A single point mutation in the Plasmodium falciparum FtsH1 metalloprotease confers actinonin resistance

  1. Christopher D Goodman  Is a corresponding author
  2. Taher Uddin
  3. Natalie J Spillman
  4. Geoffrey I McFadden
  1. School of BioSciences, University of Melbourne, Australia
1 figure, 2 tables and 2 additional files

Figures

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.
ParasiteFTSH1 Peptidase Motif
(partial amino acid sequence)
Actinonin IC50
(µM)
Tg FTSH1 WT (TGGT_259260)804 FGRDALSNGASSDI 81114a
Tg FTSH1 ActR804 FGRDALSSGASSDI 81144a
Pf 3D7 FTSH1 (PF3D7_1239700)481 FGKSETSSGASSDI 4941.99 (n = 1)b
Pf D10 FTSH1 WT481 FGKSETSSGASSDI 4942.0 ± 0.2 (n = 4)
Pf D10 FTSH1 ActR481 FGKSETSSCASSDI 49473.3 ± 2.7 (n = 4)
Pf D10 (apicoplast minus)481 FGKSETSSGASSDI 49443.1 ± 4.1 (n = 2)c
  1. 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
DesignationSource or referenceIdentifiersAdditional
information
Gene (Plasmodium falciparum)FtsH1PlasmoDB
(plasmodb.org)
PF3D7_1313200
Gene (Plasmodium falciparum)PDFPlasmoDB
(plasmodb.org)
PF3D7_0907900
Gene (Plasmodium falciparum)MAPPlasmoDB
(plasmodb.org)
PF3D7_0804400
Gene (Plasmodium falciparum)FMTPlasmoDB
(plasmodb.org)
PF3D7_1239700
Gene (Plasmodium falciparum)RINGPlasmoDB
(plasmodb.org)
PF3D7_1405700
Strain, strain background (Plasmodium falciparum)3D7MR4 - BEI Resources
(beiresources.org)
MRA-102
Strain, strain background (Plasmodium falciparum)D10MR4 - BEI Resources
(beiresources.org)
MRA-201
Strain, strain background (Plasmodium falciparum)D10 ActRThis paperTable 1
Transfected construct (Plasmodium falciparum)D10 rftsH1G489GThis paperFigure 1
Transfected construct (Plasmodium falciparum)D10 rftsH1G489CaThis paperFigure 1
Transfected construct (Plasmodium falciparum)D10 rftsH1G489CbThis paperFigure 1
Software, algorithmGraphPad Prism softwareGraphPad Prism (graphpad.com)RRID:SCR_002798
Chemical compound, drugActinoninSigmaSigma: A6671
Chemical compound, drugDSM-1SigmaSigma: 533304

Additional files

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.

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  1. Christopher D Goodman
  2. Taher Uddin
  3. Natalie J Spillman
  4. Geoffrey I McFadden
(2020)
A single point mutation in the Plasmodium falciparum FtsH1 metalloprotease confers actinonin resistance
eLife 9:e58629.
https://doi.org/10.7554/eLife.58629