Summary of Study ST003906

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.

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.

Study ID	ST003906
Study Title	Neither Plasmodium falciparum Plasmepsin Copy Number Nor Piperaquine Treatment Impact Hemoglobin Digestion
Study Summary	Global 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
Last Name	Rangel
First Name	Gabriel
Address	491 Pollock Road, University Park, PA, 16802, USA
Email	grangel0955@gmail.com
Phone	8148673527
Submit Date	2025-05-02
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-05-28
Release Version	1

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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:	SU004041
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	Genotype	Treatment
SA429922	7_12_ATQ_3	WT	10 nM Atovaquone
SA429923	6_21_ATQ_2_rerun	WT	10 nM Atovaquone
SA429924	6_30_ATQ_3	WT	10 nM Atovaquone
SA429925	6_30_ATQ_2	WT	10 nM Atovaquone
SA429926	6_30_ATQ_1	WT	10 nM Atovaquone
SA429927	7_15_ATQ_3	WT	10 nM Atovaquone
SA429928	7_15_ATQ_2	WT	10 nM Atovaquone
SA429929	7_15_ATQ_1	WT	10 nM Atovaquone
SA429930	6_21_ATQ_1_rerun	WT	10 nM Atovaquone
SA429931	7_12_ATQ_2	WT	10 nM Atovaquone
SA429932	7_12_ATQ_1	WT	10 nM Atovaquone
SA429933	7_8_ATQ_3	WT	10 nM Atovaquone
SA429934	7_8_ATQ_2	WT	10 nM Atovaquone
SA429935	7_8_ATQ_1	WT	10 nM Atovaquone
SA429936	7_1_ATQ_3	WT	10 nM Atovaquone
SA429937	7_1_ATQ_2	WT	10 nM Atovaquone
SA429938	7_1_ATQ_1	WT	10 nM Atovaquone
SA429939	6_21_ATQ_3_rerun	WT	10 nM Atovaquone
SA429940	7_15_PPQ_3	WT	140 nM Piperaquine
SA429941	7_15_PPQ_2	WT	140 nM Piperaquine
SA429942	7_15_PPQ_1	WT	140 nM Piperaquine
SA429943	7_12_PPQ_3	WT	140 nM Piperaquine
SA429944	7_12_PPQ_2	WT	140 nM Piperaquine
SA429945	7_12_PPQ_1	WT	140 nM Piperaquine
SA429946	7_8_PPQ_3	WT	140 nM Piperaquine
SA429947	7_8_PPQ_2	WT	140 nM Piperaquine
SA429948	7_8_PPQ_1	WT	140 nM Piperaquine
SA429949	7_15_ND_3	WT	None
SA429950	7_15_ND_2	WT	None
SA429951	7_15_ND_1	WT	None
SA429952	7_12_ND_3	WT	None
SA429953	7_12_ND_2	WT	None
SA429954	7_12_ND_1	WT	None
SA429955	7_8_ND_3	WT	None
SA429956	7_8_ND_2	WT	None
SA429957	7_8_ND_1	WT	None
SA429958	7_1_ND_3	WT	None
SA429959	6_21_ND_2	WT	None
SA429960	7_1_ND_2	WT	None
SA429961	6_21_ND_1	WT	None
SA429962	6_30_ND_2	WT	None
SA429963	6_21_ND_3	WT	None
SA429964	6_30_ND_3	WT	None
SA429965	7_1_ND_1	WT	None
SA429966	6_30_ND_1	WT	None
SA429967	QC11	WT	QC
SA429968	QC17	WT	QC
SA429969	QC16	WT	QC
SA429970	QC15	WT	QC
SA429971	QC14	WT	QC
SA429972	QC13	WT	QC
SA429973	QC12	WT	QC
SA429974	QC8	WT	QC
SA429975	QC10	WT	QC
SA429976	QC9	WT	QC
SA429977	QC7	WT	QC
SA429978	QC5	WT	QC
SA429979	QC4_rerun	WT	QC
SA429980	QC3_rerun	WT	QC
SA429981	QC2_rerun	WT	QC
SA429982	QC1_rerun	WT	QC
SA429983	QC6	WT	QC

Showing results 1 to 62 of 62

Showing results 1 to 62 of 62

Collection:

Collection ID:	CO004034
Collection Summary:	Each sample represents metabolites extracted from 1E8 Plasmodium falciparum infected human red blood cells (iRBCs). After treatment, the iRBCs were washed with cold PBS and metabolites were extracted using a 90% methanol solution. This solution was evaporated under nitrogen gas flow, resuspended in water plus chlorpropamide as an internal control, and run on the ThermoFisher Exactive Plus.
Sample Type:	Plasmodium cells

Treatment:

Treatment ID:	TR004050
Treatment 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. Following recovery, purified trophozoites were incubated in 6-well plates with Atovaquone (positive control) or test compound at 10XIC50, or no drug control, for 2.5 h at 37C.

Treatment ID:

TR004050

Treatment 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. Following recovery, purified trophozoites were incubated in 6-well plates with Atovaquone (positive control) or test compound at 10XIC50, or no drug control, for 2.5 h at 37C.

Sample Preparation:

Sampleprep ID:	SP004047
Sampleprep Summary:	At the time of sample collection, after recovery from purification, the parasite metabolism was quenched through addition of ice-cold PBS. Parasites were pelleted (500 g, 4°C, 7 min) and metabolites were extracted from the pellet with 1 ml ice cold 90% methanol, vortexed 30 seconds, and centrifuged for 10 min at maximum speed (16000 x g) at 4°C. Samples were treated identically and swiftly to ensure reproducible results. The methanol supernatants were stored at -80°C until analysis, when they were transferred to fresh 1.5 mL tubes, dried down completely under nitrogen gas flow, and the metabolite residues stored at -70°C.

Sampleprep ID:

SP004047

Sampleprep Summary:

At the time of sample collection, after recovery from purification, the parasite metabolism was quenched through addition of ice-cold PBS. Parasites were pelleted (500 g, 4°C, 7 min) and metabolites were extracted from the pellet with 1 ml ice cold 90% methanol, vortexed 30 seconds, and centrifuged for 10 min at maximum speed (16000 x g) at 4°C. Samples were treated identically and swiftly to ensure reproducible results. The methanol supernatants were stored at -80°C until analysis, when they were transferred to fresh 1.5 mL tubes, dried down completely under nitrogen gas flow, and the metabolite residues stored at -70°C.

Chromatography:

Chromatography ID:	CH004862
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters XSelect HSS T3 Column XP (100 x 2.1 mm, 2.5 um)
Column Temperature:	30°C
Flow Gradient:	0-5.0 min: 100% A, 0% B; 5.0-13.0 min: 80% A, 20% B; 13.0-15.0 min: 45% A, 55% B; 15.0-17.5 min: 35% A, 65% B; 17.5-21.0 min: 5% A, 95% B; 21.0-25 min: 100% A, 0% B
Flow Rate:	0.200 mL/min
Solvent A:	97% water/3% methanol; 15 mM acetic acid; 10 mM tributylamine; 2.5 µM medronic acid
Solvent B:	100% Methanol
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN006411
Analysis Type:	MS
Chromatography ID:	CH004862
Num Factors:	4
Num Metabolites:	163
Rt Units:	Minutes
Units:	blank-subtracted peak area