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antibiotic resistance; antibiofilm activity; activity-based protein profiling; fabh","Dia","fabh","antibiofilm activity"],"full_dataset_link":["https://www.ebi.ac.uk/pride/archive/projects/PXD073012"],"sample_protocol":["Fresh mid-log phase bacterial suspension (OD600nm = 0.6-0.8) in MHII btoth was pelleted and resuspended to a final volume of 1mL at a theoretical OD600nm of 20. The bacterial cells were incubated overnight at 37°C under shaking at 180 rpm with each OXyne probe (500µM final concentration), or DMSO (control). Bacteria were washed and resuspended in PBS supplemented with cOmplete Mini EDTA-free protease inhibitors (Roche Mannheim, Germany) before being lysed by mechanical disruption using a Mini-Beadbeater-96 (BioSpec, Bartlesville, OK, USA). All samples (300 µL – 0.3 mg total proteins) were subjected to click-chemistry reaction with Desthiobiotin-PEG3-N3 (Jena Bioscience, Germany, #CLK-AZ104P4–100; 40 mM in DMSO). TBTA ligand and freshly prepared TCEP solution (Sigma-Aldrich) to the cells. After gently vortexing of the samples, the cycloaddition reaction was initiated by addition of CuSO4 solution (Sigma-Aldrich). The proteins were further precipitated overnight at −20 °C by adding 4 mL ice-cold acetone, and pelletized by centrifugation. The supernatant was discarded, and the proteins were washed with 2 × 500 μL ice-cold MeOH. After centrifugation, the pellets were resuspended in 0.2% (w/v) SDS in PBS (250 μL) at room temperature by mild sonication. Affinity enrichment was performed with 25 μL Pierce High Capacity Streptavidin Agarose Resin (ThermoFischer Scientific, ref. 20359), according to the manufacturer’s instructions. The resulting captured biotinylated proteins solution was mixed with 5X Laemmli reducing sample buffer, and heated at 95 °C for 5 min. The released denatured proteins were subjected to tryptic digestion, peptide extraction, and LC-MS/MS analysis as described below. Briefly, enriched samples (n=3, biological replicates) were loaded on NuPAGE™ 4–12% Bis–Tris acrylamide gels according to the manufacturer’s instructions (Life Technologies). Protein migration was interrupted as soon as the proteins stacked in a single band. Protein-containing bands were stained with Imperial Blue (Pierce), cut from the gel and digested with high sequencing grade trypsin (Promega) before being analyzed by mass spectrometry. Briefly, gel pieces were washed and distained using few steps of 100mM NH4HCO3. Distained gel pieces were shrunk with 100 mM ammonium bicarbonate in 50% acetonitrile and dried at RT. Protein spots were then rehydrated using 10mM DTT in 25 mM ammonium bicarbonate pH 8.0 for 45 min at 56°C. This solution was replaced by 55 mM iodoacetamide in 25 mM ammonium bicarbonate pH 8.0 and the gel pieces were incubated for 30 min at room temperature in the dark. They were then washed twice in 25 mM ammonium bicarbonate and finally shrunk by incubation for 5 min with 25 mM ammonium bicarbonate in 50% acetonitrile. The resulting alkylated gel pieces were dried at room temperature. The dried gel pieces were reswollen by incubation in 25 mM ammonium bicarbonate pH 8.0 supplemented with 12.5 ng/µl trypsin (Promega) for 1h at 4°C and then incubated overnight at 37°C. Peptides were harvested by collecting the initial digestion solution and carrying out two extractions; first in 5% formic acid, then in 5% formic acid in 60% acetonitrile. Pooled extracts were dried down in a centrifugal vacuum system. Peptides were further reconstituted in 15µl water/acetonitrile/ (98/2) containing 0.05%TFA. 20% of each sample was analyzed twice by liquid chromatography (LC)-tandem MS (MS/MS) using a Q-Exactive plus Mass Spectrometer (Thermo Fisher Scientific, San Jose, CA) online with a nanoRSLC Ultimate 3000 chromatography system (Thermo Fisher Scientific, Sunnyvale, CA). First, peptides were concentrated and purified on a pre-column PepMap100 C18, 2 cm × 100 μm I.D, 100 Å pore size, 5 μm particle size in solvent A (0.1% formic acid in 2% acetonitrile). In the second step, peptides were separated on a reverse phase LC EASY-Spray C18 column PepMap RSLC C18, 50 cm × 75 μm I.D, 100 Å