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Malaria merozoite surface protein antisense oligodeoxynucleotides lack antisense activity but function as polyanions to inhibit red cell invasion
The International Journal of
PERGAMON
The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
Biochemistry & Cell Biology
Malaria merozoite surface protein antisense oligodeoxynucleotides lack antisense activity but function as polyanions to inhibit red cell invasion R. Kanagaratnam a, b, K. Misiura c, G. Rebowski c, R. Ramasamy a, * a
Division of Life Sciences, Institute of Fundamental Studies, Kandy, Sri Lanka b Faculty of Medicine, University of Jana, Jana, Sri Lanka c Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, 90-363 Lodz, Sienkiewicza 112, Poland Received 8 April 1997; accepted 21 April 1998
Abstract The eects on malaria parasite growth of antisense and sense oligodeoxynucleoside phosphorothioates based on a merozoite surface protein mRNA was examined. Speci®c antisense eects of the oligonucleotides could not be demonstrated over three cycles of schizogony or when added as a complex with cationic liposomes. Antisense and sense oligonucleotides however, inhibit merozoite invasion of red blood cells at similar concentrations to dextran sulphate, a polyanion, as determined by microscopy and [3 H]hypoxanthine incorporation into DNA. Neutralisation of the negative charge on the oligonucleotides by binding to cationic lipid liposomes, prevented the inhibition of merozoite invasion. We postulate that oligonucleotides because of their polyanionic nature interfere with the binding of merozoites to receptors on red blood cells and that polyanions may be useful in malaria therapy. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Antisense; Liposomes; Malaria; Merozoites; MSA-2; Oligonucleotides; Plasmodium falciparum; Polyanions
1. Introduction Blocking speci®c gene expression with antisense oligodeoxynucleotides (ODNs) oers a new approach to treating many diseases [1] including Abbreviations: DOTAP, N-[1-(2,3-D dioleoyloxy) propyl]N,N,N-(trimethylammonium) methyl sulphate; MSA-2, 45 kDa merozoite surface antigen; ODN, oligodeoxynucleotide; ODN-S, oligodeoxynucleoside phosphorothioate; Rbc, red blood cells. * Corresponding author. Fax: +94-1-586715; E-mail: [email protected]
malaria, where resistance to commonly used sulphonamides, dihydrofolate reductase inhibitors, and 4-aminoquinolines is now widespread. Speci®c antisense eects with oligonucleoside phosphodiesters and phosphorothioates (ODN-S) based on dihydrofolate reductase-thymidylate synthase (DHFR-TS) [2, 3], and a 185 kDa merozoite surface protein termed MSA-1 [2] have been reported in the human malaria parasite Plasmodium falciparum. Two other studies with ODNs based on DHFR-TS showed non-speci®c inhibition of parasite growth [4, 5]. Sialic acid residues on rbc membranes serve as ligands for
1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 0 5 1 - X
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R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
initial attachment of P. falciparum merozoites [6]. Antibodies to a 45 kDa merozoite surface protein, termed MSA-2, inhibit invasion [7, 8]. MSA2 is synthesised by late stage trophozoites and schizonts and is not carried by invading merozoites into rbc or synthesised by ring stages [7]. The role of MSA-2 is not known but it could serve a structural, transport or receptor function on merozoites. Proteins such as MSA-2 that are present only during a part of the cell cycle may be better targets for antisense therapy than enzymes like DHFR-TS. We therefore examined the eects of antisense ODN-S based on the MSA-2 sequence of P. falciparum (3D7 isolate) on parasite growth in vitro. 2. Materials and methods 2.1. Synthesis of oligodeoxynucleoside phosphorothioates ODN were synthesised in an automated DNA synthesiser using phosphoramidite chemistry, then stepwise sulphurised and puri®ed by HPLC followed by Na+ ion exchange chromatography as described previously [5, 9]. They were then lyophilised, reconstituted in RPMI 1640 medium at 100 mM and sterilised by ®ltration through 0.22 mm ®lters. The sequences of ®ve ODN-S used in this study are given below. The ODN-S were derived from the translation initiation site (B1, position 1±18) and an internal loop region (B2, position 83±100) predicted by secondary structure analysis of the mRNA using DNASIS software (Pharmacia). Both B1 and B2 were free of substantial self complementarity. The sense sequences corresponding to B1 and B2 were synthesised for use as controls. B2R, which was a random sequence ODN-S with the same base composition as B2K was synthesised for use as an additional control. A poly (C)18 ODN-S was used as a further control. BIK antisense TGT TTT AAT TAC CTT CAT BIA sense ATG AAG GTA ATT AAA ACA
