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Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei
Molecular and Biochemical Parasitology, 6 (1982) 181-190 Elsevier Biomedical Press
181
INVOLVEMENT OF LYSOSOMES IN THE UPTAKE OF MACROMOLECULAR MATERIAL BY BLOODSTREAM FORMS OF TR YPANOSOMA B R UCEI
FRED R. OPPERDOES and JORIS VAN ROY Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, avenue Hippocrate, 74, B-12OOBrussels, Belgium (Received 25 March 1982; accepted 14 May 1982)
To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromoleculax compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (> 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm a in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and t~-mannosidase-containing organelles of 7". brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function. Key words: Trypanosoma brucei, Lysosomes, Endocytosis, Triton WR-1339, Trypanocidal drug, Lysosomotropic drug, Cell fractionation.
INTRODUCTION A drug like suramin, which is active against Trypanosoma rhodesiense and T. gambiense, the causative agents o f sleeping sickness and T. brucei, causing nagana in lifestock, circulates in the bloodstream in close association with plasma proteins [1, 2] and it is believed that it enters the trypanosome by a mechanism of endocytosis followed by lysosomal digestion of the associated protein [3]. The recently developed trypanocidal daunorubicin-protein complexes [4] are thought to exert their action according to the same principle. This concept of lysosomotropism requires that trypanosomes are capable of taking up macromolecules by a mechanism of endocytosis or pinocytosis followed by fusion of the endocytotic vesicles with so-called primary lysosomes, resulting in the for0166-6851/82[0000-0000[$02.75 © 1982 Elsevier BiomedicalPress
182 mation of a phagolysosome. Such a phagolysosome should contain adequate hydrolases in order to free the intact drug after digestion of the carrier protein. Electron microscopy studies on several members of the Trypanosomatidae have shown that these organisms are capable of taking up macromolecular material via endocytosis. Ingestion of protein from a ferritin-containing medium was first demonstrated for Trypanosoma mega [5] and the existence of a specialized cytostome was postulated. Similar experiments demonstrated endocytosis in the case of T. rhodesiense[6], T. brucei [7] and T. cruzi [7]. Langreth and Balber [8] have carried out detailed electron microscope studies in the case of T. brucei and they have shown that macromolecular material first accumulates in the flagellar pocket and then enters into large spiny-coated vesicles by a mechanism of endocytosis or fluid pinocytosis. These vesicles subsequently become continuous with a collecting membrane system and eventually the foreign material ends up in large digestive vacuoles. Biochemical evidence to support these electron microscopic observations is still scarce. Fairlamb and Bowman [9] demonstrated that an inert macromolecule like polyvinyl pyrrolidone can be taken up by T. brucei in vitro by fluid endocytosis, albeit at a low rate, whereas in vivo the lysosomotropic drug, suramin, entered possibly by a similar mechanism but at a considerably higher rate [3]. In both cases, however, no evidence was presented on the intracellular fate of the material ingested. In previous work we have shown that T. brucei possesses a-mannosidase and acid proteinase-containing organelles resembling lysosomes [10]. However, involvement of these organelles in the uptake and digestion of exogenous macromolecules remained to be proven, since they equally well might be involved in the excretion of digestive enzymes into the lumen of the flagellar pocket. To produce evidence that macromolecules from the external medium may end up in these lysosomes, after internalization by endocytosis, we have made use of Triton WR-1339 (polyoctylphenolpolyethyleneglycol), a nondigestible macromolecular compound, known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage [11]. Part of this work has already been reported in abstract form [12]. MATERIALS AND METHODS Organism. Trypanosoma brucei brucei Stock 427 was maintained as a stabilate at -196°C. Upon intraperitoneal inoculation of 106 parasites per rat this strain produced a fulminating infection resulting in the death of the host in approximately 100 h. Growth and isolation o f trypanosomes. Trypanosomes were grown in 300 g male Wistar rats. After inoculation of the parasites infected blood was withdrawn from the animals after 92 h by cardiac puncture using heparin as an anticoagulant. The trypanosomes were separated from other blood elements by filtration through DEAE-cellulose, according to Lanham [13] followed by washing with 250 mM sucrose, 25 mM Tris/HC1 and 1 mM EDTA, pH 7.4.
