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RNA interference (RNAi) inhibits growth of Plasmodium falciparum
Molecular & Biochemical Parasitology 119 (2002) 273– 278 www.parasitology-online.com
RNA interference (RNAi) inhibits growth of Plasmodium falciparum Louisa McRobert 1, Glenn A. McConkey * School of Biology, Uni6ersity of Leeds, Leeds LS2 9JT, UK Received 14 August 2001; accepted in revised form 2 November 2001
Abstract RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P B0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P B 0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets. © 2002 Published by Elsevier Science B.V. Keywords: Plasmodium falciparum; Protozoa; Genetic interference; Dihydroorotate; Chorismate; Shikimate
1. Introduction With the continuing mortality of malaria and increasing resistance to anti-malarial drugs, new methods of control are urgently required. Data from the Plasmodium falciparum genome project provide the opportunity to discover new targets for intervention [1,2]. Novel approaches are needed to investigate the function of identified genes including those encoding known proteins and novel genes. For example, the functions of the novel chloroplast-like organelle, found to be essential for growth of the parasite, could be investigated as a potential area for chemotherapeutic attack [3 – 5]. Abbre6iations: RNAi, RNA interference; RT-PCR, reverse transcriptase polymerase chain reaction; PfDHODH, P. falciparum dihydroorotate dehydrogenase; PfAroC, P. falciparum chorismate synthase; TgDHFR, Toxoplasma gondii dihydrofolate reductasethymidylate synthase; PfCSP, P. falciparum circumsporozoite protein. * Corresponding author. Tel.: + 44-113-233-2908; fax: + 44-113233-2835. E-mail address: [email protected] (G.A. McConkey). 1 Present address: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
RNA interference (RNAi) is a method of interrupting gene expression as described in several recent reviews [6–8] and has been developed for functional genomic screening [9,10]. RNAi acts as a post-transcriptional event specifically degrading targeted mRNA that results in decreased synthesis of specific proteins. Hence, RNAi allows the roles of specific proteins to be analysed. The interference appears to be catalytic, requiring only a few molecules per cell for an effect [6]. It has been used to study gene function in a diverse range of organisms including trypanosomes and other invertebrates, Caenorhabditus elegans, Drosophila melanogaster, fungi, plants, and, to a limited extent, vertebrates [6–8,11 –14], but has yet to be applied to intracellular organisms such as malaria parasites. In this study, RNAi is applied to an enzyme in pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH). Malaria parasites, in contrast to most organisms, depend upon de novo synthesis of pyrimidines for growth [15]. Hence, disruption of DHODH expression would be predicted to interrupt parasite growth. RNAi is also applied to test the necessity for chorismate synthase (CS) in growth of malaria parasites. CS
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is involved in synthesis of chorismate, a precursor in folate biosynthesis, via the shikimate pathway. The gene encoding CS has recently been identified in P. falciparum [16] and biochemical evidence supports the presence of the shikimate pathway in Plasmodium [17].
2. Materials and methods
2.1. Cells All experiments used P. falciparum (strain 3D7) maintained as previously described, in RPMI-aro medium (deficient in the aromatic amino acids phenylalanine, tryptophan and tyrosine) containing 0.5% Albumax I (Life Technologies, Inc., Bethesda) [17].
2.2. RNA synthesis Segments of the P. falciparum dhodh (GenBank Accession number L15446) and aroC (GenBank Accession number AF008549) genes were amplified from P. falciparum genomic DNA using the following primers: 5%-GGGGGGATCCTAGATAAATGATCATATGTATCTGTACC-3% and 5%-GGGGGGATCCTTAATTAGTCAAACTGAAGCACCTGC-3%, and 5%-CCCCGGATCCTTAAACATTAGGTACACCTATTAC-3% and 5%CCCCGGATCCCCTAATTATTTAGAATCGTTGAATG-3%, respectively, following routine PCR protocols [18]. The PCR products (1.2 and 0.9 kb, respectively) were inserted into the plasmid pPCR-Script using the supplier’s kit (Stratagene, Inc., La Jolla). Control sequences Toxoplasma gondii dihydrofolate reductasethymidylate synthase (TgDHFR) and P. falciparum circumsporozoite protein (PfCSP) (from strain 3D7) (inserts 1.5 and 1.0 kb, respectively) were released from plasmids pDT.Tg23 [19] and pUC13 [unpublished] using NsiI/HindIII and EcoRI/HindIII, respectively, and inserted into the plasmid pBluescript II (Stratagene, Inc.). Purified plasmid DNA (5 mg) was linearised by two restriction enzymes, resulting in plasmid cleaved adjacent to each end of the sequence, providing templates for run-off transcription by T7 or T3 RNA polymerase. dsRNA was synthesised as described [11]. The quality of the dsRNA product was checked immediately prior to the experiment by gel electrophoresis.
