The effect of the host lymphocyte lysosomal system on the developing intracellular Theileria annulata sporozoites

Veterinary Parasitology, 26 (1988) 189-198 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

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The Effect of the Host L y m p h o c y t e L y s o s o m a l S y s t e m on the D e v e l o p i n g Intracellular T h e i l e r i a a n n u l a t a Sporozoites W.G.Z.O. JURA*

Veterinary Research Department, Kenya Agricultural Research Institute, Muguga, P.O. Box 32, Kikuyu (Kenya) (Accepted for publication 27 November 1986 )

ABSTRACT Jura, W.G.Z.0., 1988. The effect of the host lymphocyte lysosomat system on the developing intracellular Theileria annulata sporozoites. Vet. Parasitol., 26: 189-198. The fate of developing intracellular Theileria annulata sporozoites within bovine peripheral blood lymphocytes (PBL) was investigated in vitro using acid phosphatase ultracytochemistry. The intracellular sporozoites, while they developed into trophozoites, fed, and transformed into schizonts, provoked intense host lysosomal activity as early as 30 min after they were mixed with lymphocytes. Although viable developing trophozoites failed to fuse with lysosomal vesicles, thereby avoiding enzymatic digestion, the dead parasites readily formed 'phagolysosomes', resulting in lysosomal degranulation and trophozoite digestion. Possible factors responsible for the observed lack of lysosomal fusion with viable trophozoites are discussed.

INTRODUCTION

Mediterranean theileriosis in cattle is caused by the intracellular protozoan parasite, Theileria annulata, which commonly undergoes part of its cyclical development in the ixodid ticks of the genus Hyalomma, its vector. In cattle, parasite development is initiated when viable sporozoites, inoculated with saliva of an infected tick during feeding, reach and invade lymphocytes in the local drainage lymph node where they transform into schizonts. Cytochemical studies (Poore et al., 1981 ) have shown that acid phosphatase activity occurs in about 80% of normal 'resting' T lymphocytes and 45% of B cells and is most frequently observed in secondary lysosomes of varying size and content. Some lymphocytes contained reaction products for acid phosphatase within endo*Present address: International Centre of Insect Physiology and Ecology, P.O. Box 30772, Nairobi, Kenya.

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© 1988 Elsevier Science Publishers B.V.

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plasmic reticulum and the perinuclear cisterna, indicating that they are actively synthesising acid phosphatase. In phytohaemaggtutinin-stimulated lymphocytes (Hirschhorn et al., 1967) biochemical assays have demonstrated a marked net increase in two lysosomal acid hydrotases: acid phosphatase and aryl sulfatase. Lysosomal structures containing these acid hydrolases normally fuse with, and discharge their contents into, phagosomes thus producing digestive vacuoles or phagolysosomes within which degradation of ingested materials takes place. Earlier (Jura, 1983) it was demonstrated that the developing T. annulata trophozoites provoked a massive lysosomal reaction within the host lymphocytes. The trophozoites, however, circumvented the observed intense lysosomal activity and developed into the multinucleate schizont stage. How intralymphocytic stages of T. annulata evade the cytocidal attack by hydrolytic enzymes and other microbicidal factors present within the lysosomal compartment of the host lymphocyte is an intriguing question. This study uses acid phosphatase ultracytochemistry to explore the adaptive processes which allow T. annulata to evade the massive lysosomal attack and replicate within the lymphocyte. MATERIALS AND METHODS

Separation of peripheral blood lymphocytes ( PBL ) and T. annulata sporozoites suspensions and establishment of cultures PBL, separated from defibrinated blood and ground-up tick supernatant (GUTS) of T. annulata sporozoites used at a concentration of 2 ticks ml-1 were obtained by a method modified after Brown (1979) as described previously (Jura, 1984). PBL were separated from the buffer coat obtained from defibrinated blood and layered onto a Ficoll/sodium diatrizoate gradient (Ficoll-Paque, Pharmacia Fine Chemicals, Uppsala, Sweden ). The concentration was adjusted to 4 X 106 cells ml-1 of growth medium, RPMI 1640 with 20% fetal calf serum (Gibco-Europe), containing 100 i.u. ml-1 benzylpenicillin, 100/lg m l - 1 streptomycin sulphate ( Glaxo Laboratories, Greenford, England) and 2 mM m l - 1 L-glutamine ( Gibco-Europe ). GUTS of T. annulata ( Hissar ), prepared by grinding up 3-day fed, infective Hyalomma anatolicum anatolicum ticks, was centrifuged at 100×g for 5 min (1000 r.p.m., MSE Minor; MSE Scientific Instruments, Manor Royal, Crawley, Sussex, England) and the supernate filtered through a sterile 25 mm Millipore Swinnex filter holder, AP25 prefilter and 8/lm filter. The filtrate of sporozoites was diluted and used at a concentration equivalent to 2 ticks m l - 1of Eagle's minimum essential medium (MEM) containing 3.5% bovine plasma albumin, BPA (Armour fraction V, Sigma Chemical Company, St. Louis, Missouri). Fourteen cultures, seven controls and seven tests, were established in a multi-well cluster plate by mixing 0.25 ml (2X 106 cells) PBL suspension with 0.25 ml T. annulata sporozoite

