Characterization of a high molecular weight antigen of parvum micronemes possessing epitopes that are cross-reactive with all parasitic life cycle stages B Robert, H Antoine, F Dreze, P Coppe, A Collard

To cite this version:

B Robert, H Antoine, F Dreze, P Coppe, A Collard. Characterization of a high molecular weight antigen of Cryptosporidium parvum micronemes possessing epitopes that are cross-reactive with all parasitic life cycle stages. Veterinary Research, BioMed Central, 1994, 25 (4), pp.384-398. ￿hal- 00902231￿

HAL Id: hal-00902231 https://hal.archives-ouvertes.fr/hal-00902231 Submitted on 1 Jan 1994

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Original article

Characterization of a high molecular weight antigen of Cryptosporidium parvum micronemes possessing epitopes that are cross-reactive with all parasitic life cycle stages

B Robert1 H Antoine F Dreze P Coppe A Collard2

1 Département de Virologie; 2 Département d’immunologie, Centre d’Économie Rurale, 1, rue du Carmel, B-6900 Marloie, Belgium

(Received 18 November 1993; accepted 25 February 1994)

Summary ― Crossreacting antigens between life cycle stages of Cryptosporidium parvum (Proto- zoa, ) were detected using monoclonal antibodies (mAbs). Shared epitopes were demon- strated by immunoelectron microscopy, at the level of micronemes of the sporozoite and merozoite stages; some dense granules were also labelled but not so intensively. The parasitophorous vacuole membranes of all intracellular stages, the wall-forming bodies of macrogametes and the outer oocyst walls all shared these epitopes. The antigens that bear these epitopes were characterized using the whole oocyst and sporozoite stages as sources of antigenic material. Complex labelling patterns were observed on Western blots. However, all the mAbs used in this study recognized an antigen of more than 500 kDa. The glycoproteinic nature of this antigen was demonstrated by its sensitivity to pronase and periodate treatments. The expression of this high molecular weight immunoreactive antigen in the intracellular stages of C parvum was not investigated and remains to be found.

Cryptosporidium parvum / microneme / parasitophorous vacuole / wall-forming bodies / oocyst wall

Résumé ― Caractérisation d’un antigène de haut poids moléculaire localisé au niveau des micronèmes des zoites et possédant des épitopes communs avec tous les autres stades de Cryptosporidium parvum. Des antigènes communs à tous les stades du cycle parasitaire de Cryptosporidium parvum (Protozoa, Apicomplexa) ont été mis en évidence à l’aide d’anticorps monoclonaux (mAbs). Par immunoélectromiscroscopie, des épitopes communs ont été démontrés au niveau des micronèmes des stades sporozoites et mérozoites ; le marquage des granules

* Correspondence and reprints denses de ces stades a également pu être observé mais d’une manière sporadique. Ces mêmes épitopes ont également été démontrés au niveau de la membrane de la vacuole parasitophore, de tous les stades, des «wall-forming bodies» des macrogamètes et de la paroi externe de l’oocyste. Les antigènes portant ces épitopes ont été caractérisés en utilisant les stades oocystes et sporozoites comme source d’antigène. Sur western blots, des profils anügéniques complexes ont été observés ; cependant, tous les anticorps monoclonaux décrits dans cet article ont en commun la reconnaissance d’un antigène de plus de 500 kDa. Il s’agit d’une glycoprotéine comme en témoigne sa sensibilité à la pronase et au traitement par le périodate. La démonstration de la présence de cette glycoprotéine, au niveau des stades intracellulaires de Cryptosporidium parvum mériterait d’être entreprise lors d’une étude ultérieure. cryptosporidium parvum / micronèmes / vacuole parasitophore / «wall-forming bodies» / paroi de l’oocyste

