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.
Study ID | ST003642 |
Study Title | Hexosamine Biosynthesis Disruption Impairs GPI Production and Arrests Plasmodium falciparum Growth at Schizont Stages |
Study 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 |
Last Name | Rangel |
First Name | Gabriel |
Address | 491 Pollock Road, University Park, PA, 16802, USA |
grangel0955@gmail.com | |
Phone | 8148673527 |
Submit Date | 2024-12-04 |
Raw Data Available | Yes |
Raw Data File Type(s) | mzML |
Analysis Type Detail | LC-MS |
Release Date | 2025-01-06 |
Release Version | 1 |
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 |
---|---|---|---|---|
SA397809 | DMSO-1 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | DMSO |
SA397810 | DMSO-2 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | DMSO |
SA397811 | DMSO-3 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | DMSO |
SA397812 | DMSO-4 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | DMSO |
SA397813 | Rapa-3 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | Rapamycin |
SA397814 | Rapa-4 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | Rapamycin |
SA397815 | Rapa-2 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | Rapamycin |
SA397816 | Rapa-1 | Cultured Plasmodium falciparum II3 | II3 gna1-3xHA-loxP | Rapamycin |
SA397817 | Izq-QC-4 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397818 | Izq-QC-3 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397819 | Izq-QC-1 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397820 | Izq-Blank-4 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397821 | Izq-Blank-3 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397822 | Izq-Blank-2 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397823 | Izq-Blank-1 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397824 | Izq-QC-2 | Cultured Plasmodium falciparum II3 | NA | NA |
SA397825 | Parental-1 | Cultured Plasmodium falciparum II3 | WT | None |
SA397826 | Parental-2 | Cultured Plasmodium falciparum II3 | WT | None |
SA397827 | Parental-3 | Cultured Plasmodium falciparum II3 | WT | None |
SA397828 | Parental-4 | Cultured 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 |