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Mechanism of action of amphotericin B on Leishmania donovani promastigotes
Molecular and Biochemical Parasitology, 19 (1986) 195-200
195
Elsevier MBP 00664
Mechanism of action of amphotericin B on Leishmania donovani promastigotes Ahindra Kumar Saha, Tanmoy Mukherjee and Amar Bhaduri* Division of Biochemistry, Department of Pharmacy, Jadavpur University, Calcutta 700 032, India 'Received 13 August 1985; accepted 8 January 1986)
The growth of Leishmania donovani promastigotes in a liquid medium was completely inhibited by amphotericin B at a concentration of 0.3 txg m1-1 (0.3 IxM). Continuous release of small molecules that absorb at 260 nm and 280 nm was observed after contact with the drug. Uptake of [u-laC]glucose was inhibited in cells treated with the drug. An immediate release of isotopic glucose and its metabolites from preloaded cells could be demonstrated after incubation with amphotericin B (0.4 ixM). Inhibition of respiration by the drug was a comparatively slower process. All the above effects could be effectively prevented in the presence of either cholesterol or ergosterol. The primary site of action of amphotericin B on L. donovani promastigote cells appears to be membrane sterols that result in a loss of the permeability barrier to small metabolites. An interesting biochemical similarity, thus, emerges between flagellated protozoa and fungi. Key words: Leishmania donovani; Amphotericin B; Polyene antibiotics; Protozoa
Introduction
The parasitic protozoan, Leishmania donovani is the causative agent for Kalaazar, a widely prevalent disease in many parts of the tropical world. Pentavalent antimonials and pentamidine, a biguanidine compound, are the available drugs of choice for visceral leishmaniasis. Amphotericin B, a polyene antibiotic, has been widely used in several forms of dermal and subcutaneous leishmaniasis with considerable success [1]. Recently, New et al. [2] have shown that amphotericin B, when trapped into multilamellar liposomes, can become significantly effective against visceral leishmaniasis with a tolerable range of toxicity in a rodent model. The drug has also been found to be extremely potent against the amastigote or the host form of both L. donovani and L. tropica in an in vitro human monocyte-derived macrophage screening system [3]. Surprisingly, as emphasized by New et al. [2], in spite of its obvious importance as an anti-leishmanial drug, no "Present address: Indian Institute of Chemical Biology, 4, Raja, S.C. Mallick Road, Jadavpur, Calcutta 700 032, India.
systematic work has been done on the mechanism of the action of amphotericin B on Leishmania spp. This is very relevant information both for further drug development against leishmaniasis and also for comparative biochemistry. Recently, Langreth et al. [4] have observed that when the amastigote form growing in a human macrophage system is exposed to amphotericin B, parasitic cytoplasm appeared pycnotic, and ribosomes were more closely packed, before a general disintegration set in. Since the introduction of amphotericin B as an antifungal antibiotic, extensive work has been done on its mode of action on fungi. Polyene antibiotics such as amphotericin B increase the permeability of phospholipid bilayer membranes towards ions and small molecules by specifically interacting with membrane sterols [5,6]. However, the exact molecular mechanism of this interaction as studied with model systems still remains to be elucidated [7,8]. In the present paper, we demonstrate that, as in fungi, the primary site of action of the drug on the promastigote or culture form of the organism is on its membrane and the lethal effect is brought about by disruption of
0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
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the permeability barrier formed by the membrane. This work further suggests some interesting biochemical similarities between fungi and this class of protozoa. Materials and Methods
All the biochemicals were purchased from Sigma Chemical Co. Amphotericin B and pentamidine were obtained as generous gifts from May and Baker Research Division, U.K. For all experiments, amphotericin B and sterols were dissolved in dimethyl sulphoxide. [U-14C[Glucose was purchased from Bhaba Atomic Research Centre, India. The organism used for this work was L. donovani UR-6, a strain which is a recent clinical isolate and was obtained by us from Dr. D.K. Ghosh of the Indian Institute of Chemical Biology, Calcutta. The cells were grown at 22°C on blood agar slants, 100 ml of which contained 1 g glucose, 0.6 g sodium chloride, 1 g Oxoid peptone, 1.5 g agar, 50 ml beef heart extract and 5 ml rabbit blood (obtained by heart puncture). The cells were maintained by subcultures made at intervals of 72 h and occasionally passed through NNM medium [9] for vigorous growth. The liquid medium used for growth experiments was a semisynthetic one, recently developed by Chowdhuri et al. [10]. One litre of the medium contains 10 g glucose, 10 g peptone (Oxoid), 3 g choline chloride, 6 g sodium chloride, 1 mg folic acid, 10 mg sodium bicarbonate, 10 mg magnesium sulphate, 200 mg potassium dihydrogen phosphate, and 1 mg haemin. Cells were counted in a Neubauer haemocytometer. 1 × 106 cells as counted by this method contained 0.8 mg of cell protein, when determined by the biurate method [11]. Only fully motile cell preparations were used for drug experiments. After growth for 60 h on solid blood-agar medium, the cells were scraped from the surface, washed by centrifugation and resuspended in phosphate saline buffer, pH 7.2. The phosphate saline buffer consisted of 8 g sodium chloride, 0.2 g potassium chloride, 1.15 g dis0dium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 0.29 g magnesium sulphate, per litre of glass distilled water. Respiration of the organism was studied by
conventional manometric techniques in a Warburg apparatus [12]. Each Warburg flask contained 0.2 ml of 10 % potassium hydroxide in the central well. The main reservoir initially contained (in a total volume of 2.8 ml of phosphate saline buffer) 1.25 × 107 cells approximately equivalent to 10 mg of cell protein and requisite amount of glucose where indicated. After equilibration at 37°C for 15 min, amphotericin B and cholesterol, both in dimethyl sulphoxide solution, were added within 30 s of each other in quick succession when needed. The final volume was made upto 3 ml in the reservoir with phosphate saline buffer and allowed to equilibrate for another 5 min. The vessels were then closed to the atmosphere and the reading taken at this time was counted as the zero time. Air was used as gas phase; shaking spead was 120 strokes min -1. Transport studies were done according to Schaefer and Mukkada [13] with some modifications. The uptake of [U-14C]glucose was used as a measure of glucose transport. Twice washed promastigotes were resuspended in the basal salts solution (5.2 g NaCI, 0.5 g KC1, 10.3 g Na2HPO 4 in 1 1 glass distilled water, pH 7.0) to give a final cell concentration of 0.3 mg cell protein m1-1. The suspensions were equilibrated at 28°C for 10 min. The concentration of the labelled sugar and of other additions in various experiments are indicated in the results section. Aliquots of 1-ml samples were removed at appropriate intervals and were filtered immediately through cellulose acetate filters (0.45 Ixm porosity; Millipore Corp., New Bedford, MA) and washed with 20 ml basal salts solution. The filters with the cells were transferred directly to scintillation vials containing 5 ml Bray's scintillation fluids [14] and radioactivity was determined in an LKB-Wallac, 1217, Rackbeta liquid scintillation counter. The results are expressed as nmol sugar (mg cell protein)-1. Results
Leishmanicidal activity of amphotericin B. Amphotericin B was found to have a profound growth inhibitory effect on the promastigotes form of this strain of L. donovani. At a concentration of 0.3 IxM, the drug inhibited cell division and the lytic effect became very pronounced at higher
197
concentrations (Fig. 1). Vehicle control using solvent at the same concentrations had no effect on growth. Under identical growth conditions, 2 ~g ml-1 (6 ~M) of pentamidine was needed to completely arrest growth (data not shown).
