Parasitnlngy Research

Parasitol Res (1991) 77:59 64 Parasitnlngy Research 9 Springer-Verlag 1991 Monoclonal antibodies identify micronemes and a new population of cytoplasmic granules cross-reacting with micronemes of cystozoites of Sarcocystis muris* R. Entzeroth 1, A. K~inig 1, and J.-F. Dubremetz 2 1 Zoologisches Institut, Poppelsdorfer Schloss, D-5300 Bonn 1, Federal Republic of Germany 2 INSERM, U 42, 369 Rue Jules-Guesde, F-59650 Villeneuve-D'Ascq, France Accepted July 15, 1990 Abstract. Micronemes of cystozoites of Sarcocystis muris were isolated after subcellular fractionation and used for immunization of BALB/C mice. After spleen cells of immunized mice were fused with SP20 myeloma cells, ten different monoclonal antibodies (mabs) were isolated. These antibodies reacted with antigens whose molecular weight ranged from 16 to >90 kda. Six mabs recognized granules of 150-400 nm that were located in the vicinity of the Golgi complex but were not identical with dense granules. Two mabs (2A3, 3A8) were specific for micronemes of cystozoites as demonstrated by immunoelectron microscopy. However, these antibodies also recognized the population of granules near the Golgi complex. Cross-reactivity between micronemes and a dense granule population has not previously been reported. Host cells that had been contacted by cystozoites showed patchy fluorescence when probed with mab 2A3. This suggests that microneme antigens could be transferred to the host-cell surface during parasitehost cell interactions. The motile stages of Coccidia (Apicomplexa) are characterized by a pellicle, a conoid, polar rings and micropores and by electron-dense inclusions in the apical cell region: rhoptries, micronemes and dense granules (microspheres). Rhoptries have been shown to release their contents during invasion of Plasmodium knowlesi (Bannister and Mitchell 1989; Sam-Yellowe et al. 1988) and Toxoplasrna (Kimata and Tanabe 1987; Nichols etal. 1983). Dense granules (microspheres in Plasmodium) are exocytosed into a secondary parasitophorous vacuole formed by Sarcocystis muris after invasion (Entzeroth 1985; Entzeroth et al. 1986). The function of micronemes in apicomplexan protozoa is not clear. Micronemes might be involved in inva- * Dedicated to Prof. Dr. G. Piekarski on the occasion of his 80th birthday Offprint requests to: R. Entzeroth sion; however, it has also been shown that micronemederived antigen may be transported across the host cell to the host-cell surface in P. brasilianum (Torii et al. 1989). It has been recently shown that micronemes play a role in the release of gamonts in PIasrnodium (Quakyi et al. 1989). Micronemes of S. tenella and S. muris have been purified by subcellular fractionation (Dubremetz and Dissous 1980; Pohl et al. 1989), and in both cases the two major proteins of 20/22 and 16/17 kda were characterized. In Eimeria nieschulzi, micronemes contain two major proteins of 220 and 94 kda, both of which are present in sporozoites and merozoites (Dubremetz et al. 1989). In the present study we report on the characterization of other microneme proteins of S. muris by monoclonal antibodies. We also obtained monoclonal antibodies to a new population of granules that crossreact to some extent with micronemes. Materials and methods Parasites and antigen preparations from Sarcocystis muris cystozoites were obtained from skeletal muscles of mice according to the method of Pohl et al. (1989). Purified cystozoites were suspended in homogenization medium [250 mm sucrose, 1 mm ethylenediaminetetraacetic acid (EDTA), 5 mm triethanolamine-hc1 (ph 7.5)] at a concentration of 108 cells/ml. Cells were disrupted in a French pressure cell (Amico) operated at 50 kg/cm 2. The homogenate was centrifuged at 500 g for 10 rain to