Validation of Plasmodium vivax centromere and promoter activities using Plasmodium yoelii

Autoři: Kittisak Thawnashom aff001;  Miho Kaneko aff001;  Phonepadith Xangsayarath aff001;  Nattawat Chaiyawong aff001;  Kazuhide Yahata aff001;  Masahito Asada aff001;  John H. Adams aff004;  Osamu Kaneko aff001
Působiště autorů: Department of Protozoology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Sakamoto, Nagasaki, Japan aff001;  Department of Medical Technology, Faculty of Allied Health Sciences, Naresuan University, Mueang, Phitsanulok, Thailand aff002;  Leading Program, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki, Japan aff003;  Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, Tampa, Florida, United States of America aff004
Vyšlo v časopise: PLoS ONE 14(12)
Kategorie: Research Article


Plasmodium vivax is the leading cause of malaria outside Africa and represents a significant health and economic burden on affected countries. A major obstacle for P. vivax eradication is the dormant hypnozoite liver stage that causes relapse infections and the limited antimalarial drugs that clear this stage. Advances in studying the hypnozoite and other unique biological aspects of this parasite are hampered by the lack of a continuous in vitro laboratory culture system and poor availability of molecular tools for genetic manipulation. In this study, we aim to develop molecular tools that can be used for genetic manipulation of P. vivax. A putative P. vivax centromere sequence (PvCEN) was cloned and episomal centromere based plasmids expressing a GFP marker were constructed. Centromere activity was evaluated using a rodent malaria parasite Plasmodium yoelii. A plasmid carrying PvCEN was stably maintained in asexual-stage parasites in the absence of drug pressure, and approximately 45% of the parasites retained the plasmid four weeks later. The same retention rate was observed in parasites possessing a native P. yoelii centromere (PyCEN)-based control plasmid. The segregation efficiency of the plasmid per nuclear division was > 99% in PvCEN parasites, compared to ~90% in a control parasite harboring a plasmid without a centromere. In addition, we observed a clear GFP signal in both oocysts and salivary gland sporozoites isolated from mosquitoes. In blood-stage parasites after liver stage development, GFP positivity in PvCEN parasites was comparable to control PyCEN parasites. Thus, PvCEN plasmids were maintained throughout the parasite life cycle. We also validated several P. vivax promoter activities and showed that hsp70 promoter (~1 kb) was active throughout the parasite life cycle. This is the first data for the functional characterization of a P. vivax centromere that can be used in future P. vivax biological research.

Klíčová slova:

Centromeres – Malarial parasites – Parasitic diseases – Parasitic life cycles – Plasmid construction – Plasmodium – Sporozoites – Plasmodium yoelii


1. World Health Organization. World Malaria Report 2018. Geneva: World Health Organization; 2018. ISBN: 978-92-4-156565-3.

2. Adams JH and Mueller I. The Biology of Plasmodium vivax. Cold Spring Harb Perspect Med. 2017;7(9). pii: a02558 doi: 10.1101/cshperspect.a025585 28490540

3. Watson J, Taylor WRJ, Bancone G, Chu CS, Jittamala P, White NJ. Implications of current therapeutic restrictions for primaquine and tafenoquine in the radical cure of vivax malaria. PLOS Negl Trop Dis. 2018;12(4):e0006440. doi: 10.1371/journal.pntd.0006440 29677199

4. Thomas D, Tazerouni H, Sundararaj KG, Cooper JC. Therapeutic failure of primaquine and need for new medicines in radical cure of Plasmodium vivax. Acta Trop. 2016;160:35–38. doi: 10.1016/j.actatropica.2016.04.009 27109040

5. Bennett JW, Pybus BS, Yadava A, Tosh D, Sousa JC, McCarthy WF, et al. Primaquine failure and cytochrome P-450 2D6 in Plasmodium vivax malaria. N Engl J Med. 2013;369(14):1381–1382. doi: 10.1056/NEJMc1301936 24088113

6. Pybus BS, Marcsisin SR, Jin X, Deye G, Sousa JC, Li Q, et al. The metabolism of primaquine to its active metabolite is dependent on CYP 2D6. Malar J. 2013;12:212. doi: 10.1186/1475-2875-12-212 23782898

7. Potter BM, Xie LH, Vuong C, Zhang J, Zhang P, Duan D, et al. Differential CYP 2D6 metabolism alters primaquine pharmacokinetics. Antimicrob Agents Chemother. 2015;59(4):2380–2387. doi: 10.1128/AAC.00015-15 25645856

8. World Health Organization. Control and Elimination of Plasmodium vivax Malaria: a technical brief. Geneva: World Health Organization; 2015. ISBN: 978-92-4-150924-4.

