{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T17:51:37Z","timestamp":1785433897754,"version":"3.56.0"},"reference-count":115,"publisher":"American Society for Microbiology","issue":"9","license":[{"start":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T00:00:00Z","timestamp":1759190400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T00:00:00Z","timestamp":1759190400000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/100010663","name":"H2020 European Research Council","doi-asserted-by":"publisher","award":["865175 - KineTic"],"award-info":[{"award-number":["865175 - KineTic"]}],"id":[{"id":"10.13039\/100010663","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["mSphere"],"published-print":{"date-parts":[[2025,9,30]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title\/>\n                    <jats:p>\n                      Bacterial persisters are a subpopulation of cells that exhibit a transient non-susceptible phenotype in the presence of bactericidal antibiotic concentrations. This phenotype can lead to the survival and regrowth of bacteria after treatment, resulting in relapse of infections. It is also a contributing factor to antibacterial resistance. Multiple processes are believed to cause persister formation; however, identifying the proteins expressed during the induction of persistence is challenging because the persister state is rare, transient, and does not result in genetic changes. In this study, we used Bio-Orthogonal Non-Canonical Amino Acid Tagging (BONCAT) to label and retrieve the proteome expressed during persistence and recovery for two strains of\n                      <jats:italic toggle=\"yes\">Staphylococcus aureus<\/jats:italic>\n                      exposed to \u03b2-lactam and fluoroquinolone antibiotics. After incubating antibiotic-exposed bacteria with the methionine ortholog L-azidohomoalanine to label the proteins of persister cells, we retrieved labeled proteins using click chemistry-pulldown methodology. Analysis of the retrieved proteome of persisters with Label-Free Quantification-Liquid chromatography mass spectrometry (LFQ-LCMS)-based proteomics revealed widespread changes in translation. Our analysis uncovered previously identified persister genes, including, for example,\n                      <jats:italic toggle=\"yes\">relA\/spot<\/jats:italic>\n                      -system, changes in purine and amino acid metabolism, the upregulation and downregulation of transcription factors, and changes to influx and efflux pumps, thus validating our methodology. In addition, we also identified numerous novel persister-associated proteins. Few changes were conserved across the two strains and both antibiotics. Instead, results suggest that the mechanisms of persister formation vary across genotypes and the drugs to which strains are exposed. These findings provide evidence that the entry into persistence is an active process that dramatically alters the translational behavior of cells and suggest that downregulation of metabolism, by diverse but functionally similar processes, in persister cells enables cells to survive antibiotic pressure.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>In this study, we have applied a technique called \u201cBioorthogonal Non-Canonical Amino Acid-Tagging,\u201d or BONCAT, to identify which proteins are expressed when bacteria are in the persister state. Our work makes novel contributions to our understanding of persister cells, a bacterial sub-population that gives rise to recurrent infections, and establishes BONCAT as a valuable tool to study phenotypic heterogeneity in bacterial populations.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/msphere.00431-25","type":"journal-article","created":{"date-parts":[[2025,9,8]],"date-time":"2025-09-08T14:12:24Z","timestamp":1757340744000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":2,"title":["Using BONCAT to dissect the proteome of\n                    <i>S. aureus<\/i>\n                    persisters"],"prefix":"10.1128","volume":"10","author":[{"given":"Eva D. C.","family":"George Matlalcuatzi","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas","family":"Bakkum","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pooja S.","family":"Thomas","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephan","family":"Hacker","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bogdan I.","family":"Florea","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bastienne","family":"Vriesendorp","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Biology Leiden, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7772-0239","authenticated-orcid":false,"given":"Daniel E.","family":"Rozen","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Institute of Biology Leiden, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3733-818X","authenticated-orcid":true,"given":"Sander I.","family":"van Kasteren","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/027bh9e22","id-type":"ROR","asserted-by":"publisher"}],"name":"Leiden Institute of Chemistry and The Institute of Chemical Immunology, Leiden University","place":["Leiden, the