Discovery and characterization of a novel class of metabolic regulators in the malaria parasite Plasmodium falciparum

• Main Author: Guggisberg, Ann M
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2016
• Subjects: Genetics; Microbiology
• ISBN: 1369305354; 9781369305357

The malaria parasite, Plasmodium falciparum, infects hundreds of millions of people per year and causes hundreds of thousands of deaths. Within the host red blood cell, the parasite relies on glycolysis for energy and synthesis of essential biomolecules. One such anabolic fate of glucose is the synthesis of isoprenoids, a broad and essential class of compounds that participate in a variety of cellular functions. In the face of ever-evolving drug resistance, new inhibitors and better understanding of parasite metabolism are required. The antibiotic fosmidomycin (FSM) targets the methylerythritol phosphate pathway for isoprenoid synthesis and is a well-validated inhibitor of P. falciparum growth. A forward selection for FSM resistance generated a number of parasite strains with increased drug tolerance. We identify mutations in two members of the haloacid dehalogenase-like hydrolase (HAD) superfamily, PfHAD1 and PfHAD2, as causal for resistance. Enzymatic characterization and metabolic profiling reveal that these mutations are deleterious and confirm the role of PfHAD1 and PfHAD2 as novel negative regulators of glucose and isoprenoid metabolism. Despite their homology and shared role in FSM resistance, PfHAD1, a sugar phosphatase, and PfHAD2, a purine nucleotidase, appear to mediate FSM resistance via distinct enzymatic mechanisms. To further understand the role of PfHADs as metabolic regulators, we harness a growth defect in FSM-resistant PfHAD2 mutants to select for suppressors of FSM resistance. We identify suppressor mutations in the key glycolytic enzyme phosphofructokinase (PfPFK9) and describe the effect of these mutations on enzyme function and parasite metabolism. Given its safety in humans and its specificity as a MEP pathway inhibitor, FSM is a strong candidate for clinical development and is currently being evaluated in clinical trials as part of an antimalarial combination therapy. Unfortunately, previous studies have observed high rates of recrudescence following FSM treatment when paired with the antibiotic clindamycin (CLN). To understand whether any genetic changes correlate with recrudescence, we performed whole genome sequencing on patient parasite populations before and after recrudescence. We demonstrate the use of a selective amplification method to amplify and sequence parasite genomes from blood spots. Our genotyping does not reveal any genetic changes responsible for recrudescence, but rather support the hypothesis that FSM-CLN treatment failure is due to formulation or partner drug selection. We encourage further development of FSM and other MEP pathway inhibitors as antimalarial therapies.