2.3. dsRNA treatment dsRNA (1–2 mg) was added to synchronised parasites. For synchronisation, cultures were treated with 5% sorbitol [20] and cultured for 42– 44 h (yielding \ 95% ring-stage trophozoites). For experiments with late-stage trophozoites, the synchronised parasites were cultivated for a further 24 h. dsRNA was added to parasitised red blood cells (1.6×108) in incomplete
cytomix (without glutathione and ATP) and subjected to electroporation with a current of 200 V, 25 mF and 6.25 kV cm − 1 [21]. The contents of each cuvette were diluted with RPMI -aro medium containing red blood cells (5% hematocrit) and aliquoted to a 96-well plate to a final volume of 200 ml per well. Each sample was tested in triplicate with media changed every 24 h. Parasite growth was monitored by measuring incorporation of radiolabeled hypoxanthine (Amersham Life Sciences, UK) as described [22]. Plates were harvested at 24 h intervals.
2.4. Measurement of mRNA le6els The level of endogenous P. falciparum PfDHODH mRNA was measured by relative quantitative RTPCR: following dsRNA treatment as above, total RNA was extracted from 3 ml cultures with guanidinium isothiocyanate using the RNagents Total RNA Isolation kit (ProMega, Inc., Madison). RNA was treated with DNase I (Life Technologies, Inc.) and was used as a template for cDNA synthesis using random hexamers with the Superscript Preamplification System for First Strand cDNA Synthesis kit (Life Technologies, Inc.). A previously described protocol for measuring relative levels of mRNA in P. falciparum-infected erythrocytes by RT-PCR was used [3] except the primers for detection of PfDHODH were 5%-ATGGTATGAAATCAGCTGTAC-3% and 5%-ATTACATTTAAGCCCCAAAAC-3% which map near the 3%end of the gene not included in the fragment used for dsRNA treatment. The RT-PCR products were separated on a 1% agarose gel and the levels were quantitated by measuring their relative intensities using the BioRad GelDoc system.
3. Results
3.1. RNAi with PfDHODH inhibits P. falciparum growth An enzyme involved in pyrimidine biosynthesis, DHODH, was chosen as a target for RNAi because it appears to be essential for parasite growth. This enzyme converts dihydroorotate to orotate [23,24]. Substrate analogues and atovaquone inhibit DHODH activity in vitro and inhibit parasite growth, although these compounds also have other effects on parasite metabolism [25]. The inhibitory effect by dsRNA was measured by electroporating synchronised P. falciparum ring-stage trophozoite-infected erythrocytes in the presence of dsRNA encoding fragments of the dhodh gene. PfDHODH dsRNA significantly inhibited growth relative to treatment with water (60.3%, PB 0.0005; Fig. 1a). Inhibition of growth was detectable after only 24 h of
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treatment and continued through 72 h following treatment. Hence, the effect on growth is observable before the completion of one asexual cycle of growth in P. falciparum. Similar results were found in repeating the assay at least four times. To examine the specificity, sequences encoding T. gondii dihydrofolate reductase-thymidylate synthase (TgDHFR) and P. falciparum circumsporozoite protein (PfCSP) were used as negative controls. Although P. falciparum encodes dihydrofolate reductase, the P. falciparum and T. gondii nucleotide sequences only share 30% similarity and there are no stretches of conserved sequences greater than nine nucleotides long. Hence, the T. gondii gene is appropriate for testing the specificity of inhibition. The circumsporozoite protein is the
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major surface coat protein of the parasite’s sporozoite stage and has been shown by gene inactivation (in rodent malaria parasites) not to be required during blood stages [26]. There were no inhibitory effects by dsRNAs encoding TgDHFR or PfCSP (Fig. 1a, P\ 0.05). RNA interference experiments in other organisms found that the RNA is required to be double-stranded as single strands alone did not induce any effect [11,13,14]. Hence, parallel experiments were performed with P. falciparum. Neither the sense nor the antisense RNA strands alone inhibited parasite growth (Fig. 1b). There was a minor inhibition of growth by treatment with the sense strand of PfDHODH (139 7.5%) but the significance of this is unknown.
Fig. 1. Inhibition of P. falciparum growth by dsRNA. (a) The growth of synchronised ring-stage trophozoites was measured via radiolabeled hypoxanthine at 24 , 48 and 72 h following electroporation with dsRNA encoding PfDHODH, PfAroC, TgDHFR and PfCSP. Growth is plotted as a percentage of control experiments treated without dsRNA. Values are the mean of triplicate experiments with standard error noted. (b) Double-stranded , sense , and antisense RNA encoding PfDHODH and PfAroC was used to treat parasites. Growth was measured 72 h after electroporation. (C) Growth of synchronised ring-stage and late-stage trophozoites incubated with dsRNA encoding PfDHODH, PfAroC, and TgDHFR is compared with growth of ring-stage trophozoites electroporated with the same sequences, measured at 72 h post-treatment.
