Summary of Study ST003562

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 PR002196. The data can be accessed directly via it's Project DOI: 10.21228/M8RV6M 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 IDST003562
Study TitleMultiple, redundant carboxylic acid transporters support mitochondrial metabolism in Plasmodium falciparum
Study SummaryThe mitochondrion of the deadliest human malaria parasite, Plasmodium falciparum, is an essential source of cellular acetyl-CoA during the asexual blood-stage of the parasite life cycle. Indeed, blocking mitochondrial acetyl-CoA synthesis leads to a hypoacetylated proteome and parasite death. We previously determined that mitochondrial acetyl-CoA is primarily synthesized from glucose-derived pyruvate by α-ketoacid dehydrogenases. Here, we asked if inhibiting the import of glycolytic pyruvate across the mitochondrial inner membrane would affect acetyl-CoA production and, thus, could be a potential target for novel antimalarial drug development. We selected the two predicted mitochondrial pyruvate carrier proteins (MPC1 and MPC2) for genetic knockout and isotopic metabolite tracing via HPLC-MS metabolomic analysis. Surprisingly, we observed that asexual blood-stage parasites could survive the loss of either or both proteins with only minor asexual blood-stage growth defects, despite a substantial reduction in the amount of glucose-derived isotopic labelling into acetyl-CoA. Furthermore, genetic deletion of two additional mitochondrial carboxylic acid transporters – DTC (di/tricarboxylic acid carrier) and YHM2 (a putative citrate/α-ketoglutarate carrier protein) – only mildly affected asexual blood-stage replication, even in the context of MPC-deficiency. Although we observed no added impact on the incorporation of glucose carbon into acetyl-CoA in these quadruple knockout mutants, we noted a large decrease in glutamine-derived label in tricarboxylic acid cycle metabolites, suggesting that DTC and YHM2 both import glutamine derivatives into the mitochondrion. Altogether, our results expose redundant routes used to fuel the mitochondria of blood-stage malaria parasites with imported carbon from two major carbon sources – glucose and glutamine.
Institute
Pennsylvania State University
DepartmentBiochemistry and Molecular Biology
LaboratoryManuel Llinás
Last NameManuel
First NameLlinàs
Address491 Pollock Road
Emailmanuel@psu.edu
Phone8148673444
Submit Date2024-09-30
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2024-11-20
Release Version1
Llinàs Manuel Llinàs Manuel
https://dx.doi.org/10.21228/M8RV6M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002196
Project DOI:doi: 10.21228/M8RV6M
Project Title:Multiple, redundant carboxylic acid transporters support mitochondrial metabolism in Plasmodium falciparum
Project Summary:The mitochondrion of the deadliest human malaria parasite, Plasmodium falciparum, is an essential source of cellular acetyl-CoA during the asexual blood-stage of the parasite life cycle. Indeed, blocking mitochondrial acetyl-CoA synthesis leads to a hypoacetylated proteome and parasite death. We previously determined that mitochondrial acetyl-CoA is primarily synthesized from glucose-derived pyruvate by α-ketoacid dehydrogenases. Here, we asked if inhibiting the import of glycolytic pyruvate across the mitochondrial inner membrane would affect acetyl-CoA production and, thus, could be a potential target for novel antimalarial drug development. We selected the two predicted mitochondrial pyruvate carrier proteins (MPC1 and MPC2) for genetic knockout and isotopic metabolite tracing via HPLC-MS metabolomic analysis. Surprisingly, we observed that asexual blood-stage parasites could survive the loss of either or both proteins with only minor asexual blood-stage growth defects, despite a substantial reduction in the amount of glucose-derived isotopic labelling into acetyl-CoA. Furthermore, genetic deletion of two additional mitochondrial carboxylic acid transporters – DTC (di/tricarboxylic acid carrier) and YHM2 (a putative citrate/α-ketoglutarate carrier protein) – only mildly affected asexual blood-stage replication, even in the context of MPC-deficiency. Although we observed no added impact on the incorporation of glucose carbon into acetyl-CoA in these quadruple knockout mutants, we noted a large decrease in glutamine-derived label in tricarboxylic acid cycle metabolites, suggesting that DTC and YHM2 both import glutamine derivatives into the mitochondrion. Altogether, our results expose redundant routes used to fuel the mitochondria of blood-stage malaria parasites with imported carbon from two major carbon sources – glucose and glutamine.
Institute:Pennsylvania State University
Department:Biochemistry and Molecular Biology
Laboratory:Manuel Llinás
Last Name:Manuel
First Name:Llinás
Address:491 Pollock Road, Millennium Science Complex, W126
Email:manuel@psu.edu
Phone:814-867-3444