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Effect on cellular respiration. Amphotericin B at a concentration of 0.5 ~M completely inhibited the glucose-stimulated respiration within a short time of contact with the cells (Fig. 2). The cells appeared to have lost motility to a considerable extent by this time, though their morphological integrity remained unaffected throughout the course of the experiment. Simultaneous addition of amphotericin B and cholesterol in the incubation mixture resulted in a very strong protection against antibiotic activity; 80% of the original respiration rate could be restored at a molar concentration ratio of amphotericin B to cholesterol of 1:25.
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60
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( MINUTES I
Fig. 2. Effect of amphotericin B on the respiration of L. donovani promastigotes. The incubation mixture of 3 ml contained 1 ml of washed cell suspension (10 mg of protein), phosphate buffered saline (0.15 M, pH 7.2) and other additions as indicated. ( o - o ) Endogenous; ( o - o ) endogenous + glucose (2 mM); (X-X) endogenous + glucose (2 mM) + 10 gl dimethyl sulphoxide; ( & - A ) endogenous + glucose (2 mM) + amphotericin B (0.5 ~M); (A--A) endogenous + glucose (2 raM) + amphotericin B (1 p.M); ( i - n ) endogenous + glucose (2 mM) + amphotericin B (1.0 p.M) + cholesterol (12.5 ~xM); (~3-~) endogenous + glucose (2 mM) + amphotericin B (0.5 p~M) + cholesterol (12.5 ~M). 0.5 z
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Fig. 1. Inhibition of growth of L. donovani in presence of amphotericin B. The control growth curve in absence of amphotericin B is represented by o - n . D-:3, o - o and o - o indicate growth patterns in presence of 0.1, 0.3 and 1 IxM amphotericin B, respectively.
60 (MINUTES)
90
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Fig. 3. Release of 260- and 280-nm absorbing materials in the presence of amphotericin B. The incubation mixture, in a final volume of 10 ml containing cell suspension (2 mg ml 1) in phosphate buffer saline (0.15 M, pH 7.2) and amphotericin B (0.5 IxM), was incubated at 28°C. At each indicated interval, an aliquot of 1.5 ml was drawn from the flask and rapidly centrifuged. The clear supernatant was diluted suitably in phosphate buffer saline and absorbancy readings were taken. (o-o) Reading at 260 nm for cells in absence of the drug; ( ~ - A ) 280 nm and ( o - e ) 260 nm readings, respectively in presence of the drug (0.5 IxM); (:z-n) 260 nm and ( A _ A ) 280 nm readings, respectively, in presence of drug (0.5 I~M) and ergosterol (12.5 txM).
198
Release of small molecules. Fig. 3 shows the effect of the d r u g on the p e r m e a b i l i t y o f the m e m b r a n e of L. donovani p r o m a s t i g o t e s . In p r e s e n c e of t h e d r u g (0.5 ~ M ) , a c o n t i n u o u s r e l e a s e of m e t a b o l i t e s c o u l d b e o b s e r v e d for n e a r l y 90 min. M a t e r i a l s a b s o r b i n g at 260 a n d 280 n m w e r e p r o b a b l y p r i m a r i l y h e t e r o c y c l i c b a s e s a n d arom a t i c a m i n o acids, r e s p e c t i v e l y . H o w e v e r , the possibility t h a t l a r g e r m o l e c u l e s such as p r o t e i n s o r s o l u b l e R N A s w e r e also l e a k i n g o u t at s o m e stage of this e x p e r i m e n t c o u l d n o t be e l i m i n a t e d . In this case also, a d d i t i o n o f e r g o s t e r o l in a 1:25 m o l a r r a t i o r e s u l t e d in a c o m p l e t e p r o t e c t i o n of the cells a g a i n s t the d r u g action. D i m e t h y l sulp h o x i d e t r a n s f e r r e d with t h e d r u g h a d no effect on t h e m e m b r a n e . T h e r e l e a s e of m a t e r i a l s abs o r b i n g at 260 n m at the e n d of 90 m i n was a b o u t 60% of t h e t o t a l 260 n m a b s o r b i n g m a t e r i a l s that c o u l d n o t be p r e c i p i t a t e d by p e r c h l o r i c acid ( d a t a n o t s h o w n ) . N o visible effect i n d i c a t i n g significant cell d a m a g e c o u l d be o b s e r v e d u n d e r the mi-
c r o s c o p e e v e n after 70 min of i n c u b a t i o n with t h e drug.
