MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/m_MTBLS14968_LC-MS_positive_hilic_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/m_MTBLS14968_LC-MS_negative_hilic_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/a_MTBLS14968_LC-MS_positive_hilic.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/s_MTBLS14968.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968/a_MTBLS14968_LC-MS_negative_hilic.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14968SIMCAP software (Version 14.0, Umetrics, Umeå, Sweden) was used for all multivariate data analyses and modeling. Data were mean-centered using Pareto scaling. Models were built on principal component analysis (PCA), orthogonal partial least-square discriminant analysis (PLS-DA) and partial least-square discriminant analysis (OPLS-DA). All the models evaluated were tested for over fitting with methods of permutation tests.MetaboLightsPublicLiquid Chromatography MS - positive - hilicLiquid Chromatography MS - negative - hilicFor hydrophilic interaction liquid chromatography (HILIC) separation, samples were analyzed using a 2.1 mm × 100 mm ACQUIY UPLC BEH Amide 1.7 μm column (Waters, Ireland). The flow rate was 0.5 mL/min and the mobile phase contained: A: 25 mM ammonium acetate and 25 mM ammonium hydroxide in water and B: 100% acetonitrile (ACN). The gradient was 95% B for 0.5 min and was linearly reduced to 65% in 6.5 min, and then reduced to 40% in 2 min and maintained for 1 min, and then increased to 95% in 1.1 min, with 5 min reequilibration period employed.Nitrate Reductase NarGHJI Modulates Vancomycin Susceptibility by Coordinating Cell Wall Remodeling and Agmatine Metabolism in Staphylococcus aureus.Yujie LiFuyang Normal UniversitySupernatant of Staphylococcus aureusmass spectrometry assaySupernatants from separate overnight cultures of USA300 LAC and the ΔnarG mutant were collected by centrifugation (12,000 × g, 30 min).Staphylococcus aureushttps://www.ebi.ac.uk/metabolights/MTBLS14968Xiaodie Xing. Fuyang Normal University. 2025211316@stu.fynu.edu.cn.Yujie Li. Fuyang Normal University. lyj2025@fynu.edu.cn.The raw MS data were converted to MzXML files using ProteoWizard MSConvert and processed using XCMS for feature detection, retention time correction and alignment. The metabolites were identified by accuracy mass (< 25 ppm) and MS/MS data which were matched with a standards database. In the extracted-ion features, only the variables having more than 50% of the nonzero measurement values in at least one group were kept.Grouplyj2025@fynu.edu.cnSupernatants from separate overnight cultures of USA300 LAC and the ΔnarG mutant were collected by centrifugation (12,000 × g, 30 min) and submitted to Shanghai Bioprofile Technology Company, Ltd. for metabolomics analysis.MetabolomicsMetabolomicsAgmatine MetabolismVancomycin SusceptibilityNitrate Reductase NarGHJIuntargeted analysisCell Wall RemodelingAgilent 1290 Infinity LC systemStaphylococcus aureusSupernatant of Staphylococcus aureusAB SCIEX TripleTOF 5600MetabolomicsAgmatine MetabolismVancomycin SusceptibilityNitrate Reductase NarGHJIuntargeted analysisCell Wall RemodelingAgilent 1290 Infinity LC systemStaphylococcus aureusAB SCIEX TripleTOF 5600Supernatant of Staphylococcus aureusBoth electrospray ionization (ESI) positive-mode and negative mode were applied for MS data acquisition. The ESI source conditions were set as follows: Ion Source Gas 1 as 60, Ion Source Gas 2 as 60, curtain gas as 30, source temperature: 600 °C, IonSpray Voltage Floating (ISVF) ± 5500 V. In MS only acquisition, the instrument was set to acquire over the m/z range 60-1200 Da, and the accumulation time for TOF MS scanning was set at 0.15 s/spectra. In auto MS/MS acquisition, the instrument was set to acquire over the m/z range 25-1200 Da, and the accumulation time for product- ion scan was set at 0.03 s/spectra. The product-ion scan was acquired using information dependent acquisition with high sensitivity mode selected. The collisional energy was fixed at 30 V with ± 15 eV. Declustering potential was set as ± 60 V. Quality control (QC) samples were prepared by pooling aliquots of all samples that were representative of the samples under analysis, and used for data normalization. Blank samples (75 %ACN in water) and QC samples were injected every six samples during acquisition.falseNitrate Reductase NarGHJI Modulates Vancomycin Susceptibility by Coordinating Cell Wall Remodeling and Agmatine Metabolism in Staphylococcus aureusStaphylococcus aureus is a ubiquitous pathogen responsible for a wide range of severe infections. Our previous work established that the nitrate reductase NarGHJI modulates bacterial pathogenicity through regulation of the agr system, yet its role in antibiotic resistance remains poorly understood. In this study, we investigated whether NarGHJI influences susceptibility to vancomycin of S. aureus. Disruption of narG significantly reduced vancomycin susceptibility, prompting us to explore the underlying mechanisms. Phenotypic analyses revealed that the ΔnarG mutant possessed a markedly thickened cell wall and exhibited a decreased autolysis rate compared to the wild-type strain. Consistently, RT-qPCR showed a distinct transcriptional reprogramming: autolysis-related genes (lytN and ssaA) were significantly downregulated, while the cell wall synthesis gene dltA was upregulated. Furthermore, we uncovered that NarGHJI regulates vancomycin susceptibility through agmatine metabolism, which in turn reduces the net negative surface charge of S. aureus cells, ultimately impairing vancomycin binding to the cell wall. Notably, this NarGHJI-mediated resistance mechanism appeared to be strain-dependent to some extent. Collectively, our findings reveal a novel regulatory axis linking NarGHJI to vancomycin resistance, suggesting this enzyme complex as a potential therapeutic target for combating S. aureus infections.2026-07-072026-07-07MTBLS14968