecific antiserum in rocket immunoelectrophoresis. Strips carbonic anhydrase); 2, profile of radioiodinated P-1 trophozoites were incubated with horseradish peroxidase-conjugated goat anti- solubilized in SDS sample buffer. The bold arrows on the right rabbit IgG, either alone (lanes 2) or after incubation of the antigen indicate the iodinated components. (B) Lanes: 1, SDS-PAGE gel of strip with the monospecific antiserum (lane 1). The molecular a Triton X-100 extract of Iodogen-iodinated P-1 trophozoites which weights (x 103) and positions of standards ("4C methylated; from top has been stained with Coomassie blue and dried; 2, autoradiograph to bottom: myosin, phosphorylase B, BSA, gamma globulin heavy of lane 1. The molecular weights (x 103) and the positions of chain, and carbonic anhydrase) are indicated by the numbered standards are indicated by the numbered arrows on the left. The arrows on the left. The small arrow indicates the location of the dye positions in the polyacrylamide gel of some molecules detected by front. autoradiography are indicated by double-headed arrows (1 and 2). 380 EINFELD AND STIBBS INFECT. IMMUN.

of trophozoites were subjected to various treatments before analysis in crossed immunoelectrophoresis against the *1A monospecific antiserum. Sonicates were incubated for 2, 12, and 24 h at 37°C at a concentration of 2 mg of protein per ml in 10 mM Tris-hydrochloride (pH 7.5) containing 10 mM CaC12 and 0.50 mg of pronase (Calbiochem-Behring, La a Jolla, Calif.) per ml. Other sonicates were incubated at a concentration of 1.5 mg/ml with periodate (100 mM sodium metaperiodate in 20 mM acetate buffer, pH 4.5) for 24 h at 4°C in the dark. Controls were incubated in the acetate buffer .w~ ~ ~ alone. To test heat stability, sonicates were placed in a boiling water bath for 10 min.

FIG. 4. Crossed immunoelectrophoresis of a Triton X-100 ex- RESULTS tract of lactoperoxidase-iodinated P-1 trophozoites. The polyspeci- Crossed immunoelectrophoresis and adsorption studies. fic antibody bed (1) was separated from the first-dimension gel strip (3) by an intermediate gel lacking antiserum (2). The plate was dried, The crossed immunoelectrophoretic profile of a Triton X-100 stained with Coomassie blue (A), and autoradiographed (B). The sonicate of strain P-1 trophozoites tested against anti-P-1 arrow in A indicates the stained precipitate corresponding to the antiserum showed 14 peaks ranging in mobility (migration peak detected by autoradiography. distance relative to that of bovine serum albumin, assigned a value of 1.0) from 0.05 to 1.14 (Fig. 1). No significant differences were seen among the four strains when tested in crossed immunoelectrophoresis with either anti-P-1 or anti- Molectlar weight standards were obtained from Pharmacia WB antiserum. Four clones from the P-1 strains were tested Fine Chemicals, Inc., Piscataway, N.J., and New England with polyspecific anti-P-1 antiserum in crossed immunoelec- Nuclear Corp., Boston, Mass. (methyl-'4C-methylated proteins). Immunoblotting. Immunoblotting (Western blotting) was employed to determine the molecular size of antigens after 1 2 3 4 5 6 7 8 9 separation by SDS-PAGE. Electrophoretic transfer of material from SDS-PAGE gels to nitrocellulose membranes was performed as described by Burnette (5), with the modification that the transfer buffer contained 25 mM Tris and 192 mM glycine. A constant current of 150 mA was applied for 12 to 16 h to the gel-nitrocellulose sandwich in a Bio-Rad Transblot apparatus (Bio-Rad Laboratories, Richmond, Calif.). Protein blots to be reacted with antisera were first incubated in 10 mM TES [N-tris(hydroxymethyl-2-amino- _- _~~~~~~4 ethanesulfonic acid)] buffer (pH 7.3) containing 0.15 M NaCl, 5 mM EDTA, 0.25% (wt/vol) gelatin (Sigma; type 1), and 0.05% (vol/vol) Tween 20 for 3 h at 37°C. A dilution of antiserum in this same buffer was incubated with the nitrocellulose strip for 45 min at room temperature. After six washes over a period of 45 min in TES-saline (5 mM TES [pH 7.3], 0.15 M NaCl) containing 0.05% Tween 20, the strip was incubated for 45 min with horseradish peroxidase- conjugated goat anti-rabbit IgG (Antibodies Incorporated, Davis, Calif.). The strip was washed