Acta Tropica, 50(1992)67-78
67
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00168
Surface coat synthesis and turnover from epimastigote to bloodstream forms of Trypanosoma brucei U.-P. Modespacher*, W. Rudin and H. Hecker Swiss Tropical Institute, Basel, Switzerland
(Received 7 March 1991; accepted 28 May 1991) Monoclonal antibodies to metacyclic surface coat glycoproteins of Trypanosoma brucei brucei STIB 247LG were produced for a study of the synthesis of metacyclic variable surface glycoproteins (VSGs) within the salivary gland of Glossina morsitans morsitans, and of the first exchange of the surface glycoproteins after infection in mice. Immunofluorescence antibody tests and protein A-gold labelling r6vealed that the VSGs are continuously integrated into the whole surface of the trypanosome while it is still attached to the gland epithelium. A pool of 8 antibodies recognized about 50% of the metacyclic forms present in the saliva of an infected tsetse fly, which confirmed the heterogeneity of the metacyclic VSG-generation. The labelling experiments showed that the integration of the first VSG-generation into the surface of bloodstream forms takes place in the same way as in the metacyclics. This process started on day 3 after infection and was finished on day 6. Key words: Trypanosoma brucei; Variant surface glycoprotein; Monoclonal antibody; Cryo-ultramicrotomy
Introduction A f r i c a n t r y p a n o s o m e s , h a e m o f l a g e l l a t e s p a r a s i t i c in m a n a n d d o m e s t i c animals, cause c h r o n i c a n d , sometimes, lethal infections. These p r o t o z o a n s are t r a n s m i t t e d by tsetse fies, in which p a r t o f the p a r a s i t i c life-cycle takes place. In the m a m m a l i a n host they can escape the i m m u n e response o f the h o s t by varying, b i o c h e m i c a l l y a n d antigenically, their g l y c o p r o t e i n surface c o a t (VSG: v a r i a b l e surface g l y c o p r o t e i n ) ( D o n e l s o n a n d R i c e - F i c h t , 1985; Borst, 1986). The V S G s c o v e r the entire surface o f the t r y p a n o s o m a l cell d u r i n g the course o f infection in the m a m m a l i a n host. T h e y are lost in the digestive t r a c t o f the v e c t o r after the u p t a k e o f the p a r a s i t e s with an infective b l o o d m e a l , a n d are synthesized a g a i n in the metacyclic forms in the salivary glands, at the end o f the cycle in the tsetse fly. A l t h o u g h a single metacyclic t r y p a n o s o m e expresses o n l y one type o f V S G at a time, the whole metacyclic p o p u l a t i o n within the salivary g l a n d is h e t e r o g e n e o u s with respect to the V S G s expressed (Barry et al., 1979; H a j d u k et al., 1981, N a n t u l y a et al., 1983). Correspondence address." PD Dr. W. Rudin, Swiss Tropical Institute, Postfach, CH-4002 Basel, Swit-
zerland. *Present address. F. Hoffmann-La Roche LTD, CH-4002 Basel, Switzerland.
68 Synthesis and intracellular transport of VSGs have been intensively investigated in bloodstream forms both biochemically and ultrastructurally (Lheureux et al., 1979; Bangs et al., 1985; Bangs et al., 1986; Boothroyd et al., 1980; Boothroyd et al., 1981; Ferguson et al., 1986; Holder, 1985; McConnell et al., 1983; Duszenko et al., 1988; Grab et al., 1984; Steiger, 1973; Vickerman and Luckins, 1969; Webster, 1989). It could be shown that VSGs are synthesized by the rough endoplasmic reticulum, where signal peptide cleavage and some cotranslational glycosylation of oligosaccharides occur, including the addition of a glycan-phosphatidylinositol anchor (Cross, 1990). Further protein processing steps and the incorporation of various sugar residues take place in the Golgi apparatus and in the tubulovesicular network (Webster, 1989). This network consists of tubular structures and flattened cisternae on the trans side of the Golgi apparatus, and is located between the Golgi apparatus and the flagellar pocket. From there the VSGs are transported by coated vesicles to the flagellar pocket, where they are integrated into the surface coat. Some days after the metacyclic trypanosomes have been transferred to a mammalian host by the bite of an infective fly, they transform to bloodstream forms. This transformation includes not only a change of the morphology of the trypanosomes, but also a first exchange of the VSGs on the surface. The aim of the present work was to investigate the time of appearance and the intracellular transport of metacyclic VSGs in trypanosomes in the salivary gland of the tsetse fly, and also the exchange of metacyclic VSGs for the first generation of bloodstream VSGs in the mammalian host. For this purpose immunocytochemical labelling experiments with protein A-gold on ultrathin cryosections of trypanosomes at different stages were carried out, using monoclonal antibodies (mAb) raised against metacyclic VSGs of Trypanosoma brucei brucei STIB 247 LG.
Material and Methods
Trypanosomes. The experiments were carried out with Trypanosoma brucei brucei STIB 247 LG (recloned STIB 247) (Geigy and Kaufmann, 1973). Trypanosoma brucei rhodesiense STIB 386 AA (Richner and Jenni, 1986) was used as a control for the specificity of the monoclonal antibodies to STIB 247 LG in labelling experiments. The procyclic parasites were cultured and maintained according to Brun and Sch6nenberger (1979) and Brunet al. (1981). Flies and mice. Glossina morsitans morsitans were received from the Tsetse Research Laboratory, Langford/Bristol colonies and Glossina morsitans centralis from ILRAD, Nairobi/Kenya. Female Swiss ICR mice were used for infection by intraperitoneal injection of reactivated stabilates (Lanham and Godfrey, 1970) to produce bloodstream forms. Female C57/bl mice were immunized for raising antisera to metacyclics and Balb/C mice for harvesting spleen cells for production of monoclonal antibodies. Polyclonal antiserum to metacyelic trypanosomes. C57/bl mice were infected by infectious bites of 2 tsetse flies per mouse. The same 2 flies were fed a second time on the same mouse after another 2 days. 6 h later the mice were treated with a single dose (25 mg/kg body weight) of Berenil. After treatment, the mice received booster infections by allowing the same flies to feed on them 5 more times at 2-day intervals.