eliminate unbroken cells; the supernatant was collected and subjected to 12000 g in an SW 28 rotor. The resulting supernatant was then centrifuged at 72000 g for 30 rain. A pellet highly enriched with micronemes was obtained, which was frozen at -20 ~ C. The contents of each fraction were determined by electron microscopy as previously described (Entzeroth et al. 1986). Immunization of mice and production of hybridomas Monoclonal antibodies (mabs) were obtained by fusion of SP2/o myeloma cells with splenocytes of BALB/c mice immunized with S. muris. The immunization procedure included three intraperitoneal injections of 108 cell equivalents of a microneme fraction emulsified in Freund's complete (first injection) or incomplete (second

60 R. Entzeroth et al. : Monoclonal antibodies against S. muris micronemes Figs. 1--6. Immunofluorescence micrographs of air-dried cystozoites of Sareocystis muris incubated with 1 mab 1G12, 2 mab 1B5, 3 mab 2B3, 4 mab 2A9, 5 mab 1E3, 6 mab 1A4 and third injections) adjuvant, given 1 month apart and followed 2 weeks after the last injection by an intravenous boost with the same amount of material in phosphate-buffered saline [PBS: 50 mm sodium phosphate buffer (ph 7.4), 150 mm NaC1]. The fusion was performed 3 days after the last boost according to Galfre et al. (1977). Screening of hybridoma supernatants was done by immunofluorescence assay (IFA) and Western blotting. Positive hybridomas were cloned by limited dilution. Western blotting Cystozoites of S. muris were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970) (2 x 106 cystozoites per 1-cm-wide slot) and then electrophoretically transferred to nitrocellulose (100 ma, 1 h) according to Towbin et al. (1978). The nitrocellulose sheet was saturated for 30 min in 5% nonfat dry milk in buffer [140 mm NaCI, 0.5 % Tween in 15 mm TRIS-HC1 (ph 8): TNT] and then incubated in mab for 1 h at 37 ~ C. After being washed, the sheet was incubated in alkaline phosphatase-conjugated mouse IgG serum (Sigma A-5153) diluted in TNT and then revealed with 5-bromo-4- chloro-3-indolyl phosphate (Sigma B-8503) and 4-nitrotetrazoliumchloride blue-hydrate (Sigma 6876). CeH culture Madin-Darby canine kidney (MDCK) cells were grown on glass slides using Flexiperm (Heraeus) reusable culture chambers at 37 ~ C in an atmosphere containing 5% COz. Minimum essential medium (MEM, Gibco) containing 10% fetal calf serum served as the culture medium. Approximately 300 x 10 a cystozoites of S. muris were concentrated in MEM without serum, which was added to the culture chambers containing nearly confluent cell monolayers. Monolayers that had been inoculated with cystozoites for 10, 30, 60 and 120 min were fixed for 10 rain in 2% paraformaldehyde in PBS, rinsed with PBS for 2 10 min and stored at -20 ~ C until labelling with antibodies. Immunofluorescence assay Purified cystozoites of S. muris were washed three times with PBS and dried on standard IFA slides (H61zel, Dorfen), which were stored at -20 ~ C. IFA was performed at 37 ~ C in a moist chamber after 10 min fixation in cold acetone using undiluted cell-culture supernatant and anti-mouse IgG, IgA and H+ L-fluorescein isothiocyanate (FITC, Nordic). Observations were done using a Zeiss ICM 405 equipped with epifluorescence. Photographs were taken on Agfapan 400 film. Immunoelectron microscopy Purified cystozoites or cultured MDCK cells taken at 30 rain and at 1, 4 and 10 h after inoculation of cystozoites were fixed in 2.5% paraformaldehyde with 0.1% glutaraldehyde in PBS, then embedded in Lowicryl K4M according to Roth et al. (1981) and sectioned with glass knives on a Reichert Ultracut microtome. Thin sections were collected on Formvar carbon-coated nickel grids and floated for 30 min on 2% ovalbumin in PBS (PBSO), then transferred onto undiluted hybridoma culture medium for 1 h. After being washed with PBSO, the grids were floated on rabbit anti-mouse