B2K antisense CAT TGT TTA TGA ATG TGT B2A sense ACA CAT TCA TAA ACA ATG B2R random TGT TAG TTC ATG TGA TAT
2.2. Parasite growth inhibition assays P. falciparum 3D7 isolate was cultured in 0+ human rbc in RPMI 1640 medium containing 10% human AB+ serum, 22 mM Hepes, 24 mM sodium bicarbonate, 10 mg mlÿ1 gentamycin and 48 h growth inhibition assays performed as described previously [5, 10]. However, for the experiments described in Tables 4 and 5, parasites were grown in medium containing RPMI 1640 to which 44 mg mlÿ1 hypoxanthine had been added to facilitate growth. Brie¯y, synchronous late stage parasites (trophozoites and schizonts) obtained by gelatine sedimentation, at 0.4±0.6% parasitaemia and 5% haematocrit were resuspended in medium containing ODN-S at dierent concentrations. Replicate 100 ml cultures were prepared in 96 well plates. Twenty four hours later, [3 H]-hypoxanthine (Amersham) in 100 ml RPMI 1640 was added to each well and the cells harvested at 48 h for determining radioactivity incorporated into DNA. Parallel parasite cultures to which [3 H]-hypoxanthine was not added were used to determine parasitaemia after Giemsa staining. Dilutions of ODN-S were added with [3 H]-hypoxanthine in 100 ml RPMI 1640 at 24 h, instead of at the beginning of the culture, to investigate the eects of ODN-S on the ring to late stage maturation of parasites. For 6 d growth inhibition assays, late stage parasites at 0.05% parasitaemia and 5% haematocrit were cultured in 1 ml complete medium in 24 well tissue culture plates in the absence or presence of ODN-S. The medium containing ODN-S was changed every 24 h and a 1 ml aliquot of cells was removed every 24 h for Giemsa staining and microscopic examination. The average parasitaemia of duplicate cultures per treatment was determined for 6 d.
R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
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2.3. Dextran sulphate as inhibitor
3. Results
A stock 10 mg mlÿ1 solution of dextran sulphate (Sigma) was made in RPMI 1640 and dialysed extensively against RPMI 1640. Dilutions of the stock were then made in RPMI 1640 for preparation of complete medium and use in parasite growth inhibition assays as for ODN-S.
Addition of ODN-S to synchronous cultures of late stage parasites inhibited parasite growth in a sequence-independent but dose-dependent manner (Table 1). Dextran sulphate produced a similar dose-dependent inhibition of parasite growth, with almost complete inhibition being achieved at 100 mg mlÿ1 (Table 1). The inhibitory eects were observed 24 h after initiation of culture suggesting that invasion of rbc by merozoites released by schizont rupture was aected. To con®rm this, we examined the eects of adding ODN-S and dextran sulphate at the highest concentrations (10 and 100 mg mlÿ1, respectively) 24 h after initiation of the culture at which time the reinvasion of rbc by merozoites is substantially complete. The ODN-S and dextran sulphate did not similarly inhibit parasite growth under these conditions (Table 2). Since MSA-2 is synthesised only in late stage parasites [7], it was possible that speci®c antisense eects were not observed due to the limited period of exposure to antisense ODN-S. However, exposure to 0.1 and 1 mM ODN-S over six days in culture involving three
2.4. Use of liposome±ODN-S complexes as inhibitors Pre-formed complexes of ODN-S with liposomes of the cationic lipid N-[1-(2,3-D dioleoyloxy) propyl]-N,N,N-(trimethylammonium) methyl sulphate (DOTAP, Boehringer) at varying ratios of ODN-S to DOTAP were prepared according to the manufacturer's instructions, except that RPMI 1640 was used as a diluent. These were then added to cultures at ®nal concentrations of 1 and 10 mM ODN-S under the same conditions as ODN-S alone.
Table 1 Eects of ODN-S and dextran sulphate added at 0 h to late stages of P. falciparum Inhibitor
Concentration (mM)
Total Parasitemia at 24 h (%)
[3 H]hypoxanthine incorporation at 24±48 h (mean cpm2 sd)
B1K B1A B2K B2A B1K B1A B2K B2A B1K B1A B2K B2A Dextran sulphate Dextran sulphate Dextran sulphate Control
0.1 0.1 0.1 0.1 1.0 1.0 1.0 1.0 10 10 10 10 100 mg mlÿ1 10 mg mlÿ1 1 mg mlÿ1 ÿ
2.4 2.3 2.4 2.4 1.9 2.2 2.0 2.1 0.6 0.8 0.5 0.4 0.03 1.8 2.0 2.4
3924927655 3927925461 4733927716 3657528048 4144023708 4433924087 4018025269 4188525778 80402711 51032686 58122625 45742470 27732470 3939422951 4258625967 4044023517
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviation of six replicates. At 24 h, 1 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 7 Ci per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.5% late stages.