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Fig. 2. PfDHODH mRNA levels in samples from dsRNA-treated parasites Semi-quantitative RT-PCR of P. falciparum treated with dsRNA encoding a fragment of PfDHODH versus control cultures. The products from amplification with ribosomal RNA primers (lanes 2 –4) or PfDHODH primers (lanes 5 –7) of material from parasites treated with dsRNA encoding a fragment of PfDHODH (lanes 2 and 5) or controls (lanes 3 and 6) resolved on an ethidium bromide-stained agarose gel are shown. Primers for PCR of PfDHODH map outside the region of the gene targeted by the dsRNA. Genomic DNA served as a positive control for PCR (lanes 4, 7). The marker (lane 1) is a 100 bp DNA ladder.
In the experiments described above, infected erythrocytes were electroporated in the presence of dsRNA. The requirement for electroporation as a delivery method for the system was tested. Synchronised ringstage and late-stage trophozoite-infected red cells were incubated with dsRNA encoding PfDHODH, PfAroC or TgDHFR and the effect on growth was measured. Both stages were tested to ensure that during the time frame of the experiment, the extracellular merozoite stages released from the red cells would be exposed to dsRNA. There was no detectable effect on parasite growth by any of the dsRNAs (Fig. 1c, P \ 0.1) without electroporation. Hence, passive transfer of dsRNA does not promote RNAi in P. falciparum, even during the extracellular stages. There was also no non-specific inhibition of merozoites as has been observed with antisense oligonucleotides [27].
3.2. Correlation of growth with mRNA le6el for RNAi The effect of dsRNA treatment of parasites on endogenous mRNA was assessed. Specific degradation of homologous mRNA targeted by the dsRNA is the hallmark of RNAi [6 – 8]. Due to the small amounts of material recoverable from the electroporated parasites, relative quantitative RT-PCR was used to assess mRNA levels, following the procedure described in earlier studies [3]. The primers used amplify a region of the PfDHODH mRNA outside the dsRNA sequence. Reactions were standardised with rRNA for comparison of equal amounts of RNA. PfDHODH mRNA levels in parasites treated with dsRNA encoding a
fragment of PfDHODH are 46% of control cultures at 24 h (Fig. 2). The reduction of PfDHODH mRNA correlates well with the observed inhibition of growth (compare Fig. 1a and Fig. 2).
3.3. Effect of targeting chorismate synthase with dsRNA RNAi in P. falciparum will be useful for determining whether gene products are required for parasite growth; valuable information for selecting new targets for chemotherapy. Part of the sequence of the aroC gene encoding chorismate synthase (CS) was selected for interruption. CS is involved in folate synthesis in other organisms, catalysing chorismate synthesis from 5enolpyruvylshikimate phosphate (in the common shikimate pathway) which is further metabolised in parasites to produce the essential cofactor folate [16,17,22]. Shikimate analogues and the herbicide glyphosate (which disrupt the shikimate pathway in other organisms) inhibit P. falciparum growth, an effect that can be abrogated with exogenous folates. Here, P. falciparum were treated with dsRNA encoding a 900 bp fragment of aroC as described above and growth was monitored. Parasite growth was reduced (44%, PB 0.005; Fig. 1a) relative to water-treated controls in repeated experiments. Hence, the product of aroC, namely CS is required for normal growth of P. falciparum, suggesting that it may be a viable target for chemotherapy. Experiments are on-going to abrogate the inhibition with folate as in previous experiments with inhibitors [17].
