Robust inducible Cre recombinase activity in the human malaria parasite P lasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle

Abstract

Summary Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood‐stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to P lasmodium falciparum , the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site‐specific recombination in P . falciparum , capable of excising loxP ‐flanked sequences from a genomic locus with close to 100% efficiency within the time‐span of a single erythrocytic growth cycle. DiCre ‐mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P . falciparum , human dihydrofolate reductase, in the process producing constitutively DiCre ‐expressing P . falciparum clones that have broad utility for the functional analysis of essential asexual blood‐stage parasite genes.

Shown for reading. Its publisher allows quoting it, not republishing it.

References 54

1. 1.

   Agop-Nersesian C, Pfahler J, Lanzer M, Meissner M. Functional expression of ribozymes in Apicomplexa: towards exogenous control of gene expression by inducible RNA-cleavage. Int J Parasitol. 2008;38:673-681.

   Not in our library10.1016/j.ijpara.2007.10.015

2. 2.

   Andenmatten N, Egarter S, Jackson AJ, Jullien N, Herman JP, Meissner M. Conditional genome engineering in Toxoplasma gondii uncovers alternative invasion mechanisms. Nat Methods. 2013;10:125-127.

   Not in our library10.1038/nmeth.2301

3. 3.

   Arisue N, Kawai S, Hirai M, Palacpac NM, Jia M, Kaneko A, et al. Clues to evolution of the SERA multigene family in 18 Plasmodium species. PLoS ONE. 2011;6:e17775.

   Not in our library10.1371/journal.pone.0017775

4. 4.

   Armstrong CM, Goldberg DE. An FKBP destabilization domain modulates protein levels in Plasmodium falciparum. Nat Methods. 2007;4:1007-1009.

   Not in our library10.1038/nmeth1132

5. 5.

   Augagneur Y, Wesolowski D, Tae HS, Altman S, Ben Mamoun C. Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum. Proc Natl Acad Sci USA. 2012;109:6235-6240.

   Not in our library10.1073/pnas.1203516109

6. 6.

   Baum J, Papenfuss AT, Mair GR, Janse CJ, Vlachou D, Waters A, et al. Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites. Nucleic Acids Res. 2009;37:3788-3798.

   Not in our library10.1093/nar/gkp239

7. 7.

   Bayle JH, Grimley JS, Stankunas K, Gestwicki JE, Wandless TJ, Crabtree GR. Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity. Chem Biol. 2006;13:99-107.

   Not in our library10.1016/j.chembiol.2005.10.017

8. 8.

   Bell A, Wernli B, Franklin RM. Roles of peptidyl-prolyl cis-trans isomerase and calcineurin in the mechanisms of antimalarial action of cyclosporin A, FK506, and rapamycin. Biochem Pharmacol. 1994;48:495-503.

   Not in our library10.1016/0006-2952(94)90279-8

9. 9.

   Blackman MJ. Purification of Plasmodium falciparum merozoites for analysis of the processing of merozoite surface protein-1. Methods Cell Biol. 1994;45:213-220.

   Not in our library10.1016/s0091-679x(08)61853-1

10. 10.
    Malarial proteases and host cell egress: an ‘emerging’ cascade · 2008

    No notice
11. 11.

    Blackman MJ, Whittle H, Holder AA. Processing of the Plasmodium falciparum major merozoite surface protein-1: identification of a 33-kilodalton secondary processing product which is shed prior to erythrocyte invasion. Mol Biochem Parasitol. 1991;49:35-44.

    Not in our library10.1016/0166-6851(91)90128-s

12. 12.

    Brecht S, Erdhart H, Soete M, Soldati D. Genome engineering of Toxoplasma gondii using the site-specific recombinase Cre. Gene. 1999;234:239-247.

    Not in our library10.1016/s0378-1119(99)00202-4

13. 13.

    Chen J, Zheng XF, Brown EJ, Schreiber SL. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA. 1995;92:4947-4951.

    Not in our library10.1073/pnas.92.11.4947

14. 14.