69 The antiserum harvested by heart puncture 2 days after the last booster was stored at - 70°C. Monoclonal antibodies to the coat. The mAb were produced by slight modifications of the method described by Nantulya et al. (1983). Three Balb/C mice were infected with 10 flies delivering metacyclic trypanosomes in their saliva, and were treated with Berenil 24 h later (see above). Booster infections were done on day 13 and 23 with the same flies. On day 28 the spleen cells were harvested and fused with X63 (Hoechst) myeloma cells. After 2 weeks the supernatants of the cultures were checked by an indirect immunofluorescence test on fixed metacyclic trypanosomes from tsetse saliva and on fixed procyclic culture forms. The positive cell cultures were cloned by limiting dilution. Immunofluorescence antibody test (IFAT). Saliva containing metacyclic T.b. brucei was harvested by allowing infective flies to probe on a defined area of a glass slide. The saliva was dried for 30 min at 37°C followed by fixation in 100% acetone for 10 min. After air-drying they were stored at - 8 0 ° C . Procyclic culture forms in medium were mixed with an equal volume of FBS, applied to glass slides, and then treated as described above. For indirect immunofluorescence labelling the slides with the trypanosomes were rehydrated by incubation with PBS for 10 min prior to the reaction either with serial dilutions of the polyclonal antiserum or with crude hybridoma supernatants. Subsequently they were washed with PBS and then incubated with goat anti-mouse-FITC (Cappel) for 20 min, washed again, and finally air dried. As a control, the slides were incubated with PBS instead of the antiserum or supernatant. Examination was done under UV-light in a Leitz microscope. Electron microscopy. Salivary glands of infective tsetse flies were dissected under a W I L D stereomicroscope and immediately fixed for 1 h in 0.5% (v/v) glutaraldehyde in 0.2 M Pipes buffer, pH 7.4. After embedding in 8% (w/v) gelatine Type IV (Sigma), in Pipes buffer, they were cut into small pieces of about 1 mm length, postfixed in glutaraldehyde for another 30 min, and then transferred into 2.3 M sucrose in Pipes buffer for penetration with the cryoprotectant overnight at 4°C. The specimens were frozen by dropping them into liquid nitrogen after mounting on copper studs, and were stored in liquid nitrogen until cryosectioning. The blood of infected mice was collected and mixed with an equal volume of phosphate buffered saline with glucose (PSG, 6:4), pH 8.0, and centrifuged for 10 rain at 70 xg. The supernatant containing the trypanosomes was harvested and again centrifuged for 20 rain at 3700 x g. The pelleted trypanosomes were resuspended in 0.5% glutaraldehyde in 0.2 M Pipes buffer, pH 7.4, transferred into an Eppendorf tube and fixed for 1 h before another centrifugation for 1 min at 8000 ×g. The supernatant was drawn off and the pellet resuspended in a small volume of warm (45°C) 8% (w/v) gelatine in Pipes, centrifuged again and then solidified on ice. The contents of the tube were removed and transferred to glutaraldehyde for 30 min to fix the gelatine. Pyramid shaped pieces of a maximum size of 1 mm 3 were cut from the gelatine blocks and penetrated over night with 2.3 M sucrose in 0.2 M Pipes before mounting and freezing as above. Ultracryomicrotomy was done as described elsewhere (Griffith et al., 1983; Griffith et al., 1984; Tokuyasu, 1986). Immunocytochemistry. Monodisperse colloidal gold sol containing gold particles of homogeneous size (9 nm) was produced as reported in the literature (Slot and Geuze, 1985). The pH of the gold sol was adjusted to pH 6.0 with 70 mM citric acid before formation of protein A (Pharmacia)-gold complexes (Horisberger and Clerc,
70 1985). The complexes were stored in 50% (v/v) glycerol at - 30°C. Ultrathin cryosections were cut on a Reichert Ultracut E equipped with a Reichert FC 4 cryochamber. The sections were preincubated for 10 min with 10% (v/v) foetal bovine serum (FBS) in 20 m M PBS, followed by glycine (1.5 mg/ml) for 3 min, and unlabelled protein A (40 gm/ml) in PBS with 10% FBS for 15 rain. The incubations with the monoclonal antibodies were performed on 5 gl drops for 30 min. After washing intensively with PBS the sections were incubated for 15 min with 5 tal PAG diluted to A525 0.05 with PBS/FBS. Another washing with PBS and distilled H 2 0 was carried out after the incubation with the marker solution. As a control, sections were incubated with PBS/FBS instead of antibody.
Results Eighteen hybridoma cell cultures resulting from the fusion of spleen cells, harvested from a mouse infected with T.b. brucei 247 LG, and X63 myeloma cells were successfully maintained. The supernatants were tested for their reactivity against T.b. brucei from tsetse fly saliva by indirect immunofluorescence after three weeks. Six supernatants were positive for metacyclic forms of the strain 247 L G but not for T.b. rhodesiense strain 386 AA and not for procyclic culture forms. The antibodies produced in 2 other hybridoma cultures recognized procyclic forms (strain 247 LG) only. Four cultures specific for metacyclics and 2 specific for procyclics were cloned by limiting dilution (0.5 cells per well). Fourteen clones specific for metacyclic trypanosomes of the strain 247 LG and 6 specific for procyclic and epimastigote trypanosomes resulted. Each antibody specific for metacyclics recognized only a small proportion of the population present in the saliva of an infectious tsetse fly (Fig. 1), which demonstrated the heterogeneity of the antigen type expressed on the surface of the metacyclic trypanosomes. The monoclonals against procyclic culture forms recognized all procyclic and epimastigote forms (Fig. 2). A pool of 8 monoclonal antibodies to metacyclic trypanosomes of the isotype IgG 2a, identified by the Ouchterlony-double diffusion test, was used for immunocytochemical localisation of the respective antigens on ultrathin cryosections by the use of protein A-gold. The labelling on the surface of the metacyclics within the salivary gland was highly specific, and showed individual differences between cells in the labelling density (Figs. 3 and 4). I m m u n e reactions could be observed on the whole
Fig. 1. Metacyclic T.b. brucei STIB 247 LG in saliva delivered from an infective tsetse fly. Proportion of metacyclic trypanosomes recognized by one monoclonal antibody to metacyclic VSG in an immunofluorescence antibody test ( × 800). Fig. 2. Epimastigote T.b. brucei STIB 247 LG in saliva delivered from an infective tsetse fly recognized by a monoclonal antibody to procyclic trypanosomes in an immunoftuorescenceantibody test ( × 800). Fig. 3. Binding sites of a pool of 8 moabs to metacyclic VSG on ultrathin frozen sections of T.b. brucei in salivary glands of infected tsetse flies demonstrated with protein A-gold. High surface labelling density (arrowheads) ( × 70 000). Fig. 4. Same as Fig. 3 but low surface labelling density (arrowheads) ( × 72 000).
