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Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum
BBRC Biochemical and Biophysical Research Communications 348 (2006) 579–584 www.elsevier.com/locate/ybbrc
Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum q T.N.C. Ramya a, Namita Surolia b, Avadhesha Surolia b
a,c,*
a Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India c National Institute of Immunology, New Delhi 110067, India
Received 11 July 2006 Available online 31 July 2006
Abstract We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy. 2006 Elsevier Inc. All rights reserved. Keywords: Polyamine biosynthesis; Polyamine transport; Plasmodium falciparum; Putrescine; Spermidine; Spermine
The naturally occurring polyamines, spermidine and spermine, and their precursor, putrescine, are important regulators of growth and differentiation in cells. Though their exact function remains unknown, polyamines play a functional role in cell differentiation and proliferation processes probably by stabilizing the structure of newly synthesized chromatin and increasing the translational efficiency of RNA [1]. Normal erythrocytes are devoid of polyamine biosynthetic machinery and contain traces of putrescine, spermidine, and spermine. However, in Plasmodium falciparum infected erythrocytes, polyamines, and biosynthetic enzymes like ornithine decarboxylase and S-adenosyl methionine decarboxylase are known to increase [2,3]. Moreover, polyamines at certain stages
q Abbreviations: RBC, red blood cell; IRBC, Plasmodium falciparum infected red blood cell; SAM, S-adenosyl methionine; DFMO, difluoromethyl ornithine; ODC, ornithine decarboxylase; TCA, trichloroacetic acid; TLC, thin layer chromatography; CCCP, carbonyl cyanide m-chloro phenylhydrazone; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. * Corresponding author. Fax: +91 11 26717104. E-mail address: [email protected] (A. Surolia).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.127
trigger DNA synthesis as well as synthesis of certain proteins, one amongst them being the DNA polymerase itself. The activity of the cell cycle dependent, a-like malarial DNA polymerase, too, is stimulated by the cellular level of putrescine and spermidine, and inhibited by spermine. Addition of DFMO, an inhibitor of ODC, prevents the accumulation of putrescine and spermidine in parasitized erythrocytes, and arrests malaria parasite growth in the trophozoite stage prior to DNA synthesis [2]. Depleting the malaria parasite of polyamines is therefore extremely deleterious to its growth and seems an attractive target for developing antimalarial agents. It is therefore of interest to study polyamine synthesis in the malaria parasite. The precursor for polyamine biosynthesis in mammalian cells is arginine [4]. Arginine is decarboxylated by arginine decarboxylase to ornithine, which, in turn, is decarboxylated by the highly regulated enzyme activity of ornithine decarboxylase to putrescine. Spermidine and spermine are formed by the catalytic activities of the enzymes, spermidine synthase, spermine synthase, and S-adenosylmethionine (SAM) decarboxylase (Fig. 1) [4]. In some trypanosomatids, plants, and some bacteria, arginine is converted to putrescine via agmatine by arginine
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T.N.C. Ramya et al. / Biochemical and Biophysical Research Communications 348 (2006) 579–584
Fig. 1. Polyamine metabolism as speculated to exist in the malaria parasite, P. falciparum. For the sake of clarity, no subcellular organelles of the parasite are represented in the schematic. Arginine or ornithine is the precursor for the synthesis of putrescine, spermidine (and spermine).
decarboxylase, in the absence of a functional ornithine decarboxylase [5]. Arginine decarboxylase has been identified in a relative of the malaria parasite, Cryptosporidium, but not in the malaria parasite as yet [6]. The malaria parasite, however, has a bifunctional enzyme with ornithine decarboxylase and S-adenosylmethionine decarboxylase activities (Fig. 1) [7]. Importantly, the P. falciparum ornithine decarboxylase enzyme, unlike its mammalian counterpart, is not stimulated by putrescine and is very strongly feedback regulated by putrescine [7]. The malaria parasite presumably has spermidine synthase activity, but spermine synthesis is speculated to be absent [4] (Fig. 1). Current evidence does weigh in favor of a polyamine biosynthesis pathway in the malaria parasite. However, inhibitors of polyamine biosynthesis such as a-difluoromethyl ornithine, the well-known inhibitor of ornithine decarboxylase, though capable of inhibiting erythrocytic schizogony in vitro, are incapable of inhibiting parasite growth in vivo, suggesting that either the uptake of this drug is not effective or the malaria parasite satisfies its polyamine requirement by salvaging polyamines, too [8,9]. If the latter is true, a regimen of inhibitors of polyamine synthesis enzymes coupled with structural analogues of polyamines could well prove to be the ultimate campaign towards polyamine depletion and inhibition of parasite growth. We now provide biochemical proof for the presence of a functional polyamine biosynthesis pathway in the parasite. To the best of our knowledge, this is the first report of spermine synthesis in the malaria parasite. We also report the presence of new transport pathways for putrescine and spermidine in malaria parasite infected red blood cells. Experimental procedures Materials. The radioisotope [3H]-spermidine was from Perkin-Elmer. [14C]-1,4-putrescine and [3H]putrescine were from Amersham Biosciences.