    Child MA, Epp C, Bujard H, Blackman MJ. Regulated maturation of malaria merozoite surface protein-1 is essential for parasite growth. Mol Microbiol. 2010;78:187-202.

    Not in our library10.1111/j.1365-2958.2010.07324.x

15. 15.

    Choi J, Chen J, Schreiber SL, Clardy J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science. 1996;273:239-242.

    Not in our library10.1126/science.273.5272.239

16. 16.

    Clackson T, Yang W, Rozamus LW, Hatada M, Amara JF, Rollins CT, et al. Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc Natl Acad Sci USA. 1998;95:10437-10442.

    Not in our library10.1073/pnas.95.18.10437

17. 17.

    Combe A, Giovannini D, Carvalho TG, Spath S, Boisson B, Loussert C, et al. Clonal conditional mutagenesis in malaria parasites. Cell Host Microbe. 2009;5:386-396.

    Not in our library10.1016/j.chom.2009.03.008

18. 18.

    Delplace P, Dubremetz JF, Fortier B, Vernes A. A 50 kilodalton exoantigen specific to the merozoite release-reinvasion stage of Plasmodium falciparum. Mol Biochem Parasitol. 1985;17:239-251.

    Not in our library10.1016/0166-6851(85)90021-0

19. 19.

    Duraisingh MT, Triglia T, Cowman AF. Negative selection of Plasmodium falciparum reveals targeted gene deletion by double crossover recombination. Int J Parasitol. 2002;32:81-89.

    Not in our library10.1016/s0020-7519(01)00345-9

20. 20.

    Dvorin JD, Martyn DC, Patel SD, Grimley JS, Collins CR, Hopp CS, et al. A plant-like kinase in Plasmodium falciparum regulates parasite egress from erythrocytes. Science. 2010;328:910-912.

    Not in our library10.1126/science.1188191

21. 21.

    Ecker A, Lewis RE, Ekland EH, Jayabalasingham B, Fidock DA. Tricks in Plasmodium's molecular repertoire – escaping 3'UTR excision-based conditional silencing of the chloroquine resistance transporter gene. Int J Parasitol. 2012;42:969-974.

    Not in our library10.1016/j.ijpara.2012.09.003

22. 22.

    Falae A, Combe A, Amaladoss A, Carvalho T, Menard R, Bhanot P. Role of Plasmodium berghei cGMP-dependent protein kinase in late liver stage development. J Biol Chem. 2010;285:3282-3288.

    Not in our library10.1074/jbc.m109.070367

23. 23.

    Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature. 2002;419:498-511.

    Not in our library10.1038/nature01097

24. 24.

    Gilson PR, O'Donnell RA, Nebl T, Sanders PR, Wickham ME, McElwain TF, et al. MSP1(19) miniproteins can serve as targets for invasion inhibitory antibodies in Plasmodium falciparum provided they contain the correct domains for cell surface trafficking. Mol Microbiol. 2008;68:124-138.

    Not in our library10.1111/j.1365-2958.2008.06140.x

25. 25.

    Giovannini D, Spath S, Lacroix C, Perazzi A, Bargieri D, Lagal V, et al. Independent roles of apical membrane antigen 1 and rhoptry neck proteins during host cell invasion by apicomplexa. Cell Host Microbe. 2011;10:591-602.

    Not in our library10.1016/j.chom.2011.10.012

26. 26.

    Guo F, Gopaul DN, van Duyne GD. Structure of Cre recombinase complexed with DNA in a site-specific recombination synapse. Nature. 1997;389:40-46.

    Not in our library10.1038/37925

27. 27.
    Molecular Identification of a Malaria Merozoite Surface Sheddase · 2005

    No notice
28. 28.

    Herman J-P, Jullien N, Morozov A. Regulation of Cre recombinase: use of ligand-regulated and dimerizable Cre for transgenesis. Controlled Genetic Manipulations. 2012;65:1-28.

    Not in our library

29. 29.

    Jean L, Hackett F, Martin SR, Blackman MJ. Functional characterization of the propeptide of Plasmodium falciparum subtilisin-like protease-1. J Biol Chem. 2003;278:28572-28579.