Subject:

Subject ID:SU003691
Subject Type:Cultured cells
Subject Species:Plasmodium falciparum
Taxonomy ID:5833
Genotype Strain:NF54 attB
Cell Biosource Or Supplier:Prigge Lab, Johns Hopkins University

Factors:

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

mb_sample_id local_sample_id Sample source Sample type Treatment
SA38921820240315_0227-2KO-glucose-3Cultured Plasmodium falciparum 2KO glucose
SA38921920240206_2KO-glucose-1Cultured Plasmodium falciparum 2KO glucose
SA38922020240206_2KO-glucose-2Cultured Plasmodium falciparum 2KO glucose
SA38922120240206_2KO-glucose-3Cultured Plasmodium falciparum 2KO glucose
SA38922220240315_0227-2KO-glucose-2Cultured Plasmodium falciparum 2KO glucose
SA38922320240315_0227-2KO-glucose-1Cultured Plasmodium falciparum 2KO glucose
SA38922420240315_0304-2KO-glucose-1Cultured Plasmodium falciparum 2KO glucose
SA38922520240315_0304-2KO-glucose-2Cultured Plasmodium falciparum 2KO glucose
SA38922620240315_0304-2KO-glucose-3Cultured Plasmodium falciparum 2KO glucose
SA38922720240315_0227-2KO-glutamine-3Cultured Plasmodium falciparum 2KO glutamine
SA38922820240315_0304-2KO-glutamine-3Cultured Plasmodium falciparum 2KO glutamine
SA38922920240315_0304-2KO-glutamine-2Cultured Plasmodium falciparum 2KO glutamine
SA38923020240315_0304-2KO-glutamine-1Cultured Plasmodium falciparum 2KO glutamine
SA38923120240206_2KO-glutamine-1Cultured Plasmodium falciparum 2KO glutamine
SA38923220240206_2KO-glutamine-2Cultured Plasmodium falciparum 2KO glutamine
SA38923320240206_2KO-glutamine-3Cultured Plasmodium falciparum 2KO glutamine
SA38923420240315_0227-2KO-glutamine-1Cultured Plasmodium falciparum 2KO glutamine
SA38923520240315_0227-2KO-glutamine-2Cultured Plasmodium falciparum 2KO glutamine
SA38923620211215_3KO-glucose-1Cultured Plasmodium falciparum 3KO glucose
SA38923720220330_3KO-glucose-2Cultured Plasmodium falciparum 3KO glucose
SA38923820230804_3KO-glucose-3Cultured Plasmodium falciparum 3KO glucose
SA38923920230804_3KO-glucose-2Cultured Plasmodium falciparum 3KO glucose
SA38924020230804_3KO-glucose-1Cultured Plasmodium falciparum 3KO glucose
SA38924120211215_3KO-glucose-3Cultured Plasmodium falciparum 3KO glucose
SA38924220211215_3KO-glucose-2Cultured Plasmodium falciparum 3KO glucose
SA38924320220330_3KO-glucose-1Cultured Plasmodium falciparum 3KO glucose
SA38924420220429_3KO-glucose-2Cultured Plasmodium falciparum 3KO glucose
SA38924520220429_3KO-glucose-3Cultured Plasmodium falciparum 3KO glucose
SA38924620220429_3KO-glucose-1Cultured Plasmodium falciparum 3KO glucose
SA38924720220330_3KO-glucose-3Cultured Plasmodium falciparum 3KO glucose
SA38924820220429_3KO-glutamine-2Cultured Plasmodium falciparum 3KO glutamine
SA38924920220429_3KO-glutamine-1Cultured Plasmodium falciparum 3KO glutamine
SA38925020220330_3KO-glutamine-2Cultured Plasmodium falciparum 3KO glutamine
SA38925120220330_3KO-glutamine-1Cultured Plasmodium falciparum 3KO glutamine
SA38925220230804_3KO-glutamine-2Cultured Plasmodium falciparum 3KO glutamine