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suspension. The plate with cultures was then placed in a humidified plastic box, gassed with 5% C02/95% air and incubated at 37°C. Sampling for acid phosphatase ultracytochemistry was carried out at 0.5, 1, 3, 6, 18, 24, 48 and 72h.

Acid phosphatase ultracytochemistry A modification of the technique recommended by Barka and Anderson (1962) was used for the cytochemical demonstration of lysosomal activity within infected and non-infected cells. Non-infected PBL were processed immediately and examined for the presence of acid hydrolases. After appropriate incubation periods, two cultures were transferred into plastic, disposable universal bottles, centrifuged at 275xg for 10 min, (1450 r.p.m., MSE Minor at 20°C) and the supernate discarded. The pellets were resuspended in serum-free Eagle's MEM with Earle's salts and centrifuged again at 275 Xg for 5 min as above, and the resultant pellets were fixed in 1.5 % glutaraldehyde in 0.1 M sodium cacodylateHC1 buffer, pH 7.4, containing 1% sucrose at 4°C for 2 h. The pellets were washed several times in 0.1 M sodium cacodylate HC1 buffer, pH 7.4 above. They were then washed for 20 mins in sucrose-Tris/maleate buffer composed of 12.5 ml 0.2 M Tris/maleate buffer, pH 5.2, 37.5 ml deionised distilled water and 4.2 g sucrose. While one of the pair of the washed pellets was treated as a test sample, the remainder was processed as a control and incubated at 37 °C for 1 h in an incubation medium lacking the substrate, sodium-fl-glycerophosphate. The test pellet was incubated for 1 h at 37 °C in a complete incubation medium comprising 10 ml 0.2 M Tris/maleate buffer, pH 5.2, 4 ml 0.1 M sodium-fl-glycerophosphate, 6 ml 0.02 M lead nitrate, 30 ml distilled water and 4.2 g sucrose. After incubation, both control and test pellets were washed over a period of 1 h in three changes of sucrose-Tris/maleate buffer, post-fixed in a 2:1 mixture (v/v) of 1% OsO4: 2.5% glutaraldehyde in 0.1 M sodium cacodylate-HC1 buffer, pH 7.4 for 1.5 h. The pellets were then washed in three changes of 0.1 M cacodylate buffer, pH 7.4, and stained en bloc with 0.5% buffered uranyl acetate (Watson, 1958) containing 4% sucrose for 1 h at 20°C. The samples were dehydrated in graded series of ethanol and embedded in araldite. Sections for electron microscopic examination, cut with LKB glass knives on Cambridge-Huxley ultramicrotome and mounted on 200-mesh copper grids, were stained briefly (1-2 min) in lead citrate (Reynolds, 1963 ). Double staining with uranyl acetate was avoided as it would increase the density and make it difficult to recognise the reaction product. RESULTS

The acid phosphatase reaction product, lead phosphate, was demonstrated in the majority of non-infected PBL, most frequently in secondary lysosomes although in some instances it was found localised within profiles of rough en-

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Fig. 1. A non-infected bovine peripheral blood lymphocyte processed to demonstrate acid phosphatase activity. Electron-dense lead phosphate deposits showing acid phosphatase activity are localised in secondary lysosomes, occasional strands of endoplasmic reticulum and what appears to be a saccute of the Golgi complex (GC). Magnification X 53 750.

Fig. 2. T. annulata sporozoite shown advancing, with its basal end containing the nucleus, into a target lymphocyte as the latter's plasmalemma invaginates. The intraceUular parasite is already surrounded by lysosomal vesicles (LYV) after only 30 min incubation. Magnification X 44 132.