as before, except the final wash was in TES-saline without detergent. A 20-ml sample of a solution of TES-saline containing 16.5% metha- nol and 10 mg 4-chloro-1-naphthol was added to the strip, and bound conjugate was detected upon the addition of 40 RI of H202. FIG. 5. Autoradiograph of protein blot from SDS-PAGE show- Radioimmunoprecipitation. Triton X-100 extracts of ra- ing the results of radioimmunoprecipitation. Lanes: 1 and 2, frac- dioiodinated trophozoites (40 S.l, 4 mg/ml) were reacted with tions of a Triton X-100 extract from iodinated (lodogen) LT tropho- 50 p,l of a 1:5 dilution of the monospecific antiserum or zoites bound to protein A-Sepharose by preimmune serum and the preimmune serum for 2 h on ice and precipitated with an monospecific antiserum, respectively; 3, Triton X-100 extract of LT excess of protein A-Sepharose (Pharmacia) after a 30-min trophozoites; 4, LT trophozoites solubilized in SDS sample buffer; incubation on ice. Pelleted beads were washed three times 5, 14C-methylated molecular weight standards (myosin, 200,000 with 1 ml of PBS containing 0.5% Triton X-100. [faint]; phosphorylase b, 92,500; BSAi 69,000; gamma globulin Autoradiography. Autoradiographs of protein blots, dried heavy chain, 53,000; carbonic anhydrase, 30,000 [doublet]); 6, 12511 SDS-PAGE gels, or crossed immunoelectrophoresis plates labeled (lodogen) P-1 trophozoites solubilized in SDS sample buffer; 7, Triton X-100 extract of P-1; 8 and 9, fractions of the Triton were exposure to AR at P-1 obtained by Kodak X-Omat film extract bound to protein A-Sepharose in the presence of monospeci- -70°C. Cronex Lightning Plus screens (E. I. du Pont de fic antiserum and preimmune serum, respectively. The arrows in Nemours & Co., Wilmington, Del.) were used with iodinated lane 5 indicate the top of the separating gel and the dye front. The materials. arrowheads in lanes 2 and 8 indicate labeled material in addition to Physicochemical characterization. Triton X-100 sonicates the major band at 82,000 Mr. VOL. 46, 1984 SURFACE ANTIGEN OF G. LAMBLIA 381 trophoresis. No antigenic differences were detected among with monospecific antiserum, antigens migrating at M, the clones. Since both the immunogen and the antigen were 145,000 and 82,000 were detected (data not shown). The grown in medium supplemented with calf serum, this repre- 145,000-Mr was not always detected. The monospecific sented a potential source of contaminating antigens. No antiserum was negative in immunoblots when tested against peaks, however, were detected when G. lamblia antigen was a bovine lipoprotein preparation (supernatant from auto- tested against rabbit anti-bovine serum and rabbit anti-fetal claved bovine serum) and bovine IgG (Sigma). The quantity calf serum. of each of these antigens was in 10-fold excess of that which To determine whether antigens detected in crossed immu- reacted strongly in immunoblots with the homologous anti- noelectrophoresis were exposed on the surface of the tro- serum prepared against these antigens in rabbits. In addition, phozoites, polyspecific anti-P-1 antiserum was adsorbed the 145,000- and 82,000-Me antigens were not detected by with live or formaldehyde-fixed P-1 trophozoites. That the anti-bovine serum, anti-bovine lipoprotein, or antibovine polyspecific antiserum contained antibodies to parasite sur- IgG antisera when these were reacted with G. lamblia face antigens was indicated by its specific ability to aggluti- antigen in immunoblots. The monospecific antiserum was nate live and fixed trophozoites when compared with preim- also negative in immunoblots against T. vaginalis grown in mune serum. Antigens that adsorb antibodies can be the TYI-S33 medium. identified in crossed immunoelectrophoresis since the area Radioiodination. SDS-PAGE analysis of Iodogen-iodinat- of these antigen peaks will be increased or the peaks will ed P-1 trophozoites revealed a prominently radiolabeled disappear completely (19). The most striking effect of ad- component having an Mr of 82,000 (Fig. 3A). Other less sorption was on peak 0.05 (Fig. 1). Antiserum adsorbed with heavily labeled components (arrows) were detected at live cells did not precipitate this antigen, and only a faint, 180,000, 