The Coenzyme A biosynthesis pathway of the human malaria parasite Plasmodium falciparum and Toxoplasma gondii

Abstract

Malaria, caused by the apicomplexan parasite of the genus Plasmodium, was responsible for 608,000 deaths in 2022. Most of these cases occurred in Africa. Despite the existence of a vaccine, the disease still affects millions of people. Strategies such as bed nets and insecticide spraying are employed to reduce malaria infections. Treatment of infections is managed by the current best class of compounds artemisinins. Unfortunately, there is parasite resistance to these compounds, highlighting a need for novel compounds targeting the parasite.

The biosynthesis of coenzyme A (CoA) in five enzyme-mediated steps from pantothenate, and the utilization of CoA in essential pathways, represent promising antimalarial targets. This study focuses on characterizing pantothenate utilization and the CoA biosynthesis pathway in both Plasmodium falciparum, and Toxoplasma gondii - an apicomplexan parasite that is often used as a model organism for Plasmodium.

One aim was to identify pantothenate kinase (PanK) in T. gondii, the first enzyme, which is responsible for the phosphorylation of pantothenate, in CoA biosynthesis. All PanKs from other organisms that have been characterised thus far form homodimers. In both P. falciparum and T. gondii there are two PanKs, PanK1 and PanK2. Previous work identified the PanKs in P. falciparum and discovered that both are likely to be required for PanK activity. I used CRISPR and the mAID system to tag proteins of interest and conditionally knock them down, allowing for the characterisation of the PanKs in T. gondii. This allowed me to answer some specific unresolved questions in P. falciparum. I found that both TgPanK1 and TgPanK2 were essential for pantothenate phosphorylation, and that neither could substitute for the other. I also found that parasite proliferation was restored when either TgPanK1 or TgPanK2 were knocked down but PanK activity had been complemented by the Staphylococcus aureus PanK, confirming that both TgPanK1 and TgPanK2 are required for PanK activity. I concluded that in both P. falciparum and T. gondii PanK1 and PanK2 are essential for parasite proliferation and form part of a heterodimeric complex, which is unique to apicomplexan parasites. In addition, I investigated, in P. falciparum, a series of 19 mimics of pantothenamides, amide derivatives of pantothenate which have potential as antimalarials, but are broken down by a ubiquitous serum enzyme called pantetheinase, rendering them inactive in vivo. This series of compounds incorporated a stable triazole group instead of the labile amide which successfully prevented their metabolism by pantetheinase. These compounds were active against the intraerythrocytic stage of P. falciparum, with two of the compounds possessing IC50 values of approximately 50 nM. The compounds were found to target CoA biosynthesis and, importantly, were not toxic to human cells.

Finally, I explored the feasibility of targeting the pantothenate biosynthesis pathway in T. gondii. Unlike P. falciparum, T. gondii possesses a putative pantothenate synthetase gene. Pantothenate synthetases generate pantothenate from the precursors beta-alanine and pantoate. A previous paper reported that the pantothenate synthetase of T. gondii could be targeted by compounds that had been developed against Mycobacterium tuberculosis (M. tuberculosis), resulting in parasite death. Using CRISPR I knocked out the T. gondii pantothenate synthetase knockout and showed that this was not detrimental for parasite proliferation, even in the absence of extracellular pantothenate. I also confirmed that one of the compounds (SW314) used in the previous study had no effect on the proliferation of T. gondii, despite potent activity against the M. tuberculosis pantothenate synthetase. My results are consistent with the T. gondii pantothenate synthesis gene not being essential for parasite proliferation during the tachyzoite stage and therefore not a suitable drug target. Show more