Netherlands"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-019-0196-3"},{"key":"e_1_3_2_3_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.aaj2191"},{"key":"e_1_3_2_4_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.ppat.1007301"},{"key":"e_1_3_2_5_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1229858"},{"key":"e_1_3_2_6_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.02267-16"},{"key":"e_1_3_2_7_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01651-09"},{"key":"e_1_3_2_8_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1244705"},{"key":"e_1_3_2_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/nmicrobiol.2016.51"},{"key":"e_1_3_2_10_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-024-01866-5"},{"key":"e_1_3_2_11_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fux001"},{"key":"e_1_3_2_12_2","doi-asserted-by":"publisher","DOI":"10.1080\/14789450.2019.1575207"},{"key":"e_1_3_2_13_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-0716-1621-5_1"},{"key":"e_1_3_2_14_2","doi-asserted-by":"publisher","DOI":"10.1128\/aac.00850-23"},{"key":"e_1_3_2_15_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00243-13"},{"key":"e_1_3_2_16_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.00378"},{"key":"e_1_3_2_17_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.3001194"},{"key":"e_1_3_2_18_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.1099390"},{"key":"e_1_3_2_19_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00303-18"},{"key":"e_1_3_2_20_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01617-16"},{"key":"e_1_3_2_21_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00134-14"},{"key":"e_1_3_2_22_2","doi-asserted-by":"publisher","DOI":"10.1080\/21505594.2021.1878688"},{"key":"e_1_3_2_23_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-023-00852-y"},{"key":"e_1_3_2_24_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms241814311"},{"key":"e_1_3_2_25_2","doi-asserted-by":"publisher","DOI":"10.1128\/IAI.01439-09"},{"key":"e_1_3_2_26_2","doi-asserted-by":"publisher","DOI":"10.1128\/jb.00162-22"},{"key":"e_1_3_2_27_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics11121702"},{"key":"e_1_3_2_28_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2021.657753"},{"key":"e_1_3_2_29_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-13021-8"},{"key":"e_1_3_2_30_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.isci.2024.110002"},{"key":"e_1_3_2_31_2","doi-asserted-by":"publisher","DOI":"10.3390\/ijms221810059"},{"key":"e_1_3_2_32_2","doi-asserted-by":"publisher","DOI":"10.1128\/aac.00850-23"},{"key":"e_1_3_2_33_2","doi-asserted-by":"publisher","DOI":"10.1038\/s43586-021-00028-z"},{"key":"e_1_3_2_34_2","doi-asserted-by":"publisher","DOI":"10.1021\/jacs.2c03743"},{"key":"e_1_3_2_35_2","doi-asserted-by":"publisher","DOI":"10.1039\/d2cb00174h"},{"key":"e_1_3_2_36_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.1c00729"},{"key":"e_1_3_2_37_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.0c00928"},{"key":"e_1_3_2_38_2","doi-asserted-by":"publisher","DOI":"10.3354\/ame01698"},{"key":"e_1_3_2_39_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.bioconjchem.1c00461"},{"key":"e_1_3_2_40_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2023.112828"},{"key":"e_1_3_2_41_2","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202207640"},{"key":"e_1_3_2_42_2","doi-asserted-by":"publisher","DOI":"10.1111\/1462-2920.12436"},{"key":"e_1_3_2_43_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0601637103"},{"key":"e_1_3_2_44_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.012583299"},{"key":"e_1_3_2_45_2","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-7574-7_8"},{"key":"e_1_3_2_46_2","doi-asserted-by":"publisher","DOI":"10.1021\/ar200144y"},{"key":"e_1_3_2_47_2","doi-asserted-by":"publisher","DOI":"10.1021\/acschembio.8b00355"},{"key":"e_1_3_2_48_2","doi-asserted-by":"publisher","DOI":"10.1039\/c6cc01321j"},{"key":"e_1_3_2_49_2","doi-asserted-by":"publisher","DOI":"10.1038\/s43705-022-00205-5"},{"key":"e_1_3_2_50_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-39063-7"},{"key":"e_1_3_2_51_2","doi-asserted-by":"publisher","DOI":"10.1093\/database\/baaa062"},{"key":"e_1_3_2_52_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-7-99"},{"key":"e_1_3_2_53_2","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciu043"},{"key":"e_1_3_2_54_2","volume-title":"StatPearls","author":"Humma ZE","year":"2024","unstructured":"Humma ZE, Patel P. 2024. Moxifloxacin. In StatPearls. StatPearls Publishing LLC.: Treasure Island (FL)."},{"key":"e_1_3_2_55_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41597-019-0051-4"},{"key":"e_1_3_2_56_2","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.9b00096"},{"key":"e_1_3_2_57_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.521"},{"key":"e_1_3_2_58_2","volume-title":"Methods for antimicrobial susceptibility testing for human mycoplasmas","author":"Waites KB","year":"2011","unstructured":"Waites KB. 2011. CLSI standards: guidelines for health care excellence. In Methods for antimicrobial susceptibility testing for human mycoplasmas. Clinical and Laboratory Standards Institute: Wayne (PA)."