Fig 1.
Isoprenoid sources and distribution in malaria parasites.
The figure illustrates the sources and distribution of isoprenoids in malaria parasites. The figure includes the biosynthesis of isoprenoids via the MEP pathway, starting with the condensation of glyceraldehyde-3-phosphate (G3P) and pyruvate, and leads to the formation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), and their subsequent condensation to form geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP). These longer isoprenoids are essential for the biosynthesis of ubiquinone, dolichol, and for protein prenylation. The figure also shows the targets of fosmidomycin and ribosome inhibitors in the parasite. The chemical structures of FOH and GGOH are represented.
Fig 2.
PfPolK phylogenetic analysis and structural model.
A) Overall phylogenetic dendrogram of prenol binding proteins generated using maximum likelihood method (see Methods). Branch support values (SH2-like) are displayed as numbers for the most relevant clade separation, as well as colours (from the highest scores, in blue, to the lowest values, in red) and thickness of the branches. Root-external group of DolK and DolK-like containing representatives of each Eukaryote super groups, and the other group of PhyK/FolK enzymes. The discussed groups are highlighted by coloured boxes as follows: a large group with two copies of each Euviriplantae (dark green), PhyK/FolK enzymes from unicellular algae (in green light green), a monophyletic group composed by Dinoflagelata’s (yellow), Stramenopila (turquoise) and the specific Apicomplexa monophyletic clade arises (in red). Supporting information provides the full phylogenetic tree with all values for branch support and labelled taxa, as well as a key-taxa conversion table. B) AlphaFold 2 PfPolK model displays eight conserved transmembrane helices (TM1-8, coloured) and a potential prenol binding pocket, depicting in the bottom the nucleotide-binding site with the Thre144 and Glu179 composing a hinge region. This model was used to generate the potential binding mode for FOH (green, C) and GGOH (blue, D) by a combination of flexible docking and long molecular dynamics simulations (5x1 μs for each system in explicit solvent and membrane), E) the MD trajectories were utilized to infer the substrates predicted binding energy (Kcal/mol.HAC, where HAC—heavy atom count), suggesting from the median values of the violin-plot displayed distribution that GGOH would have a lower potential binding energy. Dotted lines describe the first quartile amplitude.
Fig 3.
Farnesol and geranylgeraniol kinase activities.
Autoradiographs of the PolK enzymatic activity assays using [3H] FOH or [3H] GGOH as substrates and chromatographed by TLC. The enzyme source of these assays came from whole extracts of yeast strains transformed with either the empty vector (p416-GPD) or p416-PfPolK. Compounds added to the enzymatic reaction are indicated under the TLC autoradiography image. The retention of different standards is also indicated. These experiments were repeated three times with similar results.
Fig 4.
Conditional knockout of P. falciparum PolK gene.
(A) Diagram depicting the edition of single-exon gene P. falciparum PolK. Using Cas9-assisted genome editing, all 612 bp of the native PfPolK (WT) open reading frame were replaced by a recodonized sequence (PfPolK recod) and a 3x-HA sequence (yellow box) in the 5’ end of the sequence and all flanked by two loxP sites (orange boxes). The position of the 20-nucleotide region targeted by the single guide RNA (sgRNA) is indicated (purple box). (B) Diagram of the rapamycin-induced site-specific excision. Recombination between the loxP sites removes the entire recodonized gene (green box) and 3-HA sequence; (C) PCR detection of transgenic parasites. Wild type DNA (NF54 DiCre control) was confirmed using PolK-WT-forward and reverse primers, amplifying a fragment of 383 bp. Using PolK-recod-forward and reverse primers it was detected a fragment of 541 bp corresponding to the redoconized version of PolK in transgenic parasites (Clones E3, C3 and G9) and in a plasmid containing the recodonized PolK (pUC19 polk recod) as a control. It was used as a standard the GeneRuler 1 kb DNA Ladder (#SM0311, Thermo Scientific). (D) PCR assessment of the genome integration of the construct in the PfPolK locus. Wild type DNA (NF54 DiCre control) was confirmed using Int-control-forward and Int-WT-reverse primers, amplifying a fragment of 1053 bp. Using Int-control-forward and Int-recod-reverse primers it was detected a fragment of 972 bp corresponding to the integrated locus. It was used as a standard the GeneRuler 1 kb Plus DNA Ladder (#MK-130, Cellco) (E) Confirmation of the rapamycin-induced PfPolK gene excision in the clone PolK-loxP E3. The deletion was confirmed by PCR 24, 48, 96 and 144 h after treatment with DMSO (D) or rapamycin (R) using primers PolK-HR1-forward and PolK-HR2-reverse (in red and blue on panel B, respectively). Excision reduces the amplicon from 1840 bp to 799 bp, disrupting PfPolK. It was used as a standard the GeneRuler 1 kb DNA Ladder (#SM0311, Thermo Scientific). (F) Figure shows the 144h evolution of parasitemia in different clones in which PfPolK gene was excised (parasites exposed to rapamycin) or not (parasites exposed to DMSO). (G) Western blot of transgenic parasites (left) and the respective Comassie stained gel (right). Western blot was performed to analyse the HA-tagged PfPolK of parasites in which PfPolK was excised (lane 1, parasites exposed to rapamycin) or preserved (lane 2, parasites exposed to DMSO). (H) Immunofluorescence analysis of HA-tagged PfPolK of parasites in which PfPolK was excised (parasites exposed to rapamycin, Δ-Polk) or not (control parasites, exposed to DMSO). HA-tagged PfPolK is marked in red, the apicoplast is green and the nucleus in blue. For all experiments, the excision of PolK-loxP parasites was initiated by adding 50 nM rapamycin or DMSO (used as a vehicle control) to synchronized ring-stage cultures. The cells underwent a 24-hour treatment period, followed by a washout step. At this point, we considered it as time zero, and the parasites were cultured again without rapamycin. For Western blot and IF analysis, parasites were utilized at the trophozoite/shizont stage (at 24 hours). In the case of PCR analysis and the assessment of parasitemia evolution, parasites were collected at various time points, as indicated in the figure. (I) Quantitative pixel analysis of immunofluorescence images. The table presents data from the analysis of pixel overlap between the red (anti-HA antibody staining) and green (anti-FRN antibody staining) channels in five individual parasites. The count of red, green, and overlapping pixels was performed using the ImageJ software. The "Percentage of Overlap" was calculated as the ratio of overlapping pixels to the total red channel pixels, multiplied by 100, for each parasite replicate. The average and standard deviation (SD) of the percentage overlap across the five replicates are also provided.
