- Email: [email protected]
Characterization of intracellular metabolites of axenic amastigotes of Leishmania donovani by 1H NMR spectroscopy
Acta Tropica 73 (1999) 121 – 133 www.elsevier.com/locate/actatropica
Characterization of intracellular metabolites of axenic amastigotes of Leishmania dono6ani by 1 H NMR spectroscopy Nidhi Gupta a, Neena Goyal a, U.K. Singha a, Vinod Bhakuni b, Raja Roy c, A.K. Rastogi a,* a
Di6ision of Biochemistry, Central Drug Research Institute, Lucknow 226001, India b Membrane Biology, Central Drug Research Institute, Lucknow 226001, India c Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
Received 15 June 1998; received in revised form 20 January 1999; accepted 10 March 1999
Abstract The intracellular metabolites of long-term in vitro cultured axenic amastigotes of Leishmania dono6ani (strain Dd8) were determined and compared with those of promastigotes and intracellular amastigotes, employing proton NMR spectroscopy. The presence of two new metabolites, i.e. betaine and b-hydroxybutyrate were reported. Betaine was detected in all the three stages being highest in the promastigotes while b-hydroxybutyrate could be detected only in promastigotes and axenic amastigotes. Among other metabolites, succinate and valine were found in higher quantities in intracellular amastigotes and axenic amastigotes than in promastigotes. Acetoacetate was present only in axenic and intracellular amastigotes. The comparative metabolite profile of different parasite forms reveals that axenic amastigotes seem to represent an intermediate stage between promastigotes and intracellular amastigotes in spite of their strong resemblance to intracellular amastigotes in morphology, infectivity, biochemical studies and even in the manifestation of amastigote specific A2 protein. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Transformation; Axenic amastigotes; Metabolites; Nuclear magnetic resonance; Intracellular amastigotes
* Corresponding author. Fax: + 91-522-223405. 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 0 - 0
122
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
1. Introduction Parasitic protozoan members of genus Leishmania are responsible for a variety of diseases in humans including the most severe pathologies associated with visceral leishmaniasis caused by Leishmania dono6ani. Leishmania exist as extracellular, flagellated promastigotes in the gut of the sandfly vector and are transformed into intracellular amastigotes in the macrophage phagolysosomes of the mammalian host (Molyneaux and Killick-Kendrick, 1987). Promastigotes can be cultured axenically at temperatures below 28°C in defined or semidefined commercially available media (Hendricks et al., 1978), thus explaining the numerous biochemical, immunological and molecular studies on this form of the parasite. This contrasts with the relatively fragmentary data available on the amastigote, which is the ultimate causative form for all the clinical manifestations of leishmaniasis. Studies on amastigote have been hampered due to difficulties in obtaining/isolating the purified parasites from animal lesion or macrophage cultures, and the inability to culture them axenically (Bates, 1993). Several attempt have been made to culture amastigote-like forms of different Leishmania species (Doyle et al., 1991; Pan et al., 1993; Castilla et al., 1995; Hodgkinson et al., 1996). These workers have used mostly temperature and in some cases, pH as the trigger for in vitro parasite transformation. Earlier studies from our laboratory have shown the biochemical changes in the heat-stressed promastigotes of L. dono6ani (Goyal et al., 1995). Recently, we have been successful in establishing an in vitro axenic culture of L. dono6ani (Strain Dd8) amastigotes, the causative agent of Indian kala-azar (Gupta et al., 1996a) and have characterized several of their membrane parameters (Gupta et al., 1996b). The results revealed a remarkable similarity between the axenic and intracellular amastigotes. There is little information regarding the energy metabolism of Leishmania amastigotes. Previous studies indicate quantitative rather than qualitative differences in the energy metabolism of the two forms (Hart and Coombs, 1982; Rainey and Mackenzie, 1991), because of differences in their natural environment, i.e. promastigotes reside in the digestive tract of the sandflies, whereas amastigotes reside within the parasitophorous vacuoles of host macrophages. Furthermore, promastigotes are regarded as aerobic organisms, but amastigotes are anaerobic (Mukkada, 1985). Recently, NMR spectroscopic techniques have been utilised to determine the metabolic profile of promastigotes and axenic amastigotes of different species of L. dono6ani. Singha et al., (1996) have employed proton NMR spectroscopy for the metabolite mapping of different strains of L. dono6ani promastigotes, and concluded that the technique was suitable for strain identification. Castilla et al. (1995) studied the secretory metabolites of promastigotes and short-term cultured amastigote-like forms of L. dono6ani (strain LRC133). In the present investigation, intracellular metabolites of cultured amastigotes of L. dono6ani Dd8 have been characterized and compared with those of promastigotes and intracellular amastigotes, employing proton magnetic resonance spectroscopy. Knowledge regarding the comparative intermediary metabolism of both these parasite forms could lead to the identification of stage-specific metabolite(s) for further targets as chemotherapeutic agents.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
123
2. Materials and methods Parasite culture
2.0.1. Promastigotes Strains of L. dono6ani (MHOM/IN/80/Dd8) originally obtained as promastigotes from the late Professor P.C.C. Garnham (Imperial College, London) and maintained at CDRI in golden hamsters (Mesocricetus auratus) was used in the present study. Promastigotes were maintained in biphasic NNN medium at 269 1°C. 2.0.2. Axenic amastigotes These were also cultured in NNN biphasic medium at 349 1°C (Gupta et al., 1996a). Briefly, promastigotes of stationary phase culture in fresh NNN biphasic medium were transferred to medium at 349 1°C. Amastigote like forms obtained after 96 h of culture at 34 9 1°C were maintained by regular subcultures initially every 3rd day and then subsequently every 5th day. Axenic amastigotes maintained for up to 8 months were used in the present study. 2.0.3. Intracellular amastigotes (ICAs) These were isolated and purified from spleens of infected hamsters, using a percoll gradient (Hart et al., 1981). 2.1. SDS PAGE and immunoblotting 2.1.1. Preparation of antigen samples Promastigotes and axenic amastigotes (5× 108 cells) were harvested at 4000 rpm for 20 min. at 4°C and washed twice with PBS, pH 7.2. The pellets were resuspended in 50 mM Tris – HCl buffer, pH 7.4 containing 1 mM EDTA, 2 mM phenyl methyl sulphonyl fluoride (PMSF), 0.3 mM aprotinin, l mg/ml leupeptin and 0.l mM N-tosyl-L-phenyl alanine chloromethyl ketone (TPCK) and immediately sonicated for 5 × 30 s at 4°C. The resulting lysates were spun at 20 000 rpm for 60 min and the supernatants were treated with 2×SDS-PAGE sample buffer (100 mM Tris – HCI pH 6.8, 2% w/v SDS, 20% v/v glycerol, and 0.1% w/v bromophenol blue) and boiled for 5 min. The samples were recentrifuged for 10 min and the resulting supernatants were subjected to SDS-PAGE and Western blot analysis. 2.1.2. SDS-PAGE SDS-PAGE was carried out according to the method of Lammeli (1970). A gradient of 10 – 15% separating gel and 5% stacking gel was prepared and antigen samples (in the form of equal protein) were loaded. Electrophoresis (Bio-Rad Laboratories, Richmond CA,USA) was carried out at 2–3 mA/lane. Molecular weight markers were also run in parallel.