INTRODUCTION the parasitophorous vacuole by invading zoites. Cross-reactive epitopes shared by micronemes and macrogametes were also Invasive stages of Apicomplexan parasites described by Bonnin et al (1993). possess characteristic organelles which are thought to be associated with the penetra- Using monoclonal antibodies, immuno- tion / invasion of the host cell (Scholtyseck electron microscopy and Western blotting, and Mehihorn, 1970). Three types of mem- we have investigated the antigenic struc- brane-bound organelles have been ture of micronemes of zoites, and we have described: (i) rhoptries, which are amphora- tried to show that epitopes associated with shaped, secretory granules (Perkins, 1992), these membrane-bound organelle were also connected by ductules to the tip of the api- expressed at all other life cycle stages of cal complex where secreted substances Cryptosporidium parvum. can be discharged (Sinden, 1985); (ii) micronemes, which are smaller, more numerous and may occupy much of the MATERIALS AND METHODS anterior third of the parasite; (iii) dense gran- ules, which are round, densely staining bod- ies (Bannister et al, 1975). Entzeroth et al Parasite and antigen purificafion (1986) have shown that dense granule con- tent was released the into through pellicle Parasites were isolated from faecal samples of the secondary parasitophorous vacuole of naturally infected calves. Oocysts were purified muris. Dense granules have by ether and sucrose treatments as previously been described in only few species, such described (Robert etal, 1990). The preparation as knowlesi, Sarcocystis muris was sterilized by treatment with commercial and (Torii et al, 1989b). bleach (1/10 in PBS) for 10 min at 4°C, followed by 2 washes in sterile PBS (NaCl 8 g/l, KCI 0.2 Rhoptries, numerous micronemes and g/l, Na2HP04·2H0 1.15 g/I, KH2pO4 0.2 g/I, pH dense granules have been described in the 7.2). Only very clean preparations (no contami- zoites of the Cryptosporididae family (Vet- nation as seen by phase-contrast microscopy terling etal, 1971; Current and Reese, 1986; examination) were used. Sporozoites were Uni et al, 1987; Ostrovska and obtained by incubating purified oocysts in sterile Paperna, medium 0.5% sodium Bonnin have character- excystation (0.1 % trypsin, 1990). et al (1991 ) taurocholate in PBS, pH 7.6) at 37°C for 30 min ized an antigen family located in the (Wisher and Rose, 1987), and then washed by 2 micronemes. They suggest that the con- centrifugations (4°C; 10 min; 1 500 g) in sterile tents of micronemes might be released in PBS. Immunization of mice, production Labelling after embedding of monoclonal antibodies Ileum segments of OF1 mice experimentally infected in their first week of age, were taken 7 d Monoclonal antibodies were obtained (mAbs) by after oral inoculation of 10 000 oocysts. In the fusion of myeloma cells with Sp2-0-Agl4 first experiment, small ileum segments were fixed of Balb/c mice immunized with C splenocytes in 4% in 0.1 M phosphate We 3 different cellular fusions paraformaldehyde parvum. performed buffer for 4 h at 4°C, and then embedded. Osmi- (Kohler and Milstein, 1975). The first immuniza- cated tissues were prepared for a second labelling tion was realized by intraperitoneal injections of experiment: ileum pieces were fixed in 2.5% glu- at least 10! sonicated et at, oocysts (Ungar taraldehyde in 0.1 M phosphate buffer (pH 7.2) for Immunizations were on 0 1986). performed day 2 h 30 min at 4°C, then washed 3 times with phos- and on and 156. The first was days 14, 31, 149, buffer and with with Freund the phate post-fixed phosphate- given complete adjuvant (CFA), buffered 1 % osmium tetroxide for 1 h at 4°C. After others with incomplete Freund adjuvant (IFA). several rinses in buffer, the tissues were dehy- The fusion was carried out 4 d after the last drated through a grade series of ethanol to 100% booster, and mAb S1 G12 was obtained. For the and then embedded in LR-White resin (London second fusion, called Y (production of Y3A5 and Resin Co) (Bendayan et al, 1987). Thin sections Y14F3 mAbs), the mouse received 5 intraperi- were cut with a diamond knife and collected on toneal immunizations. Intact oocysts (20 x 106) nickel grids (coated, in some cases, with form- were infected, in CFA, 230 d before the fusion; 2 var) and floated for 10 min in PBS/2% BSA, then booster doses, in IFA, were 13 and 29 d given transferred onto culture later. The final boosters consisted of viable hybridomas medium, diluted (v/v) with PBS/BSA for 1 h. After wash- sporozoites (> 4 x 106) and oocysts (12 x 106) in PBS 0.01 % Tween the were suc- injected intraperitoneally, in PBS, 6 and 2 d ing 20, grids floated on rabbit anti-mouse before the fusion. For the third fusion, called AT cessively diluted to 10 in PBS/BSA (production of AT7G10, AT7F7 and AT9H6 immunoglobulin pg/ml and 15 nm gold-protein A conjugate (Amersham) mAbs), 8 x 106 intact oocysts were injected for 30 min and 1 h, The intraperitoneally in CFA 105 d prior to the fusion; respectively. gold conju- gate was diluted 1/50 in PBS/BSA with 1/20 followed 10 d later by an intraperitoneal injec- After 2 washes in PBS-0.01 % Tween 20, tion of 15 x 106 viable purified sporozoites in gelatin. were rinsed with water and stained for 20 PBS. An intrasplenic immunization (Spitz et at, grids min in 4% acetate in water. Control sec- 1984) was performed, 4 d before the fusion, using uranyl tions were in the same manner 11 x 106 sporozoites and oocysts fixed in 4% processed except that mAbs were omitted. Sections were exam- paraformaldehyde at 4°C for 1 h and washed ined with a electron twice with PBS. Screening of hybridoma super- Philips microscope. natant was performed by an indirect immuno- fluorescent assay on air-dried or acetone-fixed sporozoites and oocysts. Hybridoma specific to SDS-polyacrylamide gel electrophoresis C parvum were cloned by limiting dilution. The isotype of secreted immunoglobulines was deter- were sodium sul- mined an Antigens analyzed by dodecyl by immunoenzymatic assay (Amer- fate sham Life Science, UK). polyacrylamide gel electrophoresis (SDS- PAGE) (Laemmli, 1970). Three different antigen preparations were used for these analyses: boiled oocysts (BO Ag), sporozoites (SP Ag), and a Immunoelectron microscopy parasite extract (PE Ag) obtained from ultrason- icated oocysts. BO Ag was obtained from oocysts (164 x 10 oocysts/lul) diluted (v/v) in sample buffer Labelling before embedding (20 mM Tris, pH 7.4, 2 mM EDTA, 5% SDS, and either 5% mercapto-2-ethanol or no mercap- A mixture of sporozoites and oocysts was fixed toethanol), boiled for 15 min, centrifuged at with 0.5% glutaraldehyde in PBS for 30 min at 8 000 g for 5 min to eliminate insoluble material. 4°C and labelled according to Sibley and Sharma It was analyzed on Phastgel homogeneous 7.5% (1987). or 4-15% acrylamide gradient with a Phastsys- tem aparatus (Pharmacia Fine Chemicals, Upp- Determination of proteinic sala, Sweden). The running conditions were 250 and carbohydrate epitopes V; 10 mA; 3 W; 63, 90, or 200 Vh. SP Ag was obtained by excystation. After 2 washes with PBS, excysted sporozoites were lysed by NET PE Ag or affinity-purified antigens were exposed buffer (150 mM NaCI, 5 mM EDTA, 50 mM Tris, to periodate or to pronase (Beeley, 1985) and 0.02% sodium azide, 0.5% Nonidet P-40, 1 mM then analyzed by SDS-PAGE and Western blot- PMSF, 5 mM iodoacetamide, pH 7.4) for 30 min ting. For periodate oxidation, the preparation was at room temperature. Intact oocysts and oocyst incubated with 33 mM Na104 in 20 mM sodium walls were removed by centrifugation at 1 500 acetate buffer (pH 4.5) for 24 h in the dark. Addi- g for 15 min. The supernatant, which contained tion of ethylene glycol was followed by treatment sporozoite antigen, was analyzed by elec- with 60 mM sodium borohydride in 60 mM sodium trophoresis. PE Ag was obtained according to borate buffer, pH 8.0, in the dark for 4 h. Con- Achbarou et al (1991) with some modifications. trols were incubated in the same way except that Briefly, 2 x 108 purified oocysts were sonicated in Na’04 was omitted from the buffer. For pronase 800 pl of NET buffer without NP-40. Sonicated treatment, predigested pronase (60°C, 30 min) parasites were centrifuged at 1 500 g for 10 min; was used at the working concentration of 1 mg/ml the supernatant was mixed with 200 pl of 5% in 100 mM Tris-HCI, 1 mM CaCl2, pH 8.0. Diges- SDS in NET buffer and left 30 min under agitation tion was performed over 30 h at 37°C; the same at room temperature; after ultrasonication for 1 incubation procedure, but without pronase, was min, 1 ml of NET containing 2% Triton X-100 applied to the control antigen. A third type of con- and 2% sodium deoxycholate was added. After trol was the same antigen preparation at the cor- agitation for 30 min at room temperature, the responding dilution stored at -20°C without any preparation was ultrasonicated for 1 min and incubation. All samples were analyzed by SDS- centrifuged at 10 000 g for 15 min at 4°C. The PAGE and Western blotting. supernatant was collected and analyzed by SDS- PAGE or used for purification by chromatogra- phy. Purification of mAbs from ascitic fluids