    Not in our library10.1074/jbc.m303827200

30. 30.

    Jullien N, Sampieri F, Enjalbert A, Herman JP. Regulation of Cre recombinase by ligand-induced complementation of inactive fragments. Nucleic Acids Res. 2003;31:e131.

    Not in our library10.1093/nar/gng131

31. 31.

    Jullien N, Goddard I, Selmi-Ruby S, Fina JL, Cremer H, Herman J. Conditional transgenesis using Dimerizable Cre (DiCre). PLoS ONE. 2007;2:e1355.

    Not in our library10.1371/journal.pone.0001355

32. 32.

    Lacroix C, Giovannini D, Combe A, Bargieri DY, Spath S, Panchal D, et al. FLP/FRT-mediated conditional mutagenesis in pre-erythrocytic stages of Plasmodium berghei. Nat Protoc. 2011;6:1412-1428.

    Not in our library10.1038/nprot.2011.363

33. 33.

    Lasonder E, Ishihama Y, Andersen JS, Vermunt AM, Pain A, Sauerwein RW, et al. Analysis of the Plasmodium falciparum proteome by high-accuracy mass spectrometry. Nature. 2002;419:537-542.

    Not in our library10.1038/nature01111

34. 34.

    Le Roch KG, Johnson JR, Florens L, Zhou Y, Santrosyan A, Grainger M, et al. Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 2004;14:2308-2318.

    Not in our library10.1101/gr.2523904

35. 35.

    Liang J, Choi J, Clardy J. Refined structure of the FKBP12-rapamycin-FRB ternary complex at 2.2 A resolution. Acta Crystallogr D Biol Crystallogr. 1999;55:736-744.

    Not in our library10.1107/s0907444998014747

36. 36.

    Lin JW, Annoura T, Sajid M, Chevalley-Maurel S, Ramesar J, Klop O, et al. A novel ‘gene insertion/marker out’ (GIMO) method for transgene expression and gene complementation in rodent malaria parasites. PLoS ONE. 2011;6:e29289.

    Not in our library10.1371/journal.pone.0029289

37. 37.

    McCoubrie JE, Miller SK, Sargeant T, Good RT, Hodder AN, Speed T, et al. Evidence for a common role for the serine-type Plasmodium falciparum serine repeat antigen proteases: implications for vaccine and drug design. Infect Immun. 2007;75:5565-5574.

    Not in our library10.1128/iai.00405-07

38. 38.

    Meissner M, Brecht S, Bujard H, Soldati D. Modulation of myosin A expression by a newly established tetracycline repressor-based inducible system in Toxoplasma gondii. Nucleic Acids Res. 2001;29:E115.

    Not in our library10.1093/nar/29.22.e115

39. 39.

    Meissner M, Schluter D, Soldati D. Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science. 2002;298:837-840.

    Not in our library10.1126/science.1074553

40. 40.

    Meissner M, Krejany E, Gilson PR, de Koning-Ward TF, Soldati D, Crabb BS. Tetracycline analogue-regulated transgene expression in Plasmodium falciparum blood stages using Toxoplasma gondii transactivators. Proc Natl Acad Sci USA. 2005;102:2980-2985.

    Not in our library10.1073/pnas.0500112102

41. 41.

    Miller SK, Good RT, Drew DR, Delorenzi M, Sanders PR, Hodder AN, et al. A subset of Plasmodium falciparum SERA genes are expressed and appear to play an important role in the erythrocytic cycle. J Biol Chem. 2002;277:47524-47532.

    Not in our library10.1074/jbc.m206974200

42. 42.

    Monaghan P, Bell A. A Plasmodium falciparum FK506-binding protein (FKBP) with peptidyl-prolyl cis-trans isomerase and chaperone activities. Mol Biochem Parasitol. 2005;139:185-195.

    Not in our library10.1016/j.molbiopara.2004.10.007

43. 43.