183
Cell fractionation. Preparation of cell homogenates, cell fractionation by differential centrifugation and isopycnic centrifugation in linear sucrose gradients in a vertical Beckman VTi 50 rotor and the presentation of the results are exactly as described previously [10, 14]. The structural latency of enzymes in cytoplasmic extracts was determined in sucrose-containing isotonic media, supplemented with 0.04% (w/w) bovine serum albumin, by measuring enzyme activity in the absence and the presence of 0.1% (v/v)Triton X-100, respectively. The sedimentability of enzymes was determined by centrifugation of cytoplasmic extracts for 1 h at 139 000 × g. Treatment of infected animals with Triton WR-1339. The nonionic detergent Triton WR-1339 (Tyloxapol, Merk index 9487) was dissolved in 0.15 M NaCI and sterilized by filtration through Millipore membranes (0.22 tan pore size) prior to injection. To isolate trypanosomes from Triton-treated animals 300 g rats were given a dose of Triton WR-1339 of 0.4 g/kg body weight 70 h after infection with trypanosomes and the trypanosomes were isolated 22 h later. To study the trypanocidal effect of Triton WR-1339, female NMRI mice of approximately 25 g were infected with l0 s virulent trypanosomes at zero time and treated with different dose levels at different times. Triton WR-1339 was dissolved in 0.15 M NaC1. For each dose level a different concentration was prepared and every mouse received 1 ml of solution intraperitoneally. Control animals only received a solution of NaC1. 100 days after infection of the mice with trypanosomes the experiment was terminated and all the survivors were considered cured. Enzyme determinations, a-D-Mannosidase (EC 3.2.1.24) was measured under optimized conditions, exactly as described by Steiger et al. [10]. Acid proteinase (EC unclassified) was measured with [3H]casein as substrate in 0.1 M phosphate buffer, pH 6.5, as described previously [14, 15]. Protein was determined fluorimetrically [16] with bovine serum albumin as standard. Materials. Triton WR-1339 was purchased from Ruger Chemical Co., Irvington, NJ, U.S.A. All other chemicals were of the highest purity available. RESULTS When rats infected with Trypanosoma brucei were treated with a relatively high dose of Triton WR-1339 (2 g/kg body weight) it proved difficult to isolate trypanosomes in sufficient quantities since, firstly, trypanosomes disappeared before the rats could be bled and, secondly, trypanosomes, if still present, were very fragile and could only be eluted with low recoveries from a DEAE-cellulose column. For these reasons the dose of Triton was lowered to 0.4 g Triton WR-1339/kg. 22 h after administration trypanosomes were still present in large quantities and could be easily purified in a yield of more than 80%. Blood smears from rats treated with such a dose showed that part of the trypanosomes had developed a massive vacuole which was always located at the posterior end between
184
Fig. 1. Giernsa-stained smear of infected rat blood after treatment with Triton WR-1339. Rats were infected with 106 T. brucei intraperitoneally and treated intraperitoneally with 0.5 g Triton WR-1339/ kg 3 days later. Blood was examined 17 h after administration of Triton. Arrows point at trypanosomes containing a vacuole.
kinetoplast and nucleus. This vacuole varied in size and could be twice as big as the normal diameter of a healthy trypanosome (Fig. 1.). After purification such mixed populations of trypanosomes were subjected to homogenization by grinding with silicon carbide [16] and the latency and sedimentability of two lysosomal enzymes was monitored in cytoplasmic extracts. Fig. 2 shows that strucural latency of both enzymes from trypanosomes that had been in contact with Triton WR-1339 for 22 h was decreased with respect to the untreated controls. Moreover, a considerable part of acid proteinase was solubilized due to the treatment. This suggests that a significant part o f the lysosomes were damaged and had lost their content, a-Mannosidase remained entirely sedimentable, even after treatment. Fractionation o f post-large-granule fractions [10, 14] by isopycnic centrifugation in sucrose (Fig. 3) showed that the lysosomal markers in control cells equilibrated at densities around 1.18 g/cm 3, but that contact with Triton WR-1339 for 22 h led to a significant shift in the density o f a-D-mannosidase towards lower values (1.14 g/cm3).
185
A similar effect was observed for the particulate part of acid proteinase (shift from 1.19 towards 1.16 g/cm 3) but in addition a considerable amount of the enzyme was solubilized and remained at the top of the gradient. Since we observed that administration of an elevated dose of Triton WR-1339 could lead to a total disappearance of trypanosomes from the blood we investigated the chemotherapeutic effect of Triton on infected mice. Fig. 4 shows that complete cures (survival for more than 100 days) were obtained even at a dose as low as 0.5 g/kg body weight in 20% of the cases, provided that Triton was given early enough after infection. The best results (80% long-term survivors) were obtained with a dose of 2 g/kg, given one day after the trypanosomes. No cures at all could be obtained when Triton was given between one and three days before infection (not shown). DISCUSSION
Several investigators have reported upon the presence of lysosomes in the species Trypanozoon, but in most studies a positive reaction for acid phosphatase served as the only evidence for the lysosomal nature of such organeUes [7, 8, 17-19]. Although acid
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Fig. 2. The effect of Triton WR-1339 treatment on the intactness o f T. brucei lysosomes. (A) Latency of two lysosomal enzymes in cytoplasmic extracts expressed as that percentage of the total activity that cannot be measured in the absence o f detergent. Total activity was determined in the presence of 0.l% Triton X-100 as a detergent. (B) Sedimentability of lysosomal enzymes in cytoplasmic extracts. Total activity in pellets and supernatants were measured in the presence of 0.1% Triton X-100 to remove any structural latency. Open bars represent cytoplasmic extracts prepared from trypanosomes isolated from control rats. Closed bars represent extracts from trypanosomes isolated from rats treated 22 h before trypanosome isolation.
