The NTP generating activity of pyruvate kinase II is critical for apicoplast maintenance in Plasmodium falciparum
- Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, United States
Figures
Figure 1
Carbon metabolism in the apicoplast.
Carbon backbones are imported into the apicoplast organelle in the form of 3-carbon phosphates by the apicoplast membrane outer triose phosphate transporter (oTPT) and inner triose phosphate …
Figure 2 with 1 supplement
Characterization of the PfMev Δotpt and Δitpt parasite lines.
(A) Genotyping PCR confirming deletion of oTPT or iTPT, with amplification demonstrating integration of the drug resistance cassette at the 5’ and 3’ loci of the target gene (Δ5’ and Δ3’), and lack …
Figure 2—figure supplement 1
Schematic and details describing gene deletion experiments.
(A) Schematic representation showing how targeted genes (WT Gene) were replaced with the hDHFR drug resistance cassette. The positions of genotyping PCR products designed to identify the wild-type …
Figure 3
Characterization of the PfMev Δtpi and ΔpyrkII parasite lines.
(A) Genotyping PCR confirming deletion of TPI or PyrKII, with amplification demonstrating drug cassette integration at the 5’ and 3’ loci of the target gene (Δ5’ and Δ3’), and lack of wild-type …
Figure 4
Characterization of the PfMev Δpdh E2 and Δdxs parasite lines.
(A) Genotyping PCR confirming deletion of PDH E2 or DXS, with amplification demonstrating drug cassette integration at the 5’ and 3’ loci of the target gene (Δ5’ and Δ3’), and lack of wild-type …
Figure 5 with 1 supplement
Growth of the PfMev CLD-PyrKII line under permissive and non-permissive conditional localization conditions.
(A) Schematic representation showing how the endogenous signal sequence (SS) and transit peptide (TP) of PyrKII were replaced with the CLD using plasmid pRSng. The positions of genotyping PCR …
Figure 5—figure supplement 1
Growth of the PfMev PyrKII TetR-DOZI line under permissive and non-permissive knockdown conditions.
Synchronized PfMev PyrKII TetR-DOZI parasites were seeded at 0.5% parasitemia and cultured under permissive (with aTc) or non-permissive (no aTc) conditions for 8 days. Parasites were collected …
Figure 6 with 4 supplements
Characterization of PfMev CLD-PyrKII parasites after mislocalization of PyrKII.
(A) Live epifluorescence microscopy of the PfMev CLD-PyrKII parasite line after Shield1 treatment. This parasite line expresses api-SFG (green) and is also stained with MitoTracker (red) and DAPI …
Figure 6—figure supplement 1
Analysis of apicoplast RNA levels in PfMev CLD-PyrKII parasites after treatment with Shield1.
(A) The microarray data shown in Figure 6C are plotted showing the averages of each RNA type, indicating that mRNAs (orange) seem to decrease in abundance prior to tRNA (blue) and rRNA (green) …
Figure 6—figure supplement 2
Mevalonate dependence of apicoplast and nuclear transcripts in PfMev Δdxs parasites quantified using RT-qPCR.
The relative transcript abundance of nuclear-encoded LDH and apicoplast encoded EF-Tu, LSU, and ClpM is shown in PfMev Δdxs parasites cultured with mevalonate (filled symbols) or without mevalonate …
Figure 6—figure supplement 3
Design and validation of the PfMev CLD-PyrKII api-luciferase parasite line.
(A) Schematic representation showing how p15-p230-aFluc-mCh plasmid linearized with SalI digestion was integrated into the p230p gene. Linearization results in the homology arms (HA1 and HA2) …
Figure 6—figure supplement 4
Sequences found in plasmid p15-p230-aFluc-mCh.
(A) The firefly luciferase protein (blue) is shown with an apicoplast-targeting peptide (green), mCherry fusion protein (red) and HA tag (orange). The K549E mutation to limit peroxisomal trafficking …
Figure 7
Roles of carbon metabolism proteins in the apicoplast.