124
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
2.1.3. Immunoblotting The polypeptides were transferred onto nitrocellulose paper (NCP) using a transblot cell (Bio-Rad) at 4°C for 3 h at 4–5 V in a transfer buffer 20 mM tris base, 150 mm glycine and 20% v/v methanol (Towbin et al., 1979). Nonspecific antigen sites were blocked in BSA (5% in PBS, pH 7.2) overnight at room temperature, control strips were washed twice with PBS–Tween 20 and were then stained with 0.1% amidoblack in order to monitor complete transfer of the protein bands from the gel onto NCP, whereas the other NCP strips were probed with anti A2 monoclonal antibody C9 which was a kind gift from Dr W.W. Zhang, Canada. 2.2. Determination of intracellular metabolites by 1H NMR spectroscopy 2.2.1. Sample preparation The cells (promastigotes, axenic amastigotes and ICAs) were collected by centrifugation at 3000 rpm for 15 min at 4°C and washed twice with ice cold normal saline. Chilled 1.8 M perchloric acid (1.0 ml) was added to the cell suspension, which was vortexed and sonicated for 5 min. at 4°C followed by centrifugation for 10 min at 10 000 rpm. The pH of supernatant was adjusted to 6.8 by 5.4 M potassium hydroxide. It was kept in ice for 1 h to allow precipitation of potassium perchlorate and centrifuged (10 000 rpm for 10 min). The supernatant was lyophilized and the lyophilized powder was suspended in 1.0 ml of 2H20 and finally centrifuged (45 000× g for 20 min).The supernatant was used for the NMR studies. 2.2.2. 1H NMR experiments One dimensional 1H NMR spectra were recorded at 298 K, on a Bruker Avance DRX 300MHz FT NMR spectrometer equipped with a 5 mm multi-nuclear inverse-probe head with Z-shielded gradient. For 1NMR experiments of the PCA extract, the samples were run using the WATERGATE technique. The acquisition parameters were as follows: acquisition time 2 s, 128 FID’s were accumulated and 0.3 Hz line broadening was used prior to Fourier transformation. For two dimensional J correlation, a WATERGATE TOCSY with a spin lock time of 82.6 ms, using 256 G increments in the axenic amastigotes was acquired. The FID’s were zero filled from 256 W to 512 W and 90° shifted sine bell-window functions were used prior to double Fourier transformation. 2.2.3. Lactate estimation Lactate was estimated in perchloric acid samples of all the three stages, i.e. promastigotes, axenic amastigotes, and ICAs, according to the method of Barker (1957) using p-hydroxydiphenyl as standard.
3. Results Fig. 1 is the Western blot employing monoclonal antibody C9. There is no antigen recognition in promastigotes (lane B) but in axenic amastigotes (lane A) a
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
125
26 kDa band is visible. Fig. 2a – c presents the 1H NMR spectra of promastigotes, axenic amastigotes and ICAs, respectively. Each spectrum represents the metabolic map/profile of the intracellular metabolites of different stages of the parasite. The different intracellular metabolites that were observed are: succinate, lactate, acetoacetate, a-glycerophosphoryl choline (GPC), b-hydroxybutyrate, betaine and amino acids as arginine, alanine and valine. Fig. 3 is the two dimensional spectrum of the metabolites of the axenic amastigotes. The various metabolites (multiplet) observed in the 1H NMR spectra were identified by cross peak mapping of the two-dimensional correlation of the WATERGATE TOCSY contour plots. The conformation of betaine and a-GPC were ascertained by spiking the samples with the standard controls (Fig. 4a – c). Since the proton nucleus has a 100% natural abundance, the area under the peak in 1H NMR represents the relative concentration of that particular metabolite. Fig. 5 shows the percentage for relative abundance of several major metabolites in respect to alanine (100%) of different parasite stages. Succinate, a-GPC and betaine were found to be the major metabolites in all the three stages. The concentration of a-GPC and betaine showed a decreasing trend from promastigotes to ICAs while, succinate exhibited an increasing trend. The assignment of succinate at 2.59 in Fig. 2a and b was confirmed by spiking the sample with sodium succinate (Fig. 4). The TOCSY spectrum showed cross peaks of lactate, with its carbinol proton, at 4.35 and alanine at 4.00. This was further confirmed by spiking the sample with sodium succinate (Fig. 4). Nevertheless, the quantitative estimation of lactate was performed using a standard colorimetric method; the values showed nearly a 4-fold increase in axenic amastigotes (52.3 1 mg/ml) and ICAs (48.5 mg/ml) as compared to promastigotes (11.5 mg/ml). Among the amino acids, valine was detected in high amounts in the ICAs, lesser amounts in the axenic amastigotes and low amounts in the promastigotes. Alanine was another amino acid present in sufficient amounts in the promastigotes and axenic amastigotes which gradually decreased in the ICAs. Arginine and serine were also detected in all three forms of the parasite.
