Summary of Study ST003642

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 PR002252. The data can be accessed directly via it's Project DOI: 10.21228/M8J242 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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST003642
Study TitleHexosamine Biosynthesis Disruption Impairs GPI Production and Arrests Plasmodium falciparum Growth at Schizont Stages
Study SummaryUDP-N-acetylglucosamine (UDP-GlcNAc) is a crucial sugar nucleotide for glycan synthesis in eukaryotes. In Plasmodium falciparum, UDP-GlcNAc is synthesized via the hexosamine biosynthetic pathway (HBP) and is essential for glycosylphosphatidylinositol (GPI) anchor production, the most prominent form of protein glycosylation in this parasite. In this study, we explore a conditional knockout of glucosamine-6-phosphate N-acetyltransferase (PfGNA1), a key HBP enzyme. PfGNA1 depletion led to significant disruptions in HBP metabolites, impairing GPI biosynthesis and causing mislocalization of the merozoite surface protein 1 (MSP1), the most abundant GPI-anchored protein in the parasite. As a result, parasites were arrested at the schizont stage, exhibiting severe segmentation defects and an incomplete rupture of the parasitophorous vacuole membrane (PVM), preventing egress from host red blood cells. Our findings demonstrate the critical role of HBP and GPI biosynthesis in P. falciparum asexual development and underscore the potential of targeting these pathways as a therapeutic strategy against malaria.
Institute
Pennsylvania State University
Last NameRangel
First NameGabriel
Address491 Pollock Road, University Park, PA, 16802, USA
Emailgrangel0955@gmail.com
Phone8148673527
Submit Date2024-12-04
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-01-06
Release Version1
Gabriel Rangel Gabriel Rangel
https://dx.doi.org/10.21228/M8J242
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:


Project:

Project ID:PR002252
Project DOI:doi: 10.21228/M8J242
Project Title:Hexosamine Biosynthesis Disruption Impairs GPI Production and Arrests Plasmodium falciparum Growth at Schizont Stages
Project Summary:UDP-N-acetylglucosamine (UDP-GlcNAc) is a crucial sugar nucleotide for glycan synthesis in eukaryotes. In Plasmodium falciparum, UDP-GlcNAc is synthesized via the hexosamine biosynthetic pathway (HBP) and is essential for glycosylphosphatidylinositol (GPI) anchor production, the most prominent form of protein glycosylation in this parasite. In this study, we explore a conditional knockout of glucosamine-6-phosphate N-acetyltransferase (PfGNA1), a key HBP enzyme. PfGNA1 depletion led to significant disruptions in HBP metabolites, impairing GPI biosynthesis and causing mislocalization of the merozoite surface protein 1 (MSP1), the most abundant GPI-anchored protein in the parasite. As a result, parasites were arrested at the schizont stage, exhibiting severe segmentation defects and an incomplete rupture of the parasitophorous vacuole membrane (PVM), preventing egress from host red blood cells. Our findings demonstrate the critical role of HBP and GPI biosynthesis in P. falciparum asexual development and underscore the potential of targeting these pathways as a therapeutic strategy against malaria.
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:grangel0955@gmail.com
Phone:8148673527

Subject:

Subject ID:SU003772
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 Sample source Genotype Treatment
SA397809DMSO-1Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP DMSO
SA397810DMSO-2Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP DMSO
SA397811DMSO-3Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP DMSO
SA397812DMSO-4Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP DMSO
SA397813Rapa-3Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP Rapamycin
SA397814Rapa-4Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP Rapamycin
SA397815Rapa-2Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP Rapamycin
SA397816Rapa-1Cultured Plasmodium falciparum II3 II3 gna1-3xHA-loxP Rapamycin
SA397817Izq-QC-4Cultured Plasmodium falciparum II3 NA NA
SA397818Izq-QC-3Cultured Plasmodium falciparum II3 NA NA
SA397819Izq-QC-1Cultured Plasmodium falciparum II3 NA NA
SA397820Izq-Blank-4Cultured Plasmodium falciparum II3 NA NA
SA397821Izq-Blank-3Cultured Plasmodium falciparum II3 NA NA
SA397822Izq-Blank-2Cultured Plasmodium falciparum II3 NA NA
SA397823Izq-Blank-1Cultured Plasmodium falciparum II3 NA NA
SA397824Izq-QC-2Cultured Plasmodium falciparum II3 NA NA
SA397825Parental-1Cultured Plasmodium falciparum II3 WT None
SA397826Parental-2Cultured Plasmodium falciparum II3 WT None
SA397827Parental-3Cultured Plasmodium falciparum II3 WT None
SA397828Parental-4Cultured Plasmodium falciparum II3 WT None
Showing results 1 to 20 of 20

Collection:

Collection ID:CO003765
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:TR003781
Treatment Summary:The II3 gna1-loxP line was tightly synchronized over a 5-hour window, using sequential 70% Percoll centrifugation followed by sorbitol lysis, and immediately treated with 10 nM rapamycin or DMSO (control) for one hour. Rapamycin or DMSO were then removed from the culture medium and the parasites were incubated until 57 hours post-Percoll when a new sorbitol synchronization was performed at the beginning of cycle 1. The samples were then incubated for about 81-89 hours post-Percoll and then enriched by MACS. For High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) analysis, 1x108 parasites per replicate were replated in a 6-well plate and incubated for 2.5 hours to allow recovery.

Sample Preparation:

Sampleprep ID:SP003779
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.

Combined analysis:

Analysis ID AN005981
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 Exactive Plus Orbitrap
Ion Mode NEGATIVE
Units peak area

Chromatography:

Chromatography ID:CH004543
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:MS005694
Analysis ID:AN005981
Instrument Name:Thermo Exactive Plus Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:.raw files were converted to .mzML files using MSConvert software of the ProteoWizard package. The .mzML files were processed using El Maven software for peak picking, alignment, and annotation.
Ion Mode:NEGATIVE
  logo