72 surface; cell body and flagellum. Trypanosomes attached to the epithelium of the salivary gland were also labelled all over their surface, including the surface of the hemidesmosomes at the contact site between vector and parasite (result not shown). Furthermore, the flagellar pocket (Fig. 5), the Golgi zone, the multivesiculated network (Fig. 6) between Golgi and flagellar pocket, and coated vesicles were labelled in the same trypanosomes. The pool of monoclonal antibodies (see above) was also applied to frozen thin sections of trypanosomes isolated from mice 3, 4, 5, and 6 days after infection. All the parasites of these stages which were recognized also showed labelling over the whole surface and the lining of the flagellar pocket which varied in density between individuals (Fig. 7). In contrast to the situation with metacyclics in the salivary gland, however, the antibodies did not bind to intracellular structures (Fig. 8). An estimation of the percentage of labelled trypanosomes from 3-6 days after infection (Fig. 9) showed a massive drop between days 4 and 5, from more than 40% to only about 5%. After six days no trypanosomes were recognized by the pool of antibodies to the metacyclic coat.
Discussion The surface coat of blood trypanosomes, consisting of variable surface glycoprotein, is lost after the uptake of the parasites by tsetse flies (Barry and Vickerman, 1979; Molyneux and Ashford, 1983). A new coat is synthesized only after the migration of the trypanosomes to the salivary glands. For the last transformation step in the vector, the parasites attach to the salivary gland epithelium by differentiating hemidesmosomes (Steiger, 1973; Vickerman, 1973). It was found in the present study that coat formation takes place on the surface of attached stages. This confirms earlier observations, where these forms were described as immature stages (Tetley and Vickerman, 1985; Tetley et al., 1987). The immunocytochemical results of the present study allow some conclusions to be drawn about the synthesis of the coat. The fact that the density of the labelling varies between individuals but is regular on the whole of an individual trypanosome, suggests that a continuous formation all over the cell surface occurs. On the other hand, there is evidence that the flagellar pocket is the site of exocytosis of coat material, because endo- or exocytosis is said to be restricted to surface areas where
Fig. 5. Binding sites of a pool of 8 mAbs to metacyclicVSG on ultrathin frozen sections of T.b. brucei in salivary glands of infected tsetse flies demonstrated with protein A-gold. Binding to the flagellar pocket (fp) ( x 90 000). Fig. 6. Binding to the multivesicular network (mvn) and to the flagellum (fl) ( × 71 000). Fig. 7. T.b. brucei harvested from mouse blood 3 days after infection. All forms recognized by the pool of mAbs to metacyclic VSG are labelled on the whole surface (arrowheads). The individual labelling density varies ( x 23 000). Fig. 8. Same as Fig. 7. Lack of binding of mAbs to metacyclicVSG to intracellular structures in opposition to salivary gland forms ( x 65 000).
74
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203040 lOo
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3 4 5 Days alter infection
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Fig. 9. T.b. brucei, 247 LG bloodstream forms, 3-6 days after cyclical transmission. Percentage of goat anti-mouse-FITC labelled trypanosomes after prior reaction with mAb-pool to metacyclic forms.
no subpellicular microtubules are present (Brown et al., 1965; Langreth and Balber, 1975; Frevert and Reinwald, 1988; Webster and Grab, 1988; Webster and Fish, 1989). The presence of coated vesicles near the flagellar pocket supports this hypothesis (Steiger, 1973). In the present study, not all of the trypanosomes in the saliva of an infective tsetse fly could be immunofluorescence labelled with a single clone of monoclonal antibody to metacyclic forms, whereas a polyclonal antiserum recognized all the parasites. When a pool of 8 different monoclonal antibodies was used for immunocytochemical labelling on ultrathin cryosections of trypanosomes harvested 3 days after infection of the vertebrate host, 48.2% of the parasites were labelled. Assuming that on day 3 after infection all trypanosomes are still covered with metacyclic VSG (see below), one can calculate that approximately some twenty variant m-VSG were expressed in the population tested. This fits the published data about the m-VSG heterogeneity of T.b. brucei (Le Ray et al., 1978; Nantulya et al., 1983; Barry and Vickerman, 1979; Barry and Emery, 1984; Hajduk et al., 1981), T.b. rhodesiense (Turner et al., 1988), and T. congolense (Luckins et al., 1986; Prain and Ross, 1988). It is this heterogeneity which decreases the chance of developing a vaccine directed against the infective stage of trypanosomes, so the present trend in vaccine development is oriented towards common structural antigens (Holder and Cross, 1981), towards flagellar pocket antigens (Balber, 1989) or transmission blocking antigens (Roditi et al., 1988). The binding of monoclonal antibodies to metacyclic VSG on cryosections of T.b. brucei in salivary glands of G.m. morsitans, which was visualised by the use of protein A-gold, did not occur on the parasite surface only. There was also binding to intracellular structures (the Golgi zone, multivesiculated network and coated vesicles) and to the surface of the flagellar pocket. This corresponded to the findings of other authors with regard to the synthetic pathway of VSG molecules (Steiger, 1971; Webster and Grab, 1988; Grab et al., 1984; Webster, 1989; Webster and Fish, 1989). Trypanosomes released into the vertebrate host by a fly bite keep their metacyclic surface coat for a few more days. The present study showed that the highest transformation rate for the T.b. brucei strain STIB 247 LG in mice occurs between