Fig. 1. Immunoblot of L. dono6ani axenic amastigotes (lane A) and promastigotes (lane B) antigens probed with anti A2 monoclonal antibody C9. Numbers represent the molecular weight standard in kDa.
126
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
Fig. 2. 300MHz 1H NMR spectra of perchloric acid extract of (a) promastigotes (b) axenic amastigotes (c) Intracellular amastigotes. Peaks: 1, valine (val); 2 =leucine/isoleucine (leu/iso); 3, b-hydroxybutyrate (hyb); 4, lactate (lac); 5, alanine (ala); 6, arginine (arg); 7, acetate (ace); 8, acetoacetate (aca); 9, glutamate (glu); 10, glutamine (glu); 11, succinate (suc); 12, a-GPC (gpc); 13, betaine (bet); 14, serine (ser); 15, creatinine (crn); 16, mannose (man)? Due to the variation in pH there is a slight change in chemical shift in Fig. 3
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
127
Fig. 3. A contour plot of the two dimensional WATERGATE TOCSY of the axenic amastigotes region (0.5 –4.5 ppm). Symbols for peaks are the same as Fig. 1.
4. Discussion The authenticity of axenic amastigotes is firmly established by Western blot analysis employing a C9 monoclonal antibody raised against A2 protein which represents a unique and probably the only identified (to date) specific protein marker for L. dono6ani amastigotes (Zhang et al., 1996). Leishmania parasites have evolved to colonize two different biological niches: that of the sandfly midgut and macrophage lysosomes. There is a pronounced change in parasite metabolism during this transition process. Since promastigotes are exposed to plant sugars from the sandfly diet, they metabolize glucose at high rates (Rainey and Mackenzie, 1991) as a major source of metabolic energy. In contrast, amastigotes which do not have a ready available source of sugars, metabolize glucose at greatly reduced levels and appear to rely on degradation of fatty acids for energy production (Hart and Coombs, 1982).
128
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
Consumption of glucose by Leishmania parasites is characterized by the production of reduced products such as succinate, pyruvate, ethanol, lactate or alanine (depending on the species) not only in anaerobiasis but also during aerobic conditions (Cazullo, 1992). Among the excretory products of glucose catabolism succinate, acetate, carbon dioxide and sometimes lactate were found to be the major catabolites of Leishmania promastigotes (Janovy, 1987; Glew et al., 1988)
Fig. 4. Spiking of different metabolites with standard compounds (control): (a) promastigotes, (b) axenic amastigotes and (c) intracellular amastigotes.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
129
Fig. 4. (Continued)
where as amino acids such as alanine and glycine were the major catabolites observed in the amastigotes (Rainey and Mackenzie, 1991; Castilla et al., 1995) using the non-invasive technique of NMR employing 13C-labelled glucose and to a lesser extent, proton NMR.
130
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
In the present study we have compared the presence and relative abundance of intracellular intermediary metabolites of the three parasite stages, i.e. promastigotes, axenic amastigotes and ICAs using 1H NMR. Succinate, lactate, alanine and a-GPC were the major metabolites. However, the relative concentration of the metabolites varied depending on the parasitic stage (Fig. 5). The metabolites are in agreement with previous reports of Rainey and Mackenzie (1991) and Singha et al. (1996). We have also reported the presence of two new metabolites, i.e. betaine and p-hydroxybutyrate. For glucose metabolism the following pathways/steps were
Fig. 4. (Continued)
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
131
Fig. 5. Percentage of relative abundance of various intracellular metabolites of different parasitic stages. The samples were prepared by the perchloric acid extraction method. For calculation of relative abundance, the peaks corresponding to the different metabolites were integrated and the values obtained for each peak was normalized for the number of protons associated with that peak. These were then added together and the abundance of each metabolite was calculated with respect to the total value. The result represents the mean percentage value for three experiments. (pm) Promastigotes, (ax) axenic amastigotes and (am) intracellular amastigotes
identified (Glew et al., 1988): glucose is converted to triose phosphate by a combination of the pathways of glycolysis and the pentose phosphate shunt. Triose phosphates are metabolised to pyruvate via the intermediary metabolism of phosphoenol pyruvate. Pyruvate is then converted to oxaloacetate (most likely by the pyruvate carboxylase reaction) and then to malate, fumarate and succinate. In a recent study, Van Hellmond and Tielens (1997) showed that in L. infantum promastigotes, succinate is mainly produced via the oxidative pathway and not via fumarate reduction. This study points out that promastigotes depend on the respiratory chain for energy generation, have poor capacity for anaerobic functioning and go into metabolic arrest under anoxic conditions. The presence of high levels of a-GPC in the axenic amastigotes and ICAs are indicative of different metabolic pathways for lipid/fatty acid catabolism. A 3–4-fold increase in intracellular lactate concentration in axenic and ICAs further supports their preference for anaerobic catabolism and is in agreement with other reports (Darling et al., 1988). Amino acids are a major source of energy in amastigotes (Mukkada, 1985). Arginine is utilised by Leishmania for metabolic functions (Blum, 1992) and has been shown to induce oxygen consumption by L. dono6ani promastigotes at rates comparable to those of D-glucose (Bera, 1987). Besides arginine, alanine was also found to be a major amino acid in promastigotes and axenic amastigotes. b-Hydroxybutyrate and betaine, the two newly reported metabolites are synthesized during glycine biosynthesis from choline. The significant difference observed in the relative concentration of the two metabolites, namely betaine and a-GPC, in
132
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
axenic and intracellular amastigotes versus promastigotes may be attributed to the differences in mitochondrial structure and enzyme abundance. These metabolites could be exploited as potential chemotherapeutic targets for destroying the parasite. The in vitro cultured amastigotes have already been characterized at the ultrastructural, biological and membrane levels and were found to be similar to ICAs in all these parameters (Gupta et al., 1996a,b). Biochemical and molecular evidence (in preparation) also points to a similar conclusion. Further, the antigenic makeup of axenic amastigotes suggests them to be truly amastigote as interpreted by Western blot analysis (Fig. 1) using the monoclonal antibody C9 directed against amastigote specific A2 protein. However, the available data in the present study indicates that the metabolite profile of axenic amastigotes resembles both the ICAs and promastigotes and a sequential pattern of increase or decrease in the relative abundance of particular metabolite was observed from promastigote to intracellular amastigotes. Though the axenic amastigotes share the presence of acetoacetate and comparable lactate content with the ICAs but they do not exhibit an absolute resemblance and rather represent an intermediate stage between promastigotes and ICAs in the metabolite profile. These observations are not surprising and are explained by the fact that the culture medium used for the cultivation of axenic amastigotes differ vastly from the intracellular milieu of macrophages where the true amastigotes reside. So the nutrition available to axenic amastigotes differs greatly from ICAs and is almost the same as promastigotes (as both were cultured in NNN medium). Furthermore, axenic amastigotes do not encounter the nutritional stress experienced by ICAs inside the parasitophorous vacoule. Since this is the first study comparing the intracellular metabolite profile of axenic amastigotes with ICAs rather than only with promastigotes as was done earlier by Rainey and Mackenzie (1991), these observations clearly indicate a need to examine the axenic amastigotes of other species on the basis of this criterion before using them as models in metabolic studies. They may not represent a true amastigote in spite of having a close resemblance to ICAs even at the gene expression level.