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Fig. 3. A T. annulata trophozoite is depicted,after 18-24 h incubation,surroundedby numerous lysosomalvesicles.The parasitedisplayswelldevelopedmitochondria(MIT) and profilesof rough endoplasmicreticulum (RER), occasionallycontinuouswith the peUicle.Magnification× 65 925. doplasmic reticulum and the Golgi complex (Fig. 1 ). Samples incubated in the medium lacking sodium-fl-glycerophosphate did not manifest any lead phosphate deposits. Interiorised T. annulata sporozoites were surrounded by lysosomal vesicles as early as 30 min after commencing incubation ( Fig. 2). Samples processed at 18-24 h showed massive lysosomal activity around trophozoites (Fig. 3). Acid phosphatase ultracytochemistry demonstrated, in every instance, lack of fusion of host cell secondary lysosomes, laden with lead phosphate deposits, with the pellicular membranes of viable T. annulata trophozoites (Fig. 4). On the other hand, samples processed at 1 h of incubation showed that dead parasites readily fused with host lysosomes (Fig. 5 ) and were rapidly destroyed. Acid phosphatase activity was demonstrated in both the host lymphocyte and T. annulata trophozoites (Fig. 6), in the latter associated with perinuclear cisterna and rough endoplasmic reticulum. The schizont stage of the parasite was not normally surrounded hy lysosomes. DISCUSSION It has been demonstrated in this study that intracellular T. annulata sporozoites rapidly trigger formation of polymorphic, vacuolar structures which

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Fig. 4. An electron micrograph of developing T. annulata trophozoites within the host cell cytoplasm at 18-24 h of incubation. Host cell secondary lysosomes, laden with lead phosphate deposits are shown in the vicinity of the parasites. No fusion takes place. Magnification × 52 250

contain the degradative hydrolytic enzyme acid phosphatase within host lymphocytes, as demonstrated by the lead phosphate technique. The host lysosomal activity against T. annulata trophozoites, evident as early as 30 min following parasite interiorisation (Fig. 2), intensified around 18-24 h to circumscribe the parasites completely (Fig. 3 ). While dead parasites succumbed to the massive host lysosomal enzyme release, viable metabolising trophozoites did not, and developed to schizonts. Mechanisms by which obligate intracellular parasites overcome the diverse and formidable array of microbicidal factors present in the host lysosomal compartment are varied. In Micrococcus lysodiekticus, it has been observed that the cell walls of the lysozyme-resistant strains contain > 100-fold higher levels of o-acyl groups than the cell walls of the sensitive strain (Brumfitt and Wardlaw~ 1958), Paper chromatography undertaken by Burness and King (1958) identified the acyl groups as being principally acetyl. Brumfitt and Wardlaw (1958) demonstrated that the lysozyme-resistant strains of M. lysodiekticus were able to acetylate certain cell wall hydroxyl groups which normally combine with lysozyme substrate. These

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Fig. 5. A sample processed 1 h after incubation shows a dead T. annulata trophozoite fused with host lysosomes. Lead phosphate deposits depicting acid phosphatase activity are disseminated within the degenerating parasite. Magnification X 94 300.

groups, however, remained free in the sensitive strain. Virulent strains of Mycobacterium tuberculosis contain a group of strongly acidic glycolipid sulphates whereas the avirulent M. tuberculosis do not (Goren et al., 1976). Leishmania species, in whose infections lysosomal fusion with parasitophorous vacuoles takes place as normal (Lewis, 1975)) not only survive but continue to multiply in the phagolysosome (Alexander and Vickerman, 1975; Chang, 1981) . While Leishmania tropica amastigotes rely on an unusual superoxide dismutase to resist destruction (Meshnick and Eaton, 1981)) the resistance of Leishmania mexicana amazonensis to lysosomal enzyme degradation is attributable to surface antigenic glycoproteins (Chang, 1983)) as confirmed by the observation that antigenic glycoproteins from the surface of L. mexicana promastigotes resist degradation when subjected to treatment with solubilised rat liver lysosomes ( Chang and Fong, 1983). Trypanosoma cruzi is able to escape from the original phagosome, by lysis of the investing host membrane and live in direct contact with host cytosol (Kress et al., 1975) so avoiding the essentially vacuole-bound lysosomal enzymes. A number of survival mechanisms have been suggested for Toxoplasma go&i. Whereas Jones and Hirsch (1972) attributed

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Fig. 6. Electron-dense deposits of lead phosphate demonstrating acid phosphatase (AP) activity are illustrated associated with the parasite nuclear membrane and rough endoplasmic reticulum in a T. annulata trophozoite at 18-24 h. Host secondary lysosome,illustrated in close proximity to the parasite, has failed to fuse with it. Magnification × 120 000. failure of lysosome fusion with the parasite to the modification of the endocytic vacuolar membrane, Murray et al. (1980) believed that the ability of T. gondii to parasitise normal resident macrophages was related to its high-level resistance to hydrogen peroxide consequent on the abundance of toxic oxygen product scavengers, catalase and glutathione peroxidase, within the parasite. In the case of T. annulata trophozoites, the exact mechanisms by which the parasites operate to inhibit lysosomal fusion are not clear. It has been demonstrated previously that 30 min following interiorisation, T. annulata sporozoites lose the investing host lymphocyte membrane acquired during entry and remain in direct contact with host cytosol ( J u r a et al., 1983). In this respect, therefore,T. annulata may simulate Trypanosoma cruzi, thus evading lysosomal fusion by rapidly losing the investing host lymphocyte membrane once intracellular. Alternatively, structural and biochemical changes accompanying the dedifferentiation of the sporozoites and their transformation to trophozoites ( J u r a et al., 1983 ) could affect the pellicular characteristics significantly to abrogate