75 day 4 and day 5. The percentage of trypanosomes recognized by a pool ofmonoclonal antibodies to metacyclic VSG, which reacts with about 50% of the parasites in the salivary gland, is more or less unchanged until day 4 (42.3%), drops to a few percent (4.7%) on day 5 and to 0 on day 6. This corresponds well with the observations of Hajduk and Vickerman (1981), though it is one day later than it was found by Nantulya et al. (1983). What happens to the metacyclic VSGs when they are replaced by the first generation of bloodstream form VSGs is still under discussion. On the one hand, it has been postulated that the release takes place by vesiculation from the trypanosome surface, called capping (Barry and Vickerman, 1979; Barry, 1986; Shapiro, 1986). On the other hand, recycling by endocytosis in the flagellar pocket, as it has been postulated for bloodstream VSGs, could occur (Webster and Grab, 1988). Capping was never observed in the present study, but, in earlier experiments a release of membrane bound VSG by vesiculation occurred after pre-embedding immunolabelling when living trypanosomes were exposed to the antiserum (unpublished results). The monoclonal antibodies to metacyclic VSGs showed, if ever (aggregate on Fig. 6), very limited binding to intracellular structures in the trypanosomes isolated from mouse blood. In contrast, there was significant binding to intracellular structures in the salivary gland forms. This supports the capping theory. However, the lining of the flagellar pocket was labelled in all trypanosomes recognized by the mAb, even at day 6. This seems to fit the recycling theory for metacyclic VSGs. The presence of enzymes within the flagellar pocket (Steiger, 1971; Grab et al., 1989) which are able to cleave the VSGs could explain why endocytosis could not be shown in the present work. It is unlikely that the glycosyl-phosphatidylinositol-specificphospholipase C plays this role, being located on the outer side ofintracellular vesicles (Buelow and Overath, 1985; Buelow et al., 1989; Grab et al., 1989; Fox et al., 1986; Grab et al., 1987). However, the recycling theory on the one hand, and the circumstances under which capping occurs, on the other hand, need further investigation. Another point which also needs additional clarification is the way in which a new VSG generation is deposited on the trypanosome surface. By double immunolabelling using mAbs to two consecutive coat types it could perhaps be shown whether this process involves a continuous integration of the new molecules into the previous coat over the whole surface, as has already been shown by immunofluorescence labelling (Esser and Schoenbechler, 1985), or is the outgrowth of a fully developed new one from the flagellar pocket.
Acknowledgements The skilful technical assistance of Mrs. E. Fluri, C. Knoell, and R. Rufener is gratefully acknowledged. We further wish to thank Dr. Paul Webster (ILRAD, Nairobi, Kenya) for his support in the establishment of the cryotechniques and Mrs. J. Jenkins for English corrections. The work has partly been supported by Roche Research Foundation, Fonds fur Forderung von Lehre und Forschung, SandozStiftung zur F6rderung der med.-biol. Wissenschaften, Basel, Switzerland, and Swiss National Science Foundation Grant No. 3.530-083.
76
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77 localisation of variable surface glycoprotein phosphatidylinositol-specific phospholipase C in African trypanosomes. J. Cell Biol. 105, 737-746. Grab, D.J., Lonsdale-Eccles, J. and Webster, P. (1989) Endocytosis and the metabolic pathways of variable surface glycoproteins in the African trypanosome: possible targets for attack. ILRAD Reports, January 1989, 1-6. Griffiths, G., Simons, K., Warren, G. and Tokuyasu, K.T. (1983) Immunoelectron microscopy using thin, frozen sections: application to studies of the intracellular transport of Semliki Forest Virus spike glycoproteins. Methods Enzymol. 96, 466 485. Griffiths, G., McDowall, A., Back, R. and Dubochet, J. (1984) On the preparation of cryosections for immunocytochemistry. J+ Ultrastr. Res. 89, 65-78. Hajduk, S.L. and Vickerman, K. (1981) Antigenic variation in cyclically transmitted Trypanosoma brucei. Variable antigen type composition of the first parasitaemia in mice bitten by trypanosome-infected Glossina morsitans. Parasitology 83, 609-621. Hajduk, S.L., Cameron, C.R., Barry, J.D. and Vickerman, K. (1981) Antigenic variation in cyclically transmitted Trypanosoma brucei. Variable antigen composition of metacyclic trypanosome population from salivary glands of Glossina morsitans. Parasitology 83, 595-607. Holder, A.A. (1985) Glycosylation of the variant surface antigens of Trypanosoma brucei. Curt. Trop. Microbiol. lmmunol. 117, 57-74. Holder, A.A. and Cross, G.A.M. (1981) Glycopeptides from variant surface glycoproteins of Trypanosoma brucei. C-terminal location of antigenically cross-reacting carbohydrate moieties. Molec. Biochem. Parasito. 2. 135-150. Horisberger, M. and Clerc, M.-F. (1985) Labelling of colloidal gold with protein A. A quantitative study. Histochemistry 82, 219-223. Langreth, S.G. and Balber, A.E. (1975) Protein uptake and digestion in blood-stream and culture forms of Trypanosoma brucei. J. Protozool. 22, 40-53. Lanham, S.M. and Godfrey, D.G. (1970) Isolation of salivarian trypanosomes from man and other mammals using DEAE-cellulose. Exp. Parasitol. 28, 521-534. Le Ray, D., Barry, J.D. and Vickerman, K. (1978) Antigenic heterogeneity of metacyclic forms of Trvpanosoma brucei. Nature 273, 300-302. Lheureux, M., Lheureux, M., Vervoort, T., van Meirvenne, N. and Steinert, M. (1979) Immunological purification and partial characterization of variant-specific surface antigen messenger RNA of Trypanosoma brucei. Nucl. Acid Res. 7, 595-609. Luckins, A.G., Frame, I+A+, Gray, M.A., Crowe, J.S. and Ross, C.A. (1986) Analysis of trypanosome variable antigen types in cultures of metacyclic and mammalian forms of Trypanosoma congolense. Parasitology 93, 99 109. McConnell, J., Turner, M. and Rovis, L. (1983) Biosynthesis of Trypanosoma brucei variant surface glycoproteins - - analysis of carbohydrate heterogeneity and timing of post-translational modifications. Mol. Biochem. Parasitol. 8, 119-135. Molyneux, D.H. and Ashford, R.W. (1983) The biology of Trypanosorna and Leishmania, parasites of man and domestic animals. Taylor and Francis, London. Nantulya, V.M., Musoke, A.J., Moloo, S.K. and Ngaira, J.M. (1983) Analysis of the variable antigen composition of Trvpanosoma brucei brucei metacyclic trypanosomes using monoclonal antibodies. Acta Trop. 40, 19-24. Prain, C+J. and Ross, C.A. (1988) Trypanosoma congolense: interactions between trypanosomes expressing different metacyclic variable antigen types in vitro and in vivo. Parasitology 97, 277 286. Richner, D. and Jenni, L. (1986) Characterization of cyclically transmitted Trypanosoma (T.) brucei isolates from man. Acta Trop. 43, 21-29. Roditi, I., Dobbelaere, D., Williams, R.O., Masterson, W., Beecroft, R.P., Richardson, J.P. and Pearson, T.W. (1988) Expression of Trypanosoma brucei procyclin as a fusion protein in E. coli. Molec. Biochem. Parasitol. 34, 35 43. Shapiro, S.Z. (1986) Trvpanosoma brucei: Release of variant surface glycoprotein during the parasite life cycle. Exp. Parasitol. 61,432 437. Slot, J.W. and Geuze, H.J. (1985) A new method of preparing gold probes for multi-labelling cytochemistry. Europ. J. Cell Biol. 38, 87 93. Steiger, R. (1971) Some aspects of the surface coat formation in Trypanosoma brucei. Acta Trop. 28, 341-346. Steiger, R. (1973) On the ultrastructure of Trypanosoma brucei in the course of its life cycle and some related aspects. Acta Trop. 30, 64-168.