Acknowledgements Financial assistance to NG and UKS from the Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged.
References Bates, P.A., 1993. Axenic culture of Leishmania amastigotes. Parasitol. Today 9, 143 – 146. Barker, S.B., 1957. Preparation and colorimeteric determination of lactic acid. Methods Enzymol. 3, 241. Bera, T., 1987. The g-guanidino butyramide pathway of L-arginine catabolism in L. dono6ani promastigotes. Mol. Biochem. Parasitol. 23, 183 – 192. Blum, J.J., 1992. Arginine catabolism by L. dono6ani promastigotes. J. Protozool. 39, 613 – 618.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
133
Cazullo, J.J., 1992. Aerobic fermentation of glucose by trypanosomatids. FASEB J. 6, 3153 – 3161. Castilla, J.J., Sanchez-Moreno, M., Mesa, C., Osuna, A, 1995. Leishmania dono6ani in vitro culture and 1 H NMR characterization of amastigote like forms. Mol. Cell. Biochem. 142, 89 – 97. Darling, T.N., Balber, A.E., Blum, J.J., 1988. A comparative study of D-lactate, L-lactate and glycerol formation by 4 species of Leishmania, Trypanosoma lewisi, T. brucei gambiense. Mol. Biochem. Parasitol. 30, 253–258. Doyle, P.S., Engel, J.C., Pimenta, P.F.P., DaSilva, P.P, Dwyer, D.M., 1991. L. dono6ani: long term culture of axenic amastigotes at 37°C. Exp. Parasitol. 73, 326 – 334. Glew, R.H., Saha, A.K., Das, S., Remaley, A.T., 1988. Biochemistry of Leishmania species. Microbiol. Rev. 52, 412–432. Goyal, N., Singh, A.K., Guru, P.Y, Rastogi, A.K., 1995. Biochemical changes in heat stressed promastigotes of L. dono6ani. Ind. J. Exp. Biol. 33, 222 – 226. Gupta, N., Mittal, N., Goyal, N., Maitra, S.C., Rastogi, A.K., 1996a. Ultrastructural characterization of serially passaged amastigote like forms of Leishmania (Leishmania) dono6ani. Trop. Med. 38, 39–50. Gupta, N., Goyal, N., Kumar, R., Agarwal, A.K., Seth, P.K., Rastogi, A.K., 1996b. Membrane characterization of amastigote like forms of Leishmania dono6ani. Trop. Med. Int. Health 1, 495–502. Hart, D.T., Coombs, G.H., 1982. Energy metabolism of amastigotes and promastigotes. Exp. Parasitol. 54, 397–409. Hart, D.T., Vickerman, K., Coombs, G.H., 1981. A quick simple method for purifying L. mexicana amastigotes in large numbers. Parasitology 82, 345 – 355. Hendricks, L.D., Wood, D.E., Hajduk, M.E., 1978. Hemoflagellates. Commercially available liquid media for rapid cultivation. Parasitology 76, 309 – 316. Hodgkinson, V., Soong, L., Duboise, S.M., McMahon-Pratt, D., 1996. L. amazonensis: cultivation of axenic amastigotes like forms. Exp. Parasitol. 83, 94 – 105. Janovy, J. Jr., 1987. Physiology and biochemistry. In: Peters, W., Killick-Kendrick, R. (Eds.), The Leishmaniasis in Biology and Medicine, vol. 1. Academic Press, London, pp. 178 – 210. Lammeli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Molyneaux, D.H., Killick-Kendrick, R., 1987. Morphology and ultrastructure. In: Peters, W., KillickKendrick, R. (Eds.), The Leishmaniasis in Biology and Medicine. Academic Press, London, pp. 122–176. Mukkada, A.J., 1985. Energy metabolism in leishmania. In: Chang, K.P., Bray, R.S. (Eds.), The Leishmaniasis. Elsevier, Amsterdam, pp. 49 – 64. Pan, A.A., Duboise, S.M., Eperon, S., et al., 1993. Developmental life cycle of Leishmania: cultivation and characterization of cultured extracellular amastigotes. J. Eur. Microbiol. 40 (2), 213 – 223. Rainey, P.M., Mackenzie, N.E., 1991. A carbon 13 nuclear magnetic resonance analysis of the product of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes. Mol. Biochem. Parasitol. 45, 307–316. Singha, U.K., Bhakuni, V., Ali, V., Roy, R., 1996. Leishmania dono6ani: metabolite mapping using proton NMR spectroscopy. Mol. Cell. Biochem. 162, 17 – 22. Towbin, H., Stahelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Van Hellmond, J.J., Tielens, A.G.M., 1997. Inhibition of respiratory chain results in a reversible metabolic arrest in Leishmania promastigotes. Mol. Biochem. Parasitol. 85, 135 – 138. Zhang, W.W., Charest, H., Ghedin, E., Matleshewski, G., 1996. Identification and overexpression of A2 amastigote specific protein in Leishmania dono6ani. Mol. Biochem. Parasitol. 79, 79 – 90.
.