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phosphatase is a typical marker [20] of mammalian lysosomes, in T. brucei the bulk of this enzyme is located in the flagellar pocket and the endoplasmic reticulum [8, 10]. However, lysosome-llke organeUes containing a number of hydrolases such as acid proteinase, RNase, a-D-mannosidase and phospholipase A~, but devoid of acid phosphatase, were recently described for T. brucei [10, 21, 22]. No direct proof existed until now for
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Fig. 4. C h e m o t h e r a p e u t i c activity o f Triton WR-1339 on a T. brucei infection in mice. Triton was given intraperitoneally either on the day of infection (D + 0) or 1, 2 or 3 days later. ( . . . . . ), untreated control mice; ( ) 0.5, ( . . . . . ) 1 and ( - - - - - - ) 2 g Triton WR-1339/kg body weight.
187 the involvement of these organelles in the uptake and digestion of foreign macromolecular material. Wattiaux et al. [11] ftrst described the loading of lysosomes from rat liver with the non-ionic detergent Triton WR-1339, and since then this process has been used as a method for high-yield isolation of lysosomes with the highest degree of purity. This method exploits the dramatic decrease in buoyant density of the hepatic lysosomes due to a massive uptake of the detergent. There is little doubt that Triton WR-1339 with a molecular weight of over 100 000 and a diameter of approximately 8 nm is taken up by endocytosis and enters into the lysosomes after fusion with phagocytic vacuoles [23]. Since numerous tissues other than liver also have been found to store Triton in their lysosomes we have investigated whether such a phenomenon also occurs with the lysosome-like organelles described for T. brucei bloodstream forms [10, 22]. The results presented in this paper are similar to those by others for rat liver [11, 24]. We interpret them to indicate that Triton WR-1339, circulating in plasma in close association with lipoproteins [25], is taken up by a mechanism of endocytosis or pinocytosis by the cell, leading to the formation of phagolysosomes. Since Triton is not digested by lysosomal hydrolases this leads to a massive accumulation of the detergent and consequently to the formation of the huge vacuoles seen with the light microscope. These Triton-loaded vacuoles probably originate from pre-existing lysosomes as is suggested by the presence of essentially all a-D-mannosidase and part of the acid proteinase in organelles with a reduced buoyant density in sucrose gradients. The majority of these organelles most likely represent leaky vacuoles, since they have lost a considerable part of their acid-proteinase activity. Mannosidase, however, remained associated with them, which can be explained by the fact that the latter enzyme is probably firmly attached to the lysosomal membrane as we have shown before [10]. These results suggest that the membranes of such massive phagolysosomes are highly fragile entities which are easily ruptured, possibly already in situ, but certainly during cell fractionation. A similar leakage of lysosomal content after massive overloading of lysosomes with undigestible material has already been described by others [26]. An overall shift in buoyant density of 0.03 g/cm 3 due to accumulation of Triton WR1339 in the case of T. brucei lysosomes is rather small as compared to a value of 0.10 g/cm a routinely obtained for rat-liver lysosomes, but can be explained as follows: firstly, the trypanosomes have been in contact with Triton for only 22 h, whereas in the case of rat liver the lysosomes are normally isolated 3 4 days after administration of the detergent; secondly, the dose-level (0.4 g/kg) used in our experiments was considerably lower than that of 0.85 g/kg normally administered for the isolation of liver lysosomes and thirdly, the trypanosomes, at the low dose level used, were still actively dividing, resulting in a continuous dilution of the Triton content of the digestive vacuoles. It is a well-known fact that Triton WR-1339 affords a remarkable protection against Mycobacterium tuberculosis [27] and a similar effect was described for Leishmania donovani infections in hamsters [28]. There seems to be little doubt that in both cases the detergent owes its antimicrobial properties to its lysosomotropism, rendering the
