Summary of Study ST003904

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 PR002442. The data can be accessed directly via it's Project DOI: 10.21228/M80544 This work is supported by NIH grant, U2C- DK119886.

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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 IDST003904
Study TitlePlasmodium falciparum plasmepsin copy number and piperaquine treatment have no effect of hemoglobin digestion - Negative Mode
Study SummaryGlobal malaria control has plateaued, with drug-resistant Plasmodium falciparum posing a significant challenge. Artemisinin-based combination therapies (ACTs) are becoming less effective, especially in South-East Asia, where resistance to dihydroartemisinin-piperaquine (DHA-PPQ) is leading to treatment failures, notably in Cambodia. Genome-wide association studies link artemisinin resistance to kelch13 mutations, while decreased PPQ sensitivity is tied to higher plasmepsin II and III gene copies and mutations in the chloroquine resistance transporter. We previously showed a connection between increased plasmepsin gene copies and reduced PPQ sensitivity. In this study we try to understand the biological role of the plasmepsins in PPQ sensitivity. Therefore, we knocked out plasmepsin II and III genes in Cambodian strains using CRISPR/Cas9, and found increased PPQ sensitivity, confirming these genes' roles in resistance. Plasmepsins are proteases that participate in the hemoglobin degradation cascade in the digestive vacuole of the parasites. Protease inhibitor experiments and hemoglobin digestion studies indicate that digestive vacuole pH fluctuations affect PPQ response, highlighting the need for further research into PPQ resistance mechanisms.
Institute
Pennsylvania State University
DepartmentBiochemistry and Molecular Biology
LaboratoryManuel Llinás
Last NameRangel
First NameGabriel
Address491 Pollock Road, University Park, PA, 16802, USA
Emailgwr5170@psu.edu
Phone8148673527
Submit Date2025-05-06
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-05-28
Release Version1
Gabriel Rangel Gabriel Rangel
https://dx.doi.org/10.21228/M80544
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002442
Project DOI:doi: 10.21228/M80544
Project Title:Neither Plasmodium falciparum Plasmepsin Copy Number Nor Piperaquine Treatment Impact Hemoglobin Digestion
Project Summary:Malaria is still a major health issue in many parts of the world, particularly in tropical and subtropical regions of Africa, Asia, and Latin America. Despite significant efforts to control and eliminate the disease, malaria remains a leading cause of illness and death, mainly due to the occurrence of drug-resistant parasites to the frontline antimalarials such as dihydroartemisinin-piperaquine (DHA-PPQ). Partial artemisinin resistance has been linked to kelch13 mutations, while decreased PPQ sensitivity has been associated with higher plasmepsin II and III gene copies and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR/Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III, as well as a double KOs of both genes, in two isogenic lines of Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased the parasites' sensitivity to PPQ, evaluated by the area under the curve. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. By treating parasites with digestive vacuole (DV) function modulators, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying PPQ resistance in Cambodian parasites.
Institute:Pennsylvania State University
Department:Biochemistry and Molecular Biology
Laboratory:Manuel Llinás
Last Name:Rangel
First Name:Gabriel
Address:491 Pollock Road, University Park, PA, 16802, USA
Email:gwr5170@psu.edu
Phone:8148673527

Subject:

Subject ID:SU004039
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833
Cell Strain Details:3D7, RF7 B9, or RF7 D4

Factors:

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

mb_sample_id local_sample_id Sample source Genotype Treatment
SA42976620220923_C18_Neg_38-6.30_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42976720220923_C18_Neg_34-6.30_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42976820220923_C18_Neg_45-7.1_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42976920220923_C18_Neg_46-7.1_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977020220923_C18_Neg_47-7.1_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977120220923_C18_Neg_60-7.12_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977220220923_C18_Neg_13-6.21_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977320220923_C18_Neg_64-7.12_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977420220923_C18_Neg_9-6.21_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977520220923_C18_Neg_8-6.21_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977620220923_C18_Neg_66-7.12_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977720220923_C18_Neg_35-6.30_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977820220923_C18_Neg_92-7.15_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42977920220923_C18_Neg_98-7.15_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42978020220923_C18_Neg_101-7.15_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42978120220923_C18_Neg_110-7.8_ATQ_3Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42978220220923_C18_Neg_115-7.8_ATQ_2Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42978320220923_C18_Neg_116-7.8_ATQ_1Cultured Plasmodium falciparum 3D7 WT 10 nM Atovaquone
SA42978420220923_C18_Neg_88-7.15_PPQ_1Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42978520220923_C18_Neg_79-7.12_PPQ_2Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42978620220923_C18_Neg_75-7.12_PPQ_1Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42978720220923_C18_Neg_96-7.15_PPQ_2Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42978820220923_C18_Neg_107-7.8_PPQ_3Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42978920220923_C18_Neg_108-7.8_PPQ_1Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42979020220923_C18_Neg_121-7.8_PPQ_2Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42979120220923_C18_Neg_83-7.15_PPQ_3Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42979220220923_C18_Neg_76-7.12_PPQ_3Cultured Plasmodium falciparum 3D7 WT 140 nM Piperaquine
SA42979320220923_C18_Neg_37-6.30_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42979420220923_C18_Neg_71-7.12_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA42979520220923_C18_Neg_120-7.8_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA42979620220923_C18_Neg_114-7.8_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42979720220923_C18_Neg_42-7.1_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA42979820220923_C18_Neg_104-7.15_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA42979920220923_C18_Neg_94-7.15_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42980020220923_C18_Neg_85-7.15_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42980120220923_C18_Neg_73-7.12_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42980220220923_C18_Neg_113-7.8_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42980320220923_C18_Neg_70-7.12_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42980420220923_C18_Neg_53-7.1_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42980520220923_C18_Neg_10-6.21_ND_3Cultured Plasmodium falciparum 3D7 WT None
SA42980620220923_C18_Neg_26-6.30_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42980720220923_C18_Neg_14-6.21_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42980820220923_C18_Neg_25-6.30_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA42980920220923_C18_Neg_56-7.1_ND_2Cultured Plasmodium falciparum 3D7 WT None
SA42981020220923_C18_Neg_16-6.21_ND_1Cultured Plasmodium falciparum 3D7 WT None
SA429811Neg_RF7_B9_1_1Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429812Neg_RF7_B9_2_1Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429813Neg_RF7_B9_2_2Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429814Neg_RF7_B9_2_3Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429815Neg_RF7_B9_3_1Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429816Neg_RF7_B9_3_2Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429817Neg_RF7_B9_1_2Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429818Neg_RF7_B9_3_3Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429819Neg_RF7_B9_1_3Cultured Plasmodium falciparum RF7_B9 1 copy plasmepsin II/III None
SA429820Neg_RF7_D4_1_2Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429821Neg_RF7_D4_1_3Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429822Neg_RF7_D4_2_1Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429823Neg_RF7_D4_2_2Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429824Neg_RF7_D4_2_3Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429825Neg_RF7_D4_3_1Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429826Neg_RF7_D4_3_2Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429827Neg_RF7_D4_3_3Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA429828Neg_RF7_D4_1_1Cultured Plasmodium falciparum RF7_D4 3 copies plasmepsin II/III None
SA42982920220923_C18_Neg_6.21_QC3Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983020220923_C18_Neg_7.15_QC15Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983120220923_C18_Neg_7.12_QC10Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983220220923_C18_Neg_7.15_QC14Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983320220923_C18_Neg_6.21_QC2Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983420220923_C18_Neg_7.15_QC13Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983520220923_C18_Neg_7.12_QC12Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983620220923_C18_Neg_6.30_QC4Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983720220923_C18_Neg_7.12_QC11Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983820220923_C18_Neg_7.8_QC18Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42983920220923_C18_Neg_7.8_QC17Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984020220923_C18_Neg_7.8_QC16Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984120220923_C18_Neg_7.1_QC9Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984220220923_C18_Neg_7.1_QC8Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984320220923_C18_Neg_7.1_QC7Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984420220923_C18_Neg_6.30_QC6Pooled Cultured Plasmodium falciparum 3D7 WT NA
SA42984520220923_C18_Neg_6.21_QCPooled Cultured Plasmodium falciparum 3D7 WT NA
SA429846Neg_RF7_QC_1Pooled Cultured Plasmodium falciparum RF7_B9 and RF7_D4 NA NA
SA429847Neg_RF7_QC_2Pooled Cultured Plasmodium falciparum RF7_B9 and RF7_D4 NA NA
SA429848Neg_RF7_QC_3Pooled Cultured Plasmodium falciparum RF7_B9 and RF7_D4 NA NA
SA429849Neg_RF7_QC_4Pooled Cultured Plasmodium falciparum RF7_B9 and RF7_D4 NA NA
Showing results 1 to 84 of 84

Collection:

Collection ID:CO004032
Collection Summary:Synchronized cultures of trophozoite stage (24-28 hours post invasion) 3D7 parasites (5-10% parasitemia, 2% hematocrit) were purified from uninfected RBCs by VarioMacs Magnet using a MACS CS column. The purified parasite pellet was resuspended at 5X107 – 1X108 cells/mL in media, and allowed to recover for 1 hour at 37°C.
Sample Type:Parasite

Treatment:

Treatment ID:TR004048
Treatment Summary:Following recovery, purified trophozoites were incubated in 6-well plates with Atovaquone (positive control) or piperaquine at 10 X IC50, or no drug as a control or for the R7 line comparisons, for 2.5 h at 37°C.

Sample Preparation:

Sampleprep ID:SP004045
Sampleprep Summary:Immediately following treatment, parasite pellets were washed with 1 mL 1X ice-cold PBS, before being resuspended in 1 mL prechilled 90:10 methanol-water and placed on ice. Samples were vortexed, resuspended and centrifuged for 10 min at 15,000 rpm and 4°C. Samples were stored at -80°C before being dried down under nitrogen flow for HPLC-MS analysis. The dried metabolites were resuspended in HPLC-grade water, containing chlorpropamide as an internal control, to a concentration between 1X105 and 1X106 cell/mL, based on hemocytometer counts of purified parasites.

Chromatography:

Chromatography ID:CH004860
Instrument Name:Shimadzu Prominence 20 UFLCXR
Column Name:Waters ACQUITY UPLC BEH C18 (100 x 2.1 mm, 1.7 μm)
Column Temperature:55°C
Flow Gradient:0.0 min 3% of B, 10.0 min 45% of B, 12.0 min 75% of B, 17.5 min 75% of B, and 18.0-20.0 min: 3% of B
Flow Rate:250 μL/min
Solvent A:100% Water; 0.1% Formic acid
Solvent B:100% Acetonitrile; 0.1% Formic acid
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006409
Analysis Type:MS
Chromatography ID:CH004860
Num Factors:7
Num Metabolites:152
Units:blank-subtracted peak area
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