Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 10

<p><b>Percentage parasite proliferation of the different lines in the presence of the pantothenate analogues (a) N5-trz-C1-Pan and (b) <i>N</i>-PE-αMe-PanAm, and inhibition of [¹⁴C]pantothenate phosphorylation in parasite lysates by various pantothenate analogues: (c) PanOH, (d) CJ-15,801, (e) N5-trz-C1-Pan and (f) <i>N</i>-PE-αMe-PanAm.</b> Symbols represent Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasite lines. The Y-axes of c−f indicate the percentage of total pantothenate phosphorylation. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate for the parasite proliferation assays and duplicate for the phosphorylation assays. Error bars represent SEM and are not visible if smaller than the symbols.</p

The pantothenate requirement of the different parasite lines observed in this study.

<p>(a) Percentage proliferation of parasites grown in different pantothenate concentrations. Values are averaged from ≥ 3 independent experiments, each carried out in triplicate. Error bars represent SEM and are not visible if smaller than the symbols. For clarity, only data from the Parent (white circles), CJ-A (black diamonds), and CJ-A^+WTPfPanK1 (grey diamonds) lines are shown. (b) The pantothenate stimulatory concentration 50 (SC₅₀) values obtained for Parent, PanOH-A, PanOH-B, CJ-A, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 line parasites, which is the concentration of pantothenate required in the medium to support parasite proliferation by 50% (with 100% set to parasites grown in complete medium containing 1 <i>μ</i>M pantothenate). Errors represent SEM (n ≥ 3). An asterisk indicates that the value is significantly different from that obtained for the Parent line (CJ-A SC₅₀ value 95% CI compared to Parent: 2.7 to 30.9). No significant difference was observed between the SC₅₀ value of the Parent and those of PanOH-A, PanOH-B, Parent^+WTPfPanK1 and CJ-A^+WTPfPanK1 (95% CI compared to Parent: PanOH-A = -6.8 to 7.1, PanOH-B = -3.8 to 9.3, Parent^+WTPfPanK1 = -9.3 to 3.3 & CJ-A^+WTPfPanK1 = -7.8 to 6.6).</p

Mutations in <i>Pfpank1</i> and the affected residues within the <i>Pf</i>PanK1 protein.

<p>(a) The single nucleotide polymorphisms detected in the <i>Pfpank1</i> gene (accession number: PF3D7_1420600) of PanOH-A, PanOH-B and CJ-A, and the corresponding amino acid changes in the <i>Pf</i>PanK1 protein. (b) A three-dimensional homology model of the <i>Pf</i>PanK1 protein (pink) based on the solved structure of human PanK3 (PDB ID: 5KPR), overlaid on the human PanK3 structure in its active conformation (blue), with an ATP analogue (AMPPNP; carbon atoms coloured green) and pantothenate (carbon atoms coloured yellow) bound. <i>Pf</i>PanK1 shares 28% sequence identity with human PanK3 over the parts of the protein that have been modeled. Red spheres indicate the residues (G95 and D507) affected by the mutations in the parasite proteins. Human PanK3 has been shown to exist as a dimer. Here, individual monomers are shown in different shades of pink and blue.</p

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 2

<p><b>Percentage proliferation of parasites from the Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) lines in the presence of (a) PanOH, (b) CJ-15,801 or (c) chloroquine.</b> Drug-pressured lines were generated by exposing Parent line parasites to 11 − 13 weeks of continuous drug-pressuring with either PanOH (for PanOH-A and PanOH-B) or CJ-15,801 (for CJ-A), followed by limiting dilution cloning. Values are averaged from ≥ 4 independent experiments, each carried out in triplicate. All error bars represent SEM. Error bars are not visible if smaller than the symbols.</p

The fitness of the different mutant lines generated in this study relative to the Parent line as determined from parasite competition assays.

<p>(a) A flow chart illustrating how the competition assay was performed. For each competition culture, an equal number of parasites from the Parent and a mutant line were combined into a single flask. These mixed cultures were maintained for a period of 6 weeks. The fitness cost associated with the <i>Pfpank1</i> mutations was assessed by determining the PanOH sensitivity of the mixed cultures: (b) Parent vs PanOH-A (grey triangles), (c) Parent vs PanOH-B (grey squares) and (d) Parent vs CJ-A (grey diamonds), at Week 0 (W0; dashed lines) and Week 6 (W6; solid lines). It was expected that the greater the fitness cost to the mutant, the greater the shift of its mixed culture PanOH dose-response curve toward the Parent line curve after 6 weeks. Arrows indicate this shift between W0 and W6. The parasite proliferation curves (dotted lines) of the respective mutant clones (black symbols) and Parent line (white circles) are also shown for comparison. Values for the mixed cultures are averaged from 2 independent experiments, each carried out in triplicate. Error bars represent SEM (n ≥ 4) for the individual cultures and range/2 for the mixed cultures, and are not visible if smaller than the symbols.</p

The phosphorylation of [¹⁴C]pantothenate (2 <i>μ</i>M) over time (in minutes) by lysates generated from Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasites.