Dibutyl phthalate, putrescine, ornithine, agmatine, spermidine, spermine, dansyl chloride, D-arginine, L-arginine, poly-L-arginine, difluoromethyl ornithine, serine, leucine, aspartate, glutamine, lysine, cycloheximide, iodoacetate, N-ethyl maleimide, valinomycin, gramicidin, p-hydroxymercuribenzoate, carbonyl cyanide m-chloro phenylhydrazone (CCCP), ouabain, and calcium ionophore were of the highest quality available from Sigma. Culture medium components were from Sigma. Incorporation of [14C]putrescine into spermidine and spermine in Plasmodium falciparum infected erythrocytes and Jurkat cells. Plasmodium falciparum infected erythrocytes (10 ml) synchronized with 5% sorbitol were incubated with [14C]putrescine (50 lCi; 100 mCi/mmol) to a final concentration of 0.2 mM for 6 h. They were then washed and resuspended in RPMI without [14C]putrescine for 6 or 18 h. The parasites were then isolated from the red blood cells by saponin lysis, and the polyamines in the parasites dansylated by a modified protocol of Minocha et al. [10]. Briefly, the cell pellet was resuspended in 5% TCA to precipitate proteins. The pH of the supernatant was brought to 9 with saturated sodium carbonate, and then an equal volume of 10 mg/ml dansyl chloride in acetone was added to it. The reaction mix was vortexed and allowed to incubate in the dark at 60 C for 60 min. The reaction was stopped with 100 mg/ml of L-alanine, the acetone was evaporated, the reaction mix cooled to 4 C, and dansylated polyamines extracted with an equal volume of toluene. The upper toluene phase was transferred to a fresh tube, concentrated to 50 ll, and spotted on a Whatman silica gel 60 TLC plate along with standard dansylated polyamines (also prepared in a similar fashion). The dansylated polyamines were developed with hexane/ethyl acetate :: 6:4 and visualized under UV illumination. Putrescine and spermidine transport in uninfected and Plasmodium falciparum infected red blood cells. Transport into P. falciparum infected red blood cells was assayed by a modified protocol of Singh et al. [11]. Briefly, 900 ll uninfected or P. falciparum infected red blood cells (3 · 107 cells) were incubated in phosphate-buffered saline at the required temperature. Assay buffer, 100 ll, with 25 lCi/ml [3H]putrescine (26 Ci/mmol) or [14C]putrescine (107 mCi/mmol) or 3H-spermidine (22.5 Ci/mmol) plus or minus other test compounds at the required concentrations, was added and the transport terminated at the required time points by rapidly separating the parasites from the other assay buffer components of a 150 ll aliquot by centrifugation through 800 ll of dibutyl phthalate (specific gravity 1.04). The upper aqueous layer was removed, the oil layer washed with cold phosphate-buffered saline, and the oil layer then carefully removed by aspiration. Traces of liquid were removed from the sides of the tube with a locally available ear bud, soaked in ethyl acetate. The red blood cell pellet was lysed with water, and proteins precipitated by 10% TCA. The supernatant was blotted on to a Whatman 3
filter disc, the filter disc dried and placed in a vial with scintillation fluid. Radioactivity was counted using a Hewlett Packard liquid scintillation counter.
a
Results Polyamine biosynthesis Following incubation of malaria parasite cultures with radiolabeled putrescine, and separation of the extracted and subsequently dansylated polyamines by thin layer chromatography, radiolabeled spermidine and spermine could be detected in the malaria parasite (Fig. 2). The amounts of radiolabeled spermidine and spermine were increased by harvesting the parasites at 18 h instead of at 9 h following the incubation with radiolabeled putrescine, suggesting that the polyamine biosynthesis pathway was rather slow. A few unidentified dansylated species (polyamines) were also seen on the autoradiogram. Polyamines other than putrescine, spermidine, and spermine, such as agmatine and hypusine, are also known to exist naturally in cells. It is possible that these dansylated species are some of these lesser studied polyamines. Putrescine transport We first followed putrescine transport in synchronized red blood cell cultures containing the various stages of the malaria parasite—ring, early trophozoite, late trophozoite, and schizont. Putrescine transport seemed to increase with the maturation of the ring stage to the trophozoite stage (Fig. 3a). There was no significant difference in the putrescine transport occurring in the trophozoite and schizont infected red blood cells, however (Fig. 3a). Hence, further experiments were conducted with trophozoite infected red blood cells. We assayed parasite cultures for putrescine transport, and determined the Arrhenius kinetics of putrescine transport in uninfected and infected red blood cells (Fig. 3b).
Fig. 2. Polyamine biosynthesis in the malaria parasite. Lane 1, dansylated putrescine; lane 2, dansylated spermidine; lane 3, dansylated spermine; lane 4, radiolabeled dansylated polyamines from malaria parasites incubated for 18 h following the pulse of [14C]putrescine; lane 5, radiolabeled dansylated polyamines from malaria parasites incubated for 6 h following the pulse of [14C]putrescine.
nmol put/1010RBCs/30 min
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2
1
0
Ring ET
LT Schizont
b
RBC IRBC
c RBC IRBC
Fig. 3. (a) Putrescine transport in infected red blood cells harboring the various asexual stages of the parasite—ring, ET (early trophozoite), LT (late trophozoite) and schizont. Putrescine transport measurements were made by monitoring the amount of [14C]putrescine taken up by infected red blood cells. Transport increased significantly with the maturation of the parasite from the ring stage to the trophozoite stage. (b) Arrhenius kinetics of the putrescine transporter in uninfected (RBC) and infected RBCs (IRBC). Putrescine transport measurements were made by monitoring the amount of [3H]putrescine taken up by infected and uninfected red blood cells incubated at various temperatures—4, 25, 37, and 42 C. (c) Michaelis–Menten kinetics of putrescine transporters in uninfected and infected RBCs. Putrescine transport measurements were made by monitoring the amount of [3H]putrescine taken up by infected and uninfected red blood cells incubated with varying concentrations of putrescine.
The plot of log V versus reciprocal of temperature indicated that the maximal rate of putrescine transport is highly temperature dependent in infected red blood cells. Activation energy values were calculated from the slopes of the plots. There was a significant difference in the activation energy calculated for the uninfected and infected red blood cells, suggesting that either new putrescine transporters are employed on the red blood cell membrane by the malaria parasite upon infection or that the Arrhenius kinetics of the existing transporters were altered following biochemical changes in the membrane of the infected red blood cell.
