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The Role of the Cytoskeleton in the Motility of Coccidian Sporozoites

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The Role of the Cytoskeleton in the Motility of Coccidian Sporozoites J. Cell Set. 50, 345-359 (1981) 345 Printed in Great Britain © Company of Biologittt Limited 1981 THE ROLE OF THE CYTOSKELETON IN THE MOTILITY OF COCCIDIAN SPOROZOITES D. G. RUSSELL AND R. E. SINDEN Department of Zoology and Applied Entomology, Imperial College, London SW7 2AZ, England SUMMARY The sporozoites of Eimeria tenella and Eimeria acervulina show bending, pivoting and gliding motility. All these types of motility occur intermittently and with decreasing frequency during the life of a sporozoite. Gliding is the only locomotive action expressed by these sporozoites and is only seen when the sporozoites are in contact with the substratum. All gliding sporozoites adopt a set pattern of body ' attitudes', which suggests that locomotion involves a fixed body shape. The microtubule inhibitors, colchicine, griseofulvin, vinblastine sulphate and nocodazole, have no effect on sporozoite motility. Ultrastructural examination reveals, in addition, that they have no effect on the subpellicular microtubules. The microfilament inhibitor, cytochalasin B, completely, and reversibly, inhibits pivoting and gliding but bending is only slightly depressed by the drug. High magnesium ion concentration inhibits all motility completely. The cell membrane was readily labelled with fluorescein isothiocyanate-conjugated cationized ferritin, the label was rapidly capped and shed from the posterior of the sporozoite. This capping reaction takes place only during sporozoite locomotion. The membrane label was seen to ' move' backwards relative to the sporozoite at the same rate as the sporozoite moved forwards relative to the substratum. The substratum and the leading edge of the cap remained static relative to each other. Both capping and locomotion are sensitive to low temperature and cytochalasin B. From these results a theory of sporozoite motility is postulated. The sporozoites adhere to the substratum by surface ligands. This ligand/substratum complex is then capped along the fixed spiral of the sporozoite body by a microfilament-based contractile system. This proposed model for motility of coccidia sporozoites is consistent with all current observations on cell invasion by the sporozoa and therefore suggests that locomotion is an integral component of host cell invasion in this group of parasites. INTRODUCTION Following ingestion of oocysts, sporozoites of Eimeria species are released within the lumen of the gut of their host, from where they invade the endothelial lining of the gut wall and subsequently undergo schizogony. Schizogonic development is highly site-specific for individual species. This site specificity may result from either the directed migration of the released sporozoites (Marquardt, 1973) or a site- limited potential for development of the intracellular parasite. Despite the potential importance of motility in the coccidian life cycle the cellular basis for locomotion has been the subject of only indirect and fragmentary studies. Jensen & Edgar (1976) demonstrated that various antiphagocytic agents will inhibit the invasion of professional macrophages by sporozoites of Eimeria magna, and 12 CEL JO 346 D. G. Russell and R. E. Sinden concluded that a microfilamentous system could be active during invasion. They also speculated that the subpellicular microtubules do not function in locomotion, unless they are formed of' aggregates of contractile elements sensitive to the inhibitory action of cytochalasin B'. Dubremetz & Ferreira (1978) correlated the motility of Eimeria sporozoites with the capping of the surface-membrane marker, cationized ferritin, and found that both were inhibited by cytochalasin B and low temperatures. Vanderberg (1974) showed that the malarial sporozoite has a limited repertoire of motility, that the sporozoites can attach to substrates, and that they leave a trail behind them, which he speculates to have originated from the rhoptries that may act as a propulsive mechanism similar to a 'camphor-boat'. In contrast to the fragmentary studies on locomotion, the ultrastructure of the coccidian and malarial parasites has been extensively examined. The motile stages show a remarkably conservative organization (Roberts & Hammond, 1970; Jensen & Edgar, 1978; Porchet-Hennere & Vivier, 1971; Sinden, 1978). Structures of potential significance in locomotion are the extensive microtubular cytoskeleton, and the tri- membranous pellicle with its distinctive inner pellicular membranes bearing linear arrays of intramembranous particles (Dubremetz & Torpier, 1978; Porchet & Torpier, 1977; Dubremetz & Ferriera, 1978; D'haese, Mehlhorn & Peters, 1977). This paper analyses the motility of sporozoites of Eimeria acervulina