P. WEBSTER
International Laboratory for Research on Animal Diseases
P O Box 30709, Nairobi, Kenya
and M. MARSH
Institute of Cancer Research, Chester Beatty Laboratories
London, SW3 6JB, UK
Transferrin binding
Cell association of transferrin at 37 °C
Cell association of 59Fe-transferrin
Transferrin-gold binding and uptake
Uptake of bovine serum albumin and transferrin coupled to gold
Binding and uptake of horseradish peroxidase (HRP)
Uptake of HRP and transferrin-gold
Conclusions
References
African trypanosomes are protozoan flagellates that are responsible for extensive disease in humans and domestic animals across a large area of Africa. The success of trypanosomes lie in their ability to avoid the host immune response by varying, both biochemically and antigenically, the glycoprotein coat (variable surface glycoprotein, or VSG) covering the entire cell surface during the course of infection in the mammalian host.1,2,3
It is believed that the surface glycoproteins of trypanosomes are too antigenically diverse to be useful as a material for a vaccine.4 However, other antigenically sensitive sites vulnerable to antibody attack may be revealed as the cell biology of the organism is studied. A process that may serve as a target for antibody attack within trypanosomes is endocytosis.
Trypanosomes, like all living cells, require materials from their environment for continued growth. Many molecules enter cells by endocytosis, either by fluid phase or by absorptive or receptor-mediated endocytosis of selected molecules via clathrin-coated vesicles.5 African trypanosomes in the bloodstream of infected animals take up various particulate and soluble substances in vesicles morphologically similar to the clathrin-coated vesicles found in other eukaryotic cells.6,7,8 These vesicles bud only from the membrane of the flagellar pocket, a deep invagination of the plasma membrane where the flagellum leaves the cell, and discharge their contents into an intracellular tubular system.6
Iron, essential to all cells, is supplied to mammalian cells primarily by a major serum protein called transferrin, which, when iron loaded, will bind to receptors on the cell surface. The receptor-ligand complexes are internalized by receptor-mediated endocytosis9 through clathrin-coated pits and vesicles10,11,12 and are delivered to the endosome compartment of the endoycytic pathway.11,l3,14 Here, the low pH environment14,15,16 triggers the dissociation of iron from the transferrin17,18 and the iron is transported across the membrane. The iron-free or apotransferrin-receptor complexes recycle to the cell surface, where, at neutral pH, the apotransferrin dissociates from the receptor and the cycle is repeated.10,11,17,18
African trypanosomes also require iron for growth (R. Kaminsky, personal communication). For these cells, however, the mechanisms by which iron is acquired are not understood. We have shown that colloidal gold coupled to diferric tranferrin bind to the surface of trypanosomes and are internalized by vesicles via the flagellar pocket.8 Uptake of VSG molecules also occurs via the same vesicles.8 It has recently been demonstrated that transferrin and low-density lipoproteins enter Trypanosoma brucei by receptor mediated endocytosis.7 However, little is known about the nature of the parasite receptors, or the fate of substances internalized by trypanosomes.
This report will investigate the fate of various endocytosed substances in African trypanosomes.
All experiments were carried out on T. brucei populations isolated from infected rat blood, as previously described.19
Trypanosomes were incubated with 125I diferric transferrin on ice. At the indicated times, free ligand was washed away and the cell associated radioactivity was determined. Binding was slow and took up to 3 h to reach a plateau. The binding was saturable and was competitively inhibited by excess unlabelled differic transferrin. Binding of 125I apotransferrin was found to be only 12% efficient when compared with differic transferrin binding.
Trypanosoma brucei incubated with either 125I diferric or 125I apotransferrin were placed on ice and, at increasing time intervals, washed with cold medium to remove unbound label and transferred to fresh tubes to determine the cell associated radioactivity.
At 37 °C diferric transferrin rapidly associated with the cell and reached a maximum of cell-associated activity at 10 min after the onset of incubation at 37 °C. Subsequently, the cell associated activity declined, suggesting that the protein was released from the cells.
