Summary of Study ST003696
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 PR002294. The data can be accessed directly via it's Project DOI: 10.21228/M83R72 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 | ST003696 |
| Study Title | A lipid transport Mla Pqi Chimeric system is essential for Brucella abortus survival in macrophages |
| Study Summary | The envelope of diderm bacteria comprises of an inner membrane (IM) and an outer membrane (OM). Several pathways have been recently identified that facilitate the transport of phospholipids between the two membranes in Escherichia coli, including maintaining OM lipid asymmetry (Mla) and paraquat inducible (Pqi) systems. In this study, we report the identification and the characterization of a complex named Mpc in the intracellular pathogen Brucella abortus. Mpc is conserved in numerous species of Hyphomicrobiales and exhibits homology to both the Mla and Pqi systems. Mpc is essential for bacterial growth under conditions of envelope stress and for survival within macrophages during the early stages of infection. Analyses of protein-protein interactions and structural predictions indicate that the Mpc complex bridges IM to OM. The absence of this system results in an altered lipid composition of the OM vesicles, supporting the fact that Mpc plays a role in the transport of lipids between membranes. The discovery of a novel lipid trafficking system enhances the diversity and complexity of known lipid trafficking systems within diderm bacteria. |
| Institute | CEMBIO |
| Last Name | Garcia |
| First Name | Antonia |
| Address | Urbanización, 28668 Monteprincipe, Madrid |
| antogar@ceu.es | |
| Phone | 913724711 |
| Submit Date | 2024-10-29 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-06-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002294 |
| Project DOI: | doi: 10.21228/M83R72 |
| Project Title: | Adaptations to virulence and growth of Brucella abortus envelope |
| Project Summary: | Bacteria of the Brucella genus are collectively responsible for brucellosis, a widely spread zoonosis. Our laboratory developed molecular tools to study growth and virulence of Brucella abortus at the single cell level. Using a genetic approach, we found that B. abortus selected a hybrid Mla-Pqi system (here called Mpc) anchored in the envelope, that is necessary to resist to envelope stress and to infect macrophages. We also found that the main component of the outer membrane, the lipopolysaccharide, is displaying a surprising traffic inside bacteria (the “round trip model”) that we propose to investigate. We also constructed a strain (here called RgsE*) that is growing bipolarly instead of unipolarly and we propose to analyze it to better understand polar ageing and potential polar virulence factors. One such polar virulence factor is the type 4 secretion system VirB, that we found associated to the new pole of the bacterium during macrophage infection. Altogether, our project aims to get a better understanding of envelope growth and properties, especially related to the virulence of B. abortus, with a high potential to generate new concepts that could be applicable to other bacterial pathogens. |
| Institute: | Université de Namur |
| Last Name: | de Bolle |
| First Name: | Xavier |
| Address: | Rue de Bruxelles 61, 5000 Namur, Bélgica |
| Email: | xavier.debolle@unamur.be |
| Phone: | 690090778 |
Subject:
| Subject ID: | SU003828 |
| Subject Type: | Bacteria |
| Subject Species: | Brucella abortus |
Factors:
Subject type: Bacteria; Subject species: Brucella abortus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype | Sample source |
|---|---|---|---|
| SA403949 | BLEBS_2_pos | mcpA-knockout | OMVs |
| SA403950 | BLEBS_2_neg | mcpA-knockout | OMVs |
| SA403951 | WHOLE CELLS_8_neg | mcpA-knockout | Whole cells |
| SA403952 | WHOLE CELLS_8_pos | mcpA-knockout | Whole cells |
| SA403953 | BLEBS_3_neg | mcpD-knockout | OMVs |
| SA403954 | BLEBS_3_pos | mcpD-knockout | OMVs |
| SA403955 | WHOLE CELLS_9_pos | mcpD-knockout | Whole cells |
| SA403956 | WHOLE CELLS_9_neg | mcpD-knockout | Whole cells |
| SA403957 | BLEBS_4_pos | mcpE-knockout | OMVs |
| SA403958 | BLEBS_4_neg | mcpE-knockout | OMVs |
| SA403959 | WHOLE CELLS_10_pos | mcpE-knockout | Whole cells |
| SA403960 | WHOLE CELLS_10_neg | mcpE-knockout | Whole cells |
| SA403961 | BLEBS_5_neg | mcpF-knockout | OMVs |
| SA403962 | BLEBS_5_pos | mcpF-knockout | OMVs |
| SA403963 | WHOLE CELLS_11_pos | mcpF-knockout | Whole cells |
| SA403964 | WHOLE CELLS_11_neg | mcpF-knockout | Whole cells |
| SA403945 | BLEBS_6_pos | mcp-fullcomplex-knockout | OMVs |