R. Entzeroth et al. : Monoclonal antibodies against S. muris micronemes 61 immunoglobulins (Southern Biotech Associates) diluted 1:100 in PBSO for 1 h. They were then washed and transferred for 1 h onto 5 nm protein A-gold conjugate prepared according to Slot and Geuze (1985) and diluted I : 100 in PBSO. Sections were stained with 3% uranyl acetate in water and then observed with a Zeiss EM 9S2 electron microscope. Results MAbs raised against the enriched microneme fraction (Fig. 9) of Sarcocystis muris showed different fluorescence patterns. Three hybridoma antibodies (mabs 1B5, 1G12, 2E4) reacted with the pellicle (Figs. 1-3) and five (mabs 1DI1, 1E3, 2A3, 3A8) reacted with the apical region of zoites (Figs. 4--6). Granular inclusions located in the middle of the cell as well as antigen surrounding the cystozoites (leaking antigen, due to drying or thawing) were recognized by mab 2B3. On Western blots of whole cystozoite proteins under nonreducing conditions, hyperimmune serum from BALB/c mice (prefusion sera) recognized a major band at 22 kda and minor bands at 47, 48 and 65 kda (Fig. 7 a). Cystozoite antigens recognized by mabs ranged in molecular weight from 14 to >90 kda (Table 1). Electron microscopy of S. muris zoites revealed that most of the mabs reacted with a population of granules located in the middle region of the cells between the nucleus and the area occupied by dense granules (Figs. 8 and 11, arrows). Two of these mabs (2A3, 3A8) also clearly reacted with the micronemes (Figs. 8 and 10, Table 1). Immune polyclonal sera raised against microneme fractions of S. muris in BALB/c mice were tested for cross-reactivity with Eimeria tenella sporozoites, E. nieschulzi sporozoites and merozoites, and Toxoplasma tachyzoites using Western blotting and IFA. All Western blots were negative, whereas IFA tests were positive for E. tenella and E. nieschulzi motile stages. However, when mabs were used with these same organisms and with additional sporozoites of E. papiltata, no reaction was observed. Antibodies were tested for their reactivity on S. muris cystozoite-infected cells. At 60 rain after incubation of zoites with MDCK cells, two mabs (2A3, 3A8) still recognized the apical pole of intracellular zoites (Figs. 11, 12). However, antigen on the host-cell surface was also recognized by these antibodies (Figs. 12, 14). Controls with non-infected cells showed no reactivity, with the mabs ruling out nonspecific cross-reactions with hostcell antigen. Discussion MAbs raised against an enriched microneme fraction of Sarcocystis muris bradyzoites showed internal and surface fluorescence. Surface-specific antibodies might derive from membrane vesicles, contaminating the microneme preparation. In addition, micronemes are surrounded by a membrane that might have cross-reacting determinants with the outer zoite membrane. Two poly- Fig. 7. [mmunoblot of Sarcocystis muris zoites analyzed under nonreduced conditions and probed with hyperimmune serum of an immunized BALB/c mouse (a), with mab 1B5 (b), mab 1Dll (c), mab IE3 (d), mab 1GI2 (e), mab 1H1 (j) mab 2A3 (g), mab 2b3 (h), mab 2E4 (0, mab 3A3 (]), mab 3A8 (k), and rabbit serum against purified 16-kDa, major microneme protein (/). Molecular-weight markers (row) are shown at left (Sigma calibration kit, MW-SDS-JOL) Table 1. MAbs against the microneme fraction of Sarcocystis muris mab Western blotting IFA IEM IB5 16, 17, 26 kda S, G G 1Dll 16 kda AP - 1E3 > 90 kda AP G IG12 24, 29, 64 kda S, G G 1H1 45, 46, 50, 67, 68 kda - - 2A3 >90 kda AP MN, G 2B3 24, 45, 22, 23 kda S, G, bg G 2E4 17, 29 kda S G 3A3 22 kda G G 3A8 20, 22, 29 