Investigation of the essential nature of TCA cycle enzyme fumarate hydratase in Plasmodium falciparum

Lindsay Pomykala Kleinwaks

doi:10.17918/00000870

Plasmodium falciparum is unable to synthesize purines, relying on the purine salvage pathway (PSP). Adenylosuccinate synthase and adenylosuccinate lyase act on scavenged hypoxanthine, adenosine, and aspartate to produce AMP, generating fumarate as a byproduct. We are interested in understanding the fate of this fumarate and the putative link to TCA metabolism. A fumarate cycle has been proposed in which PSPderived fumarate is recycled to aspartate by the action of fumarate hydratase (FH), malate dehydrogenase, and aspartate aminotransferase. PSP-essentiality raises the question as to whether the fumarate cycle is also essential. Plasmodium FH is an iron-sulfur (Fe-S) containing Type I enzyme, distinct from the Type II FH present in host cells, raising the possibility of its being an attractive drug target. Previous studies have shown that the FH gene could not be genetically disrupted using traditional knockout techniques. We have carried out additional characterization studies to have additional information needed for exploration of PfFH as a drug target. Further attempts to disrupt PfFH using a CRISPR/Cas9 approach were unsuccessful, supporting that PfFH cannot be genetically ablated. We were, however, able to tag the 3'-UTR of PfFH with a glucosamineregulatable ribozyme (glmS) sequence. This has provided a means to assess consequences of conditional expression knockdown. Using glucosamine for ribozyme-mediated mRNA degradation, it was discovered that PfFH protein expression can be knocked down up to 98% without detriment to parasite viability in as little as one asexual cycle. Monitoring gene expression showed no change in PSP-related or glycolytic enzymes, suggesting the 2-5% of FH remaining is all that is required for parasite viability. Metabolomic analysis of the knockdown line showed no changes in related metabolite presence, confirming that PfFH knockdown has no effect on parasite viability. Ectopic expression of PfFH alternatively showed an 11-fold increase in FH as well as changes in expression of phosphofructokinase, triose phosphate isomerase, and aspartate aminotransferase, which may reflect a role for FH in PSP-metabolism. Taken together, the data supports that PfFH expression cannot be completely depleted by knockout and that overexpression of PfFH trigger changes in cellular metabolism, suggesting that this divergence from Type II FH might be important for the purine salvage pathway, and is likely essential for parasite viability.

Investigation of the essential nature of TCA cycle enzyme fumarate hydratase in Plasmodium falciparum

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Investigation of the essential nature of TCA cycle enzyme fumarate hydratase in Plasmodium falciparum

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