J. zyxwvutsrqponmlkjihgfe Eukuknrwr zyxwvutsrqponmlkj Mi< mhol., zyxwvutsrqponml 47( zyxwvutsrqponmlkj I), zyxwvutsrqponmlkjihgfedcba 2(WN pp. 48-56 zyxwvutsrqpon 0 2Mx) by the Society zyxwvutsrqponmlkjihgfe of Piotomologints Identification of Proteins in Encephalitozoon intestinalis, a Microsporidian Pathogen of Immunocompromised Humans: An Immunoblotting and Immunocytochemical Study ODILE PRIGNEAU, ABDERRAHIM ACHBAROU, NICOLAS BOULADOUX, DOMINIQUE MAZIER and INSERM zyxwvutsrqponm U 51 I, Immunobiologie Celluluire et MolPculaire des Infections Parasitaires. CHU Pitid-SalpEtri2re. 91 Bd zyxw de I'H8pitu1, 75013, Paris, France ISABELLE DESPORTES-LIVAGE ABSTRACT Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube-the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55-60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies. Key Words. Confocal microscopy, Encephalitozoon irzte.~tinalis, immunogold electron microscopy, microsporidia, polar tube, spore proteins, spore wall, sporogony, Westem-blot. HE microsporidia are unicellular eukaryotic parasites found T in a wide range of animals (Canning, Lom, and Hollister 1986). Microsporidian infections have also been reported in man; their pathogenicity in immunocompromised patients with HIV infection has increased the interest in these parasites. Sev- eral species, including Encephalitozoon intestinalis, were newly identified in AIDS patients (Cali, Kotler and Orenstein 1993; Hartskeerl et a1.1994; Orenstein and Getz 1997; Rayner et al. 1994). Cases of HIV-seronegative and immunocompetent pa- tients with intestinal microsporidiosis were also reported (Des- portes-Livage and Bylkn 1998; Enriquez et al.1998b; Raynaud et a1.1998; van Gool et al. 1997). Recently, E. intestinafis has been detected in asymptomatic mammals (Bornay-Llinares et al. 1998). The invasive mechanisms of these parasites are not fully understood. Microsporidia are transmitted by spores that possess a typical extrusion apparatus ensuring the inoculation of the infectious contents into the host cell. Recent ultrastruc- tural observations of macrophages invaded by E. intestinalis showed cytological events suggesting that the spore discharge was initiated by the contact between apical part of the spore and the host-cell membrane (Magaud, Achbarou and Desportes- Livage 1997). Such processes leading to a phagocytosis-like mechanism (Docampo and Moreno 1996), are usually mediated by ligand-receptor complexes expressed on both membrane sur- faces (Oliver 1996). Our objective is the characterisation of spore proteins involved in the invasion of host cells. The first approach was to produce polyclonal and monoclonal antibodies raised against E. intestinalis proteins. Different immunoanalytic techniques were then used to obtain anti-protein markers that could enable the elucidation of invasive mechanisms and the development of new targets for antiparasitic strategy. MATERIALS AND METHODS Parasites. Encephalitozoon intestinalis spores were collected from monolayers of rabbit kidney cells (RK13) as described by van Gool et al. (1994). Spores were harvested every three days, and suspensions of parasites were centrifuged at 1500 zyxwvu g, for 5 min to eliminate cellular fragments. Sieved spores were then sedimented by centrifugation at 4000 g for 20 min and washed Corresponding Author: I. Desportes-Livage-Telephone number: 33 (0)l 40 77 81 05; FAX number: 33 (0)l 45 83 88 58; E-mail: desporte @ext.jussieu.fr two times in sterile phosphate-buffered saline (PBS). Spores were stored at - 80 "C until use. Antigen preparation. Purified spores (1 X 10"') in 1 ml sterile 0.15 M NaCl were disrupted for 50 s with silicium beads (0.1 and 0.5-mm diam) using a Mini Beadbeater Apparatus@ (Bioblock Scientific, Illkirch, France). The suspension was freeze-thawed in liquid nitrogen and then warm water before being placed on ice. The disruption procedure was repeated ten times. The suspension was clarified by a centrifugation at 10,000 g for 10 min and the supernatant was stored at - 80 "C. The quantity of protein (3 mg/ml) was estimated by the method of Bradford (1996). Mice immunization and production of polyclonal and monoclonal antibodies (MAbs). Four to six-week-old BALB/ c female mice (Charles Rivers, Saint Aubin-Les-Elbeufs, France) were used for the immunization protocol, preparation of feeder cells for hybridoma culture, and ascites production. Mice were immunized intraperitoneally (IP) with 2 mg of spore proteins emulsified in a 1: 1 ratio in Freund's complete adjuvant (Sigma Chemical Co., St. Louis, MO) for the first inoculation and in Freund's incomplete adjuvant (Sigma) for the next in- oculations. Inoculations were performed at two-week intervals. Sera were collected after each inoculation, screened by indirect immunofluorescence and immunoblot using E. intestinalis spores as antigen in order to follow the parasite-specific anti- body response. Sera were stored at - 20 "C and used as positive controls during all immunoassays. Two of the immunized mice were selected and received, three days before fusion protocol, a further intravenous dose of spore proteins emulsified with poly A-poly U (Sigma) as a booster. Spleen cells were fused with SP,O myeloma cells in 5:l ratio as previously described by Kohler and Milstein (1975). Stable hybrids were selected by growth in Dulbecco essential medium (Gibco BRL, Gaithers- burg, MD) containing 20% fetal bovine serum (Seromed, Biochrom KG, Berlin), hypoxanthine, aminopterin, and thymi- dine. Hybridomas producing antibodies (Hab) were screened and selected both by indirect immunofluorescence assay and Western blotting using E. intestinalis spores as antigen. Poten- tially interesting hybridomas were cloned by limited dilution and produced in large quantities in ascites obtained from mice primed with 500 p,1 of pristane then 10 d later injected with the corresponding clone. Immunoglobulin isotyping. The MAb isotypes were deter- mined with a dipstick isotyping kit (Sigma, ISO1) according to 48