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Table 2 Eects of ODN-S and dextran sulphate added at 24 h to ring stages of P. falciparum
Inhibitor
Concentration (mM)
[3 H]hypoxanthine incorporation at 24±48 h (mean cpm2 sd)
B1K B1A B2K B2A Dextran sulphate Control
10 10 10 10 100 mg mlÿ1 ÿ
4997822524 5085624510 4815725686 5155824518 4645624046 5479623339
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated2 standard deviation of six replicates. At 24 h, 1 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 7 Ci per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.5% late stages.
cycles of rbc invasion did not demonstrate speci®c antisense eects (Table 3). Dextran sulphate at 100 mg mlÿ1 completely inhibited parasite multiplication over 6 d (Table 3) as did 10 mM anti-sense and sense ODN-S (data not shown). B2R, an ODN-S with the same base composition and length as the B2K anti-sense ODN-S, and poly (C)18 produced a similar concentration dependent inhibition of P. falciparum growth as
Table 3 Eect of 6 d exposure to ODN-S and dextran sulphate on P. falciparum growth Inhibitor
Concentration Parasitaemia (mM) at day 6 (%)
B1K B1A B1K B1A B2K B2A Dextran Dextran Dextran Control
0.1 0.1 1.0 1.0 1.0 1.0 100 mg mlÿ1 10 mg mlÿ1 1 mg mlÿ1 ÿ
7.0 7.0 5.0 5.1 5.8 5.3 0.0 4.7 4.9 7.8
Inhibition (%) 10 10 36 35 26 32 100 40 37 0
The starting parasitaemia in the cultures was 0.05% at day 0.
the sense and anti-sense ODN-S (Table 4). The decrease in the numbers of ring stage parasites at 24 h with addition of ODN-S con®rms that the inhibition of [3 H]-hypoxanthine incorporation was due to reduced reinvasion of rbc by merozoites. The data also do not show evidence for a stimulation of [3 H]-hypoxanthine incorporation in malaria parasites by added ODNs that was reported by one group [3]. The inhibition of reinvasion by ODN-S was prevented when the ODN-S was complexed to cationic liposomes composed of DOTAP before addition to the cultures (Table 5). Adding 13 mg mlÿ1 DOTAP to 1 mM ODN-S (approximately 6 mg mlÿ1 ODN-S) completely abolished the partial inhibition of reinvasion at this concentration produced with either sense or antisense ODN-S alone. Similarly an approximately 2:1 wt/wt addition of DOTAP to 10 mM ODN-S (133 mg mlÿ1 DOTAP to 60 mg mlÿ1 ODN-S) completely abolished the very marked inhibition normally seen with 10 mM ODN-S alone. However, a 1:1 wt/wt addition of DOTAP to 10 mM ODN-S (60 mg mlÿ1 DOTAP to 60 mg mlÿ1 ODN-S) only partly reversed the ODN-S mediated inhibition of reinvasion. Speci®c antisense eects of ODN-S could also not be seen in the presence of DOTAP. Additional experiments (data not presented) showed that, (1) no inhibition of [3 H]-hypoxanthine incorporation was seen when 0.5 mM sense or antisense ODN-S complexed to DOTAP liposomes at 15 mg mlÿ1 were added to parasite cultures at 24 h, after merozoite invasion had occurred, and (2) DOTAP added alone at 0 h to cultures at 13 or 133 mg mlÿ1, did not stimulate or inhibit reinvasion or [3 H]-hypoxanthine incorporation into parasites. 4. Discussion Antisense ODNs based on MSA-1 and DHFR-TS were reported to speci®cally inhibit P. falciparum growth compared to control ODNs [2]. However it was noted in these experiments that schizont to ring transition i.e. reinvasion was more inhibited (IC50 0.5±1.1 mM) than ring to schizont maturation (IC50>2.5 mM). Our results
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Table 4 Eects of control ODN-S added at 0 h to late stages of P. falciparum
ODN-S
Concentration
Parasitaemia at 24 h rings (%)
B2R poly C B2R poly C B2R poly C Nil
0.1 0.1 1.0 1.0 10 10 ÿ
[3 H] hypoxanthine incorporation at 24±48 h (mean cpm2sd) total (%)
1.6 1.5 1.4 1.5 0.2 0.5 1.4
1.7 1.7 1.5 1.6 0.3 0.7 1.4
678 253 628 231 551 267 426 227 55208 50215 692 295
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviations of ®ve replicates. At 24 h, 0.25 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 4 mCi per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.4% late stages.
with MSA-2 speci®c ODNs are clearly dierent. Dierent experimental conditions, e.g. our parasite cultures are probably more synchronous with regard to the dierent stages, may account for some of the variation. The present results with
MSA-2 ODN-S, B2R and poly (C)18 con®rm observations with DHFR-TS ODN-S [5] that merozoite invasion is inhibited in a sequenceindependent manner and that there is no signi®cant anti-sense eect on maturation of rings to
Table 5 Eects of adding ODN-S complexed to DOTAP at 0 h to late stages of P. falciparum
ODN-S (Conc)
DOTAP (Conc)
Parasitaemia at 24 h rings (%)
B2K (1 mM) B2A (1 mM) B2K (1 mM) B2A (1 mM) B2K (10 mM) B2A (10 mM) B2K (10 mM) B2A (10 mM) B2K (10 mM) B2A (10 mM) ÿ
0 0 13 (mg mlÿ1) 13 (mg mlÿ1) 0 0 60 (mg mlÿ1) 60 (mg mlÿ1) 133 (mg mlÿ1) 133 (mg mlÿ1) ÿ
[3H]hypoxanthine incorporation at 24±48 h (mean cpm2sd)
2.2 2.3 3.5 3.0 0.5 0.5 2.3 1.9 3.1 3.0 3.1
total (%) 2.4 2.5 3.7 3.3 0.9 1.0 2.5 2.2 3.3 3.1 3.3
871294 824269 1049282 1073280 369217 224229 950279 808278 1095252 1012288 9662116
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviation of four replicates. At 24 h, 0.5 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 8 mCi per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.6% late stages. Reversal by DOTAP at any concentration of the corresponding growth inhibition due to ODN-S alone was statistically signi®cant ( p < 0.05) by the Student's t test.