<p>Inset shows [¹⁴C]pantothenate (2 <i>μ</i>M supplemented with non-radioactive pantothenate to a total pantothenate concentration of 200 <i>μ</i>M) phosphorylation by CJ-A parasite lysates measured over 420 min. Values are averaged from 3 independent experiments, each performed with a different batch of lysate and carried out in duplicate. Error bars represent SEM and are not visible if smaller than the symbols.</p

Confocal micrographs showing the subcellular location of GFP-tagged <i>Pf</i>PanK1 in 3D7 strain parasites harbouring the <i>Pfpank1</i>-pGlux-1 episomal plasmid.

<p>From left to right: Brightfield, GFP-fluorescence, DAPI- (fixed cells; top) or Hoechst 33258- (live cells; bottom) fluorescence, and merged images of erythrocytes infected with trophozoite-stage <i>P</i>. <i>falciparum</i> parasites expressing <i>Pf</i>PanK1-GFP. Arrows indicate the plasma membranes of either the erythrocytes (black) or the parasites (white). Scale bar represents 5 <i>μ</i>m.</p

<i>Pf</i>PanK activity profiles derived from the initial rates of pantothenate phosphorylation by lysates generated from Parent (white circles), PanOH-A (black triangles), PanOH-B (black squares) and CJ-A (black diamonds) parasites at various pantothenate concentrations.

<p>The grey diamond indicates a data point that was outside the 95% confidence band when it was included in the non-linear regression. This data point was therefore deemed an outlier and was omitted from the data set used to generate the fitted curve shown. Values are averaged from 3 independent experiments, each performed with a different batch of lysate and carried out in duplicate. Error bars represent SEM and are not visible if smaller than the symbols. Relative specificity constant (rsc) values express the specificity constants obtained for each cell line relative to that of the Parent. The <i>V</i>_max (<i>μ</i>mol/(10¹² cells.h)), <i>K</i>_m (<i>μ</i>M) and rsc values determined from the curves are shown within each box. Errors represent SEM (n = 3). An asterisk indicates that the value is significantly different compared to the Parent line (<i>V</i>_max 95% CI compared to Parent: PanOH-A = 173 to 344, PanOH-B = 5.2 to 21.6 & CJ-A = 112 to 133; <i>K</i>_m 95% CI compared to Parent: PanOH-A = 8.8 to 13.3, PanOH-B = 8.3 to 20.7 & CJ-A = 42 to 484; rsc 95% CI compared to Parent: PanOH-B = -1.366 to -0.519 & CJ-A = -1.404 to -0.557). No significant difference was observed between the rsc of the Parent and PanOH-A (95% CI compared to Parent: -0.698 to 0.179).</p

Structures of pantothenate and the pantothenate analogues used in this study.

<p>Structures of pantothenate and the pantothenate analogues used in this study.</p

Mutations in the pantothenate kinase of <i>Plasmodium falciparum</i> confer diverse sensitivity profiles to antiplasmodial pantothenate analogues - Fig 4

<p><b>The fold change in (a) PanOH, (b) CJ-15,801 and (c) chloroquine IC₅₀ values of PanOH-A, PanOH-B and CJ-A parasite lines, in the absence (black bars) or presence (grey bars) of wild-type <i>Pf</i>PanK1 complementation, relative to that of their corresponding parental lines.</b> Each fold change value is averaged from ≥ 3 independent experiments and error bars represent SEM. An asterisk indicates that the fold change in sensitivity of the mutant is significantly altered by complementation (PanOH fold change 95% CI: PanOH-A = -5.29 to -2.05, PanOH-B = -6.25 to -2.26 & CJ-A = -13.00 to -4.62; CJ-15,801 fold change 95% CI: PanOH-A = -4.17 to -0.79, PanOH-B = -6.14 to -1.06 & CJ-A = -8.05 to -4.22). No change in chloroquine sensitivity was observed (95% CI: PanOH-A = -0.23 to 0.43, PanOH-B = -0.09 to 0.40 & CJ-A = -0.01 to 0.59). The fold change in the sensitivity to CJ-15,801 for PanOH-B and CJ-A before and after complementation was calculated using IC₃₅ values.</p