188 vacuolar apparatus of the host cell inhospitable to certain invading microorganisms [29]. T. brucei, however, is not an intracellular parasite but circulates freely in the bloodstream and tissue fluids. Therefore, the curative effect o f Triton WR-1339 described here is most likely due to an effect o f the detergent on the trypanosome itself. Triton-t'flied lysosomes in general are largely increased in size [11, 24] contain some plasma proteins at abnormally high levels [30] and exhibit an increased activity o f some lysosomal enzymes [31], whereas the lipid content may also differ from that o f normal lysosomes [32]. Henning and Plattner [23] have suggested that leakage o f the lysosomal content in situ might occur already in a very early stage after Triton WR-1339 administration. If all these effects were also to occur in the digestive vacuoles o f T. brucei, they might easily lead to an impaired lysosomal function, resulting either in an inhibition o f nutrient digestion, or a release o f digestive enzymes into the cytosol. Both effects either alone, or in combination, can provide an explanation for the trypanocidal action o f Triton WR1339. Several authors have already described the curative effect o f Triton WR-1339 in experimental infections with African trypanosomes [33] but at that time its mechanism of action was not well understood. Our experiments strongly suggest that the trypanocidal action o f Triton WR-1339 can be explained b y an interference o f the drug with lysosomal action due to an accumulation in phagolysosomes through a mechanism o f endocytosis. ACKNOWLEDGEMENTS This investigation received financial support from the UNDP/World Bank/WHO 'Special Programme for Research and Training in Tropical Diseases'. REFERENCES 1 Dangerfield, W.G., Gaunt, W.E. and Wormall, A. (1938) Studies on Bayer 205 (Germanin) and Antrypol. I. The determination of small amounts of Bayer 205 (and Antrypol). II. The persistence of Bayer 205 in the bloodstream after injection into animals. Biochem. J. 32, 59-70. 2 Muller, W.E. and Wollert, U. (1976) Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin. Biochim. Biophys. Acta 427,465-480. 3 Fairlamb, A.H., and Bowman, I.B.R. (1980) Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effects on respiration and growth rate in vivo. Mol. Biochem. Parasitol. 1, 315- 333. 4 Williamson, J., Scott-Finnigan, T.J., H~dman, M.A. and Brown, J.R. (1981) Trypanocidal activity of daunorubicln and related compounds. Nature 292,466-467. 5 Steinert, M. and Novikoff, A.B. (1960) The existence of a cytostome and the occurrence of pinocytosis in the trypanosome Trypanosorna mega. Biophys. Biochem. Cytol. 8,563-569. 6 Brown, K.N., Armstrong, J.A. and Valentine, R.C. (1965) The ingestion of protein molecules by blood forms of Trypanosoma rhodesiense. Exp. Cell Res. 39, 129-135. 7 Jadin, J.M. (1971) Cytologic et cytophysiologie des Trypanosomidae. Acta Zool. Pathol. Antverpiensia 53, 1-168. 8 Langreth, S.G. and Balber, A.E. (1975) Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei. J. Protozool. 22, 40 53.
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25 26 27 28 29
Fairlamb, A.H. and Bowman, I.B.R. (1980) Trypanosorna brucei: Maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis. Exp. Parasitol. 49, 3 6 6 - 3 8 0 . Steiger, R.F., Opperdoes, F.R. and Bontemps, J. (1980) Subcellular fractionation of Trypanosoma brucei bloodstream forms with special reference to hydrolases. Eur. J. Biochem. 105, 163-175. Wattiaux, R., Wibo, M. and Baudhuin, P. (1963) Influence of the injection of Triton WR-1339 on the properties of rat-liver lysosomes. Ciba Symposium on lysosomes (A.U.S. de Reuck and M. P. Cameron, eds.) Churchill, London, pp. 176-196. Opperdoes, F.R. and Van Roy, J. (1982) Trypanosoma brucei: Uptake into lysosomes and trypanocidal action of Triton WR-1339, Parasitol. 84, LVII. Lanham, S.M. (1968) Separation of trypanosomes from the blood of infected rats and mice by anion-exchangers. Nature 218, 1273-1274. Opperdoes, F.R., Markos, A. and Steiger, R.F. (1981) Localization of malate dehydrogenase, adenylate kinase and glycolytic enzymes in glycosomes and the threonine pathway in the mitochondrion of cultured procyclic trypomastigotes of Trypanosorna brucei. Mol. Biochem. Parasitol. 