9. Armistead JS and Adams JH. Advancing research models and technologies to overcome biological barriers to Plasmodium vivax control. Trends Parasitol. 2018;34(2):114–126. doi: 10.1016/ 29153587

10. Pfahler JM, Galinski MR, Barnwell JW, Lanzer M. Transient transfection of Plasmodium vivax blood stage parasites. Mol Biochem Parasitol. 2006;149(1):99–101. doi: 10.1016/j.molbiopara.2006.03.018 16716418

11. Moraes Barros RR, Straimer J, Sa JM, Salzman RE, Melendez-Muniz VA, Mu J, et al. Editing the Plasmodium vivax genome, using zinc-finger nucleases. J Infect Dis. 2015;211(1):125–129. doi: 10.1093/infdis/jiu423 25081932

12. van der Wel AM, Tomás AM, Kocken CH, Malhotra P, Janse CJ, Waters AP, et al. Transfection of the primate malaria parasite Plasmodium knowlesi using entirely heterologous constructs. J Exp Med. 1997;185(8):1499–1503. doi: 10.1084/jem.185.8.1499 9126931

13. van der Wel AM, Kocken CHM, Pronk TC, Franke-Fayard B, Thomas AW. New selectable markers and single crossover integration for the highly versatile Plasmodium knowlesi transfection system. Mol Biochem Parasitol. 2004;134(1):97–104. doi: 10.1016/j.molbiopara.2003.10.019 14747147

14. Lucky AB, Sakaguchi M, Katakai Y, Kawai S, Yahata K, Templeton TJ, et al. Plasmodium knowlesi skeleton-binding protein 1 localizes to the 'Sinton and Mulligan' stipplings in the cytoplasm of monkey and human erythrocytes. PLOS One. 2016;11(10):e0164272. doi: 10.1371/journal.pone.0164272 27732628

15. Ozwara H, Langermans JA, Kocken CH, van der Wel AM, van der Meide PH, Vervenne RA, et al. Transfected Plasmodium knowlesi produces bioactive host gamma interferon: a new perspective for modulating immune responses to malaria parasites. Infect Immun. 2003;71(8):4375–4381. doi: 10.1128/IAI.71.8.4375-4381.2003 12874315

16. Ozwara H, van der Wel A, Kocken CH, Thomas AW. Heterologous promoter activity in stable and transient Plasmodium knowlesi transgenes. Mol Biochem Parasitol. 2003;130(1):61–64. doi: 10.1016/s0166-6851(03)00141-5 14550898

17. Dankwa S, Lim C, Bei AK, Jiang RH, Abshire JR, Patel SD, et al. Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite. Nat Commun. 2016;7:11187. doi: 10.1038/ncomms11187 27041489

18. Kocken CH, van der Wel A, Thomas AW. Plasmodium cynomolgi: transfection of blood-stage parasites using heterologous DNA constructs. Exp Parasitol. 1999;93(1):58–60. doi: 10.1006/expr.1999.4430 10464040

19. Voorberg-van der Wel A, Zeeman AM, van Amsterdam SM, van den Berg A, Klooster EJ, Iwanaga S, et al. Transgenic fluorescent Plasmodium cynomolgi liver stages enable live imaging and purification of malaria hypnozoite-forms. PLOS One. 2013;8(1):e54888. doi: 10.1371/journal.pone.0054888 23359816

20. Azevedo MF and del Portillo HA. Promoter regions of Plasmodium vivax are poorly or not recognized by Plasmodium falciparum. Malar J. 2007;6:20. doi: 10.1186/1475-2875-6-20 17313673

21. Kooij TW, Carlton JM, Bidwell SL, Hall N, Ramesar J, Janse CJ, et al. A Plasmodium whole-genome synteny map: indels and synteny breakpoints as foci for species-specific genes. PLoS Pathog. 2005;1(4):e44. doi: 10.1371/journal.ppat.0010044 16389297