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4. Discussion We have used genetic interference via dsRNA to study the human malaria parasite, P. falciparum. This work represents the first application of RNAi to the study of intracellular organisms and presents a new approach for the study of gene function in P. falciparum, complementing current methods of gene inactivation, namely ‘knockout’ technology, and the use of antisense oligonucleotides and vectors [19,28,29]. There are numerous advantages of RNAi for studying gene function in P. falciparum: the assay can be conducted in a few days compared with months required for gene inactivation, multiple genes can be analysed simultaneously for genome screening purposes, the cost is considerably less than the synthesis of modified antisense oligonucleotides, and the transient nature of the assay may be an advantage for investigating essential genes. Disadvantages with the current methodology include the dependence on the electroporation efficiency and the lack of a marker phenotype following manipulation of this organism. Both areas of optimisation are currently under investigation. The importance of electroporation in RNAi-dependent inhibition of these intracellular parasites has been shown. Electroporation has been used for delivery of dsRNA into other invertebrates [7,14]. The requirement for electroporation in P. falciparum may be due to the necessity for the dsRNA to permeate three barriers to reach the cytoplasmic location of mRNA: the red blood cell surface membrane, the parasitophorous vacuole (in which the parasites reside) membrane and the P. falciparum surface membrane. The parasite-induced tubovesicular network of membranes (TVM) that allows nutrients to pass through the red cell to the parasitophorous vacuole may provide a means for the dsRNA to reach the parasite membrane although only small solutes (e.g. amino acids and nucleosides) have been shown to utilise the TVM [30]. The conditions used to transform large DNA plasmids (greater than 5000 kDa plasmids vs. 600 kDa dsRNA) into the nucleus were adopted in these initial studies, but optimisation of conditions for delivery of dsRNA into the P. falciparum cytoplasm may increase effectiveness of delivery of dsRNA and survival of parasites [21]. Indeed, it may be possible that parasites may take up dsRNA spontaneously from the cytoplasm of pre-electroporated uninfected erythrocytes, as has recently been demonstrated with plasmids [31]. The inhibition with dsRNA is comparable to inhibition levels observed with modified antisense oligonucleotide treatment of P. falciparum, in which a reduction in growth ranging from 50 to 90% was observed [28]. The lack of inhibition by the antisense RNA alone in our experiments may be due to low levels of antisense RNA in the parasite and the catalytic action of
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dsRNA. This contrasts with observations with antisense RNA produced from a vector transfected into P. falciparum [29]. Their observed inhibition of clag 9 may be due to the high levels of antisense RNA synthesised in the parasites or possibly from aberrant expression of both RNA strands as observed in trypanosomes [14]. RNAi may be useful for identifying targets for malaria chemotherapy. The growth inhibition data exhibited in this study support previous studies on the role of pyrimidine biosynthesis in P. falciparum, further validating DHODH as a chemotherapeutic target. A new finding from these studies is that the product of aroC, CS, is required for normal growth suggesting it may also serve as a novel target. The application of dsRNA for interrupting gene expression in Plasmodium will be useful for elucidating gene function as a step towards vaccine and antimalarial chemotherapy development. Whether the mechanism for the degradation of the targeted endogenous mRNA in P. falciparum is as described for RNAi in other organisms, will be the subject of future studies. The specificity observed suggests the possible application for therapy and functional genomic screening. The recent application of RNAi to human cells opens the avenue for the potential use of dsRNA as an antimalarial agent [32].
Acknowledgements We would like to thank R.E. Isaac and I.A. Hope, and members of their laboratories, for helpful advice and comments, and M.C. Taylor for critical reading of the manuscript. This work was sponsored by a B.B.S.R.C. studentship (L. McRobert) and a World Health Organisation Tropical Diseases Research Grant (c 970083).
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[8] Bass BL. Double-stranded RNA as a template for gene silencing. Cell 2000;101:235 – 8. [9] Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M, Sohrmann M, Ahringer J. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature 2000;408:325 – 30. [10] Gonczy P, Echeverri G, Oegema K, et al. Functional genomic analysis of cell division in C. elegans using RNAi of genes on chromosome II. Nature 2000;408:331 –6. [11] Fire A, Xu SQ, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806 –11. [12] Misquitta L, Paterson BM. Targeted disruption of gene function in Drosophila by RNA interference (RNA-i): a role for nautilus in embryonic somatic muscle formation. Proc Natl Acad Sci USA 1999;96:1451 – 6. [13] Wargelius A, Ellingson S, Fjose A. Double-stranded RNA induces specific developmental defects in zebrafish embryos. Biochem Biophys Res Comms 1999;263:156 – 61. [14] Ngoˆ H, Tschudi C, Gull K, Ullu E. Double-stranded RNA induces mRNA degradation in Trypanosoma brucei. Proc Natl Acad Sci USA 1998;95:14687 –92. [15] Sherman IW. The biochemistry of malaria: an overview. In: Coombs GH, North MJ, editors. Biochemical protozoology, vol. 2. London: Taylor & Francis, 1991:6 –34. [16] Roberts F, Roberts CW, Johnson JJ, et al. Evidence for the shikimate pathway in apicomplexan parasites. Nature 1998;393:801 – 5. [17] McConkey GA. Targeting the shikimate pathway in the malaria parasite Plasmodium falciparum. Antimicrob Agents Chemother 1999;43:175 –7. [18] Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, second ed. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. [19] Wu Y, Kirkman LA, Wellems TE. Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine. Proc Natl Acad Sci USA 1996;93:1130 –4. [20] Lambros C, Vanderberg SP. Synchronisation of Plasmodium falciparum erythrocytic stages in cultures. J Parasitol 1979;65:418 –20.