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schizonts at ODN-S concentrations ranging from 0.1 to 10 mM. Furthermore, no signi®cant antisense eects were seen with MSA-2 ODN-S (and with DHFR-TS ODN-S, our unpublished observations) over three invasion cycles. Comparable inhibition of merozoite invasion is seen with a polyanion, dextran sulphate, at similar concentrations to the ODN-S (10 mM B1 or B2 is 060 mg mlÿ1). Heparin, a sulphated glycosaminoglycan also inhibits P. falciparum merozoite invasion [11]. Dextran sulphate and fucoidin reportedly inhibit invasion of rhesus rbc by the simian malaria parasite P. knowlesi, by binding to a 135 kDa parasite protein that functions as a ligand for Duy blood group receptors on the rbc [12]. We postulate therefore that ODNs may inhibit P. falciparum reinvasion due to their polyanionic character, possibly by blocking the recognition of human rbc sialic acids by the corresponding ligands on P. falciparum merozoites. The abrogation of the ODN-S mediated invasion inhibition when the polyanionic character of the ODN-S is neutralised by the cationic lipid DOTAP further supports this hypothesis. For ODNs to enter intracellular malaria parasites, transport across three membranes viz. the rbc membrane, the parasitophorous vacuole membrane and the parasite plasma membrane is needed. Rbc unlike nucleated cells are unable to internalise ODNs by endocytosis. However, a parasitophorous duct reportedly present in P. falciparum infected rbc [13] could enable ODNs to gain access to the parasite plasma membrane where endocytosis might occur. There is also evidence for transport of lipids from the rbc membrane to intracellular P. falciparum in vesicles [14]. While DOTAP-nucleic acid complexes are routinely used for transfecting cultured nucleated cells, ODN-S complexed with DOTAP liposomes are unable to exert a speci®c antisense eect in P. falciparum. Hence our results suggest that the intracellular location of the parasite does not readily permit anti-sense therapy for malaria. On the other hand, clinically acceptable polyanions, may prove valuable for the treatment of complicated or drug resistant malaria.
Acknowledgements This investigation was supported by the State Committee for Scienti®c Research, Poland (Research grant 4POSF.023.10) and the Natural Resources, Energy and Science Authority, Sri Lanka (95/BT/03).
References [1] P.C. Zamecnik, M.L. Stephenson, Inhibition of Rous sarcoma virus replication and cell transformation by a speci®c oligonucleotide, Proc. Natl. Acad. Sci. USA 75 (1978) 280±284. [2] E. Rapaport, K. Misiura, S. Agrawal, Z. Zamecnik, Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine resistant Plasmodium falciparum, Proc. Natl. Acad. Sci. USA 89 (1992) 8577±8580. [3] R.H. Barker, V. Metelev, E. Rapaport, P. Zamecnik, Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides, Proc. Natl. Acad. Sci. USA 93 (1996) 514±518. [4] D.L. Clark, L.A. Chrisey, J.R. Campbell, E.A. Davidson, Non-sequence speci®c antimalarial activity of oligodeoxynucleotides, Mol. Biochem. Parasitol. 63 (1994) 129±134. [5] R. Ramasamy, R. Kanagaratnam, K. Misiura, G. Amerakoon, R. Amerakoon, W.J. Stec, Anti-sense oligodeoxynucleoside phosphorothioates nonspeci®cally inhibit invasion of red blood cells by malaria parasites, Biochem. Biophys. Res. Comm. 218 (1996) 930±933. [6] B.K.L. Sim, C.E. Chitnis, K. Wasniowska, T.J. Headley, L.H. Miller, Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum, Science 264 (1994) 1941±1944. [7] R. Ramasamy, Studies on glycoproteins in the human malaria parasite Plasmodium falciparum ± identi®cation of a 45 kDa glycosylated and myristilated merozoite surface antigen, Immunol. Cell Biol. 65 (1987) 419±424. [8] R. Ramasamy, G. Jones, R. Lord, Characterisation of an inhibitory monoclonal antibody de®ned epitope on a malaria vaccine candidate antigen, Immunol. Lett. 23 (1990) 305±310. [9] W.J. Stec, A. Wilk, Sterocontrolled synthesis of oligo (deoxynucleoside phosphorothiote) S, Angew. Chem. Int. Ed. Eng. 33 (1994) 709±722. [10] R. Ramasamy, R. Rajakaruna, Association of malaria with inactivation of a1,3 - galactosyl transferase in catarrhines, Biochim. Biophys. Acta 1360 (1997) 241±246. [11] A. Kulane, H.P. Ekre, P. Perlmann, L. Rombo, M. Wahlin, B. Wahlin, Eect of dierent fractions of heparin on Plasmodium falciparum merozoite invasion of red blood cells in vitro, Am. J. Trop. Med. Hyg. 46 (1992) 589±594.