78 Tetley, L. and Vickerman, K. (1985) Differentiation in Trypanosoma brucei: host-parasite cell junctions and their persistence during acquisition of the variable antigen coat. J. Cell Sci. 74, 1-19. Tetley, L., Turner, C.M.R., Barry, J.D., Crowe, J.S. and Vickerman, K. (1987) Onset of expression of the variant surface glycoproteins of Trypanosoma brucei in the tsetse fly studied using immunoelectron microscopy. J. Cell Sci. 87, 363-372. Tokuyasu, K.T. (1986) Application of cryoultramicrotomy in immunocytochemistry. J. Microsc. 143, 139-149. Turner, C.M.R., Barry, J.D., Maudlin, I. and Vickerman, K. (1988) An estimate of the size of the metycyclic variable antigen repertoire of Trypanosoma brucei rhodesiense. Parasitology 97, 269-276. Vickerman, K. (1973) The mode of attachement of Trypanosoma vivax in the proboscis of the tsetse fly Glossina fuscipes: an ultrastructural study of the epimastigote stage of the trypanosome. J. Protozool. 20, 394-404. Vickerman, K. and Luckins, A.G. (1969) Localization of variable antigens in the surface coat of Trypanosorna brucei using ferritin conjugated antibody. Nature 224, 1125-1126. Webster, P. (1989) Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense. Europ. J. Cell Biol. 49, 295-302. Webster, P. and Grab, D.J. (1988) Intracellular colocalization of variant surface glycoprotein and transferrin-gold in Trypanosoma brucei. J. Cell Biol. 106, 2")9-288. Webster, P. and Fish, W.R. (1989) Endocytosis by African trypanosomes. II. Occurrence in different life,. cycle stages and intracellular sorting. Europ. J. Cell Biol. 49, 303-310.
67
© 1991 Elsevier Science Publishers B.V. All rights reserved. 0001-706X/91/$03.50 ACTROP 00168
Surface coat synthesis and turnover from epimastigote to bloodstream forms of Trypanosoma brucei U.-P. Modespacher*, W. Rudin and H. Hecker Swiss Tropical Institute, Basel, Switzerland
(Received 7 March 1991; accepted 28 May 1991) Monoclonal antibodies to metacyclic surface coat glycoproteins of Trypanosoma brucei brucei STIB 247LG were produced for a study of the synthesis of metacyclic variable surface glycoproteins (VSGs) within the salivary gland of Glossina morsitans morsitans, and of the first exchange of the surface glycoproteins after infection in mice. Immunofluorescence antibody tests and protein A-gold labelling r6vealed that the VSGs are continuously integrated into the whole surface of the trypanosome while it is still attached to the gland epithelium. A pool of 8 antibodies recognized about 50% of the metacyclic forms present in the saliva of an infected tsetse fly, which confirmed the heterogeneity of the metacyclic VSG-generation. The labelling experiments showed that the integration of the first VSG-generation into the surface of bloodstream forms takes place in the same way as in the metacyclics. This process started on day 3 after infection and was finished on day 6. Key words: Trypanosoma brucei; Variant surface glycoprotein; Monoclonal antibody; Cryo-ultramicrotomy
Introduction A f r i c a n t r y p a n o s o m e s , h a e m o f l a g e l l a t e s p a r a s i t i c in m a n a n d d o m e s t i c animals, cause c h r o n i c a n d , sometimes, lethal infections. These p r o t o z o a n s are t r a n s m i t t e d by tsetse fies, in which p a r t o f the p a r a s i t i c life-cycle takes place. In the m a m m a l i a n host they can escape the i m m u n e response o f the h o s t by varying, b i o c h e m i c a l l y a n d antigenically, their g l y c o p r o t e i n surface c o a t (VSG: v a r i a b l e surface g l y c o p r o t e i n ) ( D o n e l s o n a n d R i c e - F i c h t , 1985; Borst, 1986). The V S G s c o v e r the entire surface o f the t r y p a n o s o m a l cell d u r i n g the course o f infection in the m a m m a l i a n host. T h e y are lost in the digestive t r a c t o f the v e c t o r after the u p t a k e o f the p a r a s i t e s with an infective b l o o d m e a l , a n d are synthesized a g a i n in the metacyclic forms in the salivary glands, at the end o f the cycle in the tsetse fly. A l t h o u g h a single metacyclic t r y p a n o s o m e expresses o n l y one type o f V S G at a time, the whole metacyclic p o p u l a t i o n within the salivary g l a n d is h e t e r o g e n e o u s with respect to the V S G s expressed (Barry et al., 1979; H a j d u k et al., 1981, N a n t u l y a et al., 1983). Correspondence address." PD Dr. W. Rudin, Swiss Tropical Institute, Postfach, CH-4002 Basel, Swit-
zerland. *Present address. F. Hoffmann-La Roche LTD, CH-4002 Basel, Switzerland.