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We also followed putrescine transport at various concentrations of the substrate, putrescine, and obtained values of km and Vmax by non-linear regression of the plot of initial rate of transport versus putrescine concentration (Fig. 3c). The Vmax value of the putrescine transporter of infected cells was slightly higher at 4.69 ± 0.28 nmol/ 30 min/1010 red blood cells as compared to that of the uninfected cells (3.78 ± 0.42 nmol/30 min/1010 red blood cells). More importantly, infected red blood cells had a lower km (276.48 ± 48.11 lM) as compared to uninfected red blood cells (545.82 ± 51.44 lM), entailing a higher affinity for putrescine in infected red blood cells. Spermidine transport in the malaria parasite Spermidine transport was not vastly increased upon invasion of the malaria parasite, but Arrhenius kinetics gave activation energy values of 39.43 ± 5.72 and 20.59 ± 1.62 kJ/mol for uninfected and infected erythrocytes, respectively, suggesting a new transporter for spermidine in infected erythrocytes (Fig. 4a). The Q10 values for the temperature range 32–42 C in uninfected and infected erythrocytes were 3.0839 and 1.8005, respectively. Spermidine transport was also studied as a function of spermidine concentration. The initial velocity of transport a
Table 1 Inhibition of putrescine uptake by polyamine analogs, amino acids and metabolic inhibitors Effector (1 mM) Control D-Arginine Poly-L-arginine Ornithine Putrescine Spermidine Spermine Agmatine Difluoromethyl ornithine Serine L-Arginine Leucine Aspartate Glutamine Lysine Iodoacetate N-Ethyl maleimide p-Hydroxymercuribenzoate Cycloheximide Valinomycin Gramicidin Ouabain Calcium ionophore CCCP
log (nmol spermidine per 1010RBCs in 30 minutes)
The activation energy of the transporter in the uninfected cell was calculated to be 14.798 ± 1.7 kJ/mol and that of the infected cell was 37.42996 ± 3.5 kJ/mol. There was a significant difference in the Q10 values of the uninfected and infected red blood cells. The Q10 value of uninfected red blood cells was 1.80 while that of infected red blood cells was 3.45 for an increase in temperature from 32 to 42 C. Putrescine uptake by infected red blood cells was not inhibited significantly by the following amino acids that have specific transport systems in mammalian cells—serine (ASC), leucine (L), glutamine or asparagine (N), aspartate (x ), ornithine, lysine or arginine (y+) (Table 1). Thus putrescine is not transported by any of these known amino acid transporters. The other polyamines, spermidine and spermine, also did not compete with the labeled putrescine for transport. Only agmatine partially inhibited putrescine uptake by infected red blood cells (Table 1). Therefore, the parasite induced putrescine transport was highly specific for putrescine. We also determined the effect of various metabolic inhibitors on putrescine transport (Table 1). –SH group blockers such as iodoacetate, N-ethyl maleimide, and p-hydroxymercuribenzoate inhibited putrescine uptake, implying –SH group involvement in putrescine transport and thereby, a carrier-mediated process of putrescine transport in infected red blood cells. Proton transporters and ionophores such as CCCP, ouabain, valinomycin, gramicidin, and calcium ionophore inhibited the transport, implying that it could be membrane potential dependent, and an active process, requiring metabolic energy (Table 1).
2 1 0
RBC IRBC
-1 -2
Percent influx of control
0.0032
100 86.3 84.3 83.9 50.1 87.2 82.1 56.4 83.9 80.1 81.6 85.1 80.1 90.2 84.2 39.4 77.8 56.8 98.9 25.6 52.1 42.6 53.0 19.9
1/(Temperature in Kelvin)
b
nmol spermidine transported per 30 min by 1010RBCs
582
0.0036
40 RBC IRBC
30 20 10 0 0
200
400
600
μM Spermidine
Fig. 4. (a) Arrhenius kinetics of the spermidine transporter in uninfected (RBC) and infected RBCs (IRBC). Spermidine transport measurements were made by monitoring the amount of [3H]spermidine taken up by infected and uninfected red blood cells incubated at various temperatures—4, 25, 37, and 42 C. (b) Michaelis–Menten kinetics of spermidine transporters in uninfected and infected RBCs. Spermidine transport measurements were made by monitoring the amount of [3H]spermidine taken up by infected and uninfected red blood cells incubated with varying concentrations of spermidine.
