Summary of Study ST001279

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000864. The data can be accessed directly via it's Project DOI: 10.21228/M80T2X This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST001279
Study TitleK13 mutations driving artemisinin resistance rewrite Plasmodium falciparum’s programmed intra-erythrocytic development and transform mitochondrial physiology
Study SummaryThe emergence of artemisinin resistance in Southeast Asia, dictated by mutations in the Plasmodium falciparum k13 gene, has compromised antimalarial efficacy and created a core vulnerability in the global malaria elimination campaign. Applying quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we observe that K13 mutations reprogram multiple aspects of intra-erythrocytic parasite biology. These changes impact its cell cycle periodicity, the unfolded protein response and protein degradation, vesicular trafficking and endocytosis, and mitochondrial functions including the TCA cycle, the electron transport chain, and redox regulation. Ring-stage artemisinin resistance mediated by the K13 R539T mutation was neutralized using atovaquone, an electron transport chain inhibitor. Our data suggest that modification of mitochondrial physiology, accompanied by other processes to reduce artemisinin’s proteotoxic effects, help protect parasites against this pro-oxidant drug, allowing resumption of growth once the rapidly-cleared artemisinins have reached sub-therapeutic levels.
Institute
Pennsylvania State University
Last NameLlinás
First NameManuel
AddressW126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
Emailmul27@psu.edu
Phone(814) 867-3527
Submit Date2019-11-18
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2020-06-01
Release Version1
Manuel Llinás Manuel Llinás
https://dx.doi.org/10.21228/M80T2X
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR000864
Project DOI:doi: 10.21228/M80T2X
Project Title:K13 mutations driving artemisinin resistance rewrite Plasmodium falciparum’s programmed intra-erythrocytic development and transform mitochondrial physiology
Project Summary:The emergence of artemisinin resistance in Southeast Asia, dictated by mutations in the Plasmodium falciparum k13 gene, has compromised antimalarial efficacy and created a core vulnerability in the global malaria elimination campaign. Applying quantitative transcriptomics, proteomics, and metabolomics to a panel of isogenic K13 mutant or wild-type P. falciparum lines, we observe that K13 mutations reprogram multiple aspects of intra-erythrocytic parasite biology. These changes impact its cell cycle periodicity, the unfolded protein response and protein degradation, vesicular trafficking and endocytosis, and mitochondrial functions including the TCA cycle, the electron transport chain, and redox regulation. Ring-stage artemisinin resistance mediated by the K13 R539T mutation was neutralized using atovaquone, an electron transport chain inhibitor. Our data suggest that modification of mitochondrial physiology, accompanied by other processes to reduce artemisinin’s proteotoxic effects, help protect parasites against this pro-oxidant drug, allowing resumption of growth once the rapidly-cleared artemisinins have reached sub-therapeutic levels.
Institute:Pennsylvania State University
Last Name:Llinás
First Name:Manuel
Address:W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA
Email:mul27@psu.edu
Phone:(814) 867-3527

Subject:

Subject ID:SU001351
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833

Factors:

Subject type: Cultured cells; Subject species: Plasmodium falciparum (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA093379MTZC2350.1_t4350nM DHA
SA093380C2350.3_t7350nM DHA
SA093381H4350.1_t8350nM DHA
SA093382MTZC2350.2_t4350nM DHA
SA093383C2350_t6350nM DHA
SA093384MTZC2350.3_t4350nM DHA
SA093385H4350_t6350nM DHA
SA093386C2350.1_t7350nM DHA
SA093387C2350.2_t7350nM DHA
SA093388H4350.2_t8350nM DHA
SA093389H4350.3_t8350nM DHA
SA093390H4350.1_t7350nM DHA
SA093391MTZH4350.1_t3350nM DHA
SA093392C2350.1_t8350nM DHA
SA093393C2350.2_t8350nM DHA
SA093394C2350.3_t8350nM DHA
SA093395MTZH4350.2_t3350nM DHA
SA093396MTZH4350.3_t3350nM DHA
SA093397H4350.3_t7350nM DHA
SA093398H4350.2_t7350nM DHA
SA093399C270.2_t770nM DHA
SA093400C270.1_t770nM DHA
SA093401C270.3_t770nM DHA
SA093402H470.2_t870nM DHA
SA093403C270.2_t870nM DHA
SA093404C270.3_t870nM DHA
SA093405C270.1_t870nM DHA
SA093406H470.3_t870nM DHA
SA093407H470.3_t770nM DHA
SA093408H470.1_t870nM DHA
SA093409H470.2_t770nM DHA
SA093410MTZH470.3_t370nM DHA
SA093411MTZC270.2_t470nM DHA
SA093412MTZC270.3_t470nM DHA
SA093413MTZH470.2_t370nM DHA
SA093414MTZH470.1_t370nM DHA
SA093415H470.1_t770nM DHA
SA093416MTZC270.1_t470nM DHA
SA093417MTZC2DM.2_t4DMSO
SA093418MTZC2DM.3_t4DMSO
SA093419MTZC2DM.1_t4DMSO
SA093420C2DM.3_t8DMSO
SA093421MTZH4DM.2_t3DMSO
SA093422MTZH4DM.3_t3DMSO
SA093423C2DM.2_t8DMSO
SA093424C2DM.1_t8DMSO
SA093425H4DM.3_t8DMSO
SA093426C2DM.3_t7DMSO
SA093427H4DM.1_t7DMSO
SA093428C2DM.2_t7DMSO
SA093429C2DM.1_t7DMSO
SA093430H4DM.3_t7DMSO
SA093431H4DM.2_t7DMSO
SA093432MTZH4DM.1_t3DMSO
SA093433C2DM_t6DMSO
SA093434H4DM.1_t8DMSO
SA093435H4DM.2_t8DMSO
SA093436H4DM_t6DMSO
SA093437H4ND_t6None
SA093438MTZC2ND.3_t4None
SA093439MTZC2ND.2_t4None
SA093440MTZC2ND.1_t4None
SA093441C2ND_t6None
Showing results 1 to 63 of 63