105,000, 63,000, 55,000, 37,000, 30,000, and 24,000 greatly enlarged peak occurred when the antiserum had been Mr. Radioiodination with lactoperoxidase, both on ice and at adsorbed with fixed cells. Other peaks that disappeared room temperature, gave results very similar to those ob- upon adsorption of the antiserum were 0.89 (very faint), tained with Iodogen. Comparison of the SDS-PAGE profile 0.19, a, and b. of a Triton X-100 extract of iodinated P-1 trophozoites with Monospecific antiserum. Monospecific antiserum was em- the corresponding autoradiograph revealed no prominent ployed as a selective probe for identifying antigen peak Coomassie blue-stained band in the region of the major 0.05 in immunoblots of trophozoite antigen and for iodinated band (Fig. 3B, double arrow 1). In contrast, the immunoprecipitating this antigen from extracts of radioio- label at approximately 180,000 Mr correlated with a Coomas- dinated trophozoites. An antiserum monospecific for antigen sie blue-stained doublet in the gel (double arrow 2). peak 0.05 was obtained by immunizing rabbits with peak A i% Triton X-100 extract of lactoperoxidase-iodinated P- 0.05 material excised from plates containing intermediate 1 trophozoites was analyzed in crossed immunoelectropho- gels similar to that in Fig. 4A. The term monospecific used in resis to determine whether antigens precipitated by the describing this antiserum may not be completely accurate, polyspecific antiserum were labeled (Fig. 4). In initial experi- since in crossed immunoelectrophoresis two precipitin arcs, ments a large quantity of the label was found to precipitate located very close together, are produced with trophozoite around the application well. Subsequently, a blank interme- antigen, in a manner similar to that seen in Fig. 4A. diate gel was incorporated to permit migration of this materi- Nevertheless, the results with immunoblotting and radioim- al away from the origin. (Precipitation still occurs under munoprecipitation (see below) revealed that this antiserum is these conditions for reasons discussed above.) Under these directed primarily against a single component. When the conditions it was possible to correlate this labeled material monospecific antiserum was adsorbed with formaldehyde- seen by autoradiography (Fig. 4B) with a double-banded fixed cells, with the same quantities of cells and antiserum as precipitate (peak 0.05) that stained with Coomassie blue in the polyspecific adsorptions, and then employed in (Fig. 4A, arrow). Precipitates a and b (Fig. 1A) also gave a crossed immunoelectrophoresis, the complex precipitin faint image in some autoradiographs of crossed immunoelec- peak 0.05 was not detected. The specificity of the monospe- trophoresis plates containing iodinated antigen, suggesting cific antiserum for antigen 0.05 was confirmed by using the that they, too, represent cell surface components. technique of peak suppression (47): the addition of the Radioimmunoprecipitation. Extraction of radioiodinated monospecific antiserum to the polyspecific antiserum in the trophozoites with Triton X-100 effectively solubilized the antibody bed resulted in the marked reduction in the height labeled component of 82,000 M, as well as those of 105,000, of peak 0.05 relative to its appearance when polyspecific 30,000, and 24,000 Mr (Fig. 5, lanes 3, 4, 6, and 7). Triton X- antiserum alone was used, whereas no other precipitates 100 extracts of labeled strain P-1 and LT trophozoites were were affected (data not shown). All four strains of G. lamblia also utilized as antigens in radioimmunoprecipitations. The reacted similarly with the monospecific antiserum. No pre- 82,000-Mr component was bound to protein A-Sepharose cipitates were detected when the monospecific antiserum beads by the monospecific antiserum, as were components was employed in rocket immunoelectrophoresis with yeast of 105,000, 30,000, and 24,000 Mr (Fig. 5, lanes, 2 and 8). extract (0.02 to 10.0 mg), Trypticase (0.04 to 12.5 mg, BBL), None of these components was precipitated by preimmune or calf serum (20 to 100 ,ul) as the antigen. serum (Fig. 5, lanes 1 and 9). The SDS-solubilized prepara- Immunoblotting. The molecular specificity of the