Characterization of intracellular metabolites of axenic amastigotes of Leishmania dono6ani by 1 H NMR spectroscopy Nidhi Gupta a, Neena Goyal a, U.K. Singha a, Vinod Bhakuni b, Raja Roy c, A.K. Rastogi a,* a
Di6ision of Biochemistry, Central Drug Research Institute, Lucknow 226001, India b Membrane Biology, Central Drug Research Institute, Lucknow 226001, India c Medicinal Chemistry, Central Drug Research Institute, Lucknow 226001, India
Received 15 June 1998; received in revised form 20 January 1999; accepted 10 March 1999
Abstract The intracellular metabolites of long-term in vitro cultured axenic amastigotes of Leishmania dono6ani (strain Dd8) were determined and compared with those of promastigotes and intracellular amastigotes, employing proton NMR spectroscopy. The presence of two new metabolites, i.e. betaine and b-hydroxybutyrate were reported. Betaine was detected in all the three stages being highest in the promastigotes while b-hydroxybutyrate could be detected only in promastigotes and axenic amastigotes. Among other metabolites, succinate and valine were found in higher quantities in intracellular amastigotes and axenic amastigotes than in promastigotes. Acetoacetate was present only in axenic and intracellular amastigotes. The comparative metabolite profile of different parasite forms reveals that axenic amastigotes seem to represent an intermediate stage between promastigotes and intracellular amastigotes in spite of their strong resemblance to intracellular amastigotes in morphology, infectivity, biochemical studies and even in the manifestation of amastigote specific A2 protein. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Transformation; Axenic amastigotes; Metabolites; Nuclear magnetic resonance; Intracellular amastigotes
* Corresponding author. Fax: + 91-522-223405. 0001-706X/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 1 - 7 0 6 X ( 9 9 ) 0 0 0 2 0 - 0
122
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
1. Introduction Parasitic protozoan members of genus Leishmania are responsible for a variety of diseases in humans including the most severe pathologies associated with visceral leishmaniasis caused by Leishmania dono6ani. Leishmania exist as extracellular, flagellated promastigotes in the gut of the sandfly vector and are transformed into intracellular amastigotes in the macrophage phagolysosomes of the mammalian host (Molyneaux and Killick-Kendrick, 1987). Promastigotes can be cultured axenically at temperatures below 28°C in defined or semidefined commercially available media (Hendricks et al., 1978), thus explaining the numerous biochemical, immunological and molecular studies on this form of the parasite. This contrasts with the relatively fragmentary data available on the amastigote, which is the ultimate causative form for all the clinical manifestations of leishmaniasis. Studies on amastigote have been hampered due to difficulties in obtaining/isolating the purified parasites from animal lesion or macrophage cultures, and the inability to culture them axenically (Bates, 1993). Several attempt have been made to culture amastigote-like forms of different Leishmania species (Doyle et al., 1991; Pan et al., 1993; Castilla et al., 1995; Hodgkinson et al., 1996). These workers have used mostly temperature and in some cases, pH as the trigger for in vitro parasite transformation. Earlier studies from our laboratory have shown the biochemical changes in the heat-stressed promastigotes of L. dono6ani (Goyal et al., 1995). Recently, we have been successful in establishing an in vitro axenic culture of L. dono6ani (Strain Dd8) amastigotes, the causative agent of Indian kala-azar (Gupta et al., 1996a) and have characterized several of their membrane parameters (Gupta et al., 1996b). The results revealed a remarkable similarity between the axenic and intracellular amastigotes. There is little information regarding the energy metabolism of Leishmania amastigotes. Previous studies indicate quantitative rather than qualitative differences in the energy metabolism of the two forms (Hart and Coombs, 1982; Rainey and Mackenzie, 1991), because of differences in their natural environment, i.e. promastigotes reside in the digestive tract of the sandflies, whereas amastigotes reside within the parasitophorous vacuoles of host macrophages. Furthermore, promastigotes are regarded as aerobic organisms, but amastigotes are anaerobic (Mukkada, 1985). Recently, NMR spectroscopic techniques have been utilised to determine the metabolic profile of promastigotes and axenic amastigotes of different species of L. dono6ani. Singha et al., (1996) have employed proton NMR spectroscopy for the metabolite mapping of different strains of L. dono6ani promastigotes, and concluded that the technique was suitable for strain identification. Castilla et al. (1995) studied the secretory metabolites of promastigotes and short-term cultured amastigote-like forms of L. dono6ani (strain LRC133). In the present investigation, intracellular metabolites of cultured amastigotes of L. dono6ani Dd8 have been characterized and compared with those of promastigotes and intracellular amastigotes, employing proton magnetic resonance spectroscopy. Knowledge regarding the comparative intermediary metabolism of both these parasite forms could lead to the identification of stage-specific metabolite(s) for further targets as chemotherapeutic agents.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
123
2. Materials and methods Parasite culture
2.0.1. Promastigotes Strains of L. dono6ani (MHOM/IN/80/Dd8) originally obtained as promastigotes from the late Professor P.C.C. Garnham (Imperial College, London) and maintained at CDRI in golden hamsters (Mesocricetus auratus) was used in the present study. Promastigotes were maintained in biphasic NNN medium at 269 1°C. 2.0.2. Axenic amastigotes These were also cultured in NNN biphasic medium at 349 1°C (Gupta et al., 1996a). Briefly, promastigotes of stationary phase culture in fresh NNN biphasic medium were transferred to medium at 349 1°C. Amastigote like forms obtained after 96 h of culture at 34 9 1°C were maintained by regular subcultures initially every 3rd day and then subsequently every 5th day. Axenic amastigotes maintained for up to 8 months were used in the present study. 2.0.3. Intracellular amastigotes (ICAs) These were isolated and purified from spleens of infected hamsters, using a percoll gradient (Hart et al., 1981). 2.1. SDS PAGE and immunoblotting 2.1.1. Preparation of antigen samples Promastigotes and axenic amastigotes (5× 108 cells) were harvested at 4000 rpm for 20 min. at 4°C and washed twice with PBS, pH 7.2. The pellets were resuspended in 50 mM Tris – HCl buffer, pH 7.4 containing 1 mM EDTA, 2 mM phenyl methyl sulphonyl fluoride (PMSF), 0.3 mM aprotinin, l mg/ml leupeptin and 0.l mM N-tosyl-L-phenyl alanine chloromethyl ketone (TPCK) and immediately sonicated for 5 × 30 s at 4°C. The resulting lysates were spun at 20 000 rpm for 60 min and the supernatants were treated with 2×SDS-PAGE sample buffer (100 mM Tris – HCI pH 6.8, 2% w/v SDS, 20% v/v glycerol, and 0.1% w/v bromophenol blue) and boiled for 5 min. The samples were recentrifuged for 10 min and the resulting supernatants were subjected to SDS-PAGE and Western blot analysis. 2.1.2. SDS-PAGE SDS-PAGE was carried out according to the method of Lammeli (1970). A gradient of 10 – 15% separating gel and 5% stacking gel was prepared and antigen samples (in the form of equal protein) were loaded. Electrophoresis (Bio-Rad Laboratories, Richmond CA,USA) was carried out at 2–3 mA/lane. Molecular weight markers were also run in parallel.