4,291-309. Stein, S., B~hlen, P., Stone, J., Dairman, W. and Udenfriend, S. (1973) Amino acid analysis with fluorescamine at the picomole level. Arch. Biochem. Biophys. 1 5 5 , 2 0 3 - 2 1 2 . Toner, J.J. and Weber, M.M. (1972) Respiratory control in mitochondria from Crithidia fasciculata. Biochem. Biophys. Res. Commun. 46, 6 5 2 - 6 6 0 . Brooker, B.E. and Vickerman, K. (1964) Acid phosphatase in trypanosomes. Trans. R. Soc. Trop. Med. Hyg. 5 8 , 2 9 3 - 2 9 4 . Seed, J.R., Byram III, J. and Gam, A.A. (1967) Characterization and localization of acid phosphatase activity of Trypanosoma gambiense. J. Protozool. 14, 117-125. Steiger, R.F. (1973) On the ultrastructure of Trypanosoma (Trypanozoon) brucei in the course of its life cycle and some related aspects. Acta Tropica 30, 6 3 - 1 6 8 . Barrett, A.J. and Heath, M.F. (1977) Lysosomal enzymes. In: Lysosomes, a Laboratory Handbook, 2nd edition, (Dingle, J.T., ed.), pp. 19-145, North-Holland, Amsterdam, New York, Oxford. Opperdoes, F.R. and Steiger, R.F. (1981) Localization of hydrolases in cultured procyclic trypomastigotes of Trypanosoma brucei Mol. Biocliem. Parasitol. 4, 3 1 1 - 3 2 3 . Opperdoes, F.R. and Van Roy, J. (1982) The phospholipases of Trypanosoma brucei bloodstream forms and cultured procyclics. Mol. Biochem. Parasitol. 5 , 3 0 9 - 3 1 9 . Henning, R. and Plattner, H. (1975) Formation of Triton WR-1339-filled rat liver lysosomes. I. Properties and intracellular distribution of [3 H]Triton WR-1339. Exp. Cell Res. 9 4 , 3 6 3 - 3 7 6 . Leighton, R., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S. and de Duve, C. (1968) The large-scale separation of peroxisomes, mitochondria and lysosomes from the livers of rats injected with Triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J. Cell Biol. 37 (1968) 4 8 2 - 5 1 3 . Scanu, A. and Oriente, P. (1961) Triton hyperlipemia in dogs. I. In vitro effects of the detergent on serum lipoproteins and chylomicrons. J. Exp. Med. 1 1 3 , 7 3 5 - 7 5 7 . de Duve, C. and Wattiaux, R. (1966) Functions of lysosomes. Ann. Rev. Physiol. 28, 4 3 5 492. Cornforth, J.W., d'Arcy Hart, P., Rees, J.W. and Stock, J.A. (1951) Antituberculous effect of certain surface-active polyoxyethylene ethers in mice. Nature 168, 150-153. Beveridge, E. (1963) Chemotherapy of Leishmaniasis. In: Experimental Chemotherapy (Schnitzer, J.R. and Hawking, F., eds.), vol. 1, pp. 2 5 7 - 2 8 7 , Academic Press, New York. de Duve, C., de Barsy, Th., Poole, B., Trouet, A., Tulkens, P. and Van Hoof, F. (1974) Lysosomotropic agents. Biochem. Pharmacol. 23, 2495-2531.
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Trouet, A. (1964) Immunisation de lapins par des lysosomes h6patiques de rats trait6s au Triton WR-1339. Arch. Int. Physiol. Biochim. 72,698. Dean, R.T. (1975) Multiple forms of lysosomal enzymes. In: Lysosomes in Biology and Pathology, (J.T. Dingle and Dean, R.T. eds.), vol. IV, pp. 3 4 9 - 3 8 2 , North-Holland, Amsterdam. Henning, R. and Heidrich, H.G. (1974) Membrane lipids of rat liver lysosomes prepared by freelow electrophoresis. Biochim. Biophys. Acta 3 4 5 , 3 2 6 - 3 3 5 . Hawking, F. (1963) Chemotherapy of Trypanosomiasis. In: Experimental Chemotherapy (Schnitzer, J.R. and Hawking, F. eds.), Vol. 1, pp. 129-256, Academic Press, New York.
181
INVOLVEMENT OF LYSOSOMES IN THE UPTAKE OF MACROMOLECULAR MATERIAL BY BLOODSTREAM FORMS OF TR YPANOSOMA B R UCEI
FRED R. OPPERDOES and JORIS VAN ROY Research Unit for Tropical Diseases, International Institute of Cellular and Molecular Pathology, avenue Hippocrate, 74, B-12OOBrussels, Belgium (Received 25 March 1982; accepted 14 May 1982)
To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromoleculax compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (> 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm a in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and t~-mannosidase-containing organelles of 7". brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function. Key words: Trypanosoma brucei, Lysosomes, Endocytosis, Triton WR-1339, Trypanocidal drug, Lysosomotropic drug, Cell fractionation.
