granules and micronemes from Sarcocystis tenella. (Dubremetz & Dissous, 1 980), and micronemes and rhoptries from Eimeria nieschulzi. (Dubremetz,. Ferreira ...
Fractionation and antigenic characterization of organelles of Eimeria tenella sporozoites U. KAWAZOE*, F. M. TOMLEYf and]. A. FRAZIER Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, Cambs PE17 2DA (Received 9 May 1991; revised 29 July 1991; accepted 29 July 1991) SUMMARY
Sporozoites of Eimeria tenella were disrupted by sonication and subcellular fractions were separated by sucrose gradient ultracentrifugation. Fractions from gradients were characterized by electron microscopical appearance and their polypeptide and antigenic profiles determined by PAGE and Western blotting with antisera to sporozoites and 1st- and 2ndgeneration merozoites. Fractions containing micronemes, rhoptries or membranes showed markedly different polypeptide content and antigenic reactivity. Microneme epitopes were strongly conserved between sporozoites and 2nd-generation merozoites whereas the majority of rhoptry epitopes and many membrane epitopes were sporozoite specific. The only polypeptide of sporozoites which was strongly recognized by antisera raised to 1st generation merozoites was a microneme antigen of molecular weight approximately 100 kDa. Key words: Eimeria tenella, sporozoites, organelles, antigens.
INTRODUCTION
Eimeria tenella is one of the seven species of Eimeria which infect fowl and is a causative agent of the economically important disease, coccidiosis. T h e antigenic composition of the sporozoite is complex (Wisher, 1986; Sutton, Shirley & Wisher, 1989) and monoclonal antibodies (mabs) have been used to determine the intracellular location, stage- and species-specificity of some epitopes (for example, Danforth & McAndrew, 1987; Augustine, Danforth & McAndrew, 1988; Danforth & Augustine, 1989; Speer, Thammnana & Schenkel, 1989; Taylor et al. 1990). Mabs have also been used to isolate genes coding for antigens which are associated with cellular structures such as the refractile body (Miller et al. 1989) and the micronemes (Tomley et al. 1991). This approach has been used for many other apicomplexans and antigens associated with organelles have been characterized for several species, for example Toxoplasma gondii (Sadak et al. 1988), Plasmodium falciparum (Bushell et al. 1988; Ridley et al. 1990), Plasmodium knowlesi (Adams et al. 1990) and Theileria parva (lams et al. 1990). An alternative method for localizing polypeptides to particular structures is to disrupt cells, fractionate the lysates and analyse purified or semi-purified organelles. This approach has been used to establish * Present address: Department of Parasitology, IB Unicamp, Caixa Postal 6.109, 13.081 Campinas, Sao Paulo, Brazil. f Reprint requests to F. M. Tomley, Institute for Animal Health, Houghton Laboratory, Houghton, Huntingdon, Cambs PE17 2DA.
the proteinaceous content of dense granules from Sarcocystis muris (Entzeroth et al. 1986), dense granules and micronemes from Sarcocystis tenella (Dubremetz & Dissous, 1 980), and micronemes and rhoptries from Eimeria nieschulzi (Dubremetz, Ferreira & Dissous, 1989). Recently, subcellular fractionation was used t o isolate and characterize rhoptries from Toxoplasma gondii and to raise mabs to rhoptry proteins as a step towards understanding the function of these organelles (Leriche & Dubremetz, 1991). In this paper, we have used subcellular fractionation of sporozoites of E. tenella to characterize parasite antigens which a r e associated with micronemes, rhoptries and membranes. In addition to identifying antigenic components of these cell structures, we have used antisera to other extracellular stages of the parasite to determine whether organellar epitopes are conserved between the asexual stages.
MATERIALS AND METHODS
Parasites Procedures for the maintenance, isolation and sporulation of oocysts of Eimeria tenella (strains Houghton, Weybridge and 5 field isolates) and for the excystation of sporozoites have been described elsewhere (Long et al. 1976; Chapman, 1978). Sporozoites were purified by anion-exchange chromatography on DE-52 cellulose columns (Schmatz, Crane & Murray, 1984) then collected by centrifugation at 1200 £ for 5 min. First-generation merozoites were
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U. Kawazoe, F. M. Tomley and J. A. Frazier
Fig. 1. Electron microscopical examination of samples from sucrose gradients following subcellular fractionation of Eimeria tenella sporozoites. All samples except (D) were fixed for 2 min in glutaraldehyde then stained with uranyl acetate. (A) White layer from the top of gradients containing 18 nm particles (ribosomes). (B) 1-2 M fraction containing pieces of membrane and 'ghosts' of sporozoites. (C) 1 4 M fraction containing 130-180 nm diameter structures which, on fixation for several hours with glutaraldehyde, were converted to (D) rod-shaped structures which resemble micronemes. (E) 2-3 M fraction containing 500-700 n m diameter structures which are thought to be rhoptries. (F) Pellet containing 420-600 x 220-350 nm ovoid structures (amvlopectin granules). isolated from the caecal mucosa of chickens 64 h after inoculation with 8 x 106 oocysts and 2ndgeneration merozoites 96 h after oral inoculation with 5x10° oocysts as described elsewhere (McDonald, Rose & Jeffers, 1986); both were purified by DE-52 anion-exchange chromatography.
Subcellular jractionation Sporozoites were suspended at 108/ml in cold homogenization medium (HM, 250 mM sucrose, 1 mM EDTA, 5 mM triethanolamine, pH 7-5, Dubremetz & Dissous, 1980) and subsequent steps
Organelles of E. tenella sporozoites
were carried out at 4 °C. The fractionation protocol was adapted from the methods of Dubremetz & Dissous (1980). Sporozoites were sonicated (Soniprep 150, MSE Scientific Instruments) at 2/tm for 40 sec, unbroken cells and debris removed by centrifuging at 700 £ for 10 min and the sonicate recentrifuged at 8500 £ for 10 min (Sorvall RC-5B centrifuge, SS-34 rotor). The 8500 £ supernatant fraction contained ribosomes, micronemes and fragments of membranes including ghosts of the sporozoites and the pellet contained rhoptries, amylopectin granules and micronemes. The 8500 £ pellet was suspended in HM and centrifuged at 5500 ,£ for 5 min. The 8500 £ and 5500£ supernatant fractions were each made up to 6 ml with H M , containing 1 mM EDTA and 5 mM triethanolamine (pH 7-5) and centrifuged at 58000 £ for 30 min in a SW40Ti rotor (Sorvall). A white layer at the top of each gradient was removed, the tube topped up with the HM and the gradients re-centrifuged at 70000 £ for 2 h. The combined pellets from the 1-0-1-6 M gradients were suspended in HM and layered onto a 6 ml pre-formed 1-9-2-5 M sucrose gradient and centrifuged at 62000 £ for 2 h. Bands were taken from gradients and diluted with 4 volumes of H M then diluted fractions were centrifuged at 130000 £ for 45 min and the pellets suspended in 200-500/*1 of HM. An aliquot of each fraction was examined by electron microscopy and the remainder stored at - 2 0 °C.
Poly aery lamide gel electrophoresis and Western blotting Samples of sporozoites (5 x 10 6 /track) and gradient fractions (20—50 /d/track) were boiled for 5 min in 50 mM Tris, pH 6 8 , 2 % S D S , 10% glycerol, 100 m.M dithiothreitol and 10/