Effect of amphotericin B on transport and retention of metabolites. Selective t r a n s p o r t o f m e t a b olites a n d t h e i r r e t e n t i o n is a c h a r a c t e r i s t i c p r o p e r t y of biological m e m b r a n e s . E x p o s u r e to a m p h o t e r i c i n B h a d a d r a m a t i c effect on the t r a n s p o r t p r o p e r t i e s of L. donovani p r o m a s t i gotes. This is e v i d e n t f r o m the results s h o w n in Fig. 4 a n d Fig. 5. T h e glucose t r a n s p o r t s y s t e m in L. tropica a n d in L. donovani h a d e a r l i e r b e e n c h a r a c t e r i z e d by Schaffer a n d M u k k a d a [13], a n d by Z i l b e r s t e i n a n d D w y e r [15], r e s p e c t i v e l y using i s o t o p i c a l l y l a b e l l e d 2-deoxy-D-glucose. In b o t h cases, c a r r i e r m e d i a t e d active t r a n s p o r t systems w e r e o p e r a t i v e . In o u r e x p e r i m e n t s , a d d i t i o n of a m p h o t e r i c i n B at a c o n c e n t r a t i o n of 0.4 ~zM h a d a p r o f o u n d effect on the u p t a k e of r a d i o a c t i v e glucose. D u r i n g the first 3-4 rain, t h e r e was upt a k e of r a d i o a c t i v e glucose into the cell, but then
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Fig. 4. Effect of amphotericin B on the uptake of glucose by L. donovani promastigotes. Washed cells were resuspended in basal salt solution (5.2 g NaC1, 0.5 g KCI, 10.3 g Na2HPO4 in 1000 ml, pH 7.0) to a cell density of 0.6 mg of cell protein m1-1. Three flasks, one containing only cell suspension (control), another containing cell suspensions and amphotericin B (0.4 ~M) and the last one, containing cell suspension, amphotericin B (0.4 IxM) and ergosterol (7.5 tzM) were incubated with gentle shaking for 10 min at 28°C. Individual aliquots of 0.5 ml of these incubated cell suspensions were added to the tubes of incubation mixtures of basal salts solution containing 0.1 mM [U24-C]glucose (2.1 x 105 cpm) in a total volume of 1 ml. At the indicated time intervals, the contents of each tube were rapidly filtered and the radioactivity retained on the Millipore filter paper was counted. (o-o) Control; (e-o), control + drug; ( A - A ) , control + drug + ergosterol.
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Fig. 5. Release of accumulated glucose as its metabolites from L. donovani promastigotes on treatment with amphotericin B. Washed cells were suspended in basal salts solution (pH 7.0) to a cell density of 0.3 mg ml 1. Cell suspensions in final volumes of 5 ml in two separate flasks were incubated with 0.1 mM [U-14C]glucose (1.1 x 106 cpm) for 30 min at 28°C. One of the flasks was used as a control. Amphotericin B (0.4 p~M) was added to the other at the end of the preincubation period. Aliquots were drawn at indicated time intervals from the both flasks and counts retained in the cells were measured by the counting procedure described in the legend of Fig. 4. (o-o) Control and (e-o) drug-treated cells, respectively.
199 the level started to go down with time. The simultaneous presence of ergosterol (1:19 molar ratio) provided nearly complete protection (Fig. 4). In this case, ergosterol was added to the incubation mixture within 20 s of addition of amphotericin B. A second experiment was also carried out where cells were first preloaded with isotopically labelled glucose, and were then exposed to amphotericin B at its growth inhibitory concentration (0.4 p,M). An immediate and continuous release of the internal glucose pool, or its degraded metabolites, was monitored over a period of 16 min (Fig. 5). Discussion
The powerful in vitro leishmanicidal effect of amphotericin B that we observed (Fig. 1) compares favourably with the results obtained in other screening systems with other strains of L. donovani and L. tropica [3]. As in the earlier cases [3], we also found the drug to be more potent than pentamidine in arresting growth. Amphotericin B had a definite effect on the permeability properties of the protozoal membrane. Rapid release of small molecules from the internal pool could be demonstrated unambiguously by both spectrophotometric (Fig. 3) and isotopic experiments (Fig. 5). The isotopic experiment increased the sensitivity of detection and an immediate release of glucose, or its metabolites, from an internal pool after contact with the drug could be clearly demonstrated (Fig. 5). Since glucose could be rapidly metabolized in the Leishmania promastigotes, the uptake experiment with [U-14C]glucose, did not reflect the true kinetics of mediated transport across the cellular membrane. However, a definite disorganization of the membrane structure with a drastic effect on the transport system was evident from the uptake experiment (Fig. 4). The effect on the glucose uptake system is probably a reflection of both the loss of the permeability barrier and also of the malfunctioning of the carrier protein in the amphotericin B-treated disorganized membrane bilayer system. Recently Zilberstein and Dwyer have convincingly demonstrated the existence of a proton-motive forcedriven, carrier-mediated active transport system for D-glucose in L. donovani [16]. It is interesting
to note that comparatively little work has been done on the effect of amphotericin B on such a transport system either in fungi or in reconstituted vesicles. Respiration was completely blocked only after a comparatively long lag period, at a higher concentration of the drug. The effect on respiration thus appears to be a secondary phenomenon as a consequence of rapid loss of energy sources from the system (Fig. 2). As with fungi, addition of sterols could largely prevent the effect of amphotericin B on the permeability of Leishmania membranes. Effects on respiration and leakage of small molecules were mostly prevented and the glucose uptake could be almost completely restored by addition of sterols. Cholesterol was almost as effective as ergosterol in this respect. The basic mechanism of protection of cells from polyene action by sterols is dependent upon a physiochemical interaction between the antibiotic and the sterol, which lowers the effective concentration of the antibiotic for interaction with the cells [17]. However, the molecular mechanism for preferential drug action of amphotericin B on fungi remains uncertain. It has been assumed to be based on the greater reactivity of the drug with ergosterol compared with cholesterol [6], the respective sterol components of fungal and mammalian" membranes. Ergosterol has been shown to occur in Leishmania promastigotes [18], though its localisation in the membrane has not been clearly demonstrated. It is likely that the chemotherapeutic basis of the preferential action of amphotericin B towards leishmanial infections is due to the presence of ergosterol in the membrane. Recent work from different laboratories has shown that selectivity of amphotericin B towards membrane ergosterol is greatly enhanced when the drug is bound to the lipid or lipoprotein components of the plasma membrane or to synthetic membrane vesicles rather than when it is dissolved in a solvent like dimethylsulphoxide for in vitro work [8,19]. In the light of this earlier work and our findings, the increased reactivity of amphotericin B in entrapped liposomes [2] is not surprising and can be exploited further for improved drug delivery systems. This work has also emphasized the close similarity in broad outlines between the mode of ac-
200 tion of amphotericin B on fungi and flagellated protozoal systems, thus confirming the original p r o p o s i t i o n o f R a g a n a n d C h a p m a n [20] o f a b i o c h e m i c a l h o m o l o g y in t h e p h y l o g e n y o f t h e s e t w o systems.