kda AP MN, G IEM, immunoelectron microscopy; AP, apical; bg, background; G, granules; MN, micronemes; S, surface peptides of 16 and 17 kda are major components of S. muris micronemes (Pohl et al. 1989). The two mabs (2A3, 3A8) found in this study recognized proteins with molecular weights of < 90 kda and 20, 22 and 29 kda, respectively. In addition to recognizing micronemes, mabs 2A3 and 3A8 recognized a population of granules in the vicinity of the area in which the Golgi complex is usually located. This finding suggests that vesicles derived from the Golgi complex might express epitopes of micronemes. What is not clear is whether these granules represent recently synthesized microneme precursors or a new population of organelles. The fact that some of the mabs were specific for these granules and

62 R. Entzeroth et al. : Monoclonal antibodies against S. muris micronemes Fig. 8. Ultrathin section through the apical region of a zoite containing a conoid (c), micronemes (ran) and a population of granules located between the nucleus and the dense granules (arrows). Lowicryl K4M, mab 3A8, protein A-gold. x 12500. Fig. 9. Electron micrograph of the enriched microneme fraction. 37500. Fig. 10. Ultrathin section through the apical region of a cystozoite contain- ing a conoid (c), rhoptries (rh) and labelled micronemes (ran). Lowicryl K4M, mab 3A8, protein A-gold. x 23000. Fig. 11. Longitudinal section through a cystozoite with a conoid (c), micronemes (mn), dense granules (g) and a nucleus (n). Note the population of labelled cytoplasmic granules (arrows). Lowicryl K4M, mab 1G12, protein A-gold. x 14000

R. Entzeroth et al. : Monoclonal antibodies against S. muris micronemes 63 Figs. 12, 13. Fluorescence (Fig. 12) and the corresponding phasecontrast micrograph (Fig. 13) of MDCK cells obtained 60 min after infection with Sarcoeystis muris zoites, followed by incubation with mab 2A3 and labelling with antimouse FITC. Note intracellular zoites (arrow) and patchy fluorescence on the host cells, x490. Figs. 14, 15. Higher magnification of a MDCK cell with an attached cystozoite (z) shows patchy fluorescence (Fig. 14) on the surface of the host cell. 1300 did not recognize micronemes favors the second hypothesis. This matter should be further investigated. Microneme epitopes could be detected by mab 2A3 in the apical part of the zoite at 1 h after inoculation of MDCK cells. It was interesting that mab 2A3 also recognized patches of antigen on the surface of infected host cells. This might be an antigen secreted during entry by the zoite from the apical pole at the surface of the host cell. A patchy fluorescence similar to that observed in the present study has also been described on the surface of Plasmodiumfalciparum-infected erythrocytes and is recognized by an mab against a 110-kDa rhoptry protein (Sam-Yellowe et al. 1988). A microneme-derived antigen of P. falciparum, RESA, is probably transferred via the rhoptries to the host-cell surface membrane (Brown et al. 1985). The connection of micronemes with rhoptries has previously been suggested by Scholtyseck and Mehlhorn (1970) and is temporarily found in S. muris (Fig. 16). Micronemes in the vicinity of the apical tip are aligned longitudinally such that they might discharge their contents directly. Another microneme-derived antigen from P. brasilianum is transported from the parasite through the cytoplasm of the red blood cell and is eventually deposited in the knobs of infected erythrocytes (Torrii et al. 1989). This indicates that micronemes might play a vital role in modifying the infected host cell, leading to Plasmodium sequestration, an important immune evasion mechanism. Micronemes would therefore be multipotent organelles involved in the invasion of cells and, subsequently, in intracellular development. Their precise function(s) should be studied further, together with their