A amount of mAbs of the G were Western large Ig isotypes blotting purified from ascitic fluids on a protein G- Sepharose 4B column (Pharmacia, Uppsala, In the case of M the ascitic Antigens were analyzed by SDS-PAGE and elec- Sweden). Ig isotype, trophoretically transferred to nitrocellulose (Tow- fluid was applied on a protein G column, and the which contained M and A iso- bin et al, 1979), on a PhastTransfert apparatus flow-through, Ig Ig (Pharmacia, Uppsala, Sweden). Low and high types, was applied on a Sepharose 4B column molecular weight markers (Pharmacia), and phos- (Pharmacia) where rat mAbs (Ref ATCC HB58) phorylase b (Sigma Chemical Co, St Louis, MO) that were specific to mouse kappa light chain For were used. The nitrocellulose sheet was satu- were covalently linked. all these chromato- buffer was 20 mM rated with a casein solution (Pearce-Pratt and graphic steps, equilibration and elution buffer Roser, 1989) for 30 min at room temperature, NaH2pO4/NaHP0 pH 7.2; then incubated in hybridoma culture medium was 100 mM glycine hydrochloride, pH 2.7. diluted (v/v) in casein for 40 min at room tem- perature. After washing in PBS-1% Tween 20, the sheet was incubated in biotinynated anti- Isolation of antigens mouse-immunoglobulins (Amersham), diluted by affinity chromatography /500 in casein for 40 min, washed again and iinally incubated in avidin-biotinynated peroxi- dase complex (Amersham) diluted 1/500 in Purified mAbs Y3A5 were covalently linked to casein. The blot was releaved by chemilumines- CNBr-activated Sepharose 4B (Pharmacia), cence using ECL (Amersham), and exposed to according to the manufacturer’s recommenda- Hyperfilm MP (Amersham). The photographic tion. Parasite extracts (oocysts and sporozoites) film was developed after 30 s to 5 min exposure in NET buffer and detergents, prepared as time. described above, were mixed with the mAbs- sepharose 4B, and left at room temperature for 1 200 column (Pharmacia) and equilibrated in 100 h under agitation. Then, the mixture was poured mM NaCl, 50 mM phosphate buffer, pH 7.4. Frac- into a column and unfixed material was removed tions (5 ml) were collected and assayed by with 20 mM phosphate buffer, pH 7.2. The fixed ELISA. material was eluted with glycine 100 mM, pH 2.7.

