Malaria is a deadly disease caused by Eukaryotic, single-cell parasites from the genus Plasmodium, with P. falciparum associated with the most deadly instances of the disease. With 228 million cases and over 400,000 deaths in 2018, the continuous rise of anti-malarial resistant P. falciparum parasites is a threat to global health. Research into the parasite’s cellular biology uncovers potential weakness that may be exploited to kill the parasite, but this research has been historically difficult due to the haploid nature of the parasite and its recalcitrance to genetic manipulation. Conditional knockdown systems, in which parasite protein expression is controlled by small molecules, and the adaptation of CRISPR/Cas9 genome editing for use in P. falciparum has recently made research into the parasite’s cellular biology more feasible. This dissertation combines these advances, presenting a detailed protocol for using CRISPR/Cas9 genome editing to introduce conditional knockdown systems into the parasite and demonstrating how those systems were used to investigate a protein exported by the parasite into its host red blood cell. Then, these same methods are used and built upon to provide a detailed investigation of PfJ2, an essential chaperone and thioredoxin-domain protein located in the parasite’s endoplasmic reticulum (ER). The P. falciparum ER is an organelle neglected in research, despite its numerous essential functions. I uncover interactions between PfJ2 and other essential proteins that reside in or pass through the ER, and I leverage the protein’s thioredoxin domain to provide new insights into the process of disulfide bond formation in the oxidative environment of the parasite’s ER. This process likely involved PfJ2 and members of the Protein Disulfide Isomerase (PDI) family, one of which—PfPDI8—I demonstrate is also essential for parasite survival. Finally, I use a chemical biology approach to show that a small molecule can disrupt the redox interactions between PfJ2, PfPDI8, and their substrates. These results suggest that these essential proteins, and the process of disulfide bond formation in the ER, are a new aspect of the parasite’s biology that could be exploited for anti-malarial drug development.