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followed saturable Michaelis–Menten kinetics, and a plot of initial transport rate vs. spermidine concentration was fit to non-linear regression analyses to yield Vmax and km values of 329 ± 6.54 nmol/30 min/1010 red blood cells and 4992 ± 101.1 lM and 58 ± 3.46 nmol/30 min/1010 red blood cells and 880 ± 23.09 lM for uninfected and infected red blood cells, respectively (Fig. 4b). Discussion Our metabolic labeling experiments confirm that polyamines are synthesized in the malaria parasite. We demonstrate that biosynthesis of spermidine and spermine from putrescine takes place in the malaria parasite. In particular, we provide biochemical evidence for the synthesis of spermine from spermidine, contrary to the accepted view. If the parasite does synthesize its own polyamines, there should be no requirement for polyamine transport. However, DFMO or dicyclohexamine inhibition of malaria parasite growth has been known to be reversed by the exogenous addition of polyamines to the culture medium, suggesting that newly expressed transporters in the parasite infected erythrocyte afford increased permeability to polyamines in the infected erythrocytes [8,9]. Our study confirms that putrescine and spermidine are indeed transported by newly recruited transporters in malaria parasite infected red blood cells. Putrescine transport has been previously characterized in Plasmodium knowlesi [11]. Putrescine transport was demonstrated to increase by almost 11-fold in monkey erythrocytes upon infection by the malaria parasite [11]. The values of km of the putrescine transporters in uninfected and infected red blood cells were the same, but Vmax was increased in infected cells, suggesting a remarkable induction of putrescine transporter synthesis in tune with the increased requirement of polyamines. However, in contrast to mammalian cell types examined for induced polyamine transport, wherein increase in the influx rate did not accompany any change in the affinities of the substrates for the transporter, the putrescine transporter in the malaria parasite infected cell was energy dependent, involved –SH groups, and could not transport spermine, spermidine or other amino acids, indicating a distinct transporter for putrescine [11]. In our studies with the malaria parasite, P. falciparum, putrescine transport increased by hardly 2-fold in the human erythrocytes upon infection. However, though the Vmax value of the putrescine transporter of infected cells was not much higher at 4.69 ± 0.28 nmol/min/1010 erythrocytes compared to uninfected cells (3.78 ± 0.42 nmol/ min/1010 erythrocytes) as in P. knowlesi, infected erythrocytes had a lower km (276.48 ± 48.11 lM) as compared to uninfected erythrocytes (545.82 ± 51.44 lM), entailing a higher affinity for putrescine in infected erythrocytes. Also, the transporter did not facilitate transport of other polyamines other than agmatine, and amino acids, and the transport was an active process requiring metabolic energy
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and –SH groups, suggesting that a different transporter was employed in infected red blood cells. The activation energy and Q10 values, determined from Arrhenius kinetics of putrescine transport, were found to be significantly higher for infected cells than for uninfected cells, thus confirming the distinct nature of the transporters in uninfected and infected cells. It is well known that the malaria parasite modifies the membrane permeability and cytosolic composition of the host red blood cell [12]. Kirk et al. have demonstrated the induction of choline and Rb+ transport in malaria parasite infected red blood cells which either use functionally distinct pathways or have variable substrate selectivity as compared to uninfected cells [13]. We envisage such a scenario in P. falciparum with regard to putrescine transport. We established spermidine transport in red blood cells infected with P. falciparum, too, to be conducted by a distinct set of transporters. The Vmax and km of the infected cells were different in malaria parasite infected and uninfected red blood cells. Arrhenius kinetics yielded lower activation energy and Q10 values for infected cells as compared to uninfected cells, again confirming the distinct nature of the transporter. Being important cellular components essential for diverse functions such as macromolecular synthesis, cell proliferation, and differentiation, polyamines have conventionally been considered attractive targets for developing antitumor agents. Though inhibitors to enzymes of polyamine biosynthesis are known, they have met with little success as antitumor chemotherapy, possibly because mammalian cells have intricate feedback mechanisms which regulate polyamine levels, and consequently also enable cells to adapt to considerable changes in intracellular polyamine levels [4]. The difficulty encountered while dealing with inhibition of polyamine synthesis in mammalian cells may very well serve a blessing in disguise for other researchers. If the malaria parasite should have different polyamine requirements or levels, or employ different methods of acquiring polyamines, then the very inhibitors which failed as antitumor agents could very well turn out to be excellent antimalarial agents [4]. Our demonstration that malaria parasite infected red blood cells have new transporters for polyamines explains the previously reported finding that inhibition of polyamine biosynthesis per se does not affect parasite growth. More importantly, our finding paves the way for the design of molecules, polyamine analogs for instance, that inhibit both polyamine biosynthesis and transport, and antimalarial agents that can be transported into the malaria parasite through these parasite specific transporters themselves to specifically inhibit parasite growth. Acknowledgments The authors thank the Department of Biotechnology, Government of India, for the grant to N.S. T.N.C.R.
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acknowledges the Council of Scientific and Industrial Research, Government of India for the senior research fellowship.
[8]
References [1] C.W. Tabor, H. Tabor, Polyamines, Annu. Rev. Biochem. 53 (1984) 749–790. [2] Y.G. Assaraf, J. Golenser, D.T. Spira, U. Bachrach, Polyamine levels and the activity of their biosynthetic enzymes in human erythrocytes infected with the malarial parasite, Plasmodium falciparum, Biochem. J. 222 (3) (1984) 815–819. [3] J.M. Whaun, N.D. Brown, Ornithine decarboxylase inhibition and the malaria-infected red cell: a model for polyamine metabolism and growth, J. Pharmacol. Exp. Ther. 233 (2) (1985) 507–511. [4] S. Muller, G.H. Coombs, R.D. Walter, Targeting polyamines of parasitic protozoa in chemotherapy, Trends Parasitol. 17 (5) (2001) 242–249. [5] S. Majumder, J.J. Wirth, A.J. Bitonti, P.P. McCann, F. Kierszenbaum, Biochemical evidence for the presence of arginine decarboxylase activity in Trypanosoma cruzi, J. Parasitol. 78 (1992) 371–374. [6] J.S. Keithly, G. Zhu, S.J. Upton, K.M. Woods, M.P. Martinez, N. Yarlett, Polyamine biosynthesis in Cryptosporidium parvum and its implications for chemotherapy, Mol. Biochem. Parasitol. 88 (1997) 35–42. [7] S. Mu¨ller, A. Da’dara, K. Luersen, C. Wrenger, R. Dasgupta, R. Madhubala, R.D. Walter, In the human malaria parasite Plasmodium
[9]
[10]
[11]
[12] [13]
falciparum, polyamines are synthesized by a bifunctional ornithine decarboxylase, S-adenosylmethionine decarboxylase, J. Biol. Chem. 275 (2000) 8097–8102. Y.G. Assaraf, J. Golenser, D.T. Spira, U. Bachrach, Plasmodium falciparum: synchronization of cultures with DL-a-difluoromethylornithine, an inhibitor of polyamine biosynthesis, Exp. Parasitol. 61 (2) (1986) 229–235. A. Kaiser, A. Gottwald, C. Wiersch, B. Lindenthal, W. Maier, H.M. Seitz, Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum, Parasitol. Res. 87 (11) (1997) 963–972. S.C. Minocha, R. Minocha, C.A. Robie, High performance liquid chromatographic method for the determination of dansyl-polyamines, J. Chromatogr. 511 (1990) 177–183. S. Singh, S.K. Puri, S.K. Singh, R. Srivastava, R.C. Gupta, V.C. Pandey, Characterization of simian malarial parasite (Plasmodium knowlesi)—induced putrescine transport in rhesus monkey erythrocytes. A novel putrescine conjugate arrests in vitro growth of simian malarial parasite (Plasmodium knowlesi) and cures multidrug resistant murine malaria (Plasmodium yoelii) infection in vivo, J. Biol. Chem. 272 (21) (1997) 13506– 13511. K. Kirk, Membrane transport in the malaria-infected erythrocyte, Physiol. Rev. 81 (2) (2001) 495–537. K. Kirk, H.Y. Wong, B.C. Elford, C.I. Newbold, J.C. Ellory, Enhanced choline and Rb+ transport in human erythrocytes infected with the malaria parasite Plasmodium falciparum, Biochem. J. 278 (1991) 521–525.
