#METABOLOMICS WORKBENCH FernandezGarcia_M_20241029_115754 DATATRACK_ID:5324 STUDY_ID:ST003696 ANALYSIS_ID:AN006065 PROJECT_ID:PR002294 VERSION 1 CREATED_ON January 28, 2025, 2:27 pm #PROJECT PR:PROJECT_TITLE Adaptations to virulence and growth of Brucella abortus envelope PR:PROJECT_SUMMARY Bacteria of the Brucella genus are collectively responsible for brucellosis, a PR:PROJECT_SUMMARY widely spread zoonosis. Our laboratory developed molecular tools to study growth PR:PROJECT_SUMMARY and virulence of Brucella abortus at the single cell level. Using a genetic PR:PROJECT_SUMMARY approach, we found that B. abortus selected a hybrid Mla-Pqi system (here called PR:PROJECT_SUMMARY Mpc) anchored in the envelope, that is necessary to resist to envelope stress PR:PROJECT_SUMMARY and to infect macrophages. We also found that the main component of the outer PR:PROJECT_SUMMARY membrane, the lipopolysaccharide, is displaying a surprising traffic inside PR:PROJECT_SUMMARY bacteria (the “round trip model”) that we propose to investigate. We also PR:PROJECT_SUMMARY constructed a strain (here called RgsE*) that is growing bipolarly instead of PR:PROJECT_SUMMARY unipolarly and we propose to analyze it to better understand polar ageing and PR:PROJECT_SUMMARY potential polar virulence factors. One such polar virulence factor is the type 4 PR:PROJECT_SUMMARY secretion system VirB, that we found associated to the new pole of the bacterium PR:PROJECT_SUMMARY during macrophage infection. Altogether, our project aims to get a better PR:PROJECT_SUMMARY understanding of envelope growth and properties, especially related to the PR:PROJECT_SUMMARY virulence of B. abortus, with a high potential to generate new concepts that PR:PROJECT_SUMMARY could be applicable to other bacterial pathogens. PR:INSTITUTE Université de Namur PR:LAST_NAME de Bolle PR:FIRST_NAME Xavier PR:ADDRESS Rue de Bruxelles 61, 5000 Namur, Bélgica PR:EMAIL xavier.debolle@unamur.be PR:PHONE 690090778 #STUDY ST:STUDY_TITLE A lipid transport Mla Pqi Chimeric system is essential for Brucella abortus ST:STUDY_TITLE survival in macrophages ST:STUDY_SUMMARY The envelope of diderm bacteria comprises of an inner membrane (IM) and an outer ST:STUDY_SUMMARY membrane (OM). Several pathways have been recently identified that facilitate ST:STUDY_SUMMARY the transport of phospholipids between the two membranes in Escherichia coli, ST:STUDY_SUMMARY including maintaining OM lipid asymmetry (Mla) and paraquat inducible (Pqi) ST:STUDY_SUMMARY systems. In this study, we report the identification and the characterization of ST:STUDY_SUMMARY a complex named Mpc in the intracellular pathogen Brucella abortus. Mpc is ST:STUDY_SUMMARY conserved in numerous species of Hyphomicrobiales and exhibits homology to both ST:STUDY_SUMMARY the Mla and Pqi systems. Mpc is essential for bacterial growth under conditions ST:STUDY_SUMMARY of envelope stress and for survival within macrophages during the early stages ST:STUDY_SUMMARY of infection. Analyses of protein-protein interactions and structural ST:STUDY_SUMMARY predictions indicate that the Mpc complex bridges IM to OM. The absence of this ST:STUDY_SUMMARY system results in an altered lipid composition of the OM vesicles, supporting ST:STUDY_SUMMARY the fact that Mpc plays a role in the transport of lipids between membranes. The ST:STUDY_SUMMARY discovery of a novel lipid trafficking system enhances the diversity and ST:STUDY_SUMMARY complexity of known lipid trafficking systems within diderm bacteria. ST:INSTITUTE CEMBIO ST:LAST_NAME Garcia ST:FIRST_NAME Antonia ST:ADDRESS Urbanización, 28668 Monteprincipe, Madrid ST:EMAIL antogar@ceu.es ST:PHONE 913724711 #SUBJECT SU:SUBJECT_TYPE Bacteria SU:SUBJECT_SPECIES Brucella abortus #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS BLEBS_1 BLEBS_1_pos Genotype:Wild-type | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_1_pos.mzML SUBJECT_SAMPLE_FACTORS BLEBS_2 BLEBS_2_pos Genotype:mcpA-knockout | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_2_pos.mzML SUBJECT_SAMPLE_FACTORS BLEBS_3 BLEBS_3_pos Genotype:mcpD-knockout | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_3_pos.mzML SUBJECT_SAMPLE_FACTORS BLEBS_4 BLEBS_4_pos Genotype:mcpE-knockout | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_4_pos.mzML SUBJECT_SAMPLE_FACTORS BLEBS_5 BLEBS_5_pos Genotype:mcpF-knockout | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_5_pos.mzML SUBJECT_SAMPLE_FACTORS BLEBS_6 BLEBS_6_pos Genotype:mcp-fullcomplex-knockout | Sample source:OMVs ESI polarity=positive; RAW_FILE_NAME(Raw file name)=BLEBS_6_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_7 WHOLE CELLS_7_pos Genotype:Wild-type | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_7_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_8 WHOLE CELLS_8_pos Genotype:mcpA-knockout | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_8_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_9 WHOLE CELLS_9_pos Genotype:mcpD-knockout | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_9_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_10 WHOLE CELLS_10_pos Genotype:mcpE-knockout | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_10_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_11 WHOLE CELLS_11_pos Genotype:mcpF-knockout | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_11_pos.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_12 WHOLE CELLS_12_pos Genotype:mcp-fullcomplex-knockout | Sample source:Whole cells ESI polarity=positive; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_12_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_1 BLEBS_1_neg Genotype:Wild-type | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_1_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_2 BLEBS_2_neg Genotype:mcpA-knockout | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_2_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_3 BLEBS_3_neg Genotype:mcpD-knockout | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_3_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_4 BLEBS_4_neg Genotype:mcpE-knockout | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_4_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_5 BLEBS_5_neg Genotype:mcpF-knockout | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_5_neg.mzML SUBJECT_SAMPLE_FACTORS BLEBS_6 BLEBS_6_neg Genotype:mcp-fullcomplex-knockout | Sample source:OMVs ESI polarity=negative; RAW_FILE_NAME(Raw file name)=BLEBS_6_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_7 WHOLE CELLS_7_neg Genotype:Wild-type | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_7_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_8 WHOLE CELLS_8_neg Genotype:mcpA-knockout | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_8_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_9 WHOLE CELLS_9_neg Genotype:mcpD-knockout | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_9_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_10 WHOLE CELLS_10_neg Genotype:mcpE-knockout | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_10_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_11 WHOLE CELLS_11_neg Genotype:mcpF-knockout | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_11_neg.mzML SUBJECT_SAMPLE_FACTORS WHOLE CELLS_12 WHOLE CELLS_12_neg Genotype:mcp-fullcomplex-knockout | Sample source:Whole cells ESI polarity=negative; RAW_FILE_NAME(Raw file name)=WHOLE CELLS_12_neg.mzML #COLLECTION CO:COLLECTION_SUMMARY Lipids were extracted using a modified procedure inspired by earlier methods CO:COLLECTION_SUMMARY (Bligh & Dyer, 1959, https://doi.org/10.1139/o59-099; Daniels et al., 1993, CO:COLLECTION_SUMMARY https://doi.org/10.1128/9781555817497.ch18). In summary, 500 µL of bacterial or CO:COLLECTION_SUMMARY outer membrane vesicle (OMV) suspensions (20 mg of lyophilized material) in CO:COLLECTION_SUMMARY deionized water were combined with 650 µL of chloroform and 1300 µL of CO:COLLECTION_SUMMARY methanol. The mixture was vortexed for 3 hours, after which 650 µL of CO:COLLECTION_SUMMARY chloroform and 650 µL of deionized water were added and gently mixed for 30 CO:COLLECTION_SUMMARY minutes. Phase separation was achieved through brief centrifugation, and the CO:COLLECTION_SUMMARY organic layer was isolated and dried under a nitrogen stream. For OMV isolation, CO:COLLECTION_SUMMARY cultures of 800 mL were grown for 48 hours, treated with 0.5% phenol to CO:COLLECTION_SUMMARY