INTRODUCTION A drug like suramin, which is active against Trypanosoma rhodesiense and T. gambiense, the causative agents o f sleeping sickness and T. brucei, causing nagana in lifestock, circulates in the bloodstream in close association with plasma proteins [1, 2] and it is believed that it enters the trypanosome by a mechanism of endocytosis followed by lysosomal digestion of the associated protein [3]. The recently developed trypanocidal daunorubicin-protein complexes [4] are thought to exert their action according to the same principle. This concept of lysosomotropism requires that trypanosomes are capable of taking up macromolecules by a mechanism of endocytosis or pinocytosis followed by fusion of the endocytotic vesicles with so-called primary lysosomes, resulting in the for0166-6851/82[0000-0000[$02.75 © 1982 Elsevier BiomedicalPress
182 mation of a phagolysosome. Such a phagolysosome should contain adequate hydrolases in order to free the intact drug after digestion of the carrier protein. Electron microscopy studies on several members of the Trypanosomatidae have shown that these organisms are capable of taking up macromolecular material via endocytosis. Ingestion of protein from a ferritin-containing medium was first demonstrated for Trypanosoma mega [5] and the existence of a specialized cytostome was postulated. Similar experiments demonstrated endocytosis in the case of T. rhodesiense[6], T. brucei [7] and T. cruzi [7]. Langreth and Balber [8] have carried out detailed electron microscope studies in the case of T. brucei and they have shown that macromolecular material first accumulates in the flagellar pocket and then enters into large spiny-coated vesicles by a mechanism of endocytosis or fluid pinocytosis. These vesicles subsequently become continuous with a collecting membrane system and eventually the foreign material ends up in large digestive vacuoles. Biochemical evidence to support these electron microscopic observations is still scarce. Fairlamb and Bowman [9] demonstrated that an inert macromolecule like polyvinyl pyrrolidone can be taken up by T. brucei in vitro by fluid endocytosis, albeit at a low rate, whereas in vivo the lysosomotropic drug, suramin, entered possibly by a similar mechanism but at a considerably higher rate [3]. In both cases, however, no evidence was presented on the intracellular fate of the material ingested. In previous work we have shown that T. brucei possesses a-mannosidase and acid proteinase-containing organelles resembling lysosomes [10]. However, involvement of these organelles in the uptake and digestion of exogenous macromolecules remained to be proven, since they equally well might be involved in the excretion of digestive enzymes into the lumen of the flagellar pocket. To produce evidence that macromolecules from the external medium may end up in these lysosomes, after internalization by endocytosis, we have made use of Triton WR-1339 (polyoctylphenolpolyethyleneglycol), a nondigestible macromolecular compound, known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage [11]. Part of this work has already been reported in abstract form [12]. MATERIALS AND METHODS Organism. Trypanosoma brucei brucei Stock 427 was maintained as a stabilate at -196°C. Upon intraperitoneal inoculation of 106 parasites per rat this strain produced a fulminating infection resulting in the death of the host in approximately 100 h. Growth and isolation o f trypanosomes. Trypanosomes were grown in 300 g male Wistar rats. After inoculation of the parasites infected blood was withdrawn from the animals after 92 h by cardiac puncture using heparin as an anticoagulant. The trypanosomes were separated from other blood elements by filtration through DEAE-cellulose, according to Lanham [13] followed by washing with 250 mM sucrose, 25 mM Tris/HC1 and 1 mM EDTA, pH 7.4.
183
Cell fractionation. Preparation of cell homogenates, cell fractionation by differential centrifugation and isopycnic centrifugation in linear sucrose gradients in a vertical Beckman VTi 50 rotor and the presentation of the results are exactly as described previously [10, 14]. The structural latency of enzymes in cytoplasmic extracts was determined in sucrose-containing isotonic media, supplemented with 0.04% (w/w) bovine serum albumin, by measuring enzyme activity in the absence and the presence of 0.1% (v/v)Triton X-100, respectively. The sedimentability of enzymes was determined by centrifugation of cytoplasmic extracts for 1 h at 139 000 × g. Treatment of infected animals with Triton WR-1339. The nonionic detergent Triton WR-1339 (Tyloxapol, Merk index 9487) was dissolved in 0.15 M NaCI and sterilized by filtration through Millipore membranes (0.22 tan pore size) prior to injection. To isolate trypanosomes from Triton-treated animals 300 g rats were given a dose of Triton WR-1339 of 0.4 g/kg body weight 70 h after infection with trypanosomes and the trypanosomes were isolated 22 h later. To study the trypanocidal effect of Triton WR-1339, female NMRI mice of approximately 25 g were infected with l0 s virulent trypanosomes at zero time and treated with different dose levels at different times. Triton WR-1339 was dissolved in 0.15 M NaC1. For each dose level a different concentration was prepared and every mouse received 1 ml of solution intraperitoneally. Control animals only received a solution of NaC1. 100 days after infection of the mice with trypanosomes the experiment was terminated and all the survivors were considered cured. Enzyme determinations, a-D-Mannosidase (EC 3.2.1.24) was measured under optimized conditions, exactly as described by Steiger et al. [10]. Acid proteinase (EC unclassified) was measured with [3H]casein as substrate in 0.1 M phosphate buffer, pH 6.5, as described previously [14, 15]. Protein was determined fluorimetrically [16] with bovine serum albumin as standard. Materials. Triton WR-1339 was purchased from Ruger Chemical Co., Irvington, NJ, U.S.A. All other chemicals were of the highest purity available. RESULTS When rats infected with Trypanosoma brucei were treated with a relatively high dose of Triton WR-1339 (2 g/kg body weight) it proved difficult to isolate trypanosomes in sufficient quantities since, firstly, trypanosomes disappeared before the rats could be bled and, secondly, trypanosomes, if still present, were very fragile and could only be eluted with low recoveries from a DEAE-cellulose column. For these reasons the dose of Triton was lowered to 0.4 g Triton WR-1339/kg. 22 h after administration trypanosomes were still present in large quantities and could be easily purified in a yield of more than 80%. Blood smears from rats treated with such a dose showed that part of the trypanosomes had developed a massive vacuole which was always located at the posterior end between
184
Fig. 1. Giernsa-stained smear of infected rat blood after treatment with Triton WR-1339. Rats were infected with 106 T. brucei intraperitoneally and treated intraperitoneally with 0.5 g Triton WR-1339/ kg 3 days later. Blood was examined 17 h after administration of Triton. Arrows point at trypanosomes containing a vacuole.
