Summary of Study ST004244

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 PR002663. The data can be accessed directly via it's Project DOI: 10.21228/M8F545 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	ST004244
Study Title	Parasites with a disrupted arginine to proline pathway can still mount an APR
Study Summary	Plasmodium falciparum evades the antimalarial activity of proline-competitive prolyl-tRNA synthetase (PfProRS) inhibitors, such as halofuginone (HFG), by a unique resistance mechanism termed the Adaptive Proline Response (APR). The APR is characterized by a marked elevation of intracellular proline following drug exposure. Contrary to initial expectations, the APR is not mediated by alterations in canonical proline metabolic pathways involving arginase (PfARG) and ornithine aminotransferase (PfOAT). Instead, we identified loss-of-function mutations in the Apicomplexan Amino acid Transporter 2 (PfApiAT2) as the primary genetic driver of this resistance phenotype. Importantly, reversion of these mutations to wildtype effectively suppresses the APR, establishing PfApiAT2 as the molecular determinant of this novel resistance mechanism. The elucidation of the APR significantly advances our understanding of antimalarial drug resistance. By delineating the role of PfApiAT2 in this process, we establish critical insights for the development of strategies to circumvent PfProRS inhibitor resistance for future antimalarial therapies. In this study, we use a custom Triplex labeled media, containing uniquely stable isotope labeled proline (+1), arginine (+2), and glutamine (+5) to validate the arginine to proline biosynthetic pathway as major driver for the elevated proline levels in APR parasites. Labeled proline levels were assessed in Dd2-OAT KO parasites (parasites with a disrupted ornithine amino-transferase locus) and Dd2-OAT KO parasites exposed to HFG to induce the APR. Induced/APR Dd2-OAT KO parasites were analyzed as a bulk population after drug treatment as well as individual clonal populations (clones 1, 2, 3) isolated from the bulk population by limiting dilution. Uninfected RBCs (uRBCs) were run as a control.
Institute	Broad Institute of MIT and Harvard
Department	Metabolomics Platform
Last Name	Clish
First Name	Clary
Address	300 Binney Street, Cambridge, MA 02142
Email	clary@broadinstitute.org
Phone	617-714-7654
Submit Date	2025-09-22
Num Groups	6
Total Subjects	36
Study Comments	Study 3 of 5
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-10-06
Release Version	1

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Project:

Project ID:	PR002663
Project DOI:	doi: 10.21228/M8F545
Project Title:	Loss of P. falciparum Amino Acid Transporter (ApiAT2) Function Increases Intracellular Proline and Confers Resistance to Prolyl-tRNA Synthetase Inhibitors
Project Summary:	Plasmodium falciparum evades the antimalarial activity of proline-competitive prolyl-tRNA synthetase (PfProRS) inhibitors, such as halofuginone (HFG), by a unique resistance mechanism termed the Adaptive Proline Response (APR). The APR is characterized by a marked elevation of intracellular proline following drug exposure. Contrary to initial expectations, the APR is not mediated by alterations in canonical proline metabolic pathways involving arginase (PfARG) and ornithine aminotransferase (PfOAT). Instead, we identified loss-of-function mutations in the Apicomplexan Amino acid Transporter 2 (PfApiAT2) as the primary genetic driver of this resistance phenotype. Importantly, reversion of these mutations to wildtype effectively suppresses the APR, establishing PfApiAT2 as the molecular determinant of this novel resistance mechanism. The elucidation of the APR significantly advances our understanding of antimalarial drug resistance. By delineating the role of PfApiAT2 in this process, we establish critical insights for the development of strategies to circumvent PfProRS inhibitor resistance for future antimalarial therapies.
Institute:	Broad Institute of MIT and Harvard
Department:	Metabolomics Platform
Last Name:	Clish
First Name:	Clary
Address:	300 Binney Street, Cambridge, MA, 02142, USA
Email:	clary@broadinstitute.org
Phone:	617-714-7654
Funding Source:	NIH-NIAID R01AI143723 and R21AI132981; NIH-NIGMS T32 GM008666 and F31AI129412; Bill and Melinda Gates Foundation OPP1132451 and OPP1086203; and the Harvard Defeating Malaria Initiative
Project Comments:	5 experiements
Contributors:	Selina Bopp, Lọla Fagbami, Amy Deik, Claudia Taccheri, Akansha Pant, Madeline Luth, Daisy Chen, Mark A. Tye, Imran Ullah, Robert Morris, Wilhelm Haas, Elizabeth A. Winzeler, Clary B. Clish, Amanda K. Lukens, Ralph Mazitschek, Dyann F. Wirth