R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985 [12] J.P. Dalton, D. Hudson, J.H. Adams, L.H. Miller, Blocking of the receptor mediated invasion of erythrocytes of Plasmodium knowlesi malaria with sulfated polysaccharides and glycosaminoglycans, Eur. J. Biochem. 195 (1991) 789±794. [13] B. Pouvelle, R. Spiegel, L. Hsiao, R.J. Howard, R.L. Thomas, A.P. Thomas, T.F. Taraschi, Direct access to
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serum macromolecules by intraerythrocytic malaria parasites, Nature 353 (1991) 73±75. [14] K. Haldar, A. de Amorim, G.A.M. Cross, Transport of ¯uorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum infected cells, J. Cell Biol. 108 (1989) 2183±2192.
PERGAMON
The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
Biochemistry & Cell Biology
Malaria merozoite surface protein antisense oligodeoxynucleotides lack antisense activity but function as polyanions to inhibit red cell invasion R. Kanagaratnam a, b, K. Misiura c, G. Rebowski c, R. Ramasamy a, * a
Division of Life Sciences, Institute of Fundamental Studies, Kandy, Sri Lanka b Faculty of Medicine, University of Jana, Jana, Sri Lanka c Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, 90-363 Lodz, Sienkiewicza 112, Poland Received 8 April 1997; accepted 21 April 1998
Abstract The eects on malaria parasite growth of antisense and sense oligodeoxynucleoside phosphorothioates based on a merozoite surface protein mRNA was examined. Speci®c antisense eects of the oligonucleotides could not be demonstrated over three cycles of schizogony or when added as a complex with cationic liposomes. Antisense and sense oligonucleotides however, inhibit merozoite invasion of red blood cells at similar concentrations to dextran sulphate, a polyanion, as determined by microscopy and [3 H]hypoxanthine incorporation into DNA. Neutralisation of the negative charge on the oligonucleotides by binding to cationic lipid liposomes, prevented the inhibition of merozoite invasion. We postulate that oligonucleotides because of their polyanionic nature interfere with the binding of merozoites to receptors on red blood cells and that polyanions may be useful in malaria therapy. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Antisense; Liposomes; Malaria; Merozoites; MSA-2; Oligonucleotides; Plasmodium falciparum; Polyanions
1. Introduction Blocking speci®c gene expression with antisense oligodeoxynucleotides (ODNs) oers a new approach to treating many diseases [1] including Abbreviations: DOTAP, N-[1-(2,3-D dioleoyloxy) propyl]N,N,N-(trimethylammonium) methyl sulphate; MSA-2, 45 kDa merozoite surface antigen; ODN, oligodeoxynucleotide; ODN-S, oligodeoxynucleoside phosphorothioate; Rbc, red blood cells. * Corresponding author. Fax: +94-1-586715; E-mail: [email protected]
malaria, where resistance to commonly used sulphonamides, dihydrofolate reductase inhibitors, and 4-aminoquinolines is now widespread. Speci®c antisense eects with oligonucleoside phosphodiesters and phosphorothioates (ODN-S) based on dihydrofolate reductase-thymidylate synthase (DHFR-TS) [2, 3], and a 185 kDa merozoite surface protein termed MSA-1 [2] have been reported in the human malaria parasite Plasmodium falciparum. Two other studies with ODNs based on DHFR-TS showed non-speci®c inhibition of parasite growth [4, 5]. Sialic acid residues on rbc membranes serve as ligands for
1357-2725/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 1 3 5 7 - 2 7 2 5 ( 9 8 ) 0 0 0 5 1 - X
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R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
initial attachment of P. falciparum merozoites [6]. Antibodies to a 45 kDa merozoite surface protein, termed MSA-2, inhibit invasion [7, 8]. MSA2 is synthesised by late stage trophozoites and schizonts and is not carried by invading merozoites into rbc or synthesised by ring stages [7]. The role of MSA-2 is not known but it could serve a structural, transport or receptor function on merozoites. Proteins such as MSA-2 that are present only during a part of the cell cycle may be better targets for antisense therapy than enzymes like DHFR-TS. We therefore examined the eects of antisense ODN-S based on the MSA-2 sequence of P. falciparum (3D7 isolate) on parasite growth in vitro. 