124
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
2.1.3. Immunoblotting The polypeptides were transferred onto nitrocellulose paper (NCP) using a transblot cell (Bio-Rad) at 4°C for 3 h at 4–5 V in a transfer buffer 20 mM tris base, 150 mm glycine and 20% v/v methanol (Towbin et al., 1979). Nonspecific antigen sites were blocked in BSA (5% in PBS, pH 7.2) overnight at room temperature, control strips were washed twice with PBS–Tween 20 and were then stained with 0.1% amidoblack in order to monitor complete transfer of the protein bands from the gel onto NCP, whereas the other NCP strips were probed with anti A2 monoclonal antibody C9 which was a kind gift from Dr W.W. Zhang, Canada. 2.2. Determination of intracellular metabolites by 1H NMR spectroscopy 2.2.1. Sample preparation The cells (promastigotes, axenic amastigotes and ICAs) were collected by centrifugation at 3000 rpm for 15 min at 4°C and washed twice with ice cold normal saline. Chilled 1.8 M perchloric acid (1.0 ml) was added to the cell suspension, which was vortexed and sonicated for 5 min. at 4°C followed by centrifugation for 10 min at 10 000 rpm. The pH of supernatant was adjusted to 6.8 by 5.4 M potassium hydroxide. It was kept in ice for 1 h to allow precipitation of potassium perchlorate and centrifuged (10 000 rpm for 10 min). The supernatant was lyophilized and the lyophilized powder was suspended in 1.0 ml of 2H20 and finally centrifuged (45 000× g for 20 min).The supernatant was used for the NMR studies. 2.2.2. 1H NMR experiments One dimensional 1H NMR spectra were recorded at 298 K, on a Bruker Avance DRX 300MHz FT NMR spectrometer equipped with a 5 mm multi-nuclear inverse-probe head with Z-shielded gradient. For 1NMR experiments of the PCA extract, the samples were run using the WATERGATE technique. The acquisition parameters were as follows: acquisition time 2 s, 128 FID’s were accumulated and 0.3 Hz line broadening was used prior to Fourier transformation. For two dimensional J correlation, a WATERGATE TOCSY with a spin lock time of 82.6 ms, using 256 G increments in the axenic amastigotes was acquired. The FID’s were zero filled from 256 W to 512 W and 90° shifted sine bell-window functions were used prior to double Fourier transformation. 2.2.3. Lactate estimation Lactate was estimated in perchloric acid samples of all the three stages, i.e. promastigotes, axenic amastigotes, and ICAs, according to the method of Barker (1957) using p-hydroxydiphenyl as standard.
3. Results Fig. 1 is the Western blot employing monoclonal antibody C9. There is no antigen recognition in promastigotes (lane B) but in axenic amastigotes (lane A) a
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
125
26 kDa band is visible. Fig. 2a – c presents the 1H NMR spectra of promastigotes, axenic amastigotes and ICAs, respectively. Each spectrum represents the metabolic map/profile of the intracellular metabolites of different stages of the parasite. The different intracellular metabolites that were observed are: succinate, lactate, acetoacetate, a-glycerophosphoryl choline (GPC), b-hydroxybutyrate, betaine and amino acids as arginine, alanine and valine. Fig. 3 is the two dimensional spectrum of the metabolites of the axenic amastigotes. The various metabolites (multiplet) observed in the 1H NMR spectra were identified by cross peak mapping of the two-dimensional correlation of the WATERGATE TOCSY contour plots. The conformation of betaine and a-GPC were ascertained by spiking the samples with the standard controls (Fig. 4a – c). Since the proton nucleus has a 100% natural abundance, the area under the peak in 1H NMR represents the relative concentration of that particular metabolite. Fig. 5 shows the percentage for relative abundance of several major metabolites in respect to alanine (100%) of different parasite stages. Succinate, a-GPC and betaine were found to be the major metabolites in all the three stages. The concentration of a-GPC and betaine showed a decreasing trend from promastigotes to ICAs while, succinate exhibited an increasing trend. The assignment of succinate at 2.59 in Fig. 2a and b was confirmed by spiking the sample with sodium succinate (Fig. 4). The TOCSY spectrum showed cross peaks of lactate, with its carbinol proton, at 4.35 and alanine at 4.00. This was further confirmed by spiking the sample with sodium succinate (Fig. 4). Nevertheless, the quantitative estimation of lactate was performed using a standard colorimetric method; the values showed nearly a 4-fold increase in axenic amastigotes (52.3 1 mg/ml) and ICAs (48.5 mg/ml) as compared to promastigotes (11.5 mg/ml). Among the amino acids, valine was detected in high amounts in the ICAs, lesser amounts in the axenic amastigotes and low amounts in the promastigotes. Alanine was another amino acid present in sufficient amounts in the promastigotes and axenic amastigotes which gradually decreased in the ICAs. Arginine and serine were also detected in all three forms of the parasite.
Fig. 1. Immunoblot of L. dono6ani axenic amastigotes (lane A) and promastigotes (lane B) antigens probed with anti A2 monoclonal antibody C9. Numbers represent the molecular weight standard in kDa.