197 lysosomal fusion. T h e actively m e t a b o l i s i n g live t r o p h o z o i t e could be releasing e x c r e t o r y factors t h a t f u n c t i o n as l y s o s o m a l e n z y m e i n h i b i t o r s as r e p o r t e d for L e i s h m a n i a species ( E l - O n et al., 1980). A l t h o u g h d e a d T. a n n u l a t a p a r a s i t e s were s h o w n in this s t u d y to fuse with lysosomal e l e m e n t s , it was n o t determ i n e d w h e t h e r t h e fusion t o o k place before or a f t e r t h e f r a g m e n t a t i o n a n d loss of t h e host l y m p h o c y t e m e m b r a n e a c q u i r e d b y t h e sporozoites d u r i n g interiorisation. T h e r e s o l u t i o n of this p o i n t would clarify the e x a c t m e c h a n i s m operating to e n s u r e i n t r a c e l l u l a r survival of Theileria p a r a s i t e s a n d m a y e v e n t u a l l y facilitate t h e c o n t r o l of t h e parasite. ACKNOWLEDGEMENTS T h e a u t h o r is i n d e b t e d to the D i r e c t o r s of the C e n t r e for T r o p i c a l V e t e r i n a r y M e d i c i n e a n d V e t e r i n a r y R e s e a r c h D e p a r t m e n t , K e n y a Agricultural R e s e a r c h I n s t i t u t e ( M u g u g a ) for t h e p r o v i s i o n o f facilities t h a t m a d e it possible to und e r t a k e this study. H e wishes to t h a n k Drs C.G.D. B r o w n a n d A.C. R o w l a n d for t h e i r i n t e r e s t a n d advice, a n d M r B r i a n K e l l y for t e c h n i c a l assistance. T h i s work was f u n d e d as p a r t of a p r o j e c t s u p p o r t e d b y the Overseas D e v e l o p m e n t A d m i n i s t r a t i o n . W.G.Z.O. J u r a was s p o n s o r e d b y the B r i t i s h Council a n d this s t u d y c o n s t i t u t e d p a r t o f his P h . D . T h e s i s project.

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198 Hirschhorn, R., Hirschhorn, K. and Weissmann, G., 1967. Appearance of hydrolase-rich granules in human lymphocytes induced by phytohaemagglutinin and antigens. Blood, 30: 84-102. Jones, T.C. and Hirsch, J.G., 1972. The interaction between T. gondii and mammalian cells. II. The absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J. Exp. Med., 136: 1173-1194. Jura, W.G.Z.O., 1983. Ultrastructural observations on host-parasite relationships of Theileria annulata and Theileria parva in vitro. Ph.D. thesis, University of Edinburgh, Scotland, 215 pp. Jura, W.G.Z.O., 1984, Factors affecting the capacity of Theileria annulata sporozoites to invade bovine peripheral blood lymphocytes. Vet. Parasitol., 16: 215-223. Jura, W.G.Z.O., Brown, C.G.D. and Kelly, B., 1983. Fine structure and invasive behaviour of the early developmental stages of Theileria annulata in vitro. Vet. Parasitol., 12: 13-44. Kress, Y., Bloom, B.R., Wittner, M., Rowen, A. and Tanowitz, H., 1975. Resistance of Trypanosoma cruzi to killing by macrophages. Nature, 257: 394-396. Lewis, D.H., 1975. In vitro studies on host/parasite interactions between L. m. mexicana and peritoneal macrophages taken from normal and sensitised mice. J. Protozool., 22: 153. Meshnick, S.R. and Eaton, J.W., 1981. Leishmanial superoxide dismutase: A possible target for chemotherapy. Biochem. Biophys. Res. Commun., 102: 970-976. Murray, H.W., Nathan, C.F. and Cohn, Z.A., 1980. Macrophage oxygen-dependent antimicrobial activity. IV. The role of endogenous scavengers of oxygen intermediates. J. Exp. Med., 152: 1601-1624. Poore, T.E., Barrett, S.G., Kadin, M.E. and Bainton, D.F., 1981. Ultrastructural localisation of acid phosphatase in rosetted T and B lymphocytes of normal human blood. Am. J. Pathol. 102: 72-83. Reynolds, E.S., 1963. The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell. Biol., 17: 208-212. Watson, M.L., 1958. Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478.