pore size, 2 μm particle size (Thermo Fisher) at 300 nL.min−1 flow rate and 40°C. After column equilibration peptides were eluted from the analytical column by a three-step linear gradient (2.5%–27.5% solvent B [80% acetonitrile 0.1% formic acid in water] for 100 min, 27.5%–40% for 20 min and a last elution 40%–90% for 2 min). For peptide ionization in the EASY-Spray nanosource in front of the mass spectrometer, the spray voltage was set at 1,9 kV and the capillary temperature at 250°C. The mass spectrometer was used in data-independent acquisition (DIA) mode with the following parameters. First, MS spectra were acquired in the Orbitrap in the range of m/z 400–1000 at a FWHM resolution of 35,000 measured at 200 m/z. AGC target was set at 1 × 106 with a Maximum Injection Time of 60 ms. MS2 spectra were acquired in the Orbitrap with a resolution of 17,500 after isolation of the parent ion in the quadrupole and fragmentation in the HCD cell under collision Energy of 27%. DIA parent ion range was from 400 to 1000 m.z−1 divided into 24 m.z−1 windows."],"repository":["Pride"],"quantification_method":[""],"modification":[""],"data_protocol":["Relative intensity-based label-free quantification (LFQ) was processed using the DIA-NN 1.8 algorithm (Demichev, V et al., 2020). Raw files were searched against the Staphylococcus aureus database extracted from UniProt on the 17th of February 2024 and containing 2889 entries with the addition of a protein contaminant blank (Frankenfield et al., 2022). The following parameters were used for searches: (i) trypsin allowing cleavage before proline; (ii) one missed cleavage was allowed; (iii) cysteine carbamidomethylation (+57.02146) as a fixed modification and methionine oxidation (+15.99491) and N-terminal acetylation (+42.0106) as variable modifications; (iv) a maximum of one variable modification per peptide allowed; and (v) minimum peptide length was 7 amino acids and a maximum of 30 amino acids. The match between runs option was enabled. The precursor false discovery was set to 1%. DIA-NN parameters were set on Single-pass mode for the Neural Network classifier, Robust LC High precision for quantification strategy and RT-dependent mode for Cross-run normalization. The library was generated using a Smart profiling setup. The main output file from DIA-NN was further filtered at 1% FDR and LFQ intensity was calculated using our DIAgui package at 1% q-value (https://github.com/marseille-proteomique/DIAgui [Gerault et al., 2024]). The statistical analysis was done with the Perseus program (version 1.6.15.0) (Tyanova & Cox, 2018). Quantifiable proteins were defined as those detected in above 70% of samples in at least one condition. Protein LFQ normalized intensities were base 2 logarithmized to obtain a normal distribution. Missing values were replaced using data imputation by randomly selecting from a normal distribution centered on the lower edge of the intensity values that simulate signals of low abundant proteins using default parameters (a downshift of 1.8 standard deviations and a width of 0.3 of the original distribution). To determine whether a given detected protein was specifically differential, a two-sample t-test was done using permutation-based FDR-controlled employing 250 permutations."],"omics_type":["Proteomics"],"labhead":["Stéphane Audebert, PhD"],"instrument_platform":[""],"labhead_affiliation":["MaP, Marseille Protéomique Centre de Recherche en Cancérologie de Marseille, CRCM Inserm UMR1068, CNRS UMR7258, Aix Marseille Université U105, Institut Paoli Calmettes 27 Boulevard Leï Roure CS30059 13273 Marseille Cedex 09 France"],"submission_type":["PARTIAL"],"species":["Staphylococcus Aureus"],"publication":["Not available"],"submitter_mail":["stephane.audebert@inserm.fr"],"submitter_affiliation":["Marseille Proteomic, Centre de Recherche en Cancérologie de Marseille, Inserm UMR1068, CNRS UMR7258, Aix Marseille Université U105, Institut Paoli Calmettes, 27 Boulevard Leï Roure CS30059 13273 Marseille Cedex 09 France"],"submitter_country":["France"],"additional_accession":[]},"is_claimable":false,"name":"Oxadiazolone derivatives block Staphylococcus aureus growth and biofilm formation by