126
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
Fig. 2. 300MHz 1H NMR spectra of perchloric acid extract of (a) promastigotes (b) axenic amastigotes (c) Intracellular amastigotes. Peaks: 1, valine (val); 2 =leucine/isoleucine (leu/iso); 3, b-hydroxybutyrate (hyb); 4, lactate (lac); 5, alanine (ala); 6, arginine (arg); 7, acetate (ace); 8, acetoacetate (aca); 9, glutamate (glu); 10, glutamine (glu); 11, succinate (suc); 12, a-GPC (gpc); 13, betaine (bet); 14, serine (ser); 15, creatinine (crn); 16, mannose (man)? Due to the variation in pH there is a slight change in chemical shift in Fig. 3
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
127
Fig. 3. A contour plot of the two dimensional WATERGATE TOCSY of the axenic amastigotes region (0.5 –4.5 ppm). Symbols for peaks are the same as Fig. 1.
4. Discussion The authenticity of axenic amastigotes is firmly established by Western blot analysis employing a C9 monoclonal antibody raised against A2 protein which represents a unique and probably the only identified (to date) specific protein marker for L. dono6ani amastigotes (Zhang et al., 1996). Leishmania parasites have evolved to colonize two different biological niches: that of the sandfly midgut and macrophage lysosomes. There is a pronounced change in parasite metabolism during this transition process. Since promastigotes are exposed to plant sugars from the sandfly diet, they metabolize glucose at high rates (Rainey and Mackenzie, 1991) as a major source of metabolic energy. In contrast, amastigotes which do not have a ready available source of sugars, metabolize glucose at greatly reduced levels and appear to rely on degradation of fatty acids for energy production (Hart and Coombs, 1982).
128
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
Consumption of glucose by Leishmania parasites is characterized by the production of reduced products such as succinate, pyruvate, ethanol, lactate or alanine (depending on the species) not only in anaerobiasis but also during aerobic conditions (Cazullo, 1992). Among the excretory products of glucose catabolism succinate, acetate, carbon dioxide and sometimes lactate were found to be the major catabolites of Leishmania promastigotes (Janovy, 1987; Glew et al., 1988)
Fig. 4. Spiking of different metabolites with standard compounds (control): (a) promastigotes, (b) axenic amastigotes and (c) intracellular amastigotes.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
129
Fig. 4. (Continued)
where as amino acids such as alanine and glycine were the major catabolites observed in the amastigotes (Rainey and Mackenzie, 1991; Castilla et al., 1995) using the non-invasive technique of NMR employing 13C-labelled glucose and to a lesser extent, proton NMR.
130
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
In the present study we have compared the presence and relative abundance of intracellular intermediary metabolites of the three parasite stages, i.e. promastigotes, axenic amastigotes and ICAs using 1H NMR. Succinate, lactate, alanine and a-GPC were the major metabolites. However, the relative concentration of the metabolites varied depending on the parasitic stage (Fig. 5). The metabolites are in agreement with previous reports of Rainey and Mackenzie (1991) and Singha et al. (1996). We have also reported the presence of two new metabolites, i.e. betaine and p-hydroxybutyrate. For glucose metabolism the following pathways/steps were
Fig. 4. (Continued)
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
131
Fig. 5. Percentage of relative abundance of various intracellular metabolites of different parasitic stages. The samples were prepared by the perchloric acid extraction method. For calculation of relative abundance, the peaks corresponding to the different metabolites were integrated and the values obtained for each peak was normalized for the number of protons associated with that peak. These were then added together and the abundance of each metabolite was calculated with respect to the total value. The result represents the mean percentage value for three experiments. (pm) Promastigotes, (ax) axenic amastigotes and (am) intracellular amastigotes
identified (Glew et al., 1988): glucose is converted to triose phosphate by a combination of the pathways of glycolysis and the pentose phosphate shunt. Triose phosphates are metabolised to pyruvate via the intermediary metabolism of phosphoenol pyruvate. Pyruvate is then converted to oxaloacetate (most likely by the pyruvate carboxylase reaction) and then to malate, fumarate and succinate. In a recent study, Van Hellmond and Tielens (1997) showed that in L. infantum promastigotes, succinate is mainly produced via the oxidative pathway and not via fumarate reduction. This study points out that promastigotes depend on the respiratory chain for energy generation, have poor capacity for anaerobic functioning and go into metabolic arrest under anoxic conditions. The presence of high levels of a-GPC in the axenic amastigotes and ICAs are indicative of different metabolic pathways for lipid/fatty acid catabolism. A 3–4-fold increase in intracellular lactate concentration in axenic and ICAs further supports their preference for anaerobic catabolism and is in agreement with other reports (Darling et al., 1988). Amino acids are a major source of energy in amastigotes (Mukkada, 1985). Arginine is utilised by Leishmania for metabolic functions (Blum, 1992) and has been shown to induce oxygen consumption by L. dono6ani promastigotes at rates comparable to those of D-glucose (Bera, 1987). Besides arginine, alanine was also found to be a major amino acid in promastigotes and axenic amastigotes. b-Hydroxybutyrate and betaine, the two newly reported metabolites are synthesized during glycine biosynthesis from choline. The significant difference observed in the relative concentration of the two metabolites, namely betaine and a-GPC, in
132
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
axenic and intracellular amastigotes versus promastigotes may be attributed to the differences in mitochondrial structure and enzyme abundance. These metabolites could be exploited as potential chemotherapeutic targets for destroying the parasite. The in vitro cultured amastigotes have already been characterized at the ultrastructural, biological and membrane levels and were found to be similar to ICAs in all these parameters (Gupta et al., 1996a,b). Biochemical and molecular evidence (in preparation) also points to a similar conclusion. Further, the antigenic makeup of axenic amastigotes suggests them to be truly amastigote as interpreted by Western blot analysis (Fig. 1) using the monoclonal antibody C9 directed against amastigote specific A2 protein. However, the available data in the present study indicates that the metabolite profile of axenic amastigotes resembles both the ICAs and promastigotes and a sequential pattern of increase or decrease in the relative abundance of particular metabolite was observed from promastigote to intracellular amastigotes. Though the axenic amastigotes share the presence of acetoacetate and comparable lactate content with the ICAs but they do not exhibit an absolute resemblance and rather represent an intermediate stage between promastigotes and ICAs in the metabolite profile. These observations are not surprising and are explained by the fact that the culture medium used for the cultivation of axenic amastigotes differ vastly from the intracellular milieu of macrophages where the true amastigotes reside. So the nutrition available to axenic amastigotes differs greatly from ICAs and is almost the same as promastigotes (as both were cultured in NNN medium). Furthermore, axenic amastigotes do not encounter the nutritional stress experienced by ICAs inside the parasitophorous vacoule. Since this is the first study comparing the intracellular metabolite profile of axenic amastigotes with ICAs rather than only with promastigotes as was done earlier by Rainey and Mackenzie (1991), these observations clearly indicate a need to examine the axenic amastigotes of other species on the basis of this criterion before using them as models in metabolic studies. They may not represent a true amastigote in spite of having a close resemblance to ICAs even at the gene expression level.