[21] Wu Y, Sifri CD, Lei H-H, Su X-Z, Wellems TE. Transfection of Plasmodium falciparum within human red blood cells. Proc Natl Acad Sci USA 1995;92:973 – 7. [22] McConkey GA, Ittarat I, Meshnick SR, McCutchan TF. Auxotrophs of Plasmodium falciparum dependent on p-aminobenzoic acid for growth. Proc Natl Acad Sci USA 1994;91:4244 –8. [23] Gero AM, Brown GV, O’Sullivan WJ. Pyrimidine de novo synthesis during the life cycle of the intraerythrocytic stage of Plasmodium falciparum. J Parasitol 1984;70:536 – 41. [24] LeBlanc SB, Wilson CM. The dihydroorotate dehydrogenase gene homologue of Plasmodium falciparum. Mol Biochem Parasitol 1993;60:349 – 51. [25] Ridley RG, Hudson AT. Chemotherapy of malaria. Curr Opin Infect Dis 1998;11:691 – 705. [26] Me´ nard R, Sultan AA, Cortes C, et al. Circumsporozoite protein is required for development of malaria sporozoites in mosquitoes. Nature 1997;385:336 – 40. [27] Ramasamy R, Kanagaratnam R, Misiura K, Rebowski G, Amerakoon R, Stec WJ. Anti-sense oligodeoxynucleoside phosphorothioates nonspecifically inhibit invasion of red blood cells by malaria parasites. Biochem Biophys Res Comms 1996;218:930 – 3. [28] Barker RH Jr, Metelev V, Zamecnik P. Plasmodium falciparum: effect of chemical structure and specificity of antisense oligonucleotides against malaria in vitro. Exp Parasitol 1998;88:51 – 9. [29] Gardiner DL, Holt DC, Thomas EA, Kemp DJ, Trenholme KR. Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA. Mol Biochem Parasitol 2000;110:33 – 41. [30] Lauer SA, Rathod PK, Ghori N, Haldar K. A membrane network for nutrient import in red cells infected with the malaria parasite. Science 1997;276:1122 – 5. [31] Deitsch KW, Driskill CL, Wellems TE. Transformation of malaria parasites by the spontaneous uptake and expression of DNA from human erythrocytes. Nucleic Acids Res 2001;29:850 – 3. [32] Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:494 –8.
RNA interference (RNAi) inhibits growth of Plasmodium falciparum Louisa McRobert 1, Glenn A. McConkey * School of Biology, Uni6ersity of Leeds, Leeds LS2 9JT, UK Received 14 August 2001; accepted in revised form 2 November 2001
Abstract RNA interference (RNAi) causes degradation of targeted endogenous RNA in many diverse organisms. Erythrocyte-infecting stages of the malaria parasite Plasmodium falciparum were treated with double-stranded RNA (dsRNA) encoding a segment of the gene encoding dihydroorotate dehydrogenase (DHODH). DHODH is an enzyme in pyrimidine biosynthesis, essential for parasite growth. A decrease in parasite growth (P B0.0005) correlated with a decrease in levels of DHODH mRNA. Control treatments with single-stranded RNA, dsRNA encoding the circumsporozoite protein (a stage-specific protein not expressed in the asexual blood stage) and dsRNA encoding a gene from the related organism Toxoplasma gondii did not inhibit growth. As a test for the RNAi assay, parasites were treated with dsRNA encoding chorismate synthase (CS), an enzyme thought to be involved in folate synthesis, to examine the requirement for this enzyme for parasite growth. Growth decreased (P B 0.001) though less markedly than by dsRNA encoding DHODH. These results demonstrate the utility of this assay in assessing requirements for gene products, and their potential as chemotherapeutic targets. © 2002 Published by Elsevier Science B.V. Keywords: Plasmodium falciparum; Protozoa; Genetic interference; Dihydroorotate; Chorismate; Shikimate
1. Introduction With the continuing mortality of malaria and increasing resistance to anti-malarial drugs, new methods of control are urgently required. Data from the Plasmodium falciparum genome project provide the opportunity to discover new targets for intervention [1,2]. Novel approaches are needed to investigate the function of identified genes including those encoding known proteins and novel genes. For example, the functions of the novel chloroplast-like organelle, found to be essential for growth of the parasite, could be investigated as a potential area for chemotherapeutic attack [3 – 5]. Abbre6iations: RNAi, RNA interference; RT-PCR, reverse transcriptase polymerase chain reaction; PfDHODH, P. falciparum dihydroorotate dehydrogenase; PfAroC, P. falciparum chorismate synthase; TgDHFR, Toxoplasma gondii dihydrofolate reductasethymidylate synthase; PfCSP, P. falciparum circumsporozoite protein. * Corresponding author. Tel.: + 44-113-233-2908; fax: + 44-113233-2835. E-mail address: [email protected] (G.A. McConkey). 1 Present address: Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
RNA interference (RNAi) is a method of interrupting gene expression as described in several recent reviews [6–8] and has been developed for functional genomic screening [9,10]. RNAi acts as a post-transcriptional event specifically degrading targeted mRNA that results in decreased synthesis of specific proteins. Hence, RNAi allows the roles of specific proteins to be analysed. The interference appears to be catalytic, requiring only a few molecules per cell for an effect [6]. It has been used to study gene function in a diverse range of organisms including trypanosomes and other invertebrates, Caenorhabditus elegans, Drosophila melanogaster, fungi, plants, and, to a limited extent, vertebrates [6–8,11 –14], but has yet to be applied to intracellular organisms such as malaria parasites. In this study, RNAi is applied to an enzyme in pyrimidine biosynthesis, dihydroorotate dehydrogenase (DHODH). Malaria parasites, in contrast to most organisms, depend upon de novo synthesis of pyrimidines for growth [15]. Hence, disruption of DHODH expression would be predicted to interrupt parasite growth. RNAi is also applied to test the necessity for chorismate synthase (CS) in growth of malaria parasites. CS
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is involved in synthesis of chorismate, a precursor in folate biosynthesis, via the shikimate pathway. The gene encoding CS has recently been identified in P. falciparum [16] and biochemical evidence supports the presence of the shikimate pathway in Plasmodium [17].