68 Synthesis and intracellular transport of VSGs have been intensively investigated in bloodstream forms both biochemically and ultrastructurally (Lheureux et al., 1979; Bangs et al., 1985; Bangs et al., 1986; Boothroyd et al., 1980; Boothroyd et al., 1981; Ferguson et al., 1986; Holder, 1985; McConnell et al., 1983; Duszenko et al., 1988; Grab et al., 1984; Steiger, 1973; Vickerman and Luckins, 1969; Webster, 1989). It could be shown that VSGs are synthesized by the rough endoplasmic reticulum, where signal peptide cleavage and some cotranslational glycosylation of oligosaccharides occur, including the addition of a glycan-phosphatidylinositol anchor (Cross, 1990). Further protein processing steps and the incorporation of various sugar residues take place in the Golgi apparatus and in the tubulovesicular network (Webster, 1989). This network consists of tubular structures and flattened cisternae on the trans side of the Golgi apparatus, and is located between the Golgi apparatus and the flagellar pocket. From there the VSGs are transported by coated vesicles to the flagellar pocket, where they are integrated into the surface coat. Some days after the metacyclic trypanosomes have been transferred to a mammalian host by the bite of an infective fly, they transform to bloodstream forms. This transformation includes not only a change of the morphology of the trypanosomes, but also a first exchange of the VSGs on the surface. The aim of the present work was to investigate the time of appearance and the intracellular transport of metacyclic VSGs in trypanosomes in the salivary gland of the tsetse fly, and also the exchange of metacyclic VSGs for the first generation of bloodstream VSGs in the mammalian host. For this purpose immunocytochemical labelling experiments with protein A-gold on ultrathin cryosections of trypanosomes at different stages were carried out, using monoclonal antibodies (mAb) raised against metacyclic VSGs of Trypanosoma brucei brucei STIB 247 LG.
Material and Methods
Trypanosomes. The experiments were carried out with Trypanosoma brucei brucei STIB 247 LG (recloned STIB 247) (Geigy and Kaufmann, 1973). Trypanosoma brucei rhodesiense STIB 386 AA (Richner and Jenni, 1986) was used as a control for the specificity of the monoclonal antibodies to STIB 247 LG in labelling experiments. The procyclic parasites were cultured and maintained according to Brun and Sch6nenberger (1979) and Brunet al. (1981). Flies and mice. Glossina morsitans morsitans were received from the Tsetse Research Laboratory, Langford/Bristol colonies and Glossina morsitans centralis from ILRAD, Nairobi/Kenya. Female Swiss ICR mice were used for infection by intraperitoneal injection of reactivated stabilates (Lanham and Godfrey, 1970) to produce bloodstream forms. Female C57/bl mice were immunized for raising antisera to metacyclics and Balb/C mice for harvesting spleen cells for production of monoclonal antibodies. Polyclonal antiserum to metacyelic trypanosomes. C57/bl mice were infected by infectious bites of 2 tsetse flies per mouse. The same 2 flies were fed a second time on the same mouse after another 2 days. 6 h later the mice were treated with a single dose (25 mg/kg body weight) of Berenil. After treatment, the mice received booster infections by allowing the same flies to feed on them 5 more times at 2-day intervals.
69 The antiserum harvested by heart puncture 2 days after the last booster was stored at - 70°C. Monoclonal antibodies to the coat. The mAb were produced by slight modifications of the method described by Nantulya et al. (1983). Three Balb/C mice were infected with 10 flies delivering metacyclic trypanosomes in their saliva, and were treated with Berenil 24 h later (see above). Booster infections were done on day 13 and 23 with the same flies. On day 28 the spleen cells were harvested and fused with X63 (Hoechst) myeloma cells. After 2 weeks the supernatants of the cultures were checked by an indirect immunofluorescence test on fixed metacyclic trypanosomes from tsetse saliva and on fixed procyclic culture forms. The positive cell cultures were cloned by limiting dilution. Immunofluorescence antibody test (IFAT). Saliva containing metacyclic T.b. brucei was harvested by allowing infective flies to probe on a defined area of a glass slide. The saliva was dried for 30 min at 37°C followed by fixation in 100% acetone for 10 min. After air-drying they were stored at - 8 0 ° C . Procyclic culture forms in medium were mixed with an equal volume of FBS, applied to glass slides, and then treated as described above. For indirect immunofluorescence labelling the slides with the trypanosomes were rehydrated by incubation with PBS for 10 min prior to the reaction either with serial dilutions of the polyclonal antiserum or with crude hybridoma supernatants. Subsequently they were washed with PBS and then incubated with goat anti-mouse-FITC (Cappel) for 20 min, washed again, and finally air dried. As a control, the slides were incubated with PBS instead of the antiserum or supernatant. Examination was done under UV-light in a Leitz microscope. Electron microscopy. Salivary glands of infective tsetse flies were dissected under a W I L D stereomicroscope and immediately fixed for 1 h in 0.5% (v/v) glutaraldehyde in 0.2 M Pipes buffer, pH 7.4. After embedding in 8% (w/v) gelatine Type IV (Sigma), in Pipes buffer, they were cut into small pieces of about 1 mm length, postfixed in glutaraldehyde for another 30 min, and then transferred into 2.3 M sucrose in Pipes buffer for penetration with the cryoprotectant overnight at 4°C. The specimens were frozen by dropping them into liquid nitrogen after mounting on copper studs, and were stored in liquid nitrogen until cryosectioning. The blood of infected mice was collected and mixed with an equal volume of phosphate buffered saline with glucose (PSG, 6:4), pH 8.0, and centrifuged for 10 rain at 70 xg. The supernatant containing the trypanosomes was harvested and again centrifuged for 20 rain at 3700 x g. The pelleted trypanosomes were resuspended in 0.5% glutaraldehyde in 0.2 M Pipes buffer, pH 7.4, transferred into an Eppendorf tube and fixed for 1 h before another centrifugation for 1 min at 8000 ×g. The supernatant was drawn off and the pellet resuspended in a small volume of warm (45°C) 8% (w/v) gelatine in Pipes, centrifuged again and then solidified on ice. The contents of the tube were removed and transferred to glutaraldehyde for 30 min to fix the gelatine. Pyramid shaped pieces of a maximum size of 1 mm 3 were cut from the gelatine blocks and penetrated over night with 2.3 M sucrose in 0.2 M Pipes before mounting and freezing as above. Ultracryomicrotomy was done as described elsewhere (Griffith et al., 1983; Griffith et al., 1984; Tokuyasu, 1986). Immunocytochemistry. Monodisperse colloidal gold sol containing gold particles of homogeneous size (9 nm) was produced as reported in the literature (Slot and Geuze, 1985). The pH of the gold sol was adjusted to pH 6.0 with 70 mM citric acid before formation of protein A (Pharmacia)-gold complexes (Horisberger and Clerc,
70 1985). The complexes were stored in 50% (v/v) glycerol at - 30°C. Ultrathin cryosections were cut on a Reichert Ultracut E equipped with a Reichert FC 4 cryochamber. The sections were preincubated for 10 min with 10% (v/v) foetal bovine serum (FBS) in 20 m M PBS, followed by glycine (1.5 mg/ml) for 3 min, and unlabelled protein A (40 gm/ml) in PBS with 10% FBS for 15 rain. The incubations with the monoclonal antibodies were performed on 5 gl drops for 30 min. After washing intensively with PBS the sections were incubated for 15 min with 5 tal PAG diluted to A525 0.05 with PBS/FBS. Another washing with PBS and distilled H 2 0 was carried out after the incubation with the marker solution. As a control, sections were incubated with PBS/FBS instead of antibody.