RESULTS Identification of antigens by ELISA

The presence of specific antigen in solution or in Monoclonal antibodies fractions obtained by chromatography was deter- mined by ELISA; immunoplates (maxisorb-Nunc, Rockilde, Denmark) were coated overnight at 4°C Molecular weights of the antigens with purified mAbs (mAbs Y3A5, S1 G12, S7H6, recognized by mAbs AT9H6) diluted in PBS-0.1% (w/v) thimerosal (Sigma) at 1 pg/well. Saturation was performed The Western blot reactivities of S1G122 with casein at room temperature for 30 min. After washing in PBS-0.1 % Tween 20, 100 pl of each (IgG3), Y14F3 (IgG3), AT7G10 (IgM), fraction was applied per well for 1 h at room tem- AT7F7 (IgG1 ), AT9H6 (IgG2a), Y3A5 (IgG3) perature. The wells were washed in PBS-0.1% and unrelated (negative control) mAbs to Tween 20; then the mAb Y3A5-HRP conjugate solubilized C parvum sporozoite antigens (Peroxidase Ref 814393 from Man- Boehringer are in 1. All these heim, Germany, coupling procedure described (SP Ag) presented figure mAbs a by the firm) was applied for 1 h at room temper- anti-Crypfosporidium recognized ature. The plates were washed and the test was high molecular weight antigen but the bands revealed using tetramethylbenzidine (TMB Sigma) were weak in the cases of mAbs Y14F3 and as a chromogen. The colour reaction was stopped AT7F7. Moreover, mAbs AT7G10 and with 100 !11 1 M H2SO4 per well and optical den- AT9H6 recognized several additional anti- sities (OD) were recorded at 450 nm. Positive gens. Different immunoblot reactivities were fractions (OD > 0.3) were pooled, concentrated by ultrafiltration using immersible cxl0 and ultra- observed for the 3 types of antigen prepa- free-Mc (Millipore Corp, USA), and dialysed ration. overnight against PBS. This purified antigen was MAb AT7G10 recognized 8 antigenic analyzed by Western blotting as done for puri- bands in BO lane lane fied oocyst preparation. Ag (fig 2a, 1; fig 2b, 1; fig 2c, lane 1 ). Due to the large number of immunoreactive bands, different prepa- Determination of the solubility rations of BO Ag were analyzed to verify of the antigen that reproducible results could be obtained. Using chemiluminescence, even a faint In order to estimate the solubility of the antigen, a immunoreactive band could be seen, but preliminary characterization was done according in this case longer exposure times were to Fround (1990). The abundance of the antigen needed and the strong immunoreactive anti- in cell extracts crude cell sol- (cell debris, extract, gen appeared as overexposed. The approx- uble cell membrane KCI-treated extract, pellet, imate molecular weight of these bands soluble extract and pellet) was assessed by ELISA and Western blotting. ranged from 50 to more than 500 kDa (fig 2b, lane 1; fig 2c, lane 1 Unreduced prepa- rations (data not shown), or PE Ag (fig 2c, Gel filtration chromatography lane 2) gave the same pattern. With mAb AT7G10, only 3 antigenic bands were PE Ag (7 ml prepared from 240 x 106 sonicated clearly observed in the case of SP Ag (fig 1, oocysts) was applied to a Hiload 26/60 Superdex lane 3; fig 2a, lane 2). Analysis of sporo- zoites isolated by DEAE-cellulose anion- MAb Y3A5 reacted with 2 antigens of dif- exchange chromatography (data not ferent molecular weight (> 500 and 340 kDa) shown), according to Riggs and Perryman when BO Ag was analyzed (fig 2a, lane 3). (1987), gave the same results as those The antigenic reactivity was lost when PE obtained with Sp Ag. Ag was tested (fig 2c, lane 3) and the > 500 kDa antigen was the only one detected by Analysis of the antigens purified mAb Y3A5 with SP Ag (fig 1, lane 6). When by affinity chromatography negative results were obtained, the immunoreactivity of mAb Y3A5 was checked Figure 3b shows the immunoblot reactivity of by an indirect immunofluorescent assay. In the molecules purified by affinity chro- spite of the absence of reactivity of mAb matography on a sepharose 4B column Y3A5 in the Western blot of PE Ag, this type linked to mAb Y3A5. To make the compar- of preparation still contained antigenic struc- ison easier, a preparation of BO Ag (fig 3a, tures recognized by an Y3A5-HRP conju- lane 1) was also run on the same blot. A gate in ELISA. more concentrated preparation of purified antigen was separated by electrophoresis The antigen of > 500 kDa was associ- using a shorter run time (fig 3c). In this case, ated with a soluble ultrasonicated oocyst all bands were clearly seen and less diffu- because it was still in the extract, present sion occurred. The reactivity against affinity- supernatant after a at centrifugation purified antigens was as follows: mAb Y3A5 135 000 for 30 min at 4°C. This g high did not recognize any band (fig 3b, lane 2; fig molecular size was corroborated weight by 3c, lane 2); while a complex labelling pat- of PE on a 200 col- separation Ag Superdex tern was obtained with mAb AT7G10 (fig umn (exclusion limit of 600 kDa). The last 3b, lane 4; fig 3c, lane 1 The antigen of and fraction of the void volume the early > 500 kDa was heavily marked and several post-void volume fractions gave the maxi- bands of the same molecular weight range mum OD in an ELISA performed with mAb as those recognized by mAb AT7G10 in BO Y3A5 conjugate. Ag (fig 3a, lane 1) were observed. Two other mAbs always recognized some bands when treatments (fig 4, lanes 1 and 4), except a tested against purified antigen: mAb S1 G122 faint band of lower molecular weight which (fig 3b, lane 1) recognized the high mole- still appeared in figure 4, lane 4. The binding cular weight antigen; and mAb Y14F3 (fig of mAb AT7G10 to purified antigen was 3b, lane 3) gave approximately the same completely abolished by periodate oxida- pattern as the one observed with mAb tion (fig 4, lane 6), whereas only one anti- AT7G10. Mab AT9H6 recognized some genic band, corresponding to the 575 kDa bands of lower molecular weight, while the antigen, was still recognized by this mAb reactivity against the > 500 kDa antigen (as after treatment of the PE Ag extract (fig 4, seen in fig 1, lane 5) was absent. lane 3). The 3 control preparations gave the same electrophoretic pattern, and only the results obtained from the control samples The effect of pronase digestion incubated during 30 h at 37°C without and periodate oxidation pronase are shown in figure 4, lane 2 and 5. The reactivity of mAb AT7G10 0 to antigenic treated in solution with sodium preparations lmmunolocalization periodate or pronase is shown in figure 4. Two types of preparation were used: PE Ag (fig 4, lanes 1, 2 and 3) and the antigen puri- In immunofluorescent assay mAb Y3A5 fied by affinity chromatography on recognized the oocyst wall and heavily sepharose-Y3A5 mAb (fig 4, lanes 4, 5 and labelled the sporozoite apex (fig 5). On LR- 6). Pronase deeply altered the antigenic White sections, mAb Y3A5 recognized the structure of these preparations since no limiting membrane of parasitophorous vac- labelling could be observed after pronase uoles of all stages of C parvum; the mero-