Subject:

Subject ID:	SU004396
Subject Type:	Cultured cells
Subject Species:	Plasmodium falciparum
Taxonomy ID:	5833
Species Group:	Unicellular parasites

Factors:

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

mb_sample_id	local_sample_id	Sample source	Parasite_type	Parasite_line	Timepoint
SA490124	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R1	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490125	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R5	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490126	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R4	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490127	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R3	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490128	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R2	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490129	Study3_Dd2∆OATAPR-bulk_iRBC_10h_R6	infected RBC (iRBC)	del OAT 6491 bulk	Dd2∆OATAPR2 bulk	10 h
SA490130	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R1	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490131	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R2	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490132	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R3	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490133	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R4	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490134	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R5	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490135	Study3_Dd2∆OATAPR-clone2_iRBC_10h_R6	infected RBC (iRBC)	del OAT D12 clone A6	Dd2∆OATAPR2 clone 2	10 h
SA490136	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R6	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490137	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R2	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490138	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R4	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490139	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R3	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490140	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R5	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490141	Study3_Dd2∆OATAPR-clone1_iRBC_10h_R1	infected RBC (iRBC)	del OAT D12 clone B9	Dd2∆OATAPR2 clone 1	10 h
SA490142	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R3	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490143	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R6	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490144	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R5	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490145	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R4	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490146	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R1	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490147	Study3_Dd2∆OATAPR-clone3_iRBC_10h_R2	infected RBC (iRBC)	del OAT D12 clone F7	Dd2∆OATAPR2 clone 3	10 h
SA490148	Study3_Dd2∆OAT-C1_iRBC_10h_R5	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490149	Study3_Dd2∆OAT-C1_iRBC_10h_R4	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490150	Study3_Dd2∆OAT-C1_iRBC_10h_R3	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490151	Study3_Dd2∆OAT-C1_iRBC_10h_R2	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490152	Study3_Dd2∆OAT-C1_iRBC_10h_R1	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490153	Study3_Dd2∆OAT-C1_iRBC_10h_R6	infected RBC (iRBC)	del OAT D12	Dd2∆OAT C1	10 h
SA490154	Study3_uRBCs_RBC_10h_R2	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h
SA490155	Study3_uRBCs_RBC_10h_R4	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h
SA490156	Study3_uRBCs_RBC_10h_R3	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h
SA490157	Study3_uRBCs_RBC_10h_R5	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h
SA490158	Study3_uRBCs_RBC_10h_R6	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h
SA490159	Study3_uRBCs_RBC_10h_R1	uninfected RBC (uRBC)	uninfected RBCs	Control RBCs	10 h

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO004389
Collection Summary:	Highly synchronous (within 4 h) late trophozoite stage parasites were magnetically purified with MACS LD columns (Miltenyi Biotec Inc., San Diego, CA, USA). Each sample was incubated for 10hr with unlabeled media, harvested by centrifugation, washed twice in PBS, and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at −80°C prior to the metabolite profiling assays.
Sample Type:	Cultured cells
Collection Location:	Harvard Chan School of Public Health, 665 Huntington Ave. Boston, MA 02115
Volumeoramount Collected:	100 uL
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR004405
Treatment Summary:	Cultures were cultured in 5% human O+ hematocrit and maintained under standard conditions [RPMI 1640 (Life Technologies) supplemented with 28 mM NaHCO3 (Sigma), 25 mM HEPES (Sigma), 50 mg/mL hypoxanthine (Sigma), 25 μg/mL gentamycin (Sigma), and 0.5% AlbuMAX II (Life Technologies)] until preparation for treatment. In preparation for treatment, 25mL synchronized parasites culture at ~5% parasitemia and 32-36hr staging at beginning of experiment were pelleted and resuspended in 4mL serum-free RPMI (Incomplete Media). Parasites were magnetically purified with an LD column (Miltenyi Biotec Inc., San Diego, CA, USA) and washed until the flow thru ran clear. The column was removed from the magnet and each sample eluted in 2mL incomplete RPMI. Samples were divided into two microfuge tubes, each receiving half (1mL) of the cell suspension. Cells were pelleted and resuspended in incomplete RPMI (unlabeled). Parasites were incubated in unlabeled media for 10h at 37ºC under standard malaria gas mixture. Samples were harvested by centrifugation, washed twice, and then polar metabolites extracted.