2. Materials and methods 2.1. Synthesis of oligodeoxynucleoside phosphorothioates ODN were synthesised in an automated DNA synthesiser using phosphoramidite chemistry, then stepwise sulphurised and puri®ed by HPLC followed by Na+ ion exchange chromatography as described previously [5, 9]. They were then lyophilised, reconstituted in RPMI 1640 medium at 100 mM and sterilised by ®ltration through 0.22 mm ®lters. The sequences of ®ve ODN-S used in this study are given below. The ODN-S were derived from the translation initiation site (B1, position 1±18) and an internal loop region (B2, position 83±100) predicted by secondary structure analysis of the mRNA using DNASIS software (Pharmacia). Both B1 and B2 were free of substantial self complementarity. The sense sequences corresponding to B1 and B2 were synthesised for use as controls. B2R, which was a random sequence ODN-S with the same base composition as B2K was synthesised for use as an additional control. A poly (C)18 ODN-S was used as a further control. BIK antisense TGT TTT AAT TAC CTT CAT BIA sense ATG AAG GTA ATT AAA ACA
B2K antisense CAT TGT TTA TGA ATG TGT B2A sense ACA CAT TCA TAA ACA ATG B2R random TGT TAG TTC ATG TGA TAT
2.2. Parasite growth inhibition assays P. falciparum 3D7 isolate was cultured in 0+ human rbc in RPMI 1640 medium containing 10% human AB+ serum, 22 mM Hepes, 24 mM sodium bicarbonate, 10 mg mlÿ1 gentamycin and 48 h growth inhibition assays performed as described previously [5, 10]. However, for the experiments described in Tables 4 and 5, parasites were grown in medium containing RPMI 1640 to which 44 mg mlÿ1 hypoxanthine had been added to facilitate growth. Brie¯y, synchronous late stage parasites (trophozoites and schizonts) obtained by gelatine sedimentation, at 0.4±0.6% parasitaemia and 5% haematocrit were resuspended in medium containing ODN-S at dierent concentrations. Replicate 100 ml cultures were prepared in 96 well plates. Twenty four hours later, [3 H]-hypoxanthine (Amersham) in 100 ml RPMI 1640 was added to each well and the cells harvested at 48 h for determining radioactivity incorporated into DNA. Parallel parasite cultures to which [3 H]-hypoxanthine was not added were used to determine parasitaemia after Giemsa staining. Dilutions of ODN-S were added with [3 H]-hypoxanthine in 100 ml RPMI 1640 at 24 h, instead of at the beginning of the culture, to investigate the eects of ODN-S on the ring to late stage maturation of parasites. For 6 d growth inhibition assays, late stage parasites at 0.05% parasitaemia and 5% haematocrit were cultured in 1 ml complete medium in 24 well tissue culture plates in the absence or presence of ODN-S. The medium containing ODN-S was changed every 24 h and a 1 ml aliquot of cells was removed every 24 h for Giemsa staining and microscopic examination. The average parasitaemia of duplicate cultures per treatment was determined for 6 d.
R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
981
2.3. Dextran sulphate as inhibitor
3. Results
A stock 10 mg mlÿ1 solution of dextran sulphate (Sigma) was made in RPMI 1640 and dialysed extensively against RPMI 1640. Dilutions of the stock were then made in RPMI 1640 for preparation of complete medium and use in parasite growth inhibition assays as for ODN-S.
Addition of ODN-S to synchronous cultures of late stage parasites inhibited parasite growth in a sequence-independent but dose-dependent manner (Table 1). Dextran sulphate produced a similar dose-dependent inhibition of parasite growth, with almost complete inhibition being achieved at 100 mg mlÿ1 (Table 1). The inhibitory eects were observed 24 h after initiation of culture suggesting that invasion of rbc by merozoites released by schizont rupture was aected. To con®rm this, we examined the eects of adding ODN-S and dextran sulphate at the highest concentrations (10 and 100 mg mlÿ1, respectively) 24 h after initiation of the culture at which time the reinvasion of rbc by merozoites is substantially complete. The ODN-S and dextran sulphate did not similarly inhibit parasite growth under these conditions (Table 2). Since MSA-2 is synthesised only in late stage parasites [7], it was possible that speci®c antisense eects were not observed due to the limited period of exposure to antisense ODN-S. However, exposure to 0.1 and 1 mM ODN-S over six days in culture involving three
2.4. Use of liposome±ODN-S complexes as inhibitors Pre-formed complexes of ODN-S with liposomes of the cationic lipid N-[1-(2,3-D dioleoyloxy) propyl]-N,N,N-(trimethylammonium) methyl sulphate (DOTAP, Boehringer) at varying ratios of ODN-S to DOTAP were prepared according to the manufacturer's instructions, except that RPMI 1640 was used as a diluent. These were then added to cultures at ®nal concentrations of 1 and 10 mM ODN-S under the same conditions as ODN-S alone.