2. Materials and methods
2.1. Cells All experiments used P. falciparum (strain 3D7) maintained as previously described, in RPMI-aro medium (deficient in the aromatic amino acids phenylalanine, tryptophan and tyrosine) containing 0.5% Albumax I (Life Technologies, Inc., Bethesda) [17].
2.2. RNA synthesis Segments of the P. falciparum dhodh (GenBank Accession number L15446) and aroC (GenBank Accession number AF008549) genes were amplified from P. falciparum genomic DNA using the following primers: 5%-GGGGGGATCCTAGATAAATGATCATATGTATCTGTACC-3% and 5%-GGGGGGATCCTTAATTAGTCAAACTGAAGCACCTGC-3%, and 5%-CCCCGGATCCTTAAACATTAGGTACACCTATTAC-3% and 5%CCCCGGATCCCCTAATTATTTAGAATCGTTGAATG-3%, respectively, following routine PCR protocols [18]. The PCR products (1.2 and 0.9 kb, respectively) were inserted into the plasmid pPCR-Script using the supplier’s kit (Stratagene, Inc., La Jolla). Control sequences Toxoplasma gondii dihydrofolate reductasethymidylate synthase (TgDHFR) and P. falciparum circumsporozoite protein (PfCSP) (from strain 3D7) (inserts 1.5 and 1.0 kb, respectively) were released from plasmids pDT.Tg23 [19] and pUC13 [unpublished] using NsiI/HindIII and EcoRI/HindIII, respectively, and inserted into the plasmid pBluescript II (Stratagene, Inc.). Purified plasmid DNA (5 mg) was linearised by two restriction enzymes, resulting in plasmid cleaved adjacent to each end of the sequence, providing templates for run-off transcription by T7 or T3 RNA polymerase. dsRNA was synthesised as described [11]. The quality of the dsRNA product was checked immediately prior to the experiment by gel electrophoresis.
2.3. dsRNA treatment dsRNA (1–2 mg) was added to synchronised parasites. For synchronisation, cultures were treated with 5% sorbitol [20] and cultured for 42– 44 h (yielding \ 95% ring-stage trophozoites). For experiments with late-stage trophozoites, the synchronised parasites were cultivated for a further 24 h. dsRNA was added to parasitised red blood cells (1.6×108) in incomplete
cytomix (without glutathione and ATP) and subjected to electroporation with a current of 200 V, 25 mF and 6.25 kV cm − 1 [21]. The contents of each cuvette were diluted with RPMI -aro medium containing red blood cells (5% hematocrit) and aliquoted to a 96-well plate to a final volume of 200 ml per well. Each sample was tested in triplicate with media changed every 24 h. Parasite growth was monitored by measuring incorporation of radiolabeled hypoxanthine (Amersham Life Sciences, UK) as described [22]. Plates were harvested at 24 h intervals.
2.4. Measurement of mRNA le6els The level of endogenous P. falciparum PfDHODH mRNA was measured by relative quantitative RTPCR: following dsRNA treatment as above, total RNA was extracted from 3 ml cultures with guanidinium isothiocyanate using the RNagents Total RNA Isolation kit (ProMega, Inc., Madison). RNA was treated with DNase I (Life Technologies, Inc.) and was used as a template for cDNA synthesis using random hexamers with the Superscript Preamplification System for First Strand cDNA Synthesis kit (Life Technologies, Inc.). A previously described protocol for measuring relative levels of mRNA in P. falciparum-infected erythrocytes by RT-PCR was used [3] except the primers for detection of PfDHODH were 5%-ATGGTATGAAATCAGCTGTAC-3% and 5%-ATTACATTTAAGCCCCAAAAC-3% which map near the 3%end of the gene not included in the fragment used for dsRNA treatment. The RT-PCR products were separated on a 1% agarose gel and the levels were quantitated by measuring their relative intensities using the BioRad GelDoc system.