Polyamine synthesis and salvage pathways in the malaria parasite Plasmodium falciparum q T.N.C. Ramya a, Namita Surolia b, Avadhesha Surolia b
a,c,*
a Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India c National Institute of Immunology, New Delhi 110067, India
Received 11 July 2006 Available online 31 July 2006
Abstract We demonstrate, for the first time, a functional polyamine biosynthetic pathway in the malaria parasite Plasmodium falciparum that culminates in the synthesis of spermine. Additionally, we also report putrescine and spermidine salvage in the malaria parasite. Putrescine and spermidine transport in P. falciparum infected red blood cells is a highly specific, carrier mediated and active process, mediated by new transporters that differ from the transporters of uninfected red blood cells in their kinetic parameters, Vmax and km, as well as in their activation energy. 2006 Elsevier Inc. All rights reserved. Keywords: Polyamine biosynthesis; Polyamine transport; Plasmodium falciparum; Putrescine; Spermidine; Spermine
The naturally occurring polyamines, spermidine and spermine, and their precursor, putrescine, are important regulators of growth and differentiation in cells. Though their exact function remains unknown, polyamines play a functional role in cell differentiation and proliferation processes probably by stabilizing the structure of newly synthesized chromatin and increasing the translational efficiency of RNA [1]. Normal erythrocytes are devoid of polyamine biosynthetic machinery and contain traces of putrescine, spermidine, and spermine. However, in Plasmodium falciparum infected erythrocytes, polyamines, and biosynthetic enzymes like ornithine decarboxylase and S-adenosyl methionine decarboxylase are known to increase [2,3]. Moreover, polyamines at certain stages
q Abbreviations: RBC, red blood cell; IRBC, Plasmodium falciparum infected red blood cell; SAM, S-adenosyl methionine; DFMO, difluoromethyl ornithine; ODC, ornithine decarboxylase; TCA, trichloroacetic acid; TLC, thin layer chromatography; CCCP, carbonyl cyanide m-chloro phenylhydrazone; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. * Corresponding author. Fax: +91 11 26717104. E-mail address: [email protected] (A. Surolia).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.07.127
trigger DNA synthesis as well as synthesis of certain proteins, one amongst them being the DNA polymerase itself. The activity of the cell cycle dependent, a-like malarial DNA polymerase, too, is stimulated by the cellular level of putrescine and spermidine, and inhibited by spermine. Addition of DFMO, an inhibitor of ODC, prevents the accumulation of putrescine and spermidine in parasitized erythrocytes, and arrests malaria parasite growth in the trophozoite stage prior to DNA synthesis [2]. Depleting the malaria parasite of polyamines is therefore extremely deleterious to its growth and seems an attractive target for developing antimalarial agents. It is therefore of interest to study polyamine synthesis in the malaria parasite. The precursor for polyamine biosynthesis in mammalian cells is arginine [4]. Arginine is decarboxylated by arginine decarboxylase to ornithine, which, in turn, is decarboxylated by the highly regulated enzyme activity of ornithine decarboxylase to putrescine. Spermidine and spermine are formed by the catalytic activities of the enzymes, spermidine synthase, spermine synthase, and S-adenosylmethionine (SAM) decarboxylase (Fig. 1) [4]. In some trypanosomatids, plants, and some bacteria, arginine is converted to putrescine via agmatine by arginine
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Fig. 1. Polyamine metabolism as speculated to exist in the malaria parasite, P. falciparum. For the sake of clarity, no subcellular organelles of the parasite are represented in the schematic. Arginine or ornithine is the precursor for the synthesis of putrescine, spermidine (and spermine).
