Moraes Barros Roberto R, Gibson Tyler J, Kite Whitney A, Sá Juliana M, Wellems Thomas E
Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8132, USA.
Mol Biochem Parasitol. 2017 Dec;218:16-22. doi: 10.1016/j.molbiopara.2017.10.001. Epub 2017 Oct 6.
Human infections from Plasmodium knowlesi present challenges to malaria control in Southeast Asia. P. knowlesi also offers a model for other human malaria species including Plasmodium vivax. P. knowlesi parasites can be cultivated in the laboratory, and their transformation is standardly performed by direct electroporation of schizont-infected red blood cells (RBCs) with plasmid DNA. Here we show that the efficiency of direct electroporation is exquisitely dependent on developmental age of the schizonts. Additionally, we show that transformation of P. knowlesi can be achieved without direct electroporation by using the parasite's ability to infect and take up DNA from plasmid-loaded RBCs. Transformation with plasmid-loaded RBCs does not require labor-intensive preparations of schizont-infected RBCs as for direct electroporation, and parasite damage from high voltage discharge is avoided. Further studies of the mechanism of spontaneous DNA uptake may suggest strategies for improved transformation and provide insights into the transport pathways of apicomplexans.
诺氏疟原虫引起的人类感染给东南亚的疟疾防控带来了挑战。诺氏疟原虫也为包括间日疟原虫在内的其他人类疟原虫物种提供了一个模型。诺氏疟原虫寄生虫可在实验室中培养，其转化通常通过用质粒DNA直接电穿孔感染裂殖体的红细胞（RBCs）来进行。在此我们表明，直接电穿孔的效率极其依赖于裂殖体的发育阶段。此外，我们表明，利用该寄生虫感染并从加载质粒的红细胞中摄取DNA的能力，无需直接电穿孔即可实现诺氏疟原虫的转化。与直接电穿孔不同，用加载质粒的红细胞进行转化不需要对感染裂殖体的红细胞进行劳动密集型制备，并且避免了高压放电对寄生虫的损伤。对自发摄取DNA机制的进一步研究可能会提出改进转化的策略，并为顶复门原虫的运输途径提供见解。