3. Results
3.1. RNAi with PfDHODH inhibits P. falciparum growth An enzyme involved in pyrimidine biosynthesis, DHODH, was chosen as a target for RNAi because it appears to be essential for parasite growth. This enzyme converts dihydroorotate to orotate [23,24]. Substrate analogues and atovaquone inhibit DHODH activity in vitro and inhibit parasite growth, although these compounds also have other effects on parasite metabolism [25]. The inhibitory effect by dsRNA was measured by electroporating synchronised P. falciparum ring-stage trophozoite-infected erythrocytes in the presence of dsRNA encoding fragments of the dhodh gene. PfDHODH dsRNA significantly inhibited growth relative to treatment with water (60.3%, PB 0.0005; Fig. 1a). Inhibition of growth was detectable after only 24 h of
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treatment and continued through 72 h following treatment. Hence, the effect on growth is observable before the completion of one asexual cycle of growth in P. falciparum. Similar results were found in repeating the assay at least four times. To examine the specificity, sequences encoding T. gondii dihydrofolate reductase-thymidylate synthase (TgDHFR) and P. falciparum circumsporozoite protein (PfCSP) were used as negative controls. Although P. falciparum encodes dihydrofolate reductase, the P. falciparum and T. gondii nucleotide sequences only share 30% similarity and there are no stretches of conserved sequences greater than nine nucleotides long. Hence, the T. gondii gene is appropriate for testing the specificity of inhibition. The circumsporozoite protein is the
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major surface coat protein of the parasite’s sporozoite stage and has been shown by gene inactivation (in rodent malaria parasites) not to be required during blood stages [26]. There were no inhibitory effects by dsRNAs encoding TgDHFR or PfCSP (Fig. 1a, P\ 0.05). RNA interference experiments in other organisms found that the RNA is required to be double-stranded as single strands alone did not induce any effect [11,13,14]. Hence, parallel experiments were performed with P. falciparum. Neither the sense nor the antisense RNA strands alone inhibited parasite growth (Fig. 1b). There was a minor inhibition of growth by treatment with the sense strand of PfDHODH (139 7.5%) but the significance of this is unknown.
Fig. 1. Inhibition of P. falciparum growth by dsRNA. (a) The growth of synchronised ring-stage trophozoites was measured via radiolabeled hypoxanthine at 24 , 48 and 72 h following electroporation with dsRNA encoding PfDHODH, PfAroC, TgDHFR and PfCSP. Growth is plotted as a percentage of control experiments treated without dsRNA. Values are the mean of triplicate experiments with standard error noted. (b) Double-stranded , sense , and antisense RNA encoding PfDHODH and PfAroC was used to treat parasites. Growth was measured 72 h after electroporation. (C) Growth of synchronised ring-stage and late-stage trophozoites incubated with dsRNA encoding PfDHODH, PfAroC, and TgDHFR is compared with growth of ring-stage trophozoites electroporated with the same sequences, measured at 72 h post-treatment.
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Fig. 2. PfDHODH mRNA levels in samples from dsRNA-treated parasites Semi-quantitative RT-PCR of P. falciparum treated with dsRNA encoding a fragment of PfDHODH versus control cultures. The products from amplification with ribosomal RNA primers (lanes 2 –4) or PfDHODH primers (lanes 5 –7) of material from parasites treated with dsRNA encoding a fragment of PfDHODH (lanes 2 and 5) or controls (lanes 3 and 6) resolved on an ethidium bromide-stained agarose gel are shown. Primers for PCR of PfDHODH map outside the region of the gene targeted by the dsRNA. Genomic DNA served as a positive control for PCR (lanes 4, 7). The marker (lane 1) is a 100 bp DNA ladder.
In the experiments described above, infected erythrocytes were electroporated in the presence of dsRNA. The requirement for electroporation as a delivery method for the system was tested. Synchronised ringstage and late-stage trophozoite-infected red cells were incubated with dsRNA encoding PfDHODH, PfAroC or TgDHFR and the effect on growth was measured. Both stages were tested to ensure that during the time frame of the experiment, the extracellular merozoite stages released from the red cells would be exposed to dsRNA. There was no detectable effect on parasite growth by any of the dsRNAs (Fig. 1c, P \ 0.1) without electroporation. Hence, passive transfer of dsRNA does not promote RNAi in P. falciparum, even during the extracellular stages. There was also no non-specific inhibition of merozoites as has been observed with antisense oligonucleotides [27].
3.2. Correlation of growth with mRNA le6el for RNAi The effect of dsRNA treatment of parasites on endogenous mRNA was assessed. Specific degradation of homologous mRNA targeted by the dsRNA is the hallmark of RNAi [6 – 8]. Due to the small amounts of material recoverable from the electroporated parasites, relative quantitative RT-PCR was used to assess mRNA levels, following the procedure described in earlier studies [3]. The primers used amplify a region of the PfDHODH mRNA outside the dsRNA sequence. Reactions were standardised with rRNA for comparison of equal amounts of RNA. PfDHODH mRNA levels in parasites treated with dsRNA encoding a
fragment of PfDHODH are 46% of control cultures at 24 h (Fig. 2). The reduction of PfDHODH mRNA correlates well with the observed inhibition of growth (compare Fig. 1a and Fig. 2).