22. Carlton JM, Adams JH, Silva JC, Bidwell SL, Lorenzi H, Caler E, et al. Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature. 2008;455(7214):757–763. doi: 10.1038/nature07327 18843361

23. Iwanaga S, Kato T, Kaneko I, Yuda M. Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum. PLoS One. 2012;7(3):e33326. doi: 10.1371/journal.pone.0033326 22479383

24. O'Donnell RA, Freitas-Junior LH, Preiser PR, Williamson DH, Duraisingh M, McElwain TF, et al. A genetic screen for improved plasmid segregation reveals a role for Rep20 in the interaction of Plasmodium falciparum chromosomes. EMBO J. 2002;21(5):1231–1239. doi: 10.1093/emboj/21.5.1231 11867551

25. Gautret P, Deharo E, Chabaud AG, Ginsburg H, Landau I. Plasmodium vinckei vinckei, P. v. lentum and P. yoelii yoelii: chronobiology of the asexual cycle in the blood. Parasite. 1994;1(3):235–239. doi: 10.1051/parasite/1994013235 9140490

26. Iwanaga S, Khan SM, Kaneko I, Christodoulou Z, Newbold C, Yuda M, et al. Functional identification of the Plasmodium centromere and generation of a Plasmodium artificial chromosome. Cell Host Microbe. 2010;7(3):245–255. doi: 10.1016/j.chom.2010.02.010 20227667

27. Mons B, Janse CJ, Boorsma EG, Van der Kaay HJ. Synchronized erythrocytic schizogony and gametocytogenesis of Plasmodium berghei in vivo and in vitro. Parasitology. 1985;91(3):423–30. doi: 10.1017/s0031182000062673 3909068

28. Muller I, Jex AR, Kappe SHI, Mikolajczak SA, Sattabongkot J, Patrapuvich R, et al. Transcriptome and histone epigenome of Plasmodium vivax salivary-gland sporozoites point to tight regulatory control and mechanisms for liver-stage differentiation in relapsing malaria. Int J Parasitol. 2019;49(7):501–513. doi: 10.1016/j.ijpara.2019.02.007 31071319

29. Balu B, Shoue DA, Fraser MJ Jr, Adams JH. High-efficiency transformation of Plasmodium falciparum by the lepidopteran transposable element piggyBac. Proc Natl Acad Sci U S A. 2005;102(45):16391–16396 doi: 10.1073/pnas.0504679102 16260745

30. van Dooren GG, Marti M, Tonkin CJ, Stimmler LM, Cowman AF, McFadden GI. Development of the endoplasmic reticulum, mitochondrion and apicoplast during the asexual life cycle of Plasmodium falciparum. Mol Microbiol. 2005;57:405–419. doi: 10.1111/j.1365-2958.2005.04699.x 15978074

31. Sakura T, Yahata K, Kaneko O. The upstream sequence segment of the C-terminal cysteine-rich domain is required for microneme trafficking of Plasmodium falciparum erythrocyte binding antigen 175. Parasitol Int. 2013;62:157–164. doi: 10.1016/j.parint.2012.12.002 23268338

32. Zhu X, Yahata K, Alexandre JSF, Tsuboi T, Kaneko O. The N-terminal segment of Plasmodium falciparum SURFIN4.1 is required for its trafficking to the red blood cell cytosol through the endoplasmic reticulum. Parasitol Int. 2013;62(2):215–229. doi: 10.1016/j.parint.2012.12.006 23287798

33. Fernandez-Becerra C, de Azevedo MF, Yamamoto MM, del Portillo HA. Plasmodium falciparum: new vector with bi-directional promoter activity to stably express transgenes. Exp Parasitol. 2003;103(1–2):88–91. doi: 10.1016/s0014-4894(03)00065-1 12810052

34. Reed MB, Saliba KJ, Caruana SR, Kirk K, Cowman AF. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature. 2000;403(6772):906–909. doi: 10.1038/35002615 10706290

35. Janse CJ, Ramesar J, Waters AP. High-efficiency transfection and drug selection of genetically transformed blood stages of the rodent malaria parasite Plasmodium berghei. Nat Protoc. 2006;1(1):346–356. doi: 10.1038/nprot.2006.53 17406255

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2019 Číslo 12
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