To determine whether the iron associated with diferric transferrin remained cell-associated, trypanosomes were incubated in 59Fe-transferrin. At increasing times the cells were washed and the cell-associated radioactivity was determined. A steady, linear accumulation of cell-associated radioactivity was seen over a 90 min period.
In the presence of ammonium chloride, the accumulation of radioactivity was initially inhibited, but within 30 min the rate of ferric 59Fe uptake was equivalent to that in the untreated samples. The results suggest that iron accumulates in trypanosomes after dissociating from transferrin in an acidic compartment. In the conditions used for these experiments, the cells rapidly acidify the medium and as a consequence NH3 is protonated, becomes less permanent to membranes and loses its ability to neutralize intracellular acidic compartments.
Trypanosomes were incubated on ice in medium containing transferrin coupled to colloidal gold particles. By electron microscopy, the gold particles were seen to bind to the surface of the trypanosomes. Occasionally gold particles were found in flagellar pockets, but most pockets did not contain the marker.
When trypanosomes were warmed to 37°C in the presence of transferrin-gold, the gold particles were found in the flagellar pocket, in pits on the flagellar pocket membrane and in intracellular vesicles in close proximity to the flagellar pocket. After a 5-min incubation at 37°C, the gold markers were found in intracellular structures with tubular profiles and large vesicular and lysosome-like structures.
If trypanosomes, incubated in transferrin-gold, were reacted with a silver intensification solution and viewed by light microscopy, a dark reaction product revealing the colloidal gold was present in the region between the nucleus and the flagellar pocket, situated at the posterior end of the cell. Reaction product was seldom found in the anterior part of the cell.
When trypanosomes were incubated in medium containing transferrin coupled to colloidal gold of one particle size and bovine serum albumin (BSA) coupled to gold of a different particle size, both markers bound to the cell surface. Both markers entered the cell via the flagellar pocket and in the same intracellular vesicles. No segregation of the two markers was seen in any of the endocytotic organelles except that some vesicular structures contained only the smaller gold particles, regardless of the coupled protein.
Trypanosomes incubated on ice in medium containing HRP were seen to have horseradish peroxidase (HRP) bound to the cell surface, after visualization with diamino-benzidene. The flagellar pockets did not contain HRP. Upon warming to 37°C, the HRP entered the cells via the flagellar pocket and after 5 min was found in many intracellular structures. Often tubular structures near to the Golgi apparatus were filled with HRP, but there was no consistent association of HRP with the Golgi.
Serial sections through trypanosomes containing endocytosed HRP produced a pseudo three-dimensional view of the endocytotic organelles and revealed them to be a complex tubulo-vesicular network.
When transferrin-gold was included in the incubation medium with HRP, the two markers co-localized in the flagellar pocket and in many intracellular organelles. Whereas the transferrin-gold always co-localized with the HRP, the HRP was found in vesicles close to the flagellar pocket and in many tubular and vesicular structures that did not contain transferrin-gold. Many of these HRP-containing structures were connected to structures that contained the colloidal gold marker.
A fractional volume analysis revealed that transferrin-gold containing structures constituted 2% of the total cell volume, whereas the HRP-containing structures constituted 5% of the total cell volume.
It appears that trypanosomes obtain iron from the blood that surrounds them in a way similar to that used by the mammalian hosts. Receptors on the surface of trypanosomes specifically bind diferric transferrin, and the receptor-ligand complexes enter the cell, where they meet an acidic environment that causes the iron to dissociate from the transferrin. The eventual fate of the internalized transferrin has not yet been determined.