Acknowledgements
8
9 10
T h i s w o r k w a s s u p p o r t e d by g r a n t s f r o m t h e Indian Council of Medical Research and University G r a n t s C o m m i s s i o n , I n d i a . W e a r e g r a t e f u l to M a y & B a k e r R e s e a r c h D i v i s i o n , U . K . f o r their samples of pure drug amphotericin B and pentamidine.
References 1 Sampaio, S.A.P., Castro, R.M., Dillon, N.L. and Martins, J.E.C. (1971) Treatment of mucocutaneous leishmaniasis with amphotericin B: report of 70 cases. Int. J. Dermatol. 10, 179-181. 2 New, R.R.C., Chance, M.L. and Heath, S. (1981) Antileishmanial activity of amphotericin and other antifungal agents entrapped in liposomes. J. Antimicrobial Chemother. 8,371-381. 3 Berman, J.D. and Wyler, D.J. (1980) An in vitro model for investigation of chemotherapeutic agents in leishmaniasis. J. Infect. Dis. 142, 83-86. 4 Langreth, S.G., Berman, J.D., Riordan, P.G. and Lee, L.S. (1983) Fine-structural alterations in L. tropica within human macrophages exposed to anti-leishmanial drugs in vitro. J. Protozool. 30,555-561. 5 Sessa, G. and Weishman, G. (1967) Effect of polyene antibiotics on phospholipid spherules containing varying amount of charged components. Biochim. Biophys. Acta. 435,416-426. 6 Hammond, S.M. (1977) Biological activity of polyene antibiotics. Prog. Med. Chem. 14, 105-179. 7 Vertrat-Croquin, A., Boland, J. and Gray-Bobo, C.M. (1984) Enhancement of amphotericin B selectively by an-
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14
15
16
17
18
19
20
tibiotic incorporation into gel state vesicles. A circular dichorism and permeability study. Biochem. Biophys. Res. Commun. 125,360-366. Braijtburg, J., Elberg, S., Bolard, J., Kobayashi, G., Levy, R.A., Ostlund, R.F., Schlessinger, D. and Medoff, G. (1984) Interaction of plasma proteins with amphotericin B. J. Infect. Dis. 149,986-997. Nicolle, Ch. (1908) Culture du Parasite du Bouton d'orient. C.R.H. Seances Acad. Sci. Paris 146, 842-843. Chowdhuri, G., Chatterjee, T.K. and Banerjee, A.B. (1982) Growth factor requirements for in vitro growth of Leishmania donovani. Ind. J. Med. Res. 76, 157-163. Gornall, A.G., Bardawill, C.G. and David, M.M. (1949) Determination of serum protein by means of the biurate reaction. J. Biol. Chem. 177,751-766. Umbrejt, A.G., Burris, R.H. and Stanffer, J.W. (1957) Manometric Techniques, pp. 64-78, Burgess Publishing Co., Minneapolis. Schaffer, F.W. and Mukkada, A.J. (1976) Specificity of the glucose transport system in Leishmania tropica promastigotes. J. Protozool. 23,446-449. Bray, G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1,279-285. Zilberstein, D. and Dwyer, D.M. (1984) Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 12, 32%336. Zilberstein, D. and Dwyer, D.M. (1985) Proton motive force-driven active transport of D-glucose and c-proline in the protozoan parasite Leishrnania donovani. Proc. Natl. Acad. Sci. U.S.A. 82, 1716-1720. Lampen, J.O., Arnow, P.M. and Safferman, R.S. (1960) Mechanism of Protection by sterols against polyene antibiotics. J. Bacteriol. 80, 200-206. Goad, L.J., George, G.H. and David, H.B. (1984) Sterols in Leishmania species. Implications for biosynthesis. Mol. Biochem. Parasitol. 10, 161-170. Mehta, R., Lopez-Berestein, G., Hopfer, R., Mills, K. and Juliano, R.L. (1984) Liposomal amphotericin B is toxic to fungal cells but not to mammalian cells. Biochim. Biophys. Acta 770,230-234. Regan, M. and Chapman, A. (1978) In: A Biochemical Phylogeny of the Protists, p. 221, Academic Press, London.