Collection:

Collection ID:CO001345
Collection Summary:P. falciparum parasites were cultured at 3% hematocrit in human O+ RBCs (Interstate blood bank, USA) and P. falciparum culture media comprising of RPMI1640 (Thermo Fisher Scientific) supplemented with 0.5% (w/v) Albumax II, 50mg/L hypoxanthine, 0.2% NaHCO3, 25mM HEPES and 10mg/L gentamycin (Fidock et al., 1998). Parasites were cultured at 37ºC in 5% O2, 5%CO2 and 90% N2. For the collection of RNA, proteins and metabolite extracts, parasite cultures at 3% hematocrit and 20 mL or 200 mL volumes were kept in T75 or T225 flasks respectively, with daily media changes. Parasite lines were genotyped by Sanger sequencing for the k13 gene to verify their identities before the start of an experiment.
Sample Type:Parasite

Treatment:

Treatment ID:TR001366
Treatment Summary:Mycoplasma-free Cam3.IIC580Y and Cam3.IIWT parasites were doubly synchronized by 5% D-Sorbitol in each generation for at least two generations. 0-3 hpi early rings of each parasite line were treated for 3h at 350 nM or 70 nM DHA along with vehicle-treated 0.05% DMSO controls in two to three independent experiments. 24 hpi trophozoites were similarly treated with DHA or DMSO control in a single experiment for each parasite line and subsequently magnetically enriched using MACS CS columns on the SuperMACS™ II Separator (Miltenyi Biotec, Inc.) to remove uninfected RBCs.

Sample Preparation:

Sampleprep ID:SP001359
Sampleprep Summary:The K13 mutant and WT ring-stage parasites were run in parallel for each metabolomic experiment to allow direct comparisons of their metabolomic states and the effects of DHA. Metabolites from saponin-lysed parasites were extracted with cold methanol along with spike-in control [13C4, 15N1]-Aspartate (Cambridge Isotope) as an internal LC/MS standard to correct for technical variation arising from sample processing in the data analysis phase, as described previously (Allman et al., 2016).Samples were injected on a Thermo Exactive Plus Orbitrap in negative ion mode (Lu, W. et al., Anal. Chem. 2010.).

Combined analysis:

Analysis ID AN002120
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Dionex Ultimate 3000
Column Phenomenex Synergi Hydro RP 100 A (100 x 2mm,2.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units Peak Area

Chromatography:

Chromatography ID:CH001553
Instrument Name:Thermo Dionex Ultimate 3000
Column Name:Phenomenex Synergi Hydro RP 100 A (100 x 2mm,2.5um)
Chromatography Type:Reversed phase

MS:

MS ID:MS001975
Analysis ID:AN002120
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Raw data files from the Thermo Exactive Plus orbitrap (.raw) were converted to a format compatible with our analysis software (.raw¡.mzXML) and spectral data (.mzXML files) were visualized in MAVEN. The labeled [13C4, 15N1]-Aspartate internal standard intensity was assessed for technical reproducibility. Peaks for each metabolite in the targeted library were identified based on proximity to standard retention time, the observed mass falling within 10 ppm of the expected m/z (calculated from the monoisotopic mass), and the signal/blank ratio (minimum, 10,000 ions). Based upon the above criteria, peaks were manually inspected and demarcated as good or bad based on peak shape. Peak areas were exported into an R working environment (http://www.R-project.org) to calculate log2 fold changes for each sample compared to an untreated control. Metabolites that were not reliably detected across 90% of all the trials were removed prior to additional analysis to minimize subsequent imputation bias. The peak areas for any remaining metabolites not detected were imputed to have 10,000 ions, and metabolites detected below background levels (negative after blank subtraction) were maintained as “0” prior to averaging and log2 calculation. Because our extraction method did not include a wash step we excluded metabolites found in the RPMI-based medium.
Ion Mode:NEGATIVE
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