A New Drug Selectable Marker in Plasmodium yoelii Malaria Parasites

Open Access
Reese, Amanda Catherine
Area of Honors:
Biochemistry and Molecular Biology
Bachelor of Science
Document Type:
Thesis Supervisors:
  • Scott Lindner, Thesis Supervisor
  • David Gilmour, Honors Advisor
  • molecular biology
  • malaria
  • plasmodium
  • gene editing
  • parasitology
Sequential and conditional gene knockout approaches are valuable tools for reverse genetics in model species. However, studies of rodent malaria parasites (e.g. Plasmodium yoelii) suffer from having only one drug-selectable marker, a variant of dihydrofolate reductase (DHFR), which is used to select for transgenic parasites in the presence of pyrimethamine. I hypothesized that variants of dihydropterotate synthase (DHPS), another enzyme in the folate pathway, could be used to convey resistance to the drug sulfadiazine, as they have conferred resistance in field isolates of human-infectious Plasmodium species. The purpose of this study is three-fold. First, to determine which variant of the DHPS sequence will provide the best resistance to sulfadiazine. Second, to determine if DHPS alone can confer resistance, as previous publications have utilized this cassette either with DHFR or with PPPK, the enzyme before DHPS in the folate pathway. Third, to determine if it is possible to co-select for both DHPS and DHFR expression using both sulfadiazine and pyrimethamine. To test the effectiveness of DHPS in conferring drug resistance, parasites were transfected with plasmids that express a field variant of DHPS and GFP separately, and were selected by administering sulfadiazine via host drinking water. Data indicates that it is possible to select for the 3D7 variant of the bi-functional PPPK-DHPS protein at a dose of 3 mg/L sulfadiazine, and that it is not possible to select for the DHPS only. Co-selection with DHFR has not been possible using this variant, but future experiments will use the full length sequence of variants with stronger sulfadiazine resistance and study the metabolic effect of drug treatment to better understand the effect it has on the folate pathway. Once optimized, this system would provide an efficient means by which doubly transgenic parasites could be selected for by a drug cocktail.