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Elsevier MBP 00664
Mechanism of action of amphotericin B on Leishmania donovani promastigotes Ahindra Kumar Saha, Tanmoy Mukherjee and Amar Bhaduri* Division of Biochemistry, Department of Pharmacy, Jadavpur University, Calcutta 700 032, India 'Received 13 August 1985; accepted 8 January 1986)
The growth of Leishmania donovani promastigotes in a liquid medium was completely inhibited by amphotericin B at a concentration of 0.3 txg m1-1 (0.3 IxM). Continuous release of small molecules that absorb at 260 nm and 280 nm was observed after contact with the drug. Uptake of [u-laC]glucose was inhibited in cells treated with the drug. An immediate release of isotopic glucose and its metabolites from preloaded cells could be demonstrated after incubation with amphotericin B (0.4 ixM). Inhibition of respiration by the drug was a comparatively slower process. All the above effects could be effectively prevented in the presence of either cholesterol or ergosterol. The primary site of action of amphotericin B on L. donovani promastigote cells appears to be membrane sterols that result in a loss of the permeability barrier to small metabolites. An interesting biochemical similarity, thus, emerges between flagellated protozoa and fungi. Key words: Leishmania donovani; Amphotericin B; Polyene antibiotics; Protozoa
Introduction
The parasitic protozoan, Leishmania donovani is the causative agent for Kalaazar, a widely prevalent disease in many parts of the tropical world. Pentavalent antimonials and pentamidine, a biguanidine compound, are the available drugs of choice for visceral leishmaniasis. Amphotericin B, a polyene antibiotic, has been widely used in several forms of dermal and subcutaneous leishmaniasis with considerable success [1]. Recently, New et al. [2] have shown that amphotericin B, when trapped into multilamellar liposomes, can become significantly effective against visceral leishmaniasis with a tolerable range of toxicity in a rodent model. The drug has also been found to be extremely potent against the amastigote or the host form of both L. donovani and L. tropica in an in vitro human monocyte-derived macrophage screening system [3]. Surprisingly, as emphasized by New et al. [2], in spite of its obvious importance as an anti-leishmanial drug, no "Present address: Indian Institute of Chemical Biology, 4, Raja, S.C. Mallick Road, Jadavpur, Calcutta 700 032, India.
systematic work has been done on the mechanism of the action of amphotericin B on Leishmania spp. This is very relevant information both for further drug development against leishmaniasis and also for comparative biochemistry. Recently, Langreth et al. [4] have observed that when the amastigote form growing in a human macrophage system is exposed to amphotericin B, parasitic cytoplasm appeared pycnotic, and ribosomes were more closely packed, before a general disintegration set in. Since the introduction of amphotericin B as an antifungal antibiotic, extensive work has been done on its mode of action on fungi. Polyene antibiotics such as amphotericin B increase the permeability of phospholipid bilayer membranes towards ions and small molecules by specifically interacting with membrane sterols [5,6]. However, the exact molecular mechanism of this interaction as studied with model systems still remains to be elucidated [7,8]. In the present paper, we demonstrate that, as in fungi, the primary site of action of the drug on the promastigote or culture form of the organism is on its membrane and the lethal effect is brought about by disruption of
0166-6851/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
196
the permeability barrier formed by the membrane. This work further suggests some interesting biochemical similarities between fungi and this class of protozoa. Materials and Methods
All the biochemicals were purchased from Sigma Chemical Co. Amphotericin B and pentamidine were obtained as generous gifts from May and Baker Research Division, U.K. For all experiments, amphotericin B and sterols were dissolved in dimethyl sulphoxide. [U-14C[Glucose was purchased from Bhaba Atomic Research Centre, India. The organism used for this work was L. donovani UR-6, a strain which is a recent clinical isolate and was obtained by us from Dr. D.K. Ghosh of the Indian Institute of Chemical Biology, Calcutta. The cells were grown at 22°C on blood agar slants, 100 ml of which contained 1 g glucose, 0.6 g sodium chloride, 1 g Oxoid peptone, 1.5 g agar, 50 ml beef heart extract and 5 ml rabbit blood (obtained by heart puncture). The cells were maintained by subcultures made at intervals of 72 h and occasionally passed through NNM medium [9] for vigorous growth. The liquid medium used for growth experiments was a semisynthetic one, recently developed by Chowdhuri et al. [10]. One litre of the medium contains 10 g glucose, 10 g peptone (Oxoid), 3 g choline chloride, 6 g sodium chloride, 1 mg folic acid, 10 mg sodium bicarbonate, 10 mg magnesium sulphate, 200 mg potassium dihydrogen phosphate, and 1 mg haemin. Cells were counted in a Neubauer haemocytometer. 1 × 106 cells as counted by this method contained 0.8 mg of cell protein, when determined by the biurate method [11]. Only fully motile cell preparations were used for drug experiments. After growth for 60 h on solid blood-agar medium, the cells were scraped from the surface, washed by centrifugation and resuspended in phosphate saline buffer, pH 7.2. The phosphate saline buffer consisted of 8 g sodium chloride, 0.2 g potassium chloride, 1.15 g dis0dium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, 0.29 g magnesium sulphate, per litre of glass distilled water. Respiration of the organism was studied by
conventional manometric techniques in a Warburg apparatus [12]. Each Warburg flask contained 0.2 ml of 10 % potassium hydroxide in the central well. The main reservoir initially contained (in a total volume of 2.8 ml of phosphate saline buffer) 1.25 × 107 cells approximately equivalent to 10 mg of cell protein and requisite amount of glucose where indicated. After equilibration at 37°C for 15 min, amphotericin B and cholesterol, both in dimethyl sulphoxide solution, were added within 30 s of each other in quick succession when needed. The final volume was made upto 3 ml in the reservoir with phosphate saline buffer and allowed to equilibrate for another 5 min. The vessels were then closed to the atmosphere and the reading taken at this time was counted as the zero time. Air was used as gas phase; shaking spead was 120 strokes min -1. Transport studies were done according to Schaefer and Mukkada [13] with some modifications. The uptake of [U-14C]glucose was used as a measure of glucose transport. Twice washed promastigotes were resuspended in the basal salts solution (5.2 g NaCI, 0.5 g KC1, 10.3 g Na2HPO 4 in 1 1 glass distilled water, pH 7.0) to give a final cell concentration of 0.3 mg cell protein m1-1. The suspensions were equilibrated at 28°C for 10 min. The concentration of the labelled sugar and of other additions in various experiments are indicated in the results section. Aliquots of 1-ml samples were removed at appropriate intervals and were filtered immediately through cellulose acetate filters (0.45 Ixm porosity; Millipore Corp., New Bedford, MA) and washed with 20 ml basal salts solution. The filters with the cells were transferred directly to scintillation vials containing 5 ml Bray's scintillation fluids [14] and radioactivity was determined in an LKB-Wallac, 1217, Rackbeta liquid scintillation counter. The results are expressed as nmol sugar (mg cell protein)-1. Results
Leishmanicidal activity of amphotericin B. Amphotericin B was found to have a profound growth inhibitory effect on the promastigotes form of this strain of L. donovani. At a concentration of 0.3 IxM, the drug inhibited cell division and the lytic effect became very pronounced at higher
197
concentrations (Fig. 1). Vehicle control using solvent at the same concentrations had no effect on growth. Under identical growth conditions, 2 ~g ml-1 (6 ~M) of pentamidine was needed to completely arrest growth (data not shown).