zoite apex was also labelled (fig 6). Gold Western blot patterns observed with these particles were also localized over the struc- mAbs suggest that different epitopes located tures, which seemed to be dense granules on a common antigen are concerned. In (fig 6, arrow). With preembedding assays, immunoelectron microscopy stronger the outer layer of the oocyst wall was labellings were observed with mAbs labelled (data not shown), but we never AT7G10 and Y3A5, so we have empha- succeeded in labelling the pellicle of sporo- sized the study of the antigens recognized zoites with any mAb described in this paper. by these mAbs. All of them exhibited a pronounced polar Each stage of C parvum carried the epi- for in reactivity air-dried sporozoites topes recognized by mAbs AT7G10 and immunofluorescent as seen for assays, Y3A5. These epitopes were clearly asso- mAb Y3A5 in 5. figure ciated with: (i) the micronemes of sporo- Mabs AT7G10 and Y3A5 showed the zoites and merozoites; (ii) the para- same labelling pattern in immunofluores- sitophorous vacuole membrane of cent and immunogold assays. Very inten- trophozoites, schizonts, microgamonts and sive labelling was observed with mAb macrogametes; (iii) the outer oocyst wall; AT7G10 on LR-White sections of tissues and (iv) the wall-forming bodies in maturat- fixed with paraformaldehyde, and it was the ing macrogametes. only mAb which was still reactive on osmi- In Western blots probed with mAb cated tissues. In this case, was labelling AT7G10, the same patterns were observed but the of the less pronounced morphology with PE Ag and BO Ag: 8 antigenic bands different structures was well In preserved. were observed. Two of these bands (> 500 merozoite, were associated gold particles kDa and 340 kDa) were recognized by mAb with micronemes No could (fig 7). labelling Y3A5 in BO Ag. Results obtained with mAb be associated with rhoptries which were Y3A5 suggest that some modifications had seen in cross-section in 7. In figure young occurred after treatment with detergents some micronemes located at trophozoites, and sonication because mAb Y3A5 gave the periphery of the cytoplasm were no signal on Western blot of PE Ag (anti- labelled (fig 7). In macrogamete, mAb gen purified by affinity chromatography using AT7G10 labelled electron-dense struc- Y3A5 as capture mAb). This antigenic mate- tures that we identified as bod- wall-forming rial still contained antigens recognized by ies (fig 8). The parasitophorous vacuole a Y3A5-HRP conjugate in ELISA but this membranes of mature not trophozoite (data was tested before dilution and in schizont boiling shown), (fig 6), macrogamete electrophoresis buffer. These last steps must and were (fig 8), microgamont (fig 9) have modified the epitopes labelled. recognized by mAb Y3A5. Otherwise, signals could be observed when blots of PE Ag were incu- bated with mAbs AT7G10, Y14F3 or S1 G12. DISCUSSION In this case the antigen of > 500 kDa was labelled. These data would suggest that Even it different patterns of reactivity were these mAbs and mAb Y3A5 recognize the observed in Western blots, all the mAbs same antigen and that the treatment with used in the present work recognized a high detergents or sonication could modify the molecular weight antigen (> 500 kDa) of C antigenic structure but not the molecular parvum and gave the same labelling pat- weight of the molecule. Surprisingly, the tern by immunoelectron microscopy and antigen purified using mAb Y3A5 and immunofluorescent assay. The different revealed by mAb AT7G10 on Western blots, showed a labelling pattern close to that molecules bear the epitopes recognized by obtained with mAb AT7G10 against BO Ag mAbs AT7G10 and Y3A5 in other stages or PE Ag. This complex pattern could be of C parvum. The explained by 3 hypotheses. (i) epitope The pronase treatment abolished the recognized by mAb Y3A5 was not only pre- reactivity of mAb AT7G10. This suggests sent on 2 of > 500 and 340 antigens kDa, that the molecule identified was a protein. but also on other antigens and these epi- Results of periodate oxidation demonstrate were altered for topes during preparation that the epitopes recognized were localized Different molecular electrophoresis. (ii) on a glycoprotein. Periodate treatment of weight antigens were physically linked to PE Ag suppressed the binding of mAb the of > 500 kDa and/or 340 kDa antigens AT7G10 with all molecules except one. so they would be copurified by affinity chro- Indeed, the high molecular weight antigen Some matography. (iii) degradations was still labelled but it seemed to migrate occurred the various during purification further into the gel, which suggests an alter- steps. ation of this molecule. It is possible that car- The comparison of Western blot patterns bohydrate moieties were cleaved by perio- obtained with SP Ag or BO Ag and mAb date treatment and that a lighter molecule Y3A5 showed that the high molecular with unchanged epitopes was generated. If weight antigen (> 500 kDa) was certainly this hypothesis is proved, the epitopes rec- located at the sporozoite level whereas the ognized are not localized on carbohydrate 340 kDa antigen was only observed when moieties. As far as the antigens of lower oocyst walls were present in the prepara- molecular weight are concerned, we hypo- tion. Of the 8 antigens that bear the epi- thesize that periodate oxidation caused a topes recognized by mAb AT7G10, only 3 big conformational change. Purified anti- of them were clearly detected with lysed gens were more susceptible to periodate sporozoites (> 500, 255 and 215 kDa). We oxidation since binding to all bands was were not able to determine whether these 3 abolished. During purification, antigens were antigens were