Acknowledgements Financial assistance to NG and UKS from the Council of Scientific and Industrial Research, New Delhi is gratefully acknowledged.
References Bates, P.A., 1993. Axenic culture of Leishmania amastigotes. Parasitol. Today 9, 143 – 146. Barker, S.B., 1957. Preparation and colorimeteric determination of lactic acid. Methods Enzymol. 3, 241. Bera, T., 1987. The g-guanidino butyramide pathway of L-arginine catabolism in L. dono6ani promastigotes. Mol. Biochem. Parasitol. 23, 183 – 192. Blum, J.J., 1992. Arginine catabolism by L. dono6ani promastigotes. J. Protozool. 39, 613 – 618.
N. Gupta et al. / Acta Tropica 73 (1999) 121–133
133
Cazullo, J.J., 1992. Aerobic fermentation of glucose by trypanosomatids. FASEB J. 6, 3153 – 3161. Castilla, J.J., Sanchez-Moreno, M., Mesa, C., Osuna, A, 1995. Leishmania dono6ani in vitro culture and 1 H NMR characterization of amastigote like forms. Mol. Cell. Biochem. 142, 89 – 97. Darling, T.N., Balber, A.E., Blum, J.J., 1988. A comparative study of D-lactate, L-lactate and glycerol formation by 4 species of Leishmania, Trypanosoma lewisi, T. brucei gambiense. Mol. Biochem. Parasitol. 30, 253–258. Doyle, P.S., Engel, J.C., Pimenta, P.F.P., DaSilva, P.P, Dwyer, D.M., 1991. L. dono6ani: long term culture of axenic amastigotes at 37°C. Exp. Parasitol. 73, 326 – 334. Glew, R.H., Saha, A.K., Das, S., Remaley, A.T., 1988. Biochemistry of Leishmania species. Microbiol. Rev. 52, 412–432. Goyal, N., Singh, A.K., Guru, P.Y, Rastogi, A.K., 1995. Biochemical changes in heat stressed promastigotes of L. dono6ani. Ind. J. Exp. Biol. 33, 222 – 226. Gupta, N., Mittal, N., Goyal, N., Maitra, S.C., Rastogi, A.K., 1996a. Ultrastructural characterization of serially passaged amastigote like forms of Leishmania (Leishmania) dono6ani. Trop. Med. 38, 39–50. Gupta, N., Goyal, N., Kumar, R., Agarwal, A.K., Seth, P.K., Rastogi, A.K., 1996b. Membrane characterization of amastigote like forms of Leishmania dono6ani. Trop. Med. Int. Health 1, 495–502. Hart, D.T., Coombs, G.H., 1982. Energy metabolism of amastigotes and promastigotes. Exp. Parasitol. 54, 397–409. Hart, D.T., Vickerman, K., Coombs, G.H., 1981. A quick simple method for purifying L. mexicana amastigotes in large numbers. Parasitology 82, 345 – 355. Hendricks, L.D., Wood, D.E., Hajduk, M.E., 1978. Hemoflagellates. Commercially available liquid media for rapid cultivation. Parasitology 76, 309 – 316. Hodgkinson, V., Soong, L., Duboise, S.M., McMahon-Pratt, D., 1996. L. amazonensis: cultivation of axenic amastigotes like forms. Exp. Parasitol. 83, 94 – 105. Janovy, J. Jr., 1987. Physiology and biochemistry. In: Peters, W., Killick-Kendrick, R. (Eds.), The Leishmaniasis in Biology and Medicine, vol. 1. Academic Press, London, pp. 178 – 210. Lammeli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Molyneaux, D.H., Killick-Kendrick, R., 1987. Morphology and ultrastructure. In: Peters, W., KillickKendrick, R. (Eds.), The Leishmaniasis in Biology and Medicine. Academic Press, London, pp. 122–176. Mukkada, A.J., 1985. Energy metabolism in leishmania. In: Chang, K.P., Bray, R.S. (Eds.), The Leishmaniasis. Elsevier, Amsterdam, pp. 49 – 64. Pan, A.A., Duboise, S.M., Eperon, S., et al., 1993. Developmental life cycle of Leishmania: cultivation and characterization of cultured extracellular amastigotes. J. Eur. Microbiol. 40 (2), 213 – 223. Rainey, P.M., Mackenzie, N.E., 1991. A carbon 13 nuclear magnetic resonance analysis of the product of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes. Mol. Biochem. Parasitol. 45, 307–316. Singha, U.K., Bhakuni, V., Ali, V., Roy, R., 1996. Leishmania dono6ani: metabolite mapping using proton NMR spectroscopy. Mol. Cell. Biochem. 162, 17 – 22. Towbin, H., Stahelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354. Van Hellmond, J.J., Tielens, A.G.M., 1997. Inhibition of respiratory chain results in a reversible metabolic arrest in Leishmania promastigotes. Mol. Biochem. Parasitol. 85, 135 – 138. Zhang, W.W., Charest, H., Ghedin, E., Matleshewski, G., 1996. Identification and overexpression of A2 amastigote specific protein in Leishmania dono6ani. Mol. Biochem. Parasitol. 79, 79 – 90.
.