60~ z
i
B
Effect on cellular respiration. Amphotericin B at a concentration of 0.5 ~M completely inhibited the glucose-stimulated respiration within a short time of contact with the cells (Fig. 2). The cells appeared to have lost motility to a considerable extent by this time, though their morphological integrity remained unaffected throughout the course of the experiment. Simultaneous addition of amphotericin B and cholesterol in the incubation mixture resulted in a very strong protection against antibiotic activity; 80% of the original respiration rate could be restored at a molar concentration ratio of amphotericin B to cholesterol of 1:25.
25
0
20
40 TLME
60
80
( MINUTES I
Fig. 2. Effect of amphotericin B on the respiration of L. donovani promastigotes. The incubation mixture of 3 ml contained 1 ml of washed cell suspension (10 mg of protein), phosphate buffered saline (0.15 M, pH 7.2) and other additions as indicated. ( o - o ) Endogenous; ( o - o ) endogenous + glucose (2 mM); (X-X) endogenous + glucose (2 mM) + 10 gl dimethyl sulphoxide; ( & - A ) endogenous + glucose (2 mM) + amphotericin B (0.5 ~M); (A--A) endogenous + glucose (2 raM) + amphotericin B (1 p.M); ( i - n ) endogenous + glucose (2 mM) + amphotericin B (1.0 p.M) + cholesterol (12.5 ~xM); (~3-~) endogenous + glucose (2 mM) + amphotericin B (0.5 p~M) + cholesterol (12.5 ~M). 0.5 z
2O
0
0.4
/
% ×
/
u_ 0.5 0
S
E
~ 0.2 a3
~o~
/
8 oa
/
0.1
O0 0
30 TIME
20
40
60
80
I00
TIME (HOURS)
Fig. 1. Inhibition of growth of L. donovani in presence of amphotericin B. The control growth curve in absence of amphotericin B is represented by o - n . D-:3, o - o and o - o indicate growth patterns in presence of 0.1, 0.3 and 1 IxM amphotericin B, respectively.
60 (MINUTES)
90
120
Fig. 3. Release of 260- and 280-nm absorbing materials in the presence of amphotericin B. The incubation mixture, in a final volume of 10 ml containing cell suspension (2 mg ml 1) in phosphate buffer saline (0.15 M, pH 7.2) and amphotericin B (0.5 IxM), was incubated at 28°C. At each indicated interval, an aliquot of 1.5 ml was drawn from the flask and rapidly centrifuged. The clear supernatant was diluted suitably in phosphate buffer saline and absorbancy readings were taken. (o-o) Reading at 260 nm for cells in absence of the drug; ( ~ - A ) 280 nm and ( o - e ) 260 nm readings, respectively in presence of the drug (0.5 IxM); (:z-n) 260 nm and ( A _ A ) 280 nm readings, respectively, in presence of drug (0.5 I~M) and ergosterol (12.5 txM).
198
Release of small molecules. Fig. 3 shows the effect of the d r u g on the p e r m e a b i l i t y o f the m e m b r a n e of L. donovani p r o m a s t i g o t e s . In p r e s e n c e of t h e d r u g (0.5 ~ M ) , a c o n t i n u o u s r e l e a s e of m e t a b o l i t e s c o u l d b e o b s e r v e d for n e a r l y 90 min. M a t e r i a l s a b s o r b i n g at 260 a n d 280 n m w e r e p r o b a b l y p r i m a r i l y h e t e r o c y c l i c b a s e s a n d arom a t i c a m i n o acids, r e s p e c t i v e l y . H o w e v e r , the possibility t h a t l a r g e r m o l e c u l e s such as p r o t e i n s o r s o l u b l e R N A s w e r e also l e a k i n g o u t at s o m e stage of this e x p e r i m e n t c o u l d n o t be e l i m i n a t e d . In this case also, a d d i t i o n o f e r g o s t e r o l in a 1:25 m o l a r r a t i o r e s u l t e d in a c o m p l e t e p r o t e c t i o n of the cells a g a i n s t the d r u g action. D i m e t h y l sulp h o x i d e t r a n s f e r r e d with t h e d r u g h a d no effect on t h e m e m b r a n e . T h e r e l e a s e of m a t e r i a l s abs o r b i n g at 260 n m at the e n d of 90 m i n was a b o u t 60% of t h e t o t a l 260 n m a b s o r b i n g m a t e r i a l s that c o u l d n o t be p r e c i p i t a t e d by p e r c h l o r i c acid ( d a t a n o t s h o w n ) . N o visible effect i n d i c a t i n g significant cell d a m a g e c o u l d be o b s e r v e d u n d e r the mi-
c r o s c o p e e v e n after 70 min of i n c u b a t i o n with t h e drug.