    Moon RW, Hall J, Rangkuti F, Shwen YH, Almond N, Mitchell GH, et al. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes. Proc Natl Acad Sci USA. 2013;110:531-536.

    Not in our library10.1073/pnas.1216457110

44. 44.

    Muralidharan V, Oksman A, Iwamoto M, Wandless TJ, Goldberg DE. Asparagine repeat function in a Plasmodium falciparum protein assessed via a regulatable fluorescent affinity tag. Proc Natl Acad Sci USA. 2011;108:4411-4416.

    Not in our library10.1073/pnas.1018449108

45. 45.

    O'Neill MT, Phuong T, Healer J, Richard D, Cowman AF. Gene deletion from Plasmodium falciparum using FLP and Cre recombinases: implications for applied site-specific recombination. Int J Parasitol. 2011;41:117-123.

    Not in our library10.1016/j.ijpara.2010.08.001

46. 46.

    Pain A, Bohme U, Berry AE, Mungall K, Finn RD, Jackson AP, et al. The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature. 2008;455:799-803.

    Not in our library10.1038/nature07306

47. 47.

    Pino P, Sebastian S, Kim EA, Bush E, Brochet M, Volkmann K, et al. A tetracycline-repressible transactivator system to study essential genes in malaria parasites. Cell Host Microbe. 2012;12:824-834.

    Not in our library10.1016/j.chom.2012.10.016

48. 48.

    Pollock R, Clackson T. Dimerizer-regulated gene expression. Curr Opin Biotechnol. 2002;13:459-467.

    Not in our library10.1016/s0958-1669(02)00373-7

49. 49.

    Ruecker A, Shea M, Hackett F, Suarez C, Hirst EM, Milutinovic K, et al. Proteolytic activation of the essential parasitophorous vacuole cysteine protease SERA6 accompanies malaria parasite egress from its host erythrocyte. J Biol Chem. 2012;287:37949-37963.

    Not in our library10.1074/jbc.m112.400820

50. 50.

    Russo I, Oksman A, Vaupel B, Goldberg DE. A calpain unique to alveolates is essential in Plasmodium falciparum and its knockdown reveals an involvement in pre-S-phase development. Proc Natl Acad Sci USA. 2009;106:1554-1559.

    Not in our library10.1073/pnas.0806926106

51. 51.

    Trager W, Jensen JB. Human malaria parasites in continuous culture. Science. 1976;193:673-675.

    Not in our library10.1126/science.781840

52. 52.

    Trepanier DJ, Gallant H, Legatt DF, Yatscoff RW. Rapamycin: distribution, pharmacokinetics and therapeutic range investigations: an update. Clin Biochem. 1998;31:345-351.

    Not in our library10.1016/s0009-9120(98)00048-4

53. 53.

    Yatscoff RW, Wang P, Chan K, Hicks D, Zimmerman J. Rapamycin: distribution, pharmacokinetics, and therapeutic range investigations. Ther Drug Monit. 1995;17:666-671.

    Not in our library10.1097/00007691-199512000-00020

54. 54.

    Yeoh S, O'Donnell RA, Koussis K, Dluzewski AR, Ansell KH, Osborne SA, et al. Subcellular discharge of a serine protease mediates release of invasive malaria parasites from host erythrocytes. Cell. 2007;131:1072-1083.

    Not in our library10.1016/j.cell.2007.10.049

Cited by papers we hold 5

• Redesigned TetR-Aptamer System To Control Gene Expression in <named-content content-type="genus-species">Plasmodium falciparum</named-content> · 2020
  Not checked
• Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9 · 2017
  No notice
• A Genome-Scale Vector Resource Enables High-Throughput Reverse Genetic Screening in a Malaria Parasite · 2015
  No notice
• Processing of Plasmodium falciparum Merozoite Surface Protein MSP1 Activates a Spectrin-Binding Function Enabling Parasite Egress from RBCs · 2015
  No notice
• Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme · 2013
  No notice

Reads

A person counts once a day per paper. We keep no cookie and no address: a reader is a hash that changes every day. Any month under five reads shows no number.