only specific to the sporo- exposed to acidic pH (glycine buffer pH 2.7), zoite stage or were also associated with and some molecular changes could have the oocyst wall. Five antigens of 340, 130, occurred during this step. 80 and 50 kDa could be demon- 100, only The description of antigen of such a high strated when the walls were oocysts pre- molecular weight is uncommon; there was sent in the sample. only one report of a > 900 kDa Crypto- It is difficult to know whether these cross- sporidium glycoprotein (Petersen et al, reacting antigens were linked together in 1992). Thus, in order to be sure that the same metabolic process. In the case molecules were well solubilized, different of Plasmodium falciparum, 4 levels of cross- procedures were used to dissociate the reactivities were described (Moelans and parasite. Even after drastic treatments with Schoenmakers, 1992). According to this detergents, the high molecular weight anti- classification, the antigens of C parvum rec- gen was still recognized in Western blot by ognized by mAbs AT7G10 and Y3A5 could some mAbs (AT7G10, S1G12, Y14F3). The be cross-reacting epitopes of type III (epi- reproductiveness of these results and the topes of different proteins that are individ- solubility of this antigen led us to believe ually expressed at different life cycle that the epitopes were not carried by an stages) or type IV (expression of the same insoluble membrane-associated molecule. antigen in different life cycle stages). Fur- The high molecular weight of this antigen ther studies are needed to find out which was confirmed by its higher concentration in the last fraction of the void volume and work, our data suggest that these events in the first post-void volume fractions of a are closely linked. By immunolocalization, Superdex 200 column (exclusion limit of 600 we observed that the same epitopes are kDa). Of course, more accurate determina- involved in separate development process, tion with molecular weight markers for gel fil- which would imply membrane synthesis. tration chromatography should be neces- These epitopes are expressed at different sary. levels by all parasitic stages of C parvum Bonnin et al (1991) described mAbs and therefore could not only be confined to HAD and TOU, which reacted with the invasive process. In sporozoites these are carried a micronemes of zoites and also with the par- epitopes always by greater asitophorous vacuole of trophozoites and than 500 kDa antigen which is localized in macrogametes. On the other hand, the the micronemes. Dense granules could also contain this because was same authors (Bonnin et al, 1993) antigen labelling described mAbs ABD, BAX and SPO, also observed on some of them, but not so which reacted with micronemes and intensively. This antigen could play a role in the colonization of the host cell since the macrogametes granules. MAbs AT7G100 vacuole membrane is a and Y3A5, which we described in this parasitophorous sack of host cell and Madara, paper, exhibited the 2 immunolabelling pat- origin (Marcial The role to microneme in terns described by Bonnin et al (1991, 1986). assigned the of vacuole 1993) with their 2 sets of mAbs. Moreover, building parasitophorous membrane of Cryptospodidium parvum is our mAbs recognized the parasitophorous similar to the function attributed to vacuole membrane of microgametes and micronemes of Plasmodium brasilianum the outer oocyst wall. The antigens identi- merozoites (Torii et al, 1989a), to dense fied by our mAbs could have some homolo- and micronemes of gies with those described by Bonnin et al granules Sarcocystis muris et al, 1986, and to (1991, 1993) (similarities in the immunola- (Entzeroth 1991) dense granules of Toxoplasma gondii (Lin- belling patterns and in the sensitivity to peri- der et al, 1992). The antigens described in odate oxidation). However there was a dis- this study appear to be shared by crepancy between the molecular weight of micronemes and dense granules but were the antigens recognized. MAb TOU, pro- not successfully demonstrated at the level of duced by Bonnin et al (1991 recognized 2 rhoptries of zoites. The antigen of > 500 major bands at 210 and 130 kDa. kDa is very immunogenic since it was rec- Bjorneby et al (1990) described epitopes ognized by several of our mAbs raised shared by sporozoites and merozoites of C according to different immunization sched- parvum. Riggs et al (1989) have reported ules and by bovine immune sera (data not the production of mAbs that can bind to shown). Further studies are needed to know sporozoites and the external wall of intact if crucial epitopes, which are the targets for oocysts. Bonnin et al (1991 ) have suggested a protective immune response, are carried that the content of microneme could be by this antigen. released in the parasitophorous vacuole by invading zoites. By electron microscopy, Current and Reese (1986) obtained evi- ACKNOWLEDGMENTS dence to suggest the participation of wall- bodies in the of the forming building oocyst We wish to thank A Vanherf for his excellent tech- wall. Bonnin described cross- ef al (1993) nical assistance, R Leloup (Histology Depart- reactive epitopes shared by micronemes ment, University of Namur, Belgium) for giving and macrogamete granules. In the present us access to his Electron Microscopy Unit where ultrathin sections were kindly prepared by C Devi- vacuole of Sarcocystis muris (Protozoa, Api- gnon. We are grateful to B Losson (Parasitology complexa). Eur J Cell Biol 41, 182-188 Department, University of Liege, Belgium) for crit- Entzeroth R, K6nig A, Dubremetz JF (1991) Mon- ical of the This work was reading manuscript. oclonal antibodies identify micronemes and a ’l’lnstitut supported by Belge pour