and E. tenella and reports the determination of the structural basis of each component of motility. A model for the cellular mechanisms of motility is proposed and its implications in studies on cell invasion are discussed. MATERIALS AND METHODS Oocysts of E. acervulina were kindly supplied by Dr J. Spelman (May & Baker, Ongar, Essex) and those of E. tenella by Dr R. Williams (Burroughs-Wellcome, Berkhamsted, Berk- shire). These were stored in 2 % potassium dichromate solution until required, whereupon they were excysted by treatment with hypochlorite, shaking with grade 7 Ballotini beads, and incubating in trypsin and bile in Hanks' buffered salts solution (HBSS) at 41 °C (Davis, 1973). A cleaned suspension of sporozoites was obtained by sieving the incubated prepara- tion through a io-/im pore bolt cloth filter. Motility studies Preliminary studies revealed a slight decline in sporozoite motility if oocysts were stored in potassium dichromate solution at 4 °C for more than 2 weeks. All studies have therefore been confined to sporozoites obtained from oocysts stored for less than this period. Motility was analysed by videotape recording of closed circuit television (CCTV) images from a Wild phase-contrast microscope with a controlled environment chamber maintained at 41 °C. Microfilament and microtubule inhibitor studies The effects of various concentrations of drugs on Sporozoite motility were recorded as described above, and ultrastructural changes were examined by electron microscopy (see below). Microrubule-assembly inhibitors included exposure to low temperature 0-4 °C and the compounds colchicine, vinblastine sulphate, griseofulvin (at concentrations of 0—200 fig/ ml) and nocodazole (at 0-20 /ig/ml). Incubation periods varied from 5 min to 6 h, the cells being incubated at either 4 °C or'41 °C. Microfilament inhibitors used included cytochalasin B (Lin, Lin & Flanagan, 1978) and magnesium salt solutions at high concentrations (5-15 mM- The cytoskeleton of coccidia 347 MgClj) (McGee-Russell & Allen, 1971; Weihing, 1976). Following solution in dimethyl sulphoxide (DMSO), cytochalasin B solutions (i-io/Jg/ml) were adjusted to contain o-i% (v/v) DMSO in HBSS. Experiments in this case were controlled by sporozoites suspended in o-i % (v/v) DMSO. No difference was observed between these controls and sporozoites in HBSS alone. Surface labelling of sporozoite plasmalemma Sporozoites were incubated with the following fluorescein-labelled reagents: the lectins, concanavalin A (Miles-Yeda) and those of Ricinus communis and peanut (Sigma chemicals): and the anionic marker, cationized ferritin (Sigma). Fluorescein-labelled cationized ferritin was prepared by the method of King & Preston (1977), briefly; 11-5 mg of cationized ferritin in 1 ml of 0-05 M-Tris-HCl buffer (pH 7-6) was mixed for 30 min at room temperature with 1-5 mg of ftuorescein isothiocyanate (FITC) on io%(w/v) Celite. Following conjugation, excess fluorochrome was removed by chromatography on Sephadex G25. The labelled eluant Table 1. Specificity of binding of membrane labels to the surface of E. acervulina and E. tenella Binding reaction with parasite surface Cell membrane label Sporozoites Sporocysts Oocysts Lectins Concanavalin A (FITC) - - + Concanavalin A (FITC) + a-methyl-D-mannoside — — — R. communis (FITC) - - + R. communis (FITC) 4- D-galactose — — — Peanut (FITC) - - + Peanut (FITC) + D-galactose - - - Anionic charge markers Cationized ferritin + + + Cationized ferritin (FITC) + + + Fixed and unfixed parasites were incubated at 4 and 41 °C with the lectins. Binding specificities are similar in all cases. The oocysts show slight autofluorescence but a marked increase in fluorescence ( + ) demonstrated binding. was diluted 1 in 5 in HBSS for use. Preparations were examined in a Leitz Orthoplan incident fluorescence microscope. Sporozoite suspensions were incubated in the diluted fluorescein- labelled preparation or with unlabelled cationized ferritin (0-575 mg/m0 at 4 °C and 41 °C in the presence and absence of cytochalasin B (10/ig/ml) or 15 mM-magnesium chloride. Sporozoite suspensions were washed 3 times in HBSS (containing cytochalasin B and magnesium ions where appropriate) and prepared for fluorescence or electron microscopy as appropriate. Lectin distribution was examined in sporozoites both before and after fixation in 0-2 % glutaraldehyde, in the latter case the parasites were incubated in 5 % (w/v) bovine serum albumin to inactivate residual, aldehyde terminal groups then washed in HBSS before labelling. All sporozoites were incubated at 4 °C and 41 °C in lectin, lectin plus the appropriate saccharide inhibitor (at 100 mM) or lectin plus cytochalasin B (10/tg/ml) for 15-40 min (see Table i), and then washed 3 times in HBSS before examination by fluorescence microscopy. Electron microscopy Sporozoite suspensions were negatively
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