kinetoplast and nucleus. This vacuole varied in size and could be twice as big as the normal diameter of a healthy trypanosome (Fig. 1.). After purification such mixed populations of trypanosomes were subjected to homogenization by grinding with silicon carbide [16] and the latency and sedimentability of two lysosomal enzymes was monitored in cytoplasmic extracts. Fig. 2 shows that strucural latency of both enzymes from trypanosomes that had been in contact with Triton WR-1339 for 22 h was decreased with respect to the untreated controls. Moreover, a considerable part of acid proteinase was solubilized due to the treatment. This suggests that a significant part o f the lysosomes were damaged and had lost their content, a-Mannosidase remained entirely sedimentable, even after treatment. Fractionation o f post-large-granule fractions [10, 14] by isopycnic centrifugation in sucrose (Fig. 3) showed that the lysosomal markers in control cells equilibrated at densities around 1.18 g/cm 3, but that contact with Triton WR-1339 for 22 h led to a significant shift in the density o f a-D-mannosidase towards lower values (1.14 g/cm3).
185
A similar effect was observed for the particulate part of acid proteinase (shift from 1.19 towards 1.16 g/cm 3) but in addition a considerable amount of the enzyme was solubilized and remained at the top of the gradient. Since we observed that administration of an elevated dose of Triton WR-1339 could lead to a total disappearance of trypanosomes from the blood we investigated the chemotherapeutic effect of Triton on infected mice. Fig. 4 shows that complete cures (survival for more than 100 days) were obtained even at a dose as low as 0.5 g/kg body weight in 20% of the cases, provided that Triton was given early enough after infection. The best results (80% long-term survivors) were obtained with a dose of 2 g/kg, given one day after the trypanosomes. No cures at all could be obtained when Triton was given between one and three days before infection (not shown). DISCUSSION
Several investigators have reported upon the presence of lysosomes in the species Trypanozoon, but in most studies a positive reaction for acid phosphatase served as the only evidence for the lysosomal nature of such organeUes [7, 8, 17-19]. Although acid
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Fig. 2. The effect of Triton WR-1339 treatment on the intactness o f T. brucei lysosomes. (A) Latency of two lysosomal enzymes in cytoplasmic extracts expressed as that percentage of the total activity that cannot be measured in the absence o f detergent. Total activity was determined in the presence of 0.l% Triton X-100 as a detergent. (B) Sedimentability of lysosomal enzymes in cytoplasmic extracts. Total activity in pellets and supernatants were measured in the presence of 0.1% Triton X-100 to remove any structural latency. Open bars represent cytoplasmic extracts prepared from trypanosomes isolated from control rats. Closed bars represent extracts from trypanosomes isolated from rats treated 22 h before trypanosome isolation.
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phosphatase is a typical marker [20] of mammalian lysosomes, in T. brucei the bulk of this enzyme is located in the flagellar pocket and the endoplasmic reticulum [8, 10]. However, lysosome-llke organeUes containing a number of hydrolases such as acid proteinase, RNase, a-D-mannosidase and phospholipase A~, but devoid of acid phosphatase, were recently described for T. brucei [10, 21, 22]. No direct proof existed until now for
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Fig. 4. C h e m o t h e r a p e u t i c activity o f Triton WR-1339 on a T. brucei infection in mice. Triton was given intraperitoneally either on the day of infection (D + 0) or 1, 2 or 3 days later. ( . . . . . ), untreated control mice; ( ) 0.5, ( . . . . . ) 1 and ( - - - - - - ) 2 g Triton WR-1339/kg body weight.