I’Encourage- new population of cytoplasmic granules cross- ment de la Recherche Scientifique dans l’lndus- reacting with micronemes of cystozoites of trie et I’Agriculture’. Sarcocystis muris. Parasitol Res 77, 59-64 Fround SJ (1990) Purification of cytochromes. In: Protein Purification Applications. A Practi- REFERENCES cal Approach (ELV Harris, S Angal, ed). IRL Press at Oxford University, UK pp 113-124 Laemmli UK (1970) Cleavage of structural pro- Achbarou A, Mercereau-Puijalon 0, Autheman teins during the assembly of the head of bac- Fortier Camus Dubremetz JF JM, B, D, (1991) teriophage T4. Nature (Lond) 227, 680-685 Characterization of microneme proteins of Toxoplasma gondii. Mol Biochem Parasitol Kohler G, Milstein C (1975) Continuous culture 47, 223-234 of fused cells secreting antibody of predefined specificity. Nature (Lond) 256, 495-497 Bannister LH, Butcher GA, Dennis ED, Mitchell GH (1975) Structure and invasive behaviour Linder E, Thors C, Edberg F, Haglund S, Von of Plasmodium knowlesi merozoites in vitro. Bondsdorff CH (1992) Generation of antibod- Parasitology 71, 483-4911 ies against Toxoplasma gondii antigen asso- ciated with dense and the para- JG and granules Beeley (1985) Glycoprotein Proteogly- sitophorous vacuole of the host cell. Parasitol can In: in techniques. Laboratory Techniques Res 78, 175-178 Biochemistry and Molecular Biology, vol 166 Marcial Madara JL (RH Burdon, PH van Knippenberg, eds). Else- MA, (1986) Cryptosporidium: vier Science Publishers BV, Amsterdam, pp cellular localization, structural analysis of cell - mem- 160-161 and pp 279-288 absorptive parasite membrane ― brane interactions in guinea pigs, and sug- Bendayan M, Nanci A, Frederick WKK (1987) gestion of protozoan transport by M cells. Gastro- Effect of tissue processing on colloidal gold enterology 90, 583-594 cytochemistry. J Histochem Cytochem 35, 983-996 Moelans I, Schoenmakers J (1992) Crossreac- tive antigens between life cycle stages of Plas- Bjorneby JM, MW, Perryman LE Riggs (1990) modium Parasitol 8, 118- merozoites share falciparum. Today Cryptosporidium parvum 123 neutralization - sensitive epitopes with sporo- zoites. J Immunol 145, 298-304 Ostrovska K, Paperna I (1990) Cryptosporidium of the starred lizard stellio: ultra- Bonnin A, Dubremetz JF, P sp Agama Camerlynck (1991) structure and life Parasitol 712- Characterization of microneme of cycle. Res 76, antigens 720 Cryptosporidium parvum (Protozoa, Apicom- plexa). Infect Immun 59, 1703-1708 Pearce-Pratt R, Roser B (1989) Comparison of for ELISA. Nunc Bulletin 7 Bonnin Dubremetz P A blocking agents A, JF, Camerlynck (1993) Dec 89 new antigen of Cryptosporidium parvum micronemes possessing epitopes cross-reac- Perkins ME (1992) Rhoptry organelles of Api- tive with macrogamete granules. Parasitol complexan parasites. Parasitol Today 8, 28-32 Res 79, 8-144 Petersen C, Gut J, Doyle PS, Crabb JH, Nelson Current WL, Reese C (1986) A comparison of RG, Leech JH (1992) Characterisation of a endogenous development of three isolates of greater than 900 000 Mr Cryptosporidium Cryptosporidium in suckling mice. J Protozool parvum, sporozoite glycoprotein recognized 33, 98-108 by protective hyperimmune bovine colostral Entzeroth R, Dubremetz JF, Hodick D, Ferreira E immunoglobulin. Infect Immun 60, 5132-5138 (1986) Immunoelectron microscopic demon- Riggs MW, Me Guire TC, Mason PH, Perryman stration of the exocytosis of dense granule LE (1989) Neutralization-sensitive epitopes contents into the secondary parasitophorous are exposed on the surface of infectious Crypto- sporidium parvum sporozoites. J Immunol modium brasilianum merozoites with knobs 143, 1340-1345 and other parasite-induced structures in host Riggs MW, Perryman LE (1987) Infectivity and erythrocytes. Infect Immun 57, 596-6011 neutralization of Cryptosporidium parvum Torii M, Adams JH, Miller LH, Aikawa M (1989b) sporozoites. Infect Immun 55, 2081-2087 Release of merozoite dense granules during Robert B, Ginter A, Antoine H, Collard A, Coppe erythrocyte invasion by Plasmodium knowlesi. P (1990) Diagnosis of bovine Cryptosporidio- Infect Immun 57, 3230-3233. sis by an enzyme-linked immunosorbent Towbin H, Staehelin T, Gordon J (1979) Electro- assay. VetParasitol37, 1-8 phoretic transfer of proteins from polyacryl- Scholtyseck E, Mehlhorn H (1970) Ultrastructural amide gels to nitrocellulose sheets: procedure study of characteristic organelles (paired and some applications. Proc Natl Acad Sci organelles, micronemes, micropores) of USA 76, 4350-4354 Sporozoa and releated organisms. Z Para- Ungar BLP, Soave R, Fayer R, Nash TE (1986) sitenk 34, 97-1211 Enzyme immunoassay detection of Sibley LD, Sharma SD (1987) Ultrastructural local- immunoglobin M and G to Cryptosporidium in ization of an intracellular Toxoplasma protein immunocompetent and immunocompromised that induces protection in mice. Infect Immun persons. J Infect Dis 153, 570-578 55, 2137-2141 Uni S, Iseki M, Maekawa T, Moriya K, Tadaka S Sinden RE (1985) A cell biologist’s view of host (1987) Ultrastructure of Cryptosporidium muris cell recognition and invasion by malarial par- (strain RN 66) parasiting the murine stomach. asites. Trans R Soc Trop Med Hyg 79,598- Parasitol Res 74, 123-132 605 Vetterling JM, Takeuchi A, Madden PA (1971) Spitz M, Spitz L, Thorpe R, Eugni E (1984) Ultrastructure of Cryptosporidium wrairi from Intrasplenic primary immunization for the pro- the guinea pig. J Protozool 18, 248-260 duction of monoclonal antibodies. J Immunol Wisher MH, Rose ME (1987) tenella Methods 70, 39-43 sporozoites: the method of excystation affects Torii M, Matsumoto Y, Kamboj KK et al (1989a) the surface membrane proteins. Parasitol95, Association of micro-neme antigens of Plas- 479-489