Treatment ID:

TR004405

Treatment Summary:

Cultures were cultured in 5% human O+ hematocrit and maintained under standard conditions [RPMI 1640 (Life Technologies) supplemented with 28 mM NaHCO3 (Sigma), 25 mM HEPES (Sigma), 50 mg/mL hypoxanthine (Sigma), 25 μg/mL gentamycin (Sigma), and 0.5% AlbuMAX II (Life Technologies)] until preparation for treatment. In preparation for treatment, 25mL synchronized parasites culture at ~5% parasitemia and 32-36hr staging at beginning of experiment were pelleted and resuspended in 4mL serum-free RPMI (Incomplete Media). Parasites were magnetically purified with an LD column (Miltenyi Biotec Inc., San Diego, CA, USA) and washed until the flow thru ran clear. The column was removed from the magnet and each sample eluted in 2mL incomplete RPMI. Samples were divided into two microfuge tubes, each receiving half (1mL) of the cell suspension. Cells were pelleted and resuspended in incomplete RPMI (unlabeled). Parasites were incubated in unlabeled media for 10h at 37ºC under standard malaria gas mixture. Samples were harvested by centrifugation, washed twice, and then polar metabolites extracted.

Sample Preparation:

Sampleprep ID:	SP004402
Sampleprep Summary:	Each sample was washed twice in PBS and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at −80°C prior to the metabolite profiling assays.

Sampleprep ID:

SP004402

Sampleprep Summary:

Each sample was washed twice in PBS and then suspended in 10 μL PBS (Life Technologies, Carlsbad, CA, USA). Polar metabolites were extracted using nine volumes of 74.9:24.9:0.2 (v/v/v) acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (0.2 ng/μL valine-d8 (Sigma Aldrich, St. Louis, MO, USA); and 0.2 ng/μL phenylalanine-d8 (Cambridge Isotope Laboratories, Tewksbury, MA, USA) and stored at −80°C prior to the metabolite profiling assays.

Chromatography:

Chromatography ID:	CH005364
Chromatography Summary:	Chromatography method 1 (HILIC-POS)
Instrument Name:	Shimadzu Nexera X2
Column Name:	Water Atlantis HILIC (150 x 2.1 mm, 3 μm)
Column Temperature:	30°C
Flow Gradient:	Chromatographic separation was performed using an isocratic elution at a flow rate of 250 μL/min with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute. This was followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes. At 10 minutes, the gradient was returned to initial isocratic conditions (5% mobile phase A) and held until 18 minutes, at which point MS acquisition was stopped. The column was equilibrated with 5% mobile phase A at a flow rate of 400 μL/min for 12 minutes, followed by a reduction to the initial flow rate of 250 μL/min for 2 minutes before the next injection.
Flow Rate:	250 μL/min
Internal Standard:	L-Phenylalanine-d8 (CIL, DLM-372-1), L-Valine-d8 (Sigma, 486027)
Solvent A:	100% Water; 10 mM Ammonium formate; 0.1% Formic acid
Solvent B:	100% Acetonitrile; 0.1% Formic acid
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN007065
Analysis Type:	MS
Chromatography ID:	CH005364
Num Factors:	6
Num Metabolites:	127
Units:	peak areas