mono- tion, the Triton X-100 extract, and the radioimmunoprecipi- specific antiserum was analyzed by immunoblotting (Fig. 2). tate of the LT strain appeared similar to those of the P-1 Precipitates from rocket immunoelectrophoresis of tropho- strain, except for a difference throughout in the relative zoite antigen into monospecific antiserum were excised and intensities of the 30,000- and 24,000-Mr components. The run in SDS-PAGE. Immunoblots of these preparations ex- WB and RS strains gave results virtually identical to those of hibited a single antigenic band at 82,000 Mr (lane 1) in LT, with more iodine incorporated at 24,000 Mr than at addition to a second band at 53,000 Mr (lanes 1 and 2) 30,000 Mr (data not shown). produced by the heavy chain of the rabbit IgG present in the Antigen characterization. Experiments aimed at prelimi- precipitate. nary characterization of the 82,000-Mr surface antigen were When immunoblots of trophozoite antigen were treated undertaken with the monospecific antiserum in crossed 382 EINFELD AND STIBBS INFECT. IMMUN. immunoelectrophoresis. Trophozoite antigen preparations have detected a greater number of antigens by this tech- (P-1 strain) were subjected to charge-shift immunoelectro- nique. It would appear that our peak 0.05 corresponds to phoresis with the anionic detergent deoxycholate. The pre- either peak 33 or 37 of Smith et al. In their report, however, cipitin peak 0.05 shifted anodically in the presence of deoxy- the P-1 strain was found to lack peak 33, which was present cholate, the mobility going from 0.10 in the presence of in the RS, LT, and WB strains. As shown above, all four of Triton X-100 alone to 0.15 in Triton X-100 plus 0.025% these strains of G. lamblia possess antigen peak 0.05. deoxycholate and to 0.25 in Triton plus 0.05% deoxycholate. Experiments aimed at characterization of the 82,000-Mr This result indicates the presence of a hydrophobic compo- antigen revealed that it is pronase and periodate modifiable nent of the antigen. After periodate oxidation of Triton X- and heat labile. The fact that pronase treatment altered the 100 sonicates of the P-1 strain, the electrophoretic mobility electrophoretic mobility of the precipitated antigen indicates of the antigen in crossed immunoelectrophoresis was in- the presence of peptide sequences. The electrophoretic creased from 0.05 to 0.50 (data not shown). This altered mobility of the antigen was also modified by periodate mobility indicates a modification of the antigen's structure oxidation, pointing to the presence of carbohydrate moi- by periodate oxidation. Controls incubated in acetate buffer eties. The heat lability of the antigen was shown by the alone showed no change in the mobility of this antigen. disappearance (or in some cases the greatly altered appear- Sonicates of trophozoites subjected to pronase digestion for ance) of the precipitate after heating. The results of these 24 h exhibited precipitates with electrophoretic mobilities of characterization experiments strongly suggest that the anti- 0.69, 0.61, and 0.34 (data not shown). These probably gen is a glycoprotein. Also, the results of the charge-shift represent fragments of the molecule precipitated at 0.05 in immunoelectrophoresis experiments indicate that the anti- the untreated sonicates. Preparations analyzed after 2 and 12 gen is hydrophobic and thus is likely to be membrane bound. h of digestion indicated that the change in mobility seen at 24 lodinatable surface proteins or glycoproteins have been h was complete by 12 h. The antigen was susceptible to reported from a variety of other parasitic protozoa, including heating at 100°C for 10 min; the precipitate either disap- the African trypanosomes (6, 48), Trypanosoma cruzi (32, peared completely or appeared as a streak extending from 44, 45), Leishmania tropica (13, 15), Entamoeba histolytica the first-dimension gel toward the top of the plate. (4), T. vaginalis (1), Toxoplasma gondii (16, 20), and Plas- modium species (8, 12, 21). More unusual antigens have also been identified on some of these parasites, such as the DISCUSSION glycopeptidophosphosphingolipid of Leishmania donovani The existence of a major antigen of 82,000 Mr on the (38), a