The pantothenate kinase of the human malaria parasite Plasmodium falciparum

Abstract

The intraerythrocytic stage of the malaria parasite Plasmodium falciparum has an absolute requirement for vitamin B5 (also known as pantothenate) in order to survive. The parasite takes up extracellular pantothenate and subsequently converts it into coenzyme A (CoA) via a series of five universal enzymatic steps. The first enzyme of the pathway is pantothenate kinase (PanK), which phosphorylates pantothenate and commits the molecule to CoA biosynthesis. There are two putative PanK genes (designated Pfpank1 and Pfpank2) in the parasite’s genome, both of which are expressed in the intraerythrocytic stage of the parasite. Many antiplasmodial pantothenate analogues, including pantothenol (PanOH), CJ-15,801, N-substituted pantothenamides (PanAms) and PanAm derivatives have been shown to inhibit P. falciparum growth by targeting its CoA biosynthesis and/or utilisation, although their exact mechanism of action in the parasite remains poorly characterised. In this study, a step-wise dose-escalating drug pressure regime with either PanOH or CJ-15,801 was used to generate resistant parasite lines. These parasite lines are cross-resistant to both PanOH and CJ-15,801, but exhibit different sensitivity profiles to the PanAm derivatives N5-trz-C1-Pan and N-PE-αMe-PanAm, consistent with these two groups of pantothenate analogues having different mechanisms of action. Whole-genome sequencing revealed that these parasites harbour mutations in Pfpank1. Some of these mutations significantly alter the activity of PfPanK, the parasite’s requirement for pantothenate and consequently their fitness compared to the Parent line. These results are consistent with PfPanK1 being the active PanK during this stage of the parasite’s lifecycle. When analysed in conjunction with what has been reported for other organisms, the results of functional enzymatic assays performed in this study revealed important information about the modes of action of these pantothenate analogues. PanOH and CJ-15,801 are predicted to inhibit PfPPCS (the second enzyme of CoA biosynthesis). Conversely, N5-trz-C1-Pan and N-PE-αMe-PanAm are hypothesised to be metabolised into CoA analogues, which subsequently inhibit downstream CoA-utilising enzymes. In order to characterise the activity, conformation and potential interacting partners of PfPanK1 and PfPanK2, green fluorescent protein (GFP)-fused copies of these proteins were expressed in P. falciparum parasites to enable their purification. Results of western blot and mass spectrometry analyses of immunoprecipitated PfPanK are consistent with the native protein being a complex that is comprised of PfPanK1, PfPanK2 and the adapter protein Pf14-3-3I. This marks the first description of a heterodimeric PanK in nature. In silico analysis of the amino acid sequence of the two PfPanKs and interrogation of existing phosphoproteomic studies suggest that Pf14-3-3I binds to PfPanK2. Taken together, the research presented in this study has extended our understanding of the P. falciparum PanKs and, therefore, provided further insight into the CoA biosynthesis pathway as an antimalarial drug target. Furthermore, the information generated in this study about the mechanisms of action of the pantothenate analogues will hopefully expedite the discovery of new antimalarials. Show more