Fig 5.
Phenotypic characterization of knockout parasites.
(A) Fosmidomycin dose-response curves after 48h in parasites maintaining a functional PfPolK (DMSO (Vehicle/control)) or Δ-PolK parasites. These parasites were cultured in RPMI medium in the presence or absence of the indicated prenols (5 μM). (B) Fosmidomycin IC50 values of the results exposed in the previous panel. (C) Clindamycin dose-response curves after 96 h in parasites maintaining a functional PfPolK (DMSO—Vehicle/control) or Δ-PolK parasites. These parasites were cultured in RPMI medium in the presence or absence of GGOH (5 μM), as indicated. (D) Clindamycin IC50 values of the results exposed in the previous panel. Statistical analysis was made using one-way ANOVA/Dunnet’s Multiple Comparison Test.*p<0.05, **p<0.01, ***p<0.001. Comparison made to Vehicle/Control data. Error bars represent standard deviation (n = 3). The excision of PolK-loxP parasites was initiated one parasitic cycle before the start of the experiments. This was achieved by adding 50 nM rapamycin or DMSO (used as a vehicle control) to synchronized cultures in the ring stage. These cells underwent a 24-hour treatment period, followed by a washout step, and then used for experiments. Parasitic growth in all experiments depicted in this figure was monitored by flow cytometry.
Fig 6.
Farnesol and isoleucine incorporation into proteins in Δ-PolK parasites.
The graph shows the levels of incorporation of 3H-FOH and 14C -isoleucine (14C -Ile) into TCA-precipitated proteins in parasites maintaining or not a functional PfPolK. Statistical analysis was made using one-way ANOVA One-way ANOVA / Tukey’s Multiple Comparison Test.*p<0.05, **p<0.01, ***p<0.001. Comparison made to between samples of parasites exposed to the same radiolabelled precursor but maintaining or not PfPolK. Error bars represent standard deviation (n = 3). The excision of PolK-loxP parasites was initiated one parasitic cycle before the start of the experiments. This was achieved by adding 50 nM rapamycin or DMSO (used as a vehicle control) to synchronized cultures in the ring stage. These cells underwent a 24-hour treatment period, followed by a washout step, and then used for experiments.
Fig 7.
Sensitivity to fosmidomycin using human plasma.
(A) The images display mass spectrometry obtained using GC-MS triple quadrupole MRM analysis of the standard of GGOH, as well as GGOH detected in human plasma and AlbuMax I. GGOH is highlighted with arrows. (B) A representative image displaying the sigmoidal dose-response effect of fosmidomycin on cultures of either control or Δ-PolK parasites grown in media supplemented with either whole plasma or delipidated plasma from donor II. (C) The image presents bar graphs and a table displaying the IC50 values of the antiplasmodial effect of fosmidomycin on Δ-PolK parasite cultures supplemented with either whole or delipidated plasma from three distinct donors. The analyses in (B) and (C) were conducted on parasites subjected to rapamycin-induced knockout or those retaining PfPolK function. The excision of PolK-loxP parasites commenced one parasitic cycle prior to the initiation of the experiments, achieved by adding 50 nM rapamycin or DMSO (serving as a vehicle control) to synchronized ring-stage cultures. These cells underwent a 24-hour treatment, followed by a washout phase, and were utilized for experiments the subsequent day. Parasite growth in all the experiments shown in this figure was assessed via SYBR Green I DNA staining at 72 hours. Statistical evaluations employed one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *p<0.05, **p<0.01, ***p<0.001 when compared to Vehicle/Control data. Error bars illustrate standard deviation (n = 3).
Table 1.
MRM transition of FOH and GGOH..
The table displays the MRM transition of the FOH and GGOH for GC–MS/MS analysis. RT: Retention time, MRM: Multiple reaction monitoring.