lipopeptidophosphoglycan of T. cruzi (27), and a surface of G. lamblia trophozoites has been demonstrated. polysaccharide of T. gondii (30). Glycoproteins identified in The cell surface location of this antigen has been shown by parasitic protozoa have in some cases been shown to func- two independent approaches. First, the antigen was the tion as receptors for interactions with host cells (12) and as major component radioiodinated on live cells and was im- antigens that can elicit a protective immune response (8, 16, munoprecipitable from Triton X-100 extracts of radioiodinat- 18, 37). These components have also been implicated in the ed trophozoites with a monospecific antiserum against the control of the parasite cell cycle (39). In the case of E. antigen. Second, live or fixed parasites were shown to histolytica, a membrane protein appears to be involved in adsorb antibodies specific for this antigen from antisera. the cytotoxicity of the parasite (50). In light of these reports, Adsorption of antibodies did not seem to require secretion of it will be of interest to determine what role the major surface the antigen, since fixed cells also adsorbed antibodies to the antigen of G. lamblia reported here might play in pathogene- antigen. sis, adhesion, and host immunity. A polydisperse pattern of radioiodinated surface components as reported by Nash et al. (31) was not seen. These ACKNOWLEDGMENTS authors reported radioiodinatable surface antigens on both We thank George E. Kenny for technical advice and for critical the WB and P-1 strains which exhibited a heterogeneous comments concerning this manuscript. distribution on SDS-PAGE ranging from 94,000 to 225,000 in This work was supported by research grants to H.H.S. from the World Health Organization (C6/181/1) and from the Graduate School Mr. These components reacted with homologous, polyspeci- Research Fund, University of fic antiserum, but not with antiserum to the heterologous Washington. strain. By contrast, we detected a discrete component of 82,000 M-, shared by all four strains. The reason for this LITERATURE CITED difference in our results is not clear. A possible explanation 1. Alderete, J. F. 1983. Identification of immunogenic and anti- is that Nash et al. supplemented the TYI-S33 medium with body-binding membrane proteins of pathogenic Trichomonas bile (23, 31). Components of the bile, such as bile salts, might vaginalis. Infect. Immun. 40:284-291. influence the molecular 2. Alexander, A. G., and G. E. Kenny. 1977. Characterization of architecture of the parasite's sur- membrane and cytoplasmic antigens of Mycoplasma ar,ginini by face; bile salts have been reported to stimulate the uptake of two-dimensional (crossed) immunoelectrophoresis. Infect. Im- phospholipid from culture medium by Giardia sp. (M. J. G. mun. 15:313-321. Farthing S. Varon, G. T. Keusch, and M. C. Carey, Gastro- 3. Alexander, A. G., and G. E. Kenny. 1978. Application of charge- enterology 84:1148, 1983). There are, however, some simi- shift electrophoresis to antigenic analysis of mycoplasmic mem- larities between our results and those of Nash et al. An branes by two-dimensional (crossed) immunoelectrophoresis. iodinated component of approximately 82,000 Mr appeared Infect. Immun. 20:861-863. in some of their autoradiographs. Furthermore, the antigens 4. Aley, S. B., W. A. Scott, and Z. A. Cohn. 1980. Plasma that we detected at 30,000 and 24,000 M, appear analogous membrane of Entamoeba histolytica. J. Exp. Med. 152:391-404. to antigens detected by those authors at 30,000 and 5. Burnette, W. N. 1981. Western blotting: electrophoretic transfer 21,000 of proteins from sodiom dodecyl sulfate-polyacrylamide gels to Mr. Finally, our 180,000- and 105,000-M,. iodinated mole- unmodified nitrocellulose and radiographic detection with anti- cules fall within the range of their polydisperse antigen. Our body and radioiodinated protein A. Anal. Biochem. 112:195- crossed immunoelectrophoretic patterns are similar to those 203. reported by Smith et al. (42). Those investigators seem to 6. Cardoso de Almeida, M. L., and M. J. Turner. 1983. The VOL. 46, 1984 SURFACE ANTIGEN OF G. LAMBLIA 383

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