Comparison of two methods for transformation of Plasmodium knowlesi: Direct schizont electroporation and spontaneous plasmid uptake from plasmid-loaded red blood cells

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.