3.3. Effect of targeting chorismate synthase with dsRNA RNAi in P. falciparum will be useful for determining whether gene products are required for parasite growth; valuable information for selecting new targets for chemotherapy. Part of the sequence of the aroC gene encoding chorismate synthase (CS) was selected for interruption. CS is involved in folate synthesis in other organisms, catalysing chorismate synthesis from 5enolpyruvylshikimate phosphate (in the common shikimate pathway) which is further metabolised in parasites to produce the essential cofactor folate [16,17,22]. Shikimate analogues and the herbicide glyphosate (which disrupt the shikimate pathway in other organisms) inhibit P. falciparum growth, an effect that can be abrogated with exogenous folates. Here, P. falciparum were treated with dsRNA encoding a 900 bp fragment of aroC as described above and growth was monitored. Parasite growth was reduced (44%, PB 0.005; Fig. 1a) relative to water-treated controls in repeated experiments. Hence, the product of aroC, namely CS is required for normal growth of P. falciparum, suggesting that it may be a viable target for chemotherapy. Experiments are on-going to abrogate the inhibition with folate as in previous experiments with inhibitors [17].
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4. Discussion We have used genetic interference via dsRNA to study the human malaria parasite, P. falciparum. This work represents the first application of RNAi to the study of intracellular organisms and presents a new approach for the study of gene function in P. falciparum, complementing current methods of gene inactivation, namely ‘knockout’ technology, and the use of antisense oligonucleotides and vectors [19,28,29]. There are numerous advantages of RNAi for studying gene function in P. falciparum: the assay can be conducted in a few days compared with months required for gene inactivation, multiple genes can be analysed simultaneously for genome screening purposes, the cost is considerably less than the synthesis of modified antisense oligonucleotides, and the transient nature of the assay may be an advantage for investigating essential genes. Disadvantages with the current methodology include the dependence on the electroporation efficiency and the lack of a marker phenotype following manipulation of this organism. Both areas of optimisation are currently under investigation. The importance of electroporation in RNAi-dependent inhibition of these intracellular parasites has been shown. Electroporation has been used for delivery of dsRNA into other invertebrates [7,14]. The requirement for electroporation in P. falciparum may be due to the necessity for the dsRNA to permeate three barriers to reach the cytoplasmic location of mRNA: the red blood cell surface membrane, the parasitophorous vacuole (in which the parasites reside) membrane and the P. falciparum surface membrane. The parasite-induced tubovesicular network of membranes (TVM) that allows nutrients to pass through the red cell to the parasitophorous vacuole may provide a means for the dsRNA to reach the parasite membrane although only small solutes (e.g. amino acids and nucleosides) have been shown to utilise the TVM [30]. The conditions used to transform large DNA plasmids (greater than 5000 kDa plasmids vs. 600 kDa dsRNA) into the nucleus were adopted in these initial studies, but optimisation of conditions for delivery of dsRNA into the P. falciparum cytoplasm may increase effectiveness of delivery of dsRNA and survival of parasites [21]. Indeed, it may be possible that parasites may take up dsRNA spontaneously from the cytoplasm of pre-electroporated uninfected erythrocytes, as has recently been demonstrated with plasmids [31]. The inhibition with dsRNA is comparable to inhibition levels observed with modified antisense oligonucleotide treatment of P. falciparum, in which a reduction in growth ranging from 50 to 90% was observed [28]. The lack of inhibition by the antisense RNA alone in our experiments may be due to low levels of antisense RNA in the parasite and the catalytic action of
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dsRNA. This contrasts with observations with antisense RNA produced from a vector transfected into P. falciparum [29]. Their observed inhibition of clag 9 may be due to the high levels of antisense RNA synthesised in the parasites or possibly from aberrant expression of both RNA strands as observed in trypanosomes [14]. RNAi may be useful for identifying targets for malaria chemotherapy. The growth inhibition data exhibited in this study support previous studies on the role of pyrimidine biosynthesis in P. falciparum, further validating DHODH as a chemotherapeutic target. A new finding from these studies is that the product of aroC, CS, is required for normal growth suggesting it may also serve as a novel target. The application of dsRNA for interrupting gene expression in Plasmodium will be useful for elucidating gene function as a step towards vaccine and antimalarial chemotherapy development. Whether the mechanism for the degradation of the targeted endogenous mRNA in P. falciparum is as described for RNAi in other organisms, will be the subject of future studies. The specificity observed suggests the possible application for therapy and functional genomic screening. The recent application of RNAi to human cells opens the avenue for the potential use of dsRNA as an antimalarial agent [32].
Acknowledgements We would like to thank R.E. Isaac and I.A. Hope, and members of their laboratories, for helpful advice and comments, and M.C. Taylor for critical reading of the manuscript. This work was sponsored by a B.B.S.R.C. studentship (L. McRobert) and a World Health Organisation Tropical Diseases Research Grant (c 970083).
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