covalently binding to strategic (Ser/Cys)-based enzymes","description":"Staphylococcus aureus is a Gram-positive opportunistic pathogen and a top-priority bacterium in the fight against antimicrobial resistance. Its high propensity to mutate, develop resistance, and become more virulent, as well as its ability to form biofilms, results in difficult-to-treat infections against which new chemical classes are urgently needed. Here, we investigated the antibacterial activity of oxadiazolone-core derivatives (OX) against planktonic and biofilm-associated S. aureus. Among the tested compounds, MpPPOX exhibits a strong bactericidal effect on extracellular bacteria with similar MIC to that of Vancomycin; iBPOX mainly inhibits intracellular bacterial growth; while HPOX strongly impair biofilm formation. Such a divergence in activity prompted us to identify their potential target enzymes via activity-based protein profiling combined with mass spectrometry. Although the most active MpPPOX inhibitor targets multiple (Ser/Cys)-based enzymes, the antibiofilm HPOX compound primarily reacts with enzymes involved in biofilm formation and virulence. Among them, the FabH protein has been confirmed as a vulnerable target of MpPPOX. Overall, this study underscores the multi-target nature of the OXs covalently bind to several (Ser/Cys)-based enzymes of interest. This binding property makes them highly versatile chemotypes that could be used as broad-spectrum antimicrobial and anti-virulence agents to potentiate otherwise ineffective or poorly active drugs.","dates":{"publication":"2026-04-01","submission":"2026-01-13"},"accession":"PXD073012","cross_references":{"TAXONOMY":["NEWT:1773","NEWT:6945","NEWT:3555","NEWT:38783","NEWT:8727","NEWT:1182590","NEWT:8726","NEWT:2","NEWT:157546","NEWT:10090","NEWT:935293","NEWT:749200","NEWT:35554","NEWT:4120","NEWT:5693","NEWT:9417","NEWT:347515","NEWT:8724","NEWT:51511","NEWT:1216979","NEWT:307972","NEWT:92867","NEWT:8723","NEWT:990346","NEWT:544496","NEWT:5334","NEWT:145953","NEWT:257309","NEWT:5180","NEWT:284812","NEWT:115104","NCBITaxon:1313","NEWT:1081927","NEWT:43330","NEWT:67825","NEWT:44544","NEWT:13076","NEWT:1249668","NEWT:373995","NEWT:544404","NEWT:3702","NEWT:8839","NEWT:317","NEWT:4232","NEWT:990119","NEWT:1736309","NEWT:4113","NEWT:7227","NEWT:11298","NEWT:7469","NEWT:885318","NEWT:171101","NEWT:4081","NEWT:876138","NEWT:554","NEWT:5691","NEWT:98334","NEWT:408170","NEWT:493760","NEWT:260710","NEWT:627025","NEWT:400772","NEWT:3708","NEWT:106592","NEWT:237561","NEWT:9913","NEWT:10036","NEWT:4100","NEWT:7574","NEWT:1351","NEWT:1076","NEWT:6763","NEWT:7215","NEWT:803","NEWT:8030","NEWT:380394","NEWT:272563","NEWT:507601","NEWT:1639","NEWT:188229","NEWT:4909","NCBITaxon:79857","NEWT:95648","NEWT:746360","NEWT:6239","NEWT:1589","NEWT:135588","NEWT:470150","NEWT:135622","NEWT:216257","NEWT:6915","NEWT:9986","NEWT:101510","NEWT:95486","NEWT:3880","NEWT:58002","NEWT:9103","NEWT:4577","NEWT:5664","NEWT:2157","NEWT:146479","NEWT:1911079","NEWT:1000589","NEWT:145943","NEWT:1902","NEWT:85962","NEWT:160488","NEWT:317447","NEWT:3635","NEWT:7955","NCBITaxon:2","NEWT:1480154","NEWT:7959","NEWT:2261","NEWT:3197","NEWT:9615","NEWT:884019","NEWT:4565","NEWT:1264690","NEWT:169963","NCBITaxon:38727","NEWT:36329","NEWT:34305","NEWT:59729","NCBITaxon:183674","NEWT:224308","NEWT:626528","NEWT:139927","NEWT:4558","NEWT:9606","NEWT:367830","NEWT:157295","NEWT:243230","NEWT:931281","NEWT:373153","NEWT:7029","NEWT:915099","NEWT:1283300","NEWT:334747","NEWT:470","NCBITaxon:79824","NCBITaxon:4563","NEWT:3218","NEWT:84023","NEWT:5759","NEWT:9838","NCBITaxon:9615","NEWT:1736231","NEWT:1193501","NEWT:3055","NEWT:6287","NEWT:2242","NEWT:6326","NEWT:9796","NEWT:2762","NEWT:5476","NEWT:725","NEWT:1174673","NEWT:562","NEWT:260707","NEWT:287","NEWT:10117","NEWT:10239","NEWT:10116","NEWT:1280","NEWT:1836","NEWT:1735272","NEWT:29760","NEWT:260705","NEWT:80863","NEWT:1148","NEWT:4932","NEWT:70448","NEWT:9825","NEWT:3603","NEWT:698936","NEWT:2759","NEWT:39946","NEWT:11676","NEWT:9823","NEWT:100226","NCBITaxon:6073","NEWT:4530","NEWT:4896","NEWT:6279","NEWT:7370","NEWT:573","NEWT:6282","NEWT:7091","NEWT:1134506"],"ORCID":["0000-0002-9409-2588"]}}