Effect of amphotericin B on transport and retention of metabolites. Selective t r a n s p o r t o f m e t a b olites a n d t h e i r r e t e n t i o n is a c h a r a c t e r i s t i c p r o p e r t y of biological m e m b r a n e s . E x p o s u r e to a m p h o t e r i c i n B h a d a d r a m a t i c effect on the t r a n s p o r t p r o p e r t i e s of L. donovani p r o m a s t i gotes. This is e v i d e n t f r o m the results s h o w n in Fig. 4 a n d Fig. 5. T h e glucose t r a n s p o r t s y s t e m in L. tropica a n d in L. donovani h a d e a r l i e r b e e n c h a r a c t e r i z e d by Schaffer a n d M u k k a d a [13], a n d by Z i l b e r s t e i n a n d D w y e r [15], r e s p e c t i v e l y using i s o t o p i c a l l y l a b e l l e d 2-deoxy-D-glucose. In b o t h cases, c a r r i e r m e d i a t e d active t r a n s p o r t systems w e r e o p e r a t i v e . In o u r e x p e r i m e n t s , a d d i t i o n of a m p h o t e r i c i n B at a c o n c e n t r a t i o n of 0.4 ~zM h a d a p r o f o u n d effect on the u p t a k e of r a d i o a c t i v e glucose. D u r i n g the first 3-4 rain, t h e r e was upt a k e of r a d i o a c t i v e glucose into the cell, but then
12 z
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0 z z~
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O
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~ o_
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..... "\."
o
z
,,~ Z
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9 10
Fig. 4. Effect of amphotericin B on the uptake of glucose by L. donovani promastigotes. Washed cells were resuspended in basal salt solution (5.2 g NaC1, 0.5 g KCI, 10.3 g Na2HPO4 in 1000 ml, pH 7.0) to a cell density of 0.6 mg of cell protein m1-1. Three flasks, one containing only cell suspension (control), another containing cell suspensions and amphotericin B (0.4 ~M) and the last one, containing cell suspension, amphotericin B (0.4 IxM) and ergosterol (7.5 tzM) were incubated with gentle shaking for 10 min at 28°C. Individual aliquots of 0.5 ml of these incubated cell suspensions were added to the tubes of incubation mixtures of basal salts solution containing 0.1 mM [U24-C]glucose (2.1 x 105 cpm) in a total volume of 1 ml. At the indicated time intervals, the contents of each tube were rapidly filtered and the radioactivity retained on the Millipore filter paper was counted. (o-o) Control; (e-o), control + drug; ( A - A ) , control + drug + ergosterol.
l z
0 o
1 4
J
e
1'2
i
,e
l
20
TIME ( M I N U T E S )
Fig. 5. Release of accumulated glucose as its metabolites from L. donovani promastigotes on treatment with amphotericin B. Washed cells were suspended in basal salts solution (pH 7.0) to a cell density of 0.3 mg ml 1. Cell suspensions in final volumes of 5 ml in two separate flasks were incubated with 0.1 mM [U-14C]glucose (1.1 x 106 cpm) for 30 min at 28°C. One of the flasks was used as a control. Amphotericin B (0.4 p~M) was added to the other at the end of the preincubation period. Aliquots were drawn at indicated time intervals from the both flasks and counts retained in the cells were measured by the counting procedure described in the legend of Fig. 4. (o-o) Control and (e-o) drug-treated cells, respectively.
199 the level started to go down with time. The simultaneous presence of ergosterol (1:19 molar ratio) provided nearly complete protection (Fig. 4). In this case, ergosterol was added to the incubation mixture within 20 s of addition of amphotericin B. A second experiment was also carried out where cells were first preloaded with isotopically labelled glucose, and were then exposed to amphotericin B at its growth inhibitory concentration (0.4 p,M). An immediate and continuous release of the internal glucose pool, or its degraded metabolites, was monitored over a period of 16 min (Fig. 5). Discussion
The powerful in vitro leishmanicidal effect of amphotericin B that we observed (Fig. 1) compares favourably with the results obtained in other screening systems with other strains of L. donovani and L. tropica [3]. As in the earlier cases [3], we also found the drug to be more potent than pentamidine in arresting growth. Amphotericin B had a definite effect on the permeability properties of the protozoal membrane. Rapid release of small molecules from the internal pool could be demonstrated unambiguously by both spectrophotometric (Fig. 3) and isotopic experiments (Fig. 5). The isotopic experiment increased the sensitivity of detection and an immediate release of glucose, or its metabolites, from an internal pool after contact with the drug could be clearly demonstrated (Fig. 5). Since glucose could be rapidly metabolized in the Leishmania promastigotes, the uptake experiment with [U-14C]glucose, did not reflect the true kinetics of mediated transport across the cellular membrane. However, a definite disorganization of the membrane structure with a drastic effect on the transport system was evident from the uptake experiment (Fig. 4). The effect on the glucose uptake system is probably a reflection of both the loss of the permeability barrier and also of the malfunctioning of the carrier protein in the amphotericin B-treated disorganized membrane bilayer system. Recently Zilberstein and Dwyer have convincingly demonstrated the existence of a proton-motive forcedriven, carrier-mediated active transport system for D-glucose in L. donovani [16]. It is interesting
to note that comparatively little work has been done on the effect of amphotericin B on such a transport system either in fungi or in reconstituted vesicles. Respiration was completely blocked only after a comparatively long lag period, at a higher concentration of the drug. The effect on respiration thus appears to be a secondary phenomenon as a consequence of rapid loss of energy sources from the system (Fig. 2). As with fungi, addition of sterols could largely prevent the effect of amphotericin B on the permeability of Leishmania membranes. Effects on respiration and leakage of small molecules were mostly prevented and the glucose uptake could be almost completely restored by addition of sterols. Cholesterol was almost as effective as ergosterol in this respect. The basic mechanism of protection of cells from polyene action by sterols is dependent upon a physiochemical interaction between the antibiotic and the sterol, which lowers the effective concentration of the antibiotic for interaction with the cells [17]. However, the molecular mechanism for preferential drug action of amphotericin B on fungi remains uncertain. It has been assumed to be based on the greater reactivity of the drug with ergosterol compared with cholesterol [6], the respective sterol components of fungal and mammalian" membranes. Ergosterol has been shown to occur in Leishmania promastigotes [18], though its localisation in the membrane has not been clearly demonstrated. It is likely that the chemotherapeutic basis of the preferential action of amphotericin B towards leishmanial infections is due to the presence of ergosterol in the membrane. Recent work from different laboratories has shown that selectivity of amphotericin B towards membrane ergosterol is greatly enhanced when the drug is bound to the lipid or lipoprotein components of the plasma membrane or to synthetic membrane vesicles rather than when it is dissolved in a solvent like dimethylsulphoxide for in vitro work [8,19]. In the light of this earlier work and our findings, the increased reactivity of amphotericin B in entrapped liposomes [2] is not surprising and can be exploited further for improved drug delivery systems. This work has also emphasized the close similarity in broad outlines between the mode of ac-
200 tion of amphotericin B on fungi and flagellated protozoal systems, thus confirming the original p r o p o s i t i o n o f R a g a n a n d C h a p m a n [20] o f a b i o c h e m i c a l h o m o l o g y in t h e p h y l o g e n y o f t h e s e t w o systems.