Table 1 Eects of ODN-S and dextran sulphate added at 0 h to late stages of P. falciparum Inhibitor
Concentration (mM)
Total Parasitemia at 24 h (%)
[3 H]hypoxanthine incorporation at 24±48 h (mean cpm2 sd)
B1K B1A B2K B2A B1K B1A B2K B2A B1K B1A B2K B2A Dextran sulphate Dextran sulphate Dextran sulphate Control
0.1 0.1 0.1 0.1 1.0 1.0 1.0 1.0 10 10 10 10 100 mg mlÿ1 10 mg mlÿ1 1 mg mlÿ1 ÿ
2.4 2.3 2.4 2.4 1.9 2.2 2.0 2.1 0.6 0.8 0.5 0.4 0.03 1.8 2.0 2.4
3924927655 3927925461 4733927716 3657528048 4144023708 4433924087 4018025269 4188525778 80402711 51032686 58122625 45742470 27732470 3939422951 4258625967 4044023517
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviation of six replicates. At 24 h, 1 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 7 Ci per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.5% late stages.
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Table 2 Eects of ODN-S and dextran sulphate added at 24 h to ring stages of P. falciparum
Inhibitor
Concentration (mM)
[3 H]hypoxanthine incorporation at 24±48 h (mean cpm2 sd)
B1K B1A B2K B2A Dextran sulphate Control
10 10 10 10 100 mg mlÿ1 ÿ
4997822524 5085624510 4815725686 5155824518 4645624046 5479623339
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated2 standard deviation of six replicates. At 24 h, 1 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 7 Ci per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.5% late stages.
cycles of rbc invasion did not demonstrate speci®c antisense eects (Table 3). Dextran sulphate at 100 mg mlÿ1 completely inhibited parasite multiplication over 6 d (Table 3) as did 10 mM anti-sense and sense ODN-S (data not shown). B2R, an ODN-S with the same base composition and length as the B2K anti-sense ODN-S, and poly (C)18 produced a similar concentration dependent inhibition of P. falciparum growth as
Table 3 Eect of 6 d exposure to ODN-S and dextran sulphate on P. falciparum growth Inhibitor
Concentration Parasitaemia (mM) at day 6 (%)
B1K B1A B1K B1A B2K B2A Dextran Dextran Dextran Control
0.1 0.1 1.0 1.0 1.0 1.0 100 mg mlÿ1 10 mg mlÿ1 1 mg mlÿ1 ÿ
7.0 7.0 5.0 5.1 5.8 5.3 0.0 4.7 4.9 7.8
Inhibition (%) 10 10 36 35 26 32 100 40 37 0
The starting parasitaemia in the cultures was 0.05% at day 0.
the sense and anti-sense ODN-S (Table 4). The decrease in the numbers of ring stage parasites at 24 h with addition of ODN-S con®rms that the inhibition of [3 H]-hypoxanthine incorporation was due to reduced reinvasion of rbc by merozoites. The data also do not show evidence for a stimulation of [3 H]-hypoxanthine incorporation in malaria parasites by added ODNs that was reported by one group [3]. The inhibition of reinvasion by ODN-S was prevented when the ODN-S was complexed to cationic liposomes composed of DOTAP before addition to the cultures (Table 5). Adding 13 mg mlÿ1 DOTAP to 1 mM ODN-S (approximately 6 mg mlÿ1 ODN-S) completely abolished the partial inhibition of reinvasion at this concentration produced with either sense or antisense ODN-S alone. Similarly an approximately 2:1 wt/wt addition of DOTAP to 10 mM ODN-S (133 mg mlÿ1 DOTAP to 60 mg mlÿ1 ODN-S) completely abolished the very marked inhibition normally seen with 10 mM ODN-S alone. However, a 1:1 wt/wt addition of DOTAP to 10 mM ODN-S (60 mg mlÿ1 DOTAP to 60 mg mlÿ1 ODN-S) only partly reversed the ODN-S mediated inhibition of reinvasion. Speci®c antisense eects of ODN-S could also not be seen in the presence of DOTAP. Additional experiments (data not presented) showed that, (1) no inhibition of [3 H]-hypoxanthine incorporation was seen when 0.5 mM sense or antisense ODN-S complexed to DOTAP liposomes at 15 mg mlÿ1 were added to parasite cultures at 24 h, after merozoite invasion had occurred, and (2) DOTAP added alone at 0 h to cultures at 13 or 133 mg mlÿ1, did not stimulate or inhibit reinvasion or [3 H]-hypoxanthine incorporation into parasites. 4. Discussion Antisense ODNs based on MSA-1 and DHFR-TS were reported to speci®cally inhibit P. falciparum growth compared to control ODNs [2]. However it was noted in these experiments that schizont to ring transition i.e. reinvasion was more inhibited (IC50 0.5±1.1 mM) than ring to schizont maturation (IC50>2.5 mM). Our results
R. Kanagaratnam et al. / The International Journal of Biochemistry & Cell Biology 30 (1998) 979±985
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Table 4 Eects of control ODN-S added at 0 h to late stages of P. falciparum
ODN-S
Concentration
Parasitaemia at 24 h rings (%)
B2R poly C B2R poly C B2R poly C Nil
0.1 0.1 1.0 1.0 10 10 ÿ
[3 H] hypoxanthine incorporation at 24±48 h (mean cpm2sd) total (%)
1.6 1.5 1.4 1.5 0.2 0.5 1.4
1.7 1.7 1.5 1.6 0.3 0.7 1.4
678 253 628 231 551 267 426 227 55208 50215 692 295
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviations of ®ve replicates. At 24 h, 0.25 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 4 mCi per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.4% late stages.
