Fig 1.
Pan and CoA biosynthesis pathways and the predicted essentiality in T. gondii, P. berghei, and P. falciparum.
Phenotypic data for T. gondii tachyzoites and P. berghei and P. falciparum asexual blood stages are derived from Sidik and colleagues [20], Bushell and colleagues [21], and Zhang and colleagues [22], respectively. Since the essentiality data for each of the species (T. gondii, P. berghei, and P. falciparum) were obtained using different genetic approaches, the most representative forms are displayed. The FS for T. gondii (circles) is an experimentally observed measure (ranging from −6.9 to +3 for the fitness cost associated with the disruption of a given gene for parasite survival. Fitness-conferring genes display a lower FS and dispensable genes display a higher FS [20]. Phenotypic data for P. falciparum genes based on their MIS, which estimates the potential mutability of a gene, are represented as diamonds [22]. For P. berghei, a gene is indicated as dispensable or essential based on the relative growth rate observed upon genetic disruption within the asexual blood stage (squares) [21]. Dashed lines represent putative routes. Pantothenamides and pantetheine can further be hydrolyzed into Pan via the action of host-encoded pantetheinases (vanins) [23]. BCAT, branched-chain amino acid transaminase; CoA, coenzyme A; DPCK, dephospho-CoA kinase; FS, fitness score; KPHMT, ketopantoate hydroxymethyltransferase; KPR, α-ketopantoate reductase; MIS, mutagenesis index score; PBAL, pantoate-β-alanine ligase; Pan, pantothenate; PANK, pantothenate kinase; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine synthetase.
Fig 2.
Genes encoding Pan and CoA biosynthesis enzymes.
Phenotypic data of T. gondii tachyzoites and P. berghei and P. falciparum asexual blood stages are derived from Sidik and colleagues [20], Bushell and colleagues [21], and Zhang and colleagues [22], respectively. Since the essentiality data for each of the species (T. gondii, P. falciparum, and P. berghei) were obtained using different genetic approaches, the scores are not comparable. The FS for T. gondii is an experimentally observed measure (ranging from −6.9 to +3) for the fitness cost associated with the disruption of a given gene for parasite survival. Fitness-conferring genes display a low FS and dispensable genes display a high FS [20]. The presented fitness score for P. falciparum is based on the MFS, which estimates the relative growth fitness cost for mutating a gene based on its normalized quantitative insertion-site sequencing reads distribution. The scores range from −4.1 to 2.8, with lower scores indicating nonmutability of a gene [22]. Experimental localization data (in black) were obtained from Oppenheim and colleagues [34], Lunghi and colleagues [16], and Barylyuk and colleagues (only PBAL is identified in this study) [17] for T. gondii, Tjhin and colleagues [35] and Swift and colleagues [36] for P. falciparum. Predicted localization is shown in gray. Variability in conservation is shown with different colors: light gray when only 1 copy of the gene is present, gray when 2 ORFs are present in a single gene, and dark gray when 3 ORFs exist in a single gene. Light orange is when more than 1 gene is identified in the whole group for the indicated catalytic activity, orange when only some species within the group encode more than 1 gene, and dark orange when the gene can be identified but has poor sequence similarity to the genes found in other species. BCAT, branched-chain amino acid transaminase; CoA, coenzyme A; DPCK, dephospho-CoA kinase; FS, fitness score; KPHMT, ketopantoate hydroxymethyltransferase; KPR, α-ketopantoate reductase; LOPIT, localization of organelle proteins by isotope tagging; MFS, mutagenesis fitness score; Pan, pantothenate; PANK, pantothenate kinase; PBAL, pantoate-β-alanine ligase; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine synthetase.
Fig 3.
Pan and CoA synthesis targeting compounds.
All IC50s are determined in P. falciparum, unless stated otherwise. CoA, coenzyme A; Pan, pantothenate.
Fig 4.
Mechanism of action of PanAms.
PanAms are converted into CoA antimetabolites (CoA-PanAms) using 3 enzymes of the CoA pathway and reduce acetyl-CoA levels. Mutations in AcAS and ACS11 determine the resistance phenotype against iPanAms, which is indicated by the increased concentration of PanAms needed to kill parasites with mutations (yellow and orange lines) compared to wild-type parasites (gray line). Parasites are more resistant when both AcAS and ACS11 are mutated (dark orange line) than parasites with a single mutation in one of these enzymes (yellow and light orange lines). CoA-PanAm blocks the activity of the CoA-utilizing enzyme AcAS (red boxes), thereby reducing acetyl-CoA levels that may lead to downstream effects on protein modification or fatty acid metabolism in asexual blood stages. It is hypothesized that CoA-PanAm cannot bind to the mutated AcAS (dashed line), resulting in a normal level of acetyl-CoA. Whether ACS11 is a target of CoA-PanAm is still unknown. AcAS, acetyl-CoA synthetase; ACS11, acyl-CoA synthetase 11; CoA, coenzyme A; CoA-PanAm, pantothenamide CoA-analog; DPCK, dephospho-CoA kinase; dP-CoA, dephospho-CoA; dPCoA-PanAm, pantothenamide dephospho-CoA-analog; iPanAms, inverted-amide PanAms; Pan, pantothenate; PanAm, pantothenamide; PanK, pantothenate kinase; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine synthetase; 4′-P-Pan, 4′-phosphopantothenate; 4′P-PanAm, 4′-phosphopantothenamide; 4′-P-PC, 4′-phosphopantothenoyl-L-cysteine; 4′P-PT, pantetheine-4′-phosphate.
Fig 5.
Phylogenetic tree of ACS and AcAS.
Phylogeny of the ACS family and AcAS in Apicomplexa. The hits identified with the iterative jackhmmer search (S1 Table; [160]) were used for the phylogenetic analysis. PfACS1b was not identified based on its homology to the query sequences in the jackhmmr search but was added based on previous literature [154]. MegaX was used to align all hits with ClustalW, followed by building an unrooted tree using a maximum likelihood analysis with a bootstrap phylogeny test. In initial analyses, 5 hits were excluded because these sequences did not cluster with any of the annotated family members. (A) Phylogenetic tree of all identified ACS and AcAS family members. Different settings were tested, including the alignment of the full amino acid sequence or the AMP-binding enzyme (PF00501) domain sequence, followed by a maximum likelihood analysis using all sites or a 50% partial deletion, all leading to comparable results. Phylogenetic tree analyses with 50% partial deletion excluded 1 hit because this alignment aligned with less than 50% of the genes. The final phylogenetic tree presented here is based on the full amino acid sequence of the identified ACS and AcAS family members, analyzed by the maximum likelihood method, 50% partial deletion with a bootstrap phylogeny test with 500 replicates. (B) Phylogenetic tree of only ACS family members. The phylogeny of the clusters of ACS family members was further tested in a maximum likelihood analysis using all different approaches as under (A). In none of the analyses, TGME49_310080 and TGME49_310150 clustered with ACS9, while the Plasmodium ACS12 cluster only once included the additional sequences from (A). The final phylogenetic tree presented here is based on the full amino acid sequence, analyzed by the maximum likelihood method, 50% partial deletion with a bootstrap phylogeny test with 100 replicates. AcAS, acetyl-CoA synthetase; ACS, acyl-CoA synthetase; FASI, type I fatty acid synthase; PKS, polyketide synthase.