Apicoplast proteins and pathways are colored according to gene deletion results (dispensable = green, essential = orange, and essential for apicoplast maintenance = red). Pyruvate kinase II (PyrKII) …
Author response image 1
Expression and localization of lactate dehydrogenase (LDH) in the apicoplast.
A) Genotyping PCR confirming insertion of CLD-LDH-mCherry (clone E7), with amplification demonstrating plasmid integration at the 5’ and 3’ loci of the P230p locus (lanes a, b), and lack of …
Tables
Table 1
Enzyme kinetics of pure recombinant PyrKII as compared to T. gondii PyrKII.
Activity of PyrKII was measured using various NDPs and dNDPs as substrates. Values and standard deviations from triplicate measurements are reported for the kinetic parameters Kcat and Km. The ratio …
| Substrate | Kcat (min−1) | Km (mM) | Kcat/Km (mM−1 min−1) | Organism |
|---|---|---|---|---|
| ADP | 793 + / - 186 | 0.25 + / - 0.11 | 3200 | P. falciparum |
| GDP | 397 + / - 45 | 0.07 + / - 0.04 | 5890 | |
| CDP | 438 + / - 69 | 1.01 + / - 0.13 | 432 | |
| UDP | 252 + / - 40 | 0.82 + / - 0.22 | 306 | |
| dADP | 401 + / - 31 | 0.38 + / - 0.04 | 1066 | |
| dGDP | 90 + / - 5 | 0.28 + / - 0.03 | 318 | |
| dCDP | 78+ / - 3 | 0.52 + / - 0.12 | 149 | |
| ADP | 2850 + / - 80 | 8.0 + / - 0.40 | 360 | T. gondii |
| GDP | 6600+ / - 240 | 0.05 + / - 0.006 | 121,320 |
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Table 1—source data 1
Nucleotide sequence of P. falciparum PyrKII codon harmonized for expression in E. coli.
The endonuclease sites used for cloning (EcoRI and HindIII) are shown in lower case italics and the stop codon is highlighted in red.
- https://cdn.elifesciences.org/articles/50807/elife-50807-table1-data1-v2.pdf
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Table 1—source data 2
Purification of recombinant P. falciparum PyrKII.
A maltose binding protein (MBP), along with a histidine (His) tag were appended to the N-terminus of harmonized PyrKII sequence and expressed in E. coli. The MBP-His-PyrkII fusion protein (126,273 Da) can be seen in lane 7, after purification using an MBP column. The TEV cleaved product, His-PyrKII (83,014 Da) can be seen in lane 8. Lane 10 shows the purified product and a minor band consistent with some covalent dimer (166 kDa).
- https://cdn.elifesciences.org/articles/50807/elife-50807-table1-data2-v2.pdf
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Table 1—source data 3
Nucleotide substrate kinetic plots for His-PyrKII.
Dose-response curves from triplicate data for seven nucleotide substrates are shown fitted with hyperbolic curves based on the Michaelis-Menton equation. Insets show the same data plotted in double reciprocal (Lineweaver-Burk) plots. Error bars represent the standard error of the mean and the program Prism (GraphPad) was used to generate the plots in this figure.
- https://cdn.elifesciences.org/articles/50807/elife-50807-table1-data3-v2.pdf
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Table 1—source data 4
Nucleotide substrate kinetic plots for His-PyrKII.
Dose-response curves from triplicate data for seven nucleotide substrates are shown fitted with hyperbolic curves based on the Michaelis-Menton equation. Insets show the same data plotted in double reciprocal (Lineweaver-Burk) plots. Error bars represent the standard error of the mean and the program Prism (GraphPad) was used to generate the plots in this figure.
- https://cdn.elifesciences.org/articles/50807/elife-50807-table1-data4-v2.pdf
Additional files
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Supplementary file 1
Oligonucleotides used in this study.
- https://cdn.elifesciences.org/articles/50807/elife-50807-supp1-v2.xls
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Supplementary file 2
Analysis of microarray data.
- https://cdn.elifesciences.org/articles/50807/elife-50807-supp2-v2.xls
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50807/elife-50807-transrepform-v2.docx