with MSA-2 speci®c ODNs are clearly dierent. Dierent experimental conditions, e.g. our parasite cultures are probably more synchronous with regard to the dierent stages, may account for some of the variation. The present results with
MSA-2 ODN-S, B2R and poly (C)18 con®rm observations with DHFR-TS ODN-S [5] that merozoite invasion is inhibited in a sequenceindependent manner and that there is no signi®cant anti-sense eect on maturation of rings to
Table 5 Eects of adding ODN-S complexed to DOTAP at 0 h to late stages of P. falciparum
ODN-S (Conc)
DOTAP (Conc)
Parasitaemia at 24 h rings (%)
B2K (1 mM) B2A (1 mM) B2K (1 mM) B2A (1 mM) B2K (10 mM) B2A (10 mM) B2K (10 mM) B2A (10 mM) B2K (10 mM) B2A (10 mM) ÿ
0 0 13 (mg mlÿ1) 13 (mg mlÿ1) 0 0 60 (mg mlÿ1) 60 (mg mlÿ1) 133 (mg mlÿ1) 133 (mg mlÿ1) ÿ
[3H]hypoxanthine incorporation at 24±48 h (mean cpm2sd)
2.2 2.3 3.5 3.0 0.5 0.5 2.3 1.9 3.1 3.0 3.1
total (%) 2.4 2.5 3.7 3.3 0.9 1.0 2.5 2.2 3.3 3.1 3.3
871294 824269 1049282 1073280 369217 224229 950279 808278 1095252 1012288 9662116
The results of the [3 H] hypoxanthine assay are the mean cpm incorporated 2standard deviation of four replicates. At 24 h, 0.5 mCi of [3 H] hypoxanthine was added in 100 ml RPMI 1640 to the cultures to give a speci®c activity of approximately 8 mCi per mmol [3 H] hypoxanthine in the medium. The starting parasitaemia in the cultures was 0.6% late stages. Reversal by DOTAP at any concentration of the corresponding growth inhibition due to ODN-S alone was statistically signi®cant ( p < 0.05) by the Student's t test.
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schizonts at ODN-S concentrations ranging from 0.1 to 10 mM. Furthermore, no signi®cant antisense eects were seen with MSA-2 ODN-S (and with DHFR-TS ODN-S, our unpublished observations) over three invasion cycles. Comparable inhibition of merozoite invasion is seen with a polyanion, dextran sulphate, at similar concentrations to the ODN-S (10 mM B1 or B2 is 060 mg mlÿ1). Heparin, a sulphated glycosaminoglycan also inhibits P. falciparum merozoite invasion [11]. Dextran sulphate and fucoidin reportedly inhibit invasion of rhesus rbc by the simian malaria parasite P. knowlesi, by binding to a 135 kDa parasite protein that functions as a ligand for Duy blood group receptors on the rbc [12]. We postulate therefore that ODNs may inhibit P. falciparum reinvasion due to their polyanionic character, possibly by blocking the recognition of human rbc sialic acids by the corresponding ligands on P. falciparum merozoites. The abrogation of the ODN-S mediated invasion inhibition when the polyanionic character of the ODN-S is neutralised by the cationic lipid DOTAP further supports this hypothesis. For ODNs to enter intracellular malaria parasites, transport across three membranes viz. the rbc membrane, the parasitophorous vacuole membrane and the parasite plasma membrane is needed. Rbc unlike nucleated cells are unable to internalise ODNs by endocytosis. However, a parasitophorous duct reportedly present in P. falciparum infected rbc [13] could enable ODNs to gain access to the parasite plasma membrane where endocytosis might occur. There is also evidence for transport of lipids from the rbc membrane to intracellular P. falciparum in vesicles [14]. While DOTAP-nucleic acid complexes are routinely used for transfecting cultured nucleated cells, ODN-S complexed with DOTAP liposomes are unable to exert a speci®c antisense eect in P. falciparum. Hence our results suggest that the intracellular location of the parasite does not readily permit anti-sense therapy for malaria. On the other hand, clinically acceptable polyanions, may prove valuable for the treatment of complicated or drug resistant malaria.
Acknowledgements This investigation was supported by the State Committee for Scienti®c Research, Poland (Research grant 4POSF.023.10) and the Natural Resources, Energy and Science Authority, Sri Lanka (95/BT/03).
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