64 R. Entzeroth et al. : Monoclonal antibodies against S. muris micronemes Fig. 16. Electron micrograph of an extracellular Sarcocystis muris cystozoite fixed a few minutes after being added to a monolayer of MDCK cells. Note the connection between micronemes (ran) and rhoptries (rh). DG, dense granules, x 12000 interactions with the secretory organelles of zoites, which comprise rhoptries and dense granules. Acknowledgements. The authors are indebted to C. Ansel and M. Mortuaire for excellent technical help. This work was funded by INSERM and by grants from the Deutsche Forschungsgemeinschaft to R. Entzeroth (DFG 153/2-1, DFG 153/3-1). References Bannister LH, Mitchell GH (1989) The fine structure of secretion by Plasmodium knowlesi merozoites during red cell invasion. J Protozool 36 : 362-367 Brown GV, Culvenor JG, Crewttier PE, Bianco AE, Coppel RL, Saint RB, Stahl MD, Kemp D J, Anders RF (1985) Localization of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium faleiparum in merozoites and ring-infected erythrocytes. J Exp Med 162:774-779 Dubremetz JF, Dissous C (1980) Characteristic proteins of micronemes and dense granules from Sarcocystis tenella zoites (Protozoa, Coccidia). Mol Biochem Parasitol 1:279-289 Dubremetz JF, Ferreira E, Dissous C (1989) Isolation and characterization of rhoptries and micronemes from Eimeria nieschulzi zoites (Sporozoa, Coecidia). Parasitol Res 75:449-455 Entzeroth R (1985) Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures. J Protozool 32:446-453 Entzeroth R, Dubremetz JF, Hodick D, Ferreira E (1986) Immunoeleetron microscopic demonstration of the exocytosis of dense granule contents into the secondary parasitophorous vacuole of Sareocystis muris (Protozoa, Apicomplexa). Eur J Cell Biol 41 : 182-188 Galfre G, Howe SC, Milstein C, Butcher GW, Howard JC (1977) Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 266:550-552 Kimata I, Tanabe K (1987) Secretion by Toxoplasma gondii of an antigen that appears to become associated with the parasitophorous vacuole membrane upon invasion of the host cell. J Cell Sci 88 : 231-239 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Nichols BA, Chiappino ML, O'Connor GR (1983) Secretion from the rhoptries of Toxoplasma gondii during host-cell invasion. J Ultrastruct Res 83:85-98 Pohl U, Dubremetz JF, Entzeroth R (1989) Characterization and immunolocalization of the protein contents of micronemes of Sareocystis muris cystozoites (Protozoa, Apicomplexa). Parasitol Res 75 : 19%205 Quakyi IA, Matsumoto Y, Carter R, Udomsangpeteh R, Sjolander A, Berzins K, Perlmann P, Aikawa M, Miller LH (1989) Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes. Infect Immun 57: 833-839 Roth JM, Bendayan E, Carlemalm W, Villinger M, Garavito M (1981) Enhancement of structural preservation and immunocytochemical staining in low-temperature embedded pancreatic tissue. J Histochem Cytochem 29 : 663-671 Sam-Yellowe TY, Shio H, Perkins ME (1988) Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes. J Cell Biol 106:1507-1513 Scholtyseck E, Mehlhorn H (1970) Ultrastructural study of characteristic organelles (paired organelles, micronemes, micropores) of Sporozoa and related organisms. Z Parasitenkd 34:97-127 Slot JM, Geuze HJ (1985) A new method of preparing gold probes for multiple-labelling cytochemistry. Eur J Cell Biol 38 : 87-93 Towbin H, Staehelin T, Gordon J (1978) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 Torii M, Matsumoto Y, Kamboj KK, Maracic M, Guo SQ (1989) Association of microneme antigens of Plasmodium brasilianum merozoites with knobs and other parasite-induced structures in host erythrocytes. Infect Immun 57:596-601