},{"key":"e_1_3_2_59_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpha.2015.11.005"},{"key":"e_1_3_2_60_2","doi-asserted-by":"publisher","DOI":"10.3390\/antibiotics12030455"},{"key":"e_1_3_2_61_2","doi-asserted-by":"publisher","DOI":"10.15252\/embr.202051034"},{"key":"e_1_3_2_62_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.587364"},{"key":"e_1_3_2_63_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15966-7"},{"key":"e_1_3_2_64_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro.2016.34"},{"key":"e_1_3_2_65_2","doi-asserted-by":"publisher","DOI":"10.1039\/c5sc02905h"},{"key":"e_1_3_2_66_2","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.201800230"},{"key":"e_1_3_2_67_2","doi-asserted-by":"publisher","DOI":"10.1021\/acscentsci.0c00539"},{"key":"e_1_3_2_68_2","doi-asserted-by":"publisher","DOI":"10.1021\/cr0783479"},{"key":"e_1_3_2_69_2","doi-asserted-by":"publisher","DOI":"10.1021\/acs.est.4c02906"},{"key":"e_1_3_2_70_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.01929"},{"key":"e_1_3_2_71_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0601167103"},{"key":"e_1_3_2_72_2","doi-asserted-by":"publisher","DOI":"10.1039\/b105185g"},{"key":"e_1_3_2_73_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41396-020-00749-1"},{"key":"e_1_3_2_74_2","doi-asserted-by":"publisher","DOI":"10.1021\/pr5002862"},{"key":"e_1_3_2_75_2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-17010-0"},{"key":"e_1_3_2_76_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.xpro.2023.102574"},{"key":"e_1_3_2_77_2","doi-asserted-by":"publisher","DOI":"10.1002\/cpz1.805"},{"key":"e_1_3_2_78_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2017.159"},{"key":"e_1_3_2_79_2","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2014.00148"},{"key":"e_1_3_2_80_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00372-13"},{"key":"e_1_3_2_81_2","doi-asserted-by":"publisher","DOI":"10.15252\/msb.20166998"},{"key":"e_1_3_2_82_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.copbio.2017.02.012"},{"key":"e_1_3_2_83_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.2014920118"},{"key":"e_1_3_2_84_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2019.01222"},{"key":"e_1_3_2_85_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.00865"},{"key":"e_1_3_2_86_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2958.2004.04368.x"},{"key":"e_1_3_2_87_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00188-19"},{"key":"e_1_3_2_88_2","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2019.1595984"},{"key":"e_1_3_2_89_2","doi-asserted-by":"publisher","DOI":"10.1111\/mmi.14404"},{"key":"e_1_3_2_90_2","doi-asserted-by":"publisher","DOI":"10.1126\/scisignal.aaf7279"},{"key":"e_1_3_2_91_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00905-08"},{"key":"e_1_3_2_92_2","doi-asserted-by":"publisher","DOI":"10.1128\/mBio.01646-20"},{"key":"e_1_3_2_93_2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.3769"},{"key":"e_1_3_2_94_2","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2019.7750"},{"key":"e_1_3_2_95_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0094358"},{"key":"e_1_3_2_96_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.00113"},{"key":"e_1_3_2_97_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03599.x"},{"key":"e_1_3_2_98_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.187.5.1815-1824.2005"},{"key":"e_1_3_2_99_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-micro-092412-155620"},{"key":"e_1_3_2_100_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1558-5646.2011.01467.x"},{"key":"e_1_3_2_101_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0084361"},{"key":"e_1_3_2_102_2","doi-asserted-by":"publisher","DOI":"10.1128\/spectrum.02701-22"},{"key":"e_1_3_2_103_2","doi-asserted-by":"publisher","DOI":"10.1080\/14789450.2020.1784731"},{"key":"e_1_3_2_104_2","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2020.00197"},{"key":"e_1_3_2_105_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuac025"},{"key":"e_1_3_2_106_2","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2180-11-16"},{"key":"e_1_3_2_107_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.micpath.2022.105734"},{"key":"e_1_3_2_108_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2020.08.005"},{"key":"e_1_3_2_109_2","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pbio.1000317"},{"key":"e_1_3_2_110_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrmicro3448"},{"key":"e_1_3_2_111_2","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01283-20"},{"key":"e_1_3_2_112_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.micro.112408.134306"},{"key":"e_1_3_2_113_2","doi-asserted-by":"publisher","DOI":"10.1128\/JB.186.24.8172-8180.2004"},{"key":"e_1_3_2_114_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(84)90782-6"},{"key":"e_1_3_2_115_2","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.261"},{"key":"e_1_3_2_116_2","doi-asserted-by":"publisher","DOI":"10.1021\/pr101065j"}],"container-title":["mSphere"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00431-25","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/journals.asm.org\/doi\/pdf\/10.1128\/msphere.00431-25","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T13:01:59Z","timestamp":1759237319000},"score":1,"resource":{"primary":{"URL":"https:\/\/journals.asm.org\/doi\/10.1128\/msphere.00431-25"}},"subtitle":[],"editor":[{"given":"James P.","family":"O'Gara","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2025,9,30]]},"references-count":115,"journal-issue":{"issue":"9","published-print":{"date-parts":[[2025,9,30]]}},"alternative-id":["10.1128\/msphere.00431-25"],"URL":"https:\/\/doi.org\/10.1128\/msphere.00431-25","relation":{},"ISSN":["2379-5042"],"issn-type":[{"value":"2379-5042","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,9,30]]},"assertion":[{"value":"2025-07-07","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-08-06","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2025-09-08","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e00431-25"}}