decarboxylase, in the absence of a functional ornithine decarboxylase [5]. Arginine decarboxylase has been identified in a relative of the malaria parasite, Cryptosporidium, but not in the malaria parasite as yet [6]. The malaria parasite, however, has a bifunctional enzyme with ornithine decarboxylase and S-adenosylmethionine decarboxylase activities (Fig. 1) [7]. Importantly, the P. falciparum ornithine decarboxylase enzyme, unlike its mammalian counterpart, is not stimulated by putrescine and is very strongly feedback regulated by putrescine [7]. The malaria parasite presumably has spermidine synthase activity, but spermine synthesis is speculated to be absent [4] (Fig. 1). Current evidence does weigh in favor of a polyamine biosynthesis pathway in the malaria parasite. However, inhibitors of polyamine biosynthesis such as a-difluoromethyl ornithine, the well-known inhibitor of ornithine decarboxylase, though capable of inhibiting erythrocytic schizogony in vitro, are incapable of inhibiting parasite growth in vivo, suggesting that either the uptake of this drug is not effective or the malaria parasite satisfies its polyamine requirement by salvaging polyamines, too [8,9]. If the latter is true, a regimen of inhibitors of polyamine synthesis enzymes coupled with structural analogues of polyamines could well prove to be the ultimate campaign towards polyamine depletion and inhibition of parasite growth. We now provide biochemical proof for the presence of a functional polyamine biosynthesis pathway in the parasite. To the best of our knowledge, this is the first report of spermine synthesis in the malaria parasite. We also report the presence of new transport pathways for putrescine and spermidine in malaria parasite infected red blood cells. Experimental procedures Materials. The radioisotope [3H]-spermidine was from Perkin-Elmer. [14C]-1,4-putrescine and [3H]putrescine were from Amersham Biosciences.
Dibutyl phthalate, putrescine, ornithine, agmatine, spermidine, spermine, dansyl chloride, D-arginine, L-arginine, poly-L-arginine, difluoromethyl ornithine, serine, leucine, aspartate, glutamine, lysine, cycloheximide, iodoacetate, N-ethyl maleimide, valinomycin, gramicidin, p-hydroxymercuribenzoate, carbonyl cyanide m-chloro phenylhydrazone (CCCP), ouabain, and calcium ionophore were of the highest quality available from Sigma. Culture medium components were from Sigma. Incorporation of [14C]putrescine into spermidine and spermine in Plasmodium falciparum infected erythrocytes and Jurkat cells. Plasmodium falciparum infected erythrocytes (10 ml) synchronized with 5% sorbitol were incubated with [14C]putrescine (50 lCi; 100 mCi/mmol) to a final concentration of 0.2 mM for 6 h. They were then washed and resuspended in RPMI without [14C]putrescine for 6 or 18 h. The parasites were then isolated from the red blood cells by saponin lysis, and the polyamines in the parasites dansylated by a modified protocol of Minocha et al. [10]. Briefly, the cell pellet was resuspended in 5% TCA to precipitate proteins. The pH of the supernatant was brought to 9 with saturated sodium carbonate, and then an equal volume of 10 mg/ml dansyl chloride in acetone was added to it. The reaction mix was vortexed and allowed to incubate in the dark at 60 C for 60 min. The reaction was stopped with 100 mg/ml of L-alanine, the acetone was evaporated, the reaction mix cooled to 4 C, and dansylated polyamines extracted with an equal volume of toluene. The upper toluene phase was transferred to a fresh tube, concentrated to 50 ll, and spotted on a Whatman silica gel 60 TLC plate along with standard dansylated polyamines (also prepared in a similar fashion). The dansylated polyamines were developed with hexane/ethyl acetate :: 6:4 and visualized under UV illumination. Putrescine and spermidine transport in uninfected and Plasmodium falciparum infected red blood cells. Transport into P. falciparum infected red blood cells was assayed by a modified protocol of Singh et al. [11]. Briefly, 900 ll uninfected or P. falciparum infected red blood cells (3 · 107 cells) were incubated in phosphate-buffered saline at the required temperature. Assay buffer, 100 ll, with 25 lCi/ml [3H]putrescine (26 Ci/mmol) or [14C]putrescine (107 mCi/mmol) or 3H-spermidine (22.5 Ci/mmol) plus or minus other test compounds at the required concentrations, was added and the transport terminated at the required time points by rapidly separating the parasites from the other assay buffer components of a 150 ll aliquot by centrifugation through 800 ll of dibutyl phthalate (specific gravity 1.04). The upper aqueous layer was removed, the oil layer washed with cold phosphate-buffered saline, and the oil layer then carefully removed by aspiration. Traces of liquid were removed from the sides of the tube with a locally available ear bud, soaked in ethyl acetate. The red blood cell pellet was lysed with water, and proteins precipitated by 10% TCA. The supernatant was blotted on to a Whatman 3
filter disc, the filter disc dried and placed in a vial with scintillation fluid. Radioactivity was counted using a Hewlett Packard liquid scintillation counter.
a
Results Polyamine biosynthesis Following incubation of malaria parasite cultures with radiolabeled putrescine, and separation of the extracted and subsequently dansylated polyamines by thin layer chromatography, radiolabeled spermidine and spermine could be detected in the malaria parasite (Fig. 2). The amounts of radiolabeled spermidine and spermine were increased by harvesting the parasites at 18 h instead of at 9 h following the incubation with radiolabeled putrescine, suggesting that the polyamine biosynthesis pathway was rather slow. A few unidentified dansylated species (polyamines) were also seen on the autoradiogram. Polyamines other than putrescine, spermidine, and spermine, such as agmatine and hypusine, are also known to exist naturally in cells. It is possible that these dansylated species are some of these lesser studied polyamines. Putrescine transport We first followed putrescine transport in synchronized red blood cell cultures containing the various stages of the malaria parasite—ring, early trophozoite, late trophozoite, and schizont. Putrescine transport seemed to increase with the maturation of the ring stage to the trophozoite stage (Fig. 3a). There was no significant difference in the putrescine transport occurring in the trophozoite and schizont infected red blood cells, however (Fig. 3a). Hence, further experiments were conducted with trophozoite infected red blood cells. We assayed parasite cultures for putrescine transport, and determined the Arrhenius kinetics of putrescine transport in uninfected and infected red blood cells (Fig. 3b).