| SA403946 | BLEBS_6_neg | mcp-fullcomplex-knockout | OMVs |
| SA403947 | WHOLE CELLS_12_pos | mcp-fullcomplex-knockout | Whole cells |
| SA403948 | WHOLE CELLS_12_neg | mcp-fullcomplex-knockout | Whole cells |
| SA403941 | BLEBS_1_pos | Wild-type | OMVs |
| SA403942 | BLEBS_1_neg | Wild-type | OMVs |
| SA403943 | WHOLE CELLS_7_pos | Wild-type | Whole cells |
| SA403944 | WHOLE CELLS_7_neg | Wild-type | Whole cells |
| Showing results 1 to 24 of 24 |
Collection:
| Collection ID: | CO003821 |
| Collection Summary: | Lipids were extracted using a modified procedure inspired by earlier methods (Bligh & Dyer, 1959, https://doi.org/10.1139/o59-099; Daniels et al., 1993, https://doi.org/10.1128/9781555817497.ch18). In summary, 500 µL of bacterial or outer membrane vesicle (OMV) suspensions (20 mg of lyophilized material) in deionized water were combined with 650 µL of chloroform and 1300 µL of methanol. The mixture was vortexed for 3 hours, after which 650 µL of chloroform and 650 µL of deionized water were added and gently mixed for 30 minutes. Phase separation was achieved through brief centrifugation, and the organic layer was isolated and dried under a nitrogen stream. For OMV isolation, cultures of 800 mL were grown for 48 hours, treated with 0.5% phenol to inactivate them, and then centrifuged at 8,200 × g for 20 minutes at 4°C. The pellet was retained, while the supernatant was processed using a Pellicon® tangential flow filtration system with a 100 kDa membrane to concentrate it. Afterward, the concentrated supernatant was centrifuged again under the same conditions to remove any solid particles. To encourage the aggregation of OMVs, the supernatant was frozen at -20°C, thawed, and then ultracentrifuged at 47,000 × g for 3 hours at 4°C. The resulting pellet, containing the OMVs, was resuspended in deionized water, dialyzed at 4°C for three days, and stored at -80°C. Both the pellet from the culture and the OMV suspension were freeze-dried using a TELSTAR CRYODOS 50 lyophilizer. For additional information of isolation of OMV or sample treatment bacteria prior to extraction refer to doi: https://doi.org/10.1101/2024.10.31.621289 |
| Sample Type: | bacterial cells, outer membrane vesicles |
Treatment:
| Treatment ID: | TR003837 |
| Treatment Summary: | No treatment, since this is a genotype study where knockouts were compared. For the methodology to generate the specific knockouts of the MPC complex, please refer to doi: https://doi.org/10.1101/2024.10.31.621289 |
Sample Preparation:
| Sampleprep ID: | SP003835 |
| Sampleprep Summary: | Lipid residues from whole bacteria or OMVs, prepared following the Bligh and Dyer method, were dissolved in 300 µL of a methanol:chloroform solution containing 25 mg/L of d17:0 sphinganine (IS1). Lipids were further extracted by vortexing the mixture at room temperature for 20 minutes. The resulting extracts were transferred to individual LC-MS vials, where 20 µL of the SPLASH Lipidomix® lipid standard mix (IS2, Avanti Polar Lipids, CA, USA) were added. Samples were then dried completely using a vacuum concentrator at 37°C. Finally, the residues were re-extracted in 100 µL of methanol:chloroform (2:1, v/v) with thorough vortexing for 30 minutes at room temperature before analysis. |
Chromatography:
| Chromatography ID: | CH004608 |
| Chromatography Summary: | The chromatographic method is the one comprehensively described in doi: 10.1016/j.jlr.2024.100671 and adapted from Agilent Lipid Annotator application note (see https://www.agilent.com/cs/library/applications/application-6546-q-tof-lipidome-5994-0775en-agilent.pdf) |
| Instrument Name: | Agilent 1290 HPLC |
| Column Name: | Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3.0mm, 2.7um) with guard column Agilent InfinityLab Poroshell 120 EC-C18 (5 x 3.0mm, 2.7um) |
| Column Temperature: | 50 |
| Flow Gradient: | Time (min)/%B: 0.00/70, 1.00/70, 3.50/86, 10.00/86 11.00/100, 17.00/100, 17.10/70, 19.00/70 |
| Flow Rate: | 0.6 mL/min |
| Solvent A: | 90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride |
| Solvent B: | 20% acetonitrile/30% methanol/50% isopropanol10 mM ammonium acetate; 0.2 mM ammonium fluoride |
| Chromatography Type: | Reversed phase |
Analysis:
| Analysis ID: | AN006064 |
| Analysis Type: | MS |
| Chromatography ID: | CH004608 |
| Has Rt: | 1 |
| Rt Units: | Minutes |
| Results File: | ST003696_AN006064_Results.txt |
| Units: | amu |
| Analysis ID: | AN006065 |
| Analysis Type: | MS |
| Chromatography ID: | CH004608 |
| Has Rt: | 1 |
| Rt Units: | Minutes |
| Results File: | ST003696_AN006065_Results.txt |
| Units: | amu |
