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Here, we used quantitative liquid chromatography-tandem mass spectrometry (LC-MS\/MS)-based proteomics to define the response of Paracoccidioides brasiliensis to acidic stress (pH 4.5) versus control pH (6.5) after 5 and 24 h. We identified and quantified 4374 proteins, including 327 and 722 differentially abundant proteins at 5 and 24 h, respectively, revealing time-dependent proteomic reprogramming. Enrichment analyses highlighted proteolysis, protein metabolism, organonitrogen metabolism, MAPK- and SNF1-like signaling, central carbon metabolism, tyrosine metabolism, and ergosterol biosynthesis as major acid-responsive processes. Complementary assays showed pH-dependent extracellular proteolytic activity, increased adhesion to A549 pulmonary epithelial cells, and dynamic ergosterol remodeling. The proteomic data further indicated increased abundance of moonlighting proteins linked to adhesion and metabolic enzymes associated with ATP generation and melanin precursor production. Together, these findings indicate that P. brasiliensis adapts to acidic environments through coordinated regulation of proteostasis, metabolism, signaling, host-cell interaction, and membrane homeostasis, supporting survival and virulence potential in acidic host microenvironments.<\/jats:p>","DOI":"10.1021\/acs.jproteome.6c00370","type":"journal-article","created":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T02:01:36Z","timestamp":1786154496000},"page":"4796-4810","source":"Crossref","is-referenced-by-count":0,"title":["Acidic Stress\nInduces Proteomic Reprogramming and\nVirulence-Associated Adaptation in\n                    <i>Paracoccidioides brasiliensis<\/i>"],"prefix":"10.1021","volume":"25","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-2420-1563","authenticated-orcid":true,"given":"Reinaldo Souza","family":"Oliveira","sequence":"first","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0009-2610-0193","authenticated-orcid":true,"given":"Maria Cecilia","family":"Gordijo","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wilson Dias","family":"Segura","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, SP, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pedro Henrique Corteletti","family":"Manfio","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonardo Brogliato","family":"de Moraes","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah Fernandes","family":"Lima","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8994-1904","authenticated-orcid":true,"given":"Solange M. T.","family":"Serrano","sequence":"additional","affiliation":[{"name":"Instituto Butantan , , , ,","place":["Sa\u0303o Paulo, SP, Brazil, 05503-900"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6017-6671","authenticated-orcid":true,"given":"Alison Felipe Alencar","family":"Chaves","sequence":"additional","affiliation":[{"name":"Instituto Butantan , , , ,","place":["Sa\u0303o Paulo, SP, Brazil, 05503-900"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0280-6828","authenticated-orcid":true,"given