SA38925320220330_3KO-glutamine-3Cultured Plasmodium falciparum 3KO glutamine
SA38925420230804_3KO-glutamine-3Cultured Plasmodium falciparum 3KO glutamine
SA38925520230804_3KO-glutamine-1Cultured Plasmodium falciparum 3KO glutamine
SA38925620211215_3KO-glutamine-2Cultured Plasmodium falciparum 3KO glutamine
SA38925720211215_3KO-glutamine-1Cultured Plasmodium falciparum 3KO glutamine
SA38925820211215_3KO-glutamine-3Cultured Plasmodium falciparum 3KO glutamine
SA38925920220429_3KO-glutamine-3Cultured Plasmodium falciparum 3KO glutamine
SA38926020230804_4KO-glucose-2Cultured Plasmodium falciparum 4KO glucose
SA38926120230830_4KO-glucose-1Cultured Plasmodium falciparum 4KO glucose
SA38926220230830_4KO-glucose-2Cultured Plasmodium falciparum 4KO glucose
SA38926320240206_4KO-glucose-2Cultured Plasmodium falciparum 4KO glucose
SA38926420230804_4KO-glucose-1Cultured Plasmodium falciparum 4KO glucose
SA38926520240206_4KO-glucose-3Cultured Plasmodium falciparum 4KO glucose
SA38926620240206_4KO-glucose-1Cultured Plasmodium falciparum 4KO glucose
SA38926720230804_4KO-glucose-3Cultured Plasmodium falciparum 4KO glucose
SA38926820230830_4KO-glucose-3Cultured Plasmodium falciparum 4KO glucose
SA38926920230804_4KO-glutamine-1Cultured Plasmodium falciparum 4KO glutamine
SA38927020230804_4KO-glutamine-2Cultured Plasmodium falciparum 4KO glutamine
SA38927120230804_4KO-glutamine-3Cultured Plasmodium falciparum 4KO glutamine
SA38927220240206_4KO-glutamine-1Cultured Plasmodium falciparum 4KO glutamine
SA38927320230830_4KO-glutamine-3Cultured Plasmodium falciparum 4KO glutamine
SA38927420230830_4KO-glutamine-2Cultured Plasmodium falciparum 4KO glutamine
SA38927520230830_4KO-glutamine-1Cultured Plasmodium falciparum 4KO glutamine
SA38927620240206_4KO-glutamine-2Cultured Plasmodium falciparum 4KO glutamine
SA38927720240206_4KO-glutamine-3Cultured Plasmodium falciparum 4KO glutamine
SA38927820220330_Blank2Cultured Plasmodium falciparum Blank NA
SA38927920220429_Blank2Cultured Plasmodium falciparum Blank NA
SA38928020230830_Blank-2Cultured Plasmodium falciparum Blank NA
SA38928120230830_Blank-1Cultured Plasmodium falciparum Blank NA
SA38928220230830_Blank-3Cultured Plasmodium falciparum Blank NA
SA38928320220429_Blank3Cultured Plasmodium falciparum Blank NA
SA38928420240315_0227-Blank-3Cultured Plasmodium falciparum Blank NA
SA38928520240315_0227-Blank-2Cultured Plasmodium falciparum Blank NA
SA38928620240315_0227-Blank-1Cultured Plasmodium falciparum Blank NA
SA38928720220330_Blank3Cultured Plasmodium falciparum Blank NA
SA38928820220330_Blank4Cultured Plasmodium falciparum Blank NA
SA38928920220330_Blank5Cultured Plasmodium falciparum Blank NA
SA38929020240206_Blank-1Cultured Plasmodium falciparum Blank NA
SA38929120240206_Blank-2Cultured Plasmodium falciparum Blank NA
SA38929220240206_Blank-3Cultured Plasmodium falciparum Blank NA
SA38929320240206_Blank-4Cultured Plasmodium falciparum Blank NA
SA38929420220330_Blank1Cultured Plasmodium falciparum Blank NA
SA38929520230804_Blank-3Cultured Plasmodium falciparum Blank NA
