A single point mutation in the Plasmodium falciparum FtsH1 metalloprotease confers actinonin resistance
- School of BioSciences, University of Melbourne, Australia
Figures
Figure 1
Allelic replacement in Pfftsh1 confers actinonin resistance.
(A) Genomic sequences of parasite lines. Upper line is 3D7 reference sequence with sgRNA (red arrow) and resistance mutation site (dark blue bar) marked. Bottom four lines are genomic sequence traces with shield and resistance mutations highlighted in light blue and predicted changes to amino acid sequence highlighted in yellow (B) Comparison of parasite growth inhibition (IC50) based on the presence of the G489C mutation. (C) Dose response curves of data presented in B. Data presented are the mean of 3–5 biological replicates. Error bars represent the standard error of the mean. P values represent two-tailed, unpaired t-test. (Full statistical analysis available in Supplementary file 1b).
Tables
Table 1
The impact of mutations in ftsh1 on parasite resistance to actinonin.
| Parasite | FTSH1 Peptidase Motif (partial amino acid sequence) | Actinonin IC50 (µM) |
|---|---|---|
| Tg FTSH1 WT (TGGT_259260) | 804 FGRDALSNGASSDI 811 | 14a |
| Tg FTSH1 ActR | 804 FGRDALSSGASSDI 811 | 44a |
| Pf 3D7 FTSH1 (PF3D7_1239700) | 481 FGKSETSSGASSDI 494 | 1.99 (n = 1)b |
| Pf D10 FTSH1 WT | 481 FGKSETSSGASSDI 494 | 2.0 ± 0.2 (n = 4) |
| Pf D10 FTSH1 ActR | 481 FGKSETSSCASSDI 494 | 73.3 ± 2.7 (n = 4) |
| Pf D10 (apicoplast minus) | 481 FGKSETSSGASSDI 494 | 43.1 ± 4.1 (n = 2)c |
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acalculated from data provided in Amberg-Johnson et al., 2017, b and c are both consistent with previously published data (Goodman and McFadden, 2014; Amberg-Johnson et al., 2017).
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Plasmodium falciparum) | FtsH1 | PlasmoDB (plasmodb.org) | PF3D7_1313200 | |
| Gene (Plasmodium falciparum) | PlasmoDB (plasmodb.org) | PF3D7_0907900 | ||
| Gene (Plasmodium falciparum) | MAP | PlasmoDB (plasmodb.org) | PF3D7_0804400 | |
| Gene (Plasmodium falciparum) | FMT | PlasmoDB (plasmodb.org) | PF3D7_1239700 | |
| Gene (Plasmodium falciparum) | RING | PlasmoDB (plasmodb.org) | PF3D7_1405700 | |
| Strain, strain background (Plasmodium falciparum) | 3D7 | MR4 - BEI Resources (beiresources.org) | MRA-102 | |
| Strain, strain background (Plasmodium falciparum) | D10 | MR4 - BEI Resources (beiresources.org) | MRA-201 | |
| Strain, strain background (Plasmodium falciparum) | D10 ActR | This paper | Table 1 | |
| Transfected construct (Plasmodium falciparum) | D10 rftsH1G489G | This paper | Figure 1 | |
| Transfected construct (Plasmodium falciparum) | D10 rftsH1G489Ca | This paper | Figure 1 | |
| Transfected construct (Plasmodium falciparum) | D10 rftsH1G489Cb | This paper | Figure 1 | |
| Software, algorithm | GraphPad Prism software | GraphPad Prism (graphpad.com) | RRID:SCR_002798 | |
| Chemical compound, drug | Actinonin | Sigma | Sigma: A6671 | |
| Chemical compound, drug | DSM-1 | Sigma | Sigma: 533304 |
Additional files
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Supplementary file 1
Supplementary Tables.
(a) Screen of D10 clones for Pfftsh1 sequence and actinonin resistance. (b) Descriptive statistics for growth inhibition trials in Figure 1. (c) Oligos for PCR amplification of potential actinonin targets. (d) Oligos for generation and sequencing of allelic replacement constructs.
- https://cdn.elifesciences.org/articles/58629/elife-58629-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/58629/elife-58629-transrepform-v2.docx
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A single point mutation in the Plasmodium falciparum FtsH1 metalloprotease confers actinonin resistance
eLife 9:e58629.
https://doi.org/10.7554/eLife.58629