Morphologically, using transferrin coupled to colloidal gold, it seems that trypanosomes internalize transferrin through coated vesicles into a tubulo-vesicular compartment similar to the endosome compartment of mammalian cells.20 The precise morphological details of the endocytotic uptake of transferrin must await analysis using monomeric protein, as it is clear the proteins coupled to colloidal gold may be directed to sites other than those reached by uncoupled proteins.21,22,23
It is clear that the organelles involved with endocytosis are morphologically complex structures when visualized by HRP and that colloidal gold particles enter only parts of the endocytic pathway. Endocytosis by African trypanosomes is important for nutrient uptake and possibly for processing of intracellular VSG. An understanding of the complex mechanisms involved may reveal new ways to control trypanosomiasis.
1. CROSS G.A.M. 1975. Parasitology 71: 393-417.
2. CROSS G.A.M. 1984. Phil. Trans. R. Soc. Lond. B. Biol. Sci. 307: 3-12.
3. TURNER, M.J. 1984. Phil. Trans. R. Soc. Lond. B. Biol. Sci. 307: 27-40.
4. SHAPIRO, S.Z. and T.M. PEARSON. 1986. In Parasite Antigens: Towards New Strategies for Vaccines. T.M. Pearson, Ed.: 215-274. Marcel Dekker, New York.
5. SILVERSTEIN, S.C., R.M. STEINMAN and Z.A. COHN. 1977. Ann. Rev. Biochem. 46: 699-772.
6. LANGRETH, S.G. and A.E. BALBER. 1975. J. Protozool. 22: 40-53.
7. COPPENS, I., F.R. OPPERDOES, P.J. COURTOY and P. BAUDHUIN. 1987. J. Protozool. 34: 465-473.
8. WEBSTER, P. and D.J. GRAB. 1988. J. Cell Biol. 106: 279-288.
9. KLAUSNER, R.D., J. VAN RENSWOUDE, G. ASHWELL, C. KEMPF, A.D. SCHECHTER and K.R. BRIDGES. 1983. J. Biol. Chem. 258: 4715-4724.
10. BLIEL, J.D. and M.S. BRETSCHER. 1982. EMBO J. 1: 351-355.
11. HOPKINS, C.R. and I.S. TROWBRIDGE. 1983. J. Cell Biol. 97: 508-521.
12. HANOVER, J.A., M.C. WILLINGHAM and I. PASTAN. 1984. Cell 39: 283-293.
13. HOPKINS, C.R. 1983. Cell 35: 321-330.
14. VAN RENSWOUDE, J., K.R. BRIDGES, J.B. HARFORD and R.D. KLAUSNER. 1982. Proc. Natl. Acad. Sci. USA 61: 6186-6190.
15. TYCKO, B. and F.R. MAXFIELD. 1982. Cell 28: 643-651.
16. MARSH, M., E. BOLZAU and A. HELENIUS. 1983. Cell 32: 931-940.
17. DAUTRY-VARSAT, A., A. CIECHANOVER and H.F. LODISH. 1983. Proc. Natl. Acad. Sci. USA 80: 2258-2262.
18. KLAUSNER, R.D., G. ASHWELL, J. VAN RENSWOUDE, J.B. HARFORD and K.R. BRIDGES. 1983. Proc. Natl. Acad. Sci. USA 80: 2263-2266.
19. GRAB. D.J., P. WEBSTER, S. ITO, W.R. FISH, Y. VERJEE and J.D. LONSDALEECCLES. 1987. J. Cell Biol. 105: 737-746.
20. HELENIUS, A., I. MELLMAN, D. WALL and A. HUBBARD. 1983. Trends in Biochem. Sci. 8: 245-250.
21. UKKONEN, P., V. LEWIS, M. MARSH, A. HELENIUS and I. MELLMAN. 1986. J. Exp. Med. 163: 952-971.
22. NEUTRA, M.R., A. CECHANOVER, I.S. OWEN and H.L. LODISH. 1985. J. Histochem. Cytochem. 33: 1134-1144.
23. VAN DEURS, B., K. SANDVIG, W.O. PETERSEN, S. OLSNES, K. SIMONS and G. GRIFFITHS. 1988. J. Cell Biol. 106: 253-267.