Fig. 2. Polyamine biosynthesis in the malaria parasite. Lane 1, dansylated putrescine; lane 2, dansylated spermidine; lane 3, dansylated spermine; lane 4, radiolabeled dansylated polyamines from malaria parasites incubated for 18 h following the pulse of [14C]putrescine; lane 5, radiolabeled dansylated polyamines from malaria parasites incubated for 6 h following the pulse of [14C]putrescine.
nmol put/1010RBCs/30 min
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2
1
0
Ring ET
LT Schizont
b
RBC IRBC
c RBC IRBC
Fig. 3. (a) Putrescine transport in infected red blood cells harboring the various asexual stages of the parasite—ring, ET (early trophozoite), LT (late trophozoite) and schizont. Putrescine transport measurements were made by monitoring the amount of [14C]putrescine taken up by infected red blood cells. Transport increased significantly with the maturation of the parasite from the ring stage to the trophozoite stage. (b) Arrhenius kinetics of the putrescine transporter in uninfected (RBC) and infected RBCs (IRBC). Putrescine transport measurements were made by monitoring the amount of [3H]putrescine taken up by infected and uninfected red blood cells incubated at various temperatures—4, 25, 37, and 42 C. (c) Michaelis–Menten kinetics of putrescine transporters in uninfected and infected RBCs. Putrescine transport measurements were made by monitoring the amount of [3H]putrescine taken up by infected and uninfected red blood cells incubated with varying concentrations of putrescine.
The plot of log V versus reciprocal of temperature indicated that the maximal rate of putrescine transport is highly temperature dependent in infected red blood cells. Activation energy values were calculated from the slopes of the plots. There was a significant difference in the activation energy calculated for the uninfected and infected red blood cells, suggesting that either new putrescine transporters are employed on the red blood cell membrane by the malaria parasite upon infection or that the Arrhenius kinetics of the existing transporters were altered following biochemical changes in the membrane of the infected red blood cell.
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We also followed putrescine transport at various concentrations of the substrate, putrescine, and obtained values of km and Vmax by non-linear regression of the plot of initial rate of transport versus putrescine concentration (Fig. 3c). The Vmax value of the putrescine transporter of infected cells was slightly higher at 4.69 ± 0.28 nmol/ 30 min/1010 red blood cells as compared to that of the uninfected cells (3.78 ± 0.42 nmol/30 min/1010 red blood cells). More importantly, infected red blood cells had a lower km (276.48 ± 48.11 lM) as compared to uninfected red blood cells (545.82 ± 51.44 lM), entailing a higher affinity for putrescine in infected red blood cells. Spermidine transport in the malaria parasite Spermidine transport was not vastly increased upon invasion of the malaria parasite, but Arrhenius kinetics gave activation energy values of 39.43 ± 5.72 and 20.59 ± 1.62 kJ/mol for uninfected and infected erythrocytes, respectively, suggesting a new transporter for spermidine in infected erythrocytes (Fig. 4a). The Q10 values for the temperature range 32–42 C in uninfected and infected erythrocytes were 3.0839 and 1.8005, respectively. Spermidine transport was also studied as a function of spermidine concentration. The initial velocity of transport a
Table 1 Inhibition of putrescine uptake by polyamine analogs, amino acids and metabolic inhibitors Effector (1 mM) Control D-Arginine Poly-L-arginine Ornithine Putrescine Spermidine Spermine Agmatine Difluoromethyl ornithine Serine L-Arginine Leucine Aspartate Glutamine Lysine Iodoacetate N-Ethyl maleimide p-Hydroxymercuribenzoate Cycloheximide Valinomycin Gramicidin Ouabain Calcium ionophore CCCP
log (nmol spermidine per 1010RBCs in 30 minutes)
The activation energy of the transporter in the uninfected cell was calculated to be 14.798 ± 1.7 kJ/mol and that of the infected cell was 37.42996 ± 3.5 kJ/mol. There was a significant difference in the Q10 values of the uninfected and infected red blood cells. The Q10 value of uninfected red blood cells was 1.80 while that of infected red blood cells was 3.45 for an increase in temperature from 32 to 42 C. Putrescine uptake by infected red blood cells was not inhibited significantly by the following amino acids that have specific transport systems in mammalian cells—serine (ASC), leucine (L), glutamine or asparagine (N), aspartate (x ), ornithine, lysine or arginine (y+) (Table 1). Thus putrescine is not transported by any of these known amino acid transporters. The other polyamines, spermidine and spermine, also did not compete with the labeled putrescine for transport. Only agmatine partially inhibited putrescine uptake by infected red blood cells (Table 1). Therefore, the parasite induced putrescine transport was highly specific for putrescine. We also determined the effect of various metabolic inhibitors on putrescine transport (Table 1). –SH group blockers such as iodoacetate, N-ethyl maleimide, and p-hydroxymercuribenzoate inhibited putrescine uptake, implying –SH group involvement in putrescine transport and thereby, a carrier-mediated process of putrescine transport in infected red blood cells. Proton transporters and ionophores such as CCCP, ouabain, valinomycin, gramicidin, and calcium ionophore inhibited the transport, implying that it could be membrane potential dependent, and an active process, requiring metabolic energy (Table 1).