SA38929620211215_Blank3Cultured Plasmodium falciparum Blank NA
SA38929720211215_Blank5Cultured Plasmodium falciparum Blank NA
SA38929820211215_Blank4Cultured Plasmodium falciparum Blank NA
SA38929920211215_Blank1Cultured Plasmodium falciparum Blank NA
SA38930020220429_Blank1Cultured Plasmodium falciparum Blank NA
SA38930120220429_Blank4Cultured Plasmodium falciparum Blank NA
SA38930220230804_Blank-2Cultured Plasmodium falciparum Blank NA
SA38930320230804_Blank-1Cultured Plasmodium falciparum Blank NA
SA38930420240315_0304-Blank-1Cultured Plasmodium falciparum Blank NA
SA38930520240315_0304-Blank-2Cultured Plasmodium falciparum Blank NA
SA38930620240315_0304-Blank-3Cultured Plasmodium falciparum Blank NA
SA38930720220429_Blank5Cultured Plasmodium falciparum Blank NA
SA38930820211215_Blank2Cultured Plasmodium falciparum Blank NA
SA38930920240315_0304-Parental-glucose-1Cultured Plasmodium falciparum Parental glucose
SA38931020220330_Parental-glucose-1Cultured Plasmodium falciparum Parental glucose
SA38931120230804_Parental-glucose-3Cultured Plasmodium falciparum Parental glucose
SA38931220240315_0304-Parental-glucose-2Cultured Plasmodium falciparum Parental glucose
SA38931320240315_0304-Parental-glucose-3Cultured Plasmodium falciparum Parental glucose
SA38931420220429_Parental-glucose-3Cultured Plasmodium falciparum Parental glucose
SA38931520220429_Parental-glucose-2Cultured Plasmodium falciparum Parental glucose
SA38931620240206_Parental-glucose-1Cultured Plasmodium falciparum Parental glucose
SA38931720240206_Parental-glucose-2Cultured Plasmodium falciparum Parental glucose
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Collection:

Collection ID:CO003684
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:Cultured cells

Treatment:

Treatment ID:TR003700
Treatment Summary:Magnetically purified Plasmodium falciparum infected human red blood cells (iRBCs) were cultured for 2.5 hours in standard RPMI media lacking normal glucose or glutamine but containing radiolabeled glucose or glutamine respectively.

Sample Preparation:

Sampleprep ID:SP003698
Sampleprep Summary:Dried metabolite residue was resuspended to 1E6 former parasites per microliter of water (usually 100 microliters) containing 1 micromolar chlorpropamide as an internal control. These samples were then run on HPLC-MS

Combined analysis:

Analysis ID AN005853
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Dionex Ultimate 3000
Column Waters XSelect HSS T3 Column XP (100 x 2.1 mm, 2.5 um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units blank-subtracted peak area

Chromatography:

Chromatography ID:CH004445
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

MS:

MS ID:MS005573
Analysis ID:AN005853
Instrument Name:Thermo Q Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:.raw files were converted to .mzML files using MSConvert softward of the ProteoWizard package. The .mzML files were processed using El Maven software for peak picking, alignment, and annotation.
Ion Mode:NEGATIVE
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