187 the involvement of these organelles in the uptake and digestion of foreign macromolecular material. Wattiaux et al. [11] ftrst described the loading of lysosomes from rat liver with the non-ionic detergent Triton WR-1339, and since then this process has been used as a method for high-yield isolation of lysosomes with the highest degree of purity. This method exploits the dramatic decrease in buoyant density of the hepatic lysosomes due to a massive uptake of the detergent. There is little doubt that Triton WR-1339 with a molecular weight of over 100 000 and a diameter of approximately 8 nm is taken up by endocytosis and enters into the lysosomes after fusion with phagocytic vacuoles [23]. Since numerous tissues other than liver also have been found to store Triton in their lysosomes we have investigated whether such a phenomenon also occurs with the lysosome-like organelles described for T. brucei bloodstream forms [10, 22]. The results presented in this paper are similar to those by others for rat liver [11, 24]. We interpret them to indicate that Triton WR-1339, circulating in plasma in close association with lipoproteins [25], is taken up by a mechanism of endocytosis or pinocytosis by the cell, leading to the formation of phagolysosomes. Since Triton is not digested by lysosomal hydrolases this leads to a massive accumulation of the detergent and consequently to the formation of the huge vacuoles seen with the light microscope. These Triton-loaded vacuoles probably originate from pre-existing lysosomes as is suggested by the presence of essentially all a-D-mannosidase and part of the acid proteinase in organelles with a reduced buoyant density in sucrose gradients. The majority of these organelles most likely represent leaky vacuoles, since they have lost a considerable part of their acid-proteinase activity. Mannosidase, however, remained associated with them, which can be explained by the fact that the latter enzyme is probably firmly attached to the lysosomal membrane as we have shown before [10]. These results suggest that the membranes of such massive phagolysosomes are highly fragile entities which are easily ruptured, possibly already in situ, but certainly during cell fractionation. A similar leakage of lysosomal content after massive overloading of lysosomes with undigestible material has already been described by others [26]. An overall shift in buoyant density of 0.03 g/cm 3 due to accumulation of Triton WR1339 in the case of T. brucei lysosomes is rather small as compared to a value of 0.10 g/cm a routinely obtained for rat-liver lysosomes, but can be explained as follows: firstly, the trypanosomes have been in contact with Triton for only 22 h, whereas in the case of rat liver the lysosomes are normally isolated 3 4 days after administration of the detergent; secondly, the dose-level (0.4 g/kg) used in our experiments was considerably lower than that of 0.85 g/kg normally administered for the isolation of liver lysosomes and thirdly, the trypanosomes, at the low dose level used, were still actively dividing, resulting in a continuous dilution of the Triton content of the digestive vacuoles. It is a well-known fact that Triton WR-1339 affords a remarkable protection against Mycobacterium tuberculosis [27] and a similar effect was described for Leishmania donovani infections in hamsters [28]. There seems to be little doubt that in both cases the detergent owes its antimicrobial properties to its lysosomotropism, rendering the
188 vacuolar apparatus of the host cell inhospitable to certain invading microorganisms [29]. T. brucei, however, is not an intracellular parasite but circulates freely in the bloodstream and tissue fluids. Therefore, the curative effect o f Triton WR-1339 described here is most likely due to an effect o f the detergent on the trypanosome itself. Triton-t'flied lysosomes in general are largely increased in size [11, 24] contain some plasma proteins at abnormally high levels [30] and exhibit an increased activity o f some lysosomal enzymes [31], whereas the lipid content may also differ from that o f normal lysosomes [32]. Henning and Plattner [23] have suggested that leakage o f the lysosomal content in situ might occur already in a very early stage after Triton WR-1339 administration. If all these effects were also to occur in the digestive vacuoles o f T. brucei, they might easily lead to an impaired lysosomal function, resulting either in an inhibition o f nutrient digestion, or a release o f digestive enzymes into the cytosol. Both effects either alone, or in combination, can provide an explanation for the trypanocidal action o f Triton WR1339. Several authors have already described the curative effect o f Triton WR-1339 in experimental infections with African trypanosomes [33] but at that time its mechanism of action was not well understood. Our experiments strongly suggest that the trypanocidal action o f Triton WR-1339 can be explained b y an interference o f the drug with lysosomal action due to an accumulation in phagolysosomes through a mechanism o f endocytosis. ACKNOWLEDGEMENTS This investigation received financial support from the UNDP/World Bank/WHO 'Special Programme for Research and Training in Tropical Diseases'. REFERENCES 1 Dangerfield, W.G., Gaunt, W.E. and Wormall, A. (1938) Studies on Bayer 205 (Germanin) and Antrypol. I. The determination of small amounts of Bayer 205 (and Antrypol). II. The persistence of Bayer 205 in the bloodstream after injection into animals. Biochem. J. 32, 59-70. 2 Muller, W.E. and Wollert, U. (1976) Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin. Biochim. Biophys. Acta 427,465-480. 3 Fairlamb, A.H., and Bowman, I.B.R. (1980) Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effects on respiration and growth rate in vivo. Mol. Biochem. Parasitol. 1, 315- 333. 4 Williamson, J., Scott-Finnigan, T.J., H~dman, M.A. and Brown, J.R. (1981) Trypanocidal activity of daunorubicln and related compounds. Nature 292,466-467. 5 Steinert, M. and Novikoff, A.B. (1960) The existence of a cytostome and the occurrence of pinocytosis in the trypanosome Trypanosorna mega. Biophys. Biochem. Cytol. 8,563-569. 6 Brown, K.N., Armstrong, J.A. and Valentine, R.C. (1965) The ingestion of protein molecules by blood forms of Trypanosoma rhodesiense. Exp. Cell Res. 39, 129-135. 7 Jadin, J.M. (1971) Cytologic et cytophysiologie des Trypanosomidae. Acta Zool. Pathol. Antverpiensia 53, 1-168. 8 Langreth, S.G. and Balber, A.E. (1975) Protein uptake and digestion in bloodstream and culture forms of Trypanosoma brucei. J. Protozool. 22, 40 53.
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