Results Eighteen hybridoma cell cultures resulting from the fusion of spleen cells, harvested from a mouse infected with T.b. brucei 247 LG, and X63 myeloma cells were successfully maintained. The supernatants were tested for their reactivity against T.b. brucei from tsetse fly saliva by indirect immunofluorescence after three weeks. Six supernatants were positive for metacyclic forms of the strain 247 L G but not for T.b. rhodesiense strain 386 AA and not for procyclic culture forms. The antibodies produced in 2 other hybridoma cultures recognized procyclic forms (strain 247 LG) only. Four cultures specific for metacyclics and 2 specific for procyclics were cloned by limiting dilution (0.5 cells per well). Fourteen clones specific for metacyclic trypanosomes of the strain 247 LG and 6 specific for procyclic and epimastigote trypanosomes resulted. Each antibody specific for metacyclics recognized only a small proportion of the population present in the saliva of an infectious tsetse fly (Fig. 1), which demonstrated the heterogeneity of the antigen type expressed on the surface of the metacyclic trypanosomes. The monoclonals against procyclic culture forms recognized all procyclic and epimastigote forms (Fig. 2). A pool of 8 monoclonal antibodies to metacyclic trypanosomes of the isotype IgG 2a, identified by the Ouchterlony-double diffusion test, was used for immunocytochemical localisation of the respective antigens on ultrathin cryosections by the use of protein A-gold. The labelling on the surface of the metacyclics within the salivary gland was highly specific, and showed individual differences between cells in the labelling density (Figs. 3 and 4). I m m u n e reactions could be observed on the whole
Fig. 1. Metacyclic T.b. brucei STIB 247 LG in saliva delivered from an infective tsetse fly. Proportion of metacyclic trypanosomes recognized by one monoclonal antibody to metacyclic VSG in an immunofluorescence antibody test ( × 800). Fig. 2. Epimastigote T.b. brucei STIB 247 LG in saliva delivered from an infective tsetse fly recognized by a monoclonal antibody to procyclic trypanosomes in an immunoftuorescenceantibody test ( × 800). Fig. 3. Binding sites of a pool of 8 moabs to metacyclic VSG on ultrathin frozen sections of T.b. brucei in salivary glands of infected tsetse flies demonstrated with protein A-gold. High surface labelling density (arrowheads) ( × 70 000). Fig. 4. Same as Fig. 3 but low surface labelling density (arrowheads) ( × 72 000).
72 surface; cell body and flagellum. Trypanosomes attached to the epithelium of the salivary gland were also labelled all over their surface, including the surface of the hemidesmosomes at the contact site between vector and parasite (result not shown). Furthermore, the flagellar pocket (Fig. 5), the Golgi zone, the multivesiculated network (Fig. 6) between Golgi and flagellar pocket, and coated vesicles were labelled in the same trypanosomes. The pool of monoclonal antibodies (see above) was also applied to frozen thin sections of trypanosomes isolated from mice 3, 4, 5, and 6 days after infection. All the parasites of these stages which were recognized also showed labelling over the whole surface and the lining of the flagellar pocket which varied in density between individuals (Fig. 7). In contrast to the situation with metacyclics in the salivary gland, however, the antibodies did not bind to intracellular structures (Fig. 8). An estimation of the percentage of labelled trypanosomes from 3-6 days after infection (Fig. 9) showed a massive drop between days 4 and 5, from more than 40% to only about 5%. After six days no trypanosomes were recognized by the pool of antibodies to the metacyclic coat.
Discussion The surface coat of blood trypanosomes, consisting of variable surface glycoprotein, is lost after the uptake of the parasites by tsetse flies (Barry and Vickerman, 1979; Molyneux and Ashford, 1983). A new coat is synthesized only after the migration of the trypanosomes to the salivary glands. For the last transformation step in the vector, the parasites attach to the salivary gland epithelium by differentiating hemidesmosomes (Steiger, 1973; Vickerman, 1973). It was found in the present study that coat formation takes place on the surface of attached stages. This confirms earlier observations, where these forms were described as immature stages (Tetley and Vickerman, 1985; Tetley et al., 1987). The immunocytochemical results of the present study allow some conclusions to be drawn about the synthesis of the coat. The fact that the density of the labelling varies between individuals but is regular on the whole of an individual trypanosome, suggests that a continuous formation all over the cell surface occurs. On the other hand, there is evidence that the flagellar pocket is the site of exocytosis of coat material, because endo- or exocytosis is said to be restricted to surface areas where
Fig. 5. Binding sites of a pool of 8 mAbs to metacyclicVSG on ultrathin frozen sections of T.b. brucei in salivary glands of infected tsetse flies demonstrated with protein A-gold. Binding to the flagellar pocket (fp) ( x 90 000). Fig. 6. Binding to the multivesicular network (mvn) and to the flagellum (fl) ( × 71 000). Fig. 7. T.b. brucei harvested from mouse blood 3 days after infection. All forms recognized by the pool of mAbs to metacyclic VSG are labelled on the whole surface (arrowheads). The individual labelling density varies ( x 23 000). Fig. 8. Same as Fig. 7. Lack of binding of mAbs to metacyclicVSG to intracellular structures in opposition to salivary gland forms ( x 65 000).