2 1 0
RBC IRBC
-1 -2
Percent influx of control
0.0032
100 86.3 84.3 83.9 50.1 87.2 82.1 56.4 83.9 80.1 81.6 85.1 80.1 90.2 84.2 39.4 77.8 56.8 98.9 25.6 52.1 42.6 53.0 19.9
1/(Temperature in Kelvin)
b
nmol spermidine transported per 30 min by 1010RBCs
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0.0036
40 RBC IRBC
30 20 10 0 0
200
400
600
μM Spermidine
Fig. 4. (a) Arrhenius kinetics of the spermidine transporter in uninfected (RBC) and infected RBCs (IRBC). Spermidine transport measurements were made by monitoring the amount of [3H]spermidine taken up by infected and uninfected red blood cells incubated at various temperatures—4, 25, 37, and 42 C. (b) Michaelis–Menten kinetics of spermidine transporters in uninfected and infected RBCs. Spermidine transport measurements were made by monitoring the amount of [3H]spermidine taken up by infected and uninfected red blood cells incubated with varying concentrations of spermidine.
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followed saturable Michaelis–Menten kinetics, and a plot of initial transport rate vs. spermidine concentration was fit to non-linear regression analyses to yield Vmax and km values of 329 ± 6.54 nmol/30 min/1010 red blood cells and 4992 ± 101.1 lM and 58 ± 3.46 nmol/30 min/1010 red blood cells and 880 ± 23.09 lM for uninfected and infected red blood cells, respectively (Fig. 4b). Discussion Our metabolic labeling experiments confirm that polyamines are synthesized in the malaria parasite. We demonstrate that biosynthesis of spermidine and spermine from putrescine takes place in the malaria parasite. In particular, we provide biochemical evidence for the synthesis of spermine from spermidine, contrary to the accepted view. If the parasite does synthesize its own polyamines, there should be no requirement for polyamine transport. However, DFMO or dicyclohexamine inhibition of malaria parasite growth has been known to be reversed by the exogenous addition of polyamines to the culture medium, suggesting that newly expressed transporters in the parasite infected erythrocyte afford increased permeability to polyamines in the infected erythrocytes [8,9]. Our study confirms that putrescine and spermidine are indeed transported by newly recruited transporters in malaria parasite infected red blood cells. Putrescine transport has been previously characterized in Plasmodium knowlesi [11]. Putrescine transport was demonstrated to increase by almost 11-fold in monkey erythrocytes upon infection by the malaria parasite [11]. The values of km of the putrescine transporters in uninfected and infected red blood cells were the same, but Vmax was increased in infected cells, suggesting a remarkable induction of putrescine transporter synthesis in tune with the increased requirement of polyamines. However, in contrast to mammalian cell types examined for induced polyamine transport, wherein increase in the influx rate did not accompany any change in the affinities of the substrates for the transporter, the putrescine transporter in the malaria parasite infected cell was energy dependent, involved –SH groups, and could not transport spermine, spermidine or other amino acids, indicating a distinct transporter for putrescine [11]. In our studies with the malaria parasite, P. falciparum, putrescine transport increased by hardly 2-fold in the human erythrocytes upon infection. However, though the Vmax value of the putrescine transporter of infected cells was not much higher at 4.69 ± 0.28 nmol/min/1010 erythrocytes compared to uninfected cells (3.78 ± 0.42 nmol/ min/1010 erythrocytes) as in P. knowlesi, infected erythrocytes had a lower km (276.48 ± 48.11 lM) as compared to uninfected erythrocytes (545.82 ± 51.44 lM), entailing a higher affinity for putrescine in infected erythrocytes. Also, the transporter did not facilitate transport of other polyamines other than agmatine, and amino acids, and the transport was an active process requiring metabolic energy
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and –SH groups, suggesting that a different transporter was employed in infected red blood cells. The activation energy and Q10 values, determined from Arrhenius kinetics of putrescine transport, were found to be significantly higher for infected cells than for uninfected cells, thus confirming the distinct nature of the transporters in uninfected and infected cells. It is well known that the malaria parasite modifies the membrane permeability and cytosolic composition of the host red blood cell [12]. Kirk et al. have demonstrated the induction of choline and Rb+ transport in malaria parasite infected red blood cells which either use functionally distinct pathways or have variable substrate selectivity as compared to uninfected cells [13]. We envisage such a scenario in P. falciparum with regard to putrescine transport. We established spermidine transport in red blood cells infected with P. falciparum, too, to be conducted by a distinct set of transporters. The Vmax and km of the infected cells were different in malaria parasite infected and uninfected red blood cells. Arrhenius kinetics yielded lower activation energy and Q10 values for infected cells as compared to uninfected cells, again confirming the distinct nature of the transporter. Being important cellular components essential for diverse functions such as macromolecular synthesis, cell proliferation, and differentiation, polyamines have conventionally been considered attractive targets for developing antitumor agents. Though inhibitors to enzymes of polyamine biosynthesis are known, they have met with little success as antitumor chemotherapy, possibly because mammalian cells have intricate feedback mechanisms which regulate polyamine levels, and consequently also enable cells to adapt to considerable changes in intracellular polyamine levels [4]. The difficulty encountered while dealing with inhibition of polyamine synthesis in mammalian cells may very well serve a blessing in disguise for other researchers. If the malaria parasite should have different polyamine requirements or levels, or employ different methods of acquiring polyamines, then the very inhibitors which failed as antitumor agents could very well turn out to be excellent antimalarial agents [4]. Our demonstration that malaria parasite infected red blood cells have new transporters for polyamines explains the previously reported finding that inhibition of polyamine biosynthesis per se does not affect parasite growth. More importantly, our finding paves the way for the design of molecules, polyamine analogs for instance, that inhibit both polyamine biosynthesis and transport, and antimalarial agents that can be transported into the malaria parasite through these parasite specific transporters themselves to specifically inhibit parasite growth. Acknowledgments The authors thank the Department of Biotechnology, Government of India, for the grant to N.S. T.N.C.R.
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acknowledges the Council of Scientific and Industrial Research, Government of India for the senior research fellowship.
[8]
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