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
doi: 10.1371/journal.pone.0226884

Souhrn

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


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2019 Číslo 12