74
% ~o
203040 lOo
I
2
.
.
.
.
.
.
3 4 5 Days alter infection
6
7
Fig. 9. T.b. brucei, 247 LG bloodstream forms, 3-6 days after cyclical transmission. Percentage of goat anti-mouse-FITC labelled trypanosomes after prior reaction with mAb-pool to metacyclic forms.
no subpellicular microtubules are present (Brown et al., 1965; Langreth and Balber, 1975; Frevert and Reinwald, 1988; Webster and Grab, 1988; Webster and Fish, 1989). The presence of coated vesicles near the flagellar pocket supports this hypothesis (Steiger, 1973). In the present study, not all of the trypanosomes in the saliva of an infective tsetse fly could be immunofluorescence labelled with a single clone of monoclonal antibody to metacyclic forms, whereas a polyclonal antiserum recognized all the parasites. When a pool of 8 different monoclonal antibodies was used for immunocytochemical labelling on ultrathin cryosections of trypanosomes harvested 3 days after infection of the vertebrate host, 48.2% of the parasites were labelled. Assuming that on day 3 after infection all trypanosomes are still covered with metacyclic VSG (see below), one can calculate that approximately some twenty variant m-VSG were expressed in the population tested. This fits the published data about the m-VSG heterogeneity of T.b. brucei (Le Ray et al., 1978; Nantulya et al., 1983; Barry and Vickerman, 1979; Barry and Emery, 1984; Hajduk et al., 1981), T.b. rhodesiense (Turner et al., 1988), and T. congolense (Luckins et al., 1986; Prain and Ross, 1988). It is this heterogeneity which decreases the chance of developing a vaccine directed against the infective stage of trypanosomes, so the present trend in vaccine development is oriented towards common structural antigens (Holder and Cross, 1981), towards flagellar pocket antigens (Balber, 1989) or transmission blocking antigens (Roditi et al., 1988). The binding of monoclonal antibodies to metacyclic VSG on cryosections of T.b. brucei in salivary glands of G.m. morsitans, which was visualised by the use of protein A-gold, did not occur on the parasite surface only. There was also binding to intracellular structures (the Golgi zone, multivesiculated network and coated vesicles) and to the surface of the flagellar pocket. This corresponded to the findings of other authors with regard to the synthetic pathway of VSG molecules (Steiger, 1971; Webster and Grab, 1988; Grab et al., 1984; Webster, 1989; Webster and Fish, 1989). Trypanosomes released into the vertebrate host by a fly bite keep their metacyclic surface coat for a few more days. The present study showed that the highest transformation rate for the T.b. brucei strain STIB 247 LG in mice occurs between
75 day 4 and day 5. The percentage of trypanosomes recognized by a pool ofmonoclonal antibodies to metacyclic VSG, which reacts with about 50% of the parasites in the salivary gland, is more or less unchanged until day 4 (42.3%), drops to a few percent (4.7%) on day 5 and to 0 on day 6. This corresponds well with the observations of Hajduk and Vickerman (1981), though it is one day later than it was found by Nantulya et al. (1983). What happens to the metacyclic VSGs when they are replaced by the first generation of bloodstream form VSGs is still under discussion. On the one hand, it has been postulated that the release takes place by vesiculation from the trypanosome surface, called capping (Barry and Vickerman, 1979; Barry, 1986; Shapiro, 1986). On the other hand, recycling by endocytosis in the flagellar pocket, as it has been postulated for bloodstream VSGs, could occur (Webster and Grab, 1988). Capping was never observed in the present study, but, in earlier experiments a release of membrane bound VSG by vesiculation occurred after pre-embedding immunolabelling when living trypanosomes were exposed to the antiserum (unpublished results). The monoclonal antibodies to metacyclic VSGs showed, if ever (aggregate on Fig. 6), very limited binding to intracellular structures in the trypanosomes isolated from mouse blood. In contrast, there was significant binding to intracellular structures in the salivary gland forms. This supports the capping theory. However, the lining of the flagellar pocket was labelled in all trypanosomes recognized by the mAb, even at day 6. This seems to fit the recycling theory for metacyclic VSGs. The presence of enzymes within the flagellar pocket (Steiger, 1971; Grab et al., 1989) which are able to cleave the VSGs could explain why endocytosis could not be shown in the present work. It is unlikely that the glycosyl-phosphatidylinositol-specificphospholipase C plays this role, being located on the outer side ofintracellular vesicles (Buelow and Overath, 1985; Buelow et al., 1989; Grab et al., 1989; Fox et al., 1986; Grab et al., 1987). However, the recycling theory on the one hand, and the circumstances under which capping occurs, on the other hand, need further investigation. Another point which also needs additional clarification is the way in which a new VSG generation is deposited on the trypanosome surface. By double immunolabelling using mAbs to two consecutive coat types it could perhaps be shown whether this process involves a continuous integration of the new molecules into the previous coat over the whole surface, as has already been shown by immunofluorescence labelling (Esser and Schoenbechler, 1985), or is the outgrowth of a fully developed new one from the flagellar pocket.
Acknowledgements The skilful technical assistance of Mrs. E. Fluri, C. Knoell, and R. Rufener is gratefully acknowledged. We further wish to thank Dr. Paul Webster (ILRAD, Nairobi, Kenya) for his support in the establishment of the cryotechniques and Mrs. J. Jenkins for English corrections. The work has partly been supported by Roche Research Foundation, Fonds fur Forderung von Lehre und Forschung, SandozStiftung zur F6rderung der med.-biol. Wissenschaften, Basel, Switzerland, and Swiss National Science Foundation Grant No. 3.530-083.
76
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