Acknowledgements
8
9 10
T h i s w o r k w a s s u p p o r t e d by g r a n t s f r o m t h e Indian Council of Medical Research and University G r a n t s C o m m i s s i o n , I n d i a . W e a r e g r a t e f u l to M a y & B a k e r R e s e a r c h D i v i s i o n , U . K . f o r their samples of pure drug amphotericin B and pentamidine.
References 1 Sampaio, S.A.P., Castro, R.M., Dillon, N.L. and Martins, J.E.C. (1971) Treatment of mucocutaneous leishmaniasis with amphotericin B: report of 70 cases. Int. J. Dermatol. 10, 179-181. 2 New, R.R.C., Chance, M.L. and Heath, S. (1981) Antileishmanial activity of amphotericin and other antifungal agents entrapped in liposomes. J. Antimicrobial Chemother. 8,371-381. 3 Berman, J.D. and Wyler, D.J. (1980) An in vitro model for investigation of chemotherapeutic agents in leishmaniasis. J. Infect. Dis. 142, 83-86. 4 Langreth, S.G., Berman, J.D., Riordan, P.G. and Lee, L.S. (1983) Fine-structural alterations in L. tropica within human macrophages exposed to anti-leishmanial drugs in vitro. J. Protozool. 30,555-561. 5 Sessa, G. and Weishman, G. (1967) Effect of polyene antibiotics on phospholipid spherules containing varying amount of charged components. Biochim. Biophys. Acta. 435,416-426. 6 Hammond, S.M. (1977) Biological activity of polyene antibiotics. Prog. Med. Chem. 14, 105-179. 7 Vertrat-Croquin, A., Boland, J. and Gray-Bobo, C.M. (1984) Enhancement of amphotericin B selectively by an-
11
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tibiotic incorporation into gel state vesicles. A circular dichorism and permeability study. Biochem. Biophys. Res. Commun. 125,360-366. Braijtburg, J., Elberg, S., Bolard, J., Kobayashi, G., Levy, R.A., Ostlund, R.F., Schlessinger, D. and Medoff, G. (1984) Interaction of plasma proteins with amphotericin B. J. Infect. Dis. 149,986-997. Nicolle, Ch. (1908) Culture du Parasite du Bouton d'orient. C.R.H. Seances Acad. Sci. Paris 146, 842-843. Chowdhuri, G., Chatterjee, T.K. and Banerjee, A.B. (1982) Growth factor requirements for in vitro growth of Leishmania donovani. Ind. J. Med. Res. 76, 157-163. Gornall, A.G., Bardawill, C.G. and David, M.M. (1949) Determination of serum protein by means of the biurate reaction. J. Biol. Chem. 177,751-766. Umbrejt, A.G., Burris, R.H. and Stanffer, J.W. (1957) Manometric Techniques, pp. 64-78, Burgess Publishing Co., Minneapolis. Schaffer, F.W. and Mukkada, A.J. (1976) Specificity of the glucose transport system in Leishmania tropica promastigotes. J. Protozool. 23,446-449. Bray, G.A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal. Biochem. 1,279-285. Zilberstein, D. and Dwyer, D.M. (1984) Glucose transport in Leishmania donovani promastigotes. Mol. Biochem. Parasitol. 12, 32%336. Zilberstein, D. and Dwyer, D.M. (1985) Proton motive force-driven active transport of D-glucose and c-proline in the protozoan parasite Leishrnania donovani. Proc. Natl. Acad. Sci. U.S.A. 82, 1716-1720. Lampen, J.O., Arnow, P.M. and Safferman, R.S. (1960) Mechanism of Protection by sterols against polyene antibiotics. J. Bacteriol. 80, 200-206. Goad, L.J., George, G.H. and David, H.B. (1984) Sterols in Leishmania species. Implications for biosynthesis. Mol. Biochem. Parasitol. 10, 161-170. Mehta, R., Lopez-Berestein, G., Hopfer, R., Mills, K. and Juliano, R.L. (1984) Liposomal amphotericin B is toxic to fungal cells but not to mammalian cells. Biochim. Biophys. Acta 770,230-234. Regan, M. and Chapman, A. (1978) In: A Biochemical Phylogeny of the Protists, p. 221, Academic Press, London.