inactivate them, and then centrifuged at 8,200 × g for 20 minutes at 4°C. The CO:COLLECTION_SUMMARY pellet was retained, while the supernatant was processed using a Pellicon® CO:COLLECTION_SUMMARY tangential flow filtration system with a 100 kDa membrane to concentrate it. CO:COLLECTION_SUMMARY Afterward, the concentrated supernatant was centrifuged again under the same CO:COLLECTION_SUMMARY conditions to remove any solid particles. To encourage the aggregation of OMVs, CO:COLLECTION_SUMMARY the supernatant was frozen at -20°C, thawed, and then ultracentrifuged at CO:COLLECTION_SUMMARY 47,000 × g for 3 hours at 4°C. The resulting pellet, containing the OMVs, was CO:COLLECTION_SUMMARY resuspended in deionized water, dialyzed at 4°C for three days, and stored at CO:COLLECTION_SUMMARY -80°C. Both the pellet from the culture and the OMV suspension were CO:COLLECTION_SUMMARY freeze-dried using a TELSTAR CRYODOS 50 lyophilizer. For additional information CO:COLLECTION_SUMMARY of isolation of OMV or sample treatment bacteria prior to extraction refer to CO:COLLECTION_SUMMARY doi: https://doi.org/10.1101/2024.10.31.621289 CO:SAMPLE_TYPE bacterial cells, outer membrane vesicles #TREATMENT TR:TREATMENT_SUMMARY No treatment, since this is a genotype study where knockouts were compared. For TR:TREATMENT_SUMMARY the methodology to generate the specific knockouts of the MPC complex, please TR:TREATMENT_SUMMARY refer to doi: https://doi.org/10.1101/2024.10.31.621289 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipid residues from whole bacteria or OMVs, prepared following the Bligh and SP:SAMPLEPREP_SUMMARY Dyer method, were dissolved in 300 µL of a methanol:chloroform solution SP:SAMPLEPREP_SUMMARY containing 25 mg/L of d17:0 sphinganine (IS1). Lipids were further extracted by SP:SAMPLEPREP_SUMMARY vortexing the mixture at room temperature for 20 minutes. The resulting extracts SP:SAMPLEPREP_SUMMARY were transferred to individual LC-MS vials, where 20 µL of the SPLASH SP:SAMPLEPREP_SUMMARY Lipidomix® lipid standard mix (IS2, Avanti Polar Lipids, CA, USA) were added. SP:SAMPLEPREP_SUMMARY Samples were then dried completely using a vacuum concentrator at 37°C. SP:SAMPLEPREP_SUMMARY Finally, the residues were re-extracted in 100 µL of methanol:chloroform (2:1, SP:SAMPLEPREP_SUMMARY v/v) with thorough vortexing for 30 minutes at room temperature before analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The chromatographic method is the one comprehensively described in doi: CH:CHROMATOGRAPHY_SUMMARY 10.1016/j.jlr.2024.100671 and adapted from Agilent Lipid Annotator application CH:CHROMATOGRAPHY_SUMMARY note (see CH:CHROMATOGRAPHY_SUMMARY https://www.agilent.com/cs/library/applications/application-6546-q-tof-lipidome-5994-0775en-agilent.pdf) CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 HPLC CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (100 x 3.0mm, 2.7um) with guard column CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC-C18 (5 x 3.0mm, 2.7um) CH:SOLVENT_A 90% water/10% methanol; 10 mM ammonium acetate; 0.2 mM ammonium fluoride CH:SOLVENT_B 20% acetonitrile/30% methanol/50% isopropanol10 mM ammonium acetate; 0.2 mM CH:SOLVENT_B ammonium fluoride CH:FLOW_GRADIENT Time (min)/%B: 0.00/70, 1.00/70, 3.50/86, 10.00/86 11.00/100, 17.00/100, CH:FLOW_GRADIENT 17.10/70, 19.00/70 CH:FLOW_RATE 0.6 mL/min CH:COLUMN_TEMPERATURE 50 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Full MS mode was employed for data acquisition, with a scan rate of 3.5 spectra MS:MS_COMMENTS per second across an m/z range of 50 to 3000 in ESI- mode. Lipid annotation was MS:MS_COMMENTS performed using Agilent MassHunter Lipid Annotator (v. 1.0), followed by manual MS:MS_COMMENTS verification and refinement of annotations in Agilent MassHunter Qualitative MS:MS_COMMENTS Analysis (v. 10.0), taking into account diagnostic ions for lipid subclasses, MS:MS_COMMENTS adduct patterns, and expected elution orders. MS:CAPILLARY_VOLTAGE 3500 V MS:DRY_GAS_FLOW 10 L/min MS:DRY_GAS_TEMP 200 ºC MS:FRAGMENT_VOLTAGE 150 V MS:NEBULIZER 50 psi MS:OCTPOLE_VOLTAGE 750 V MS:MS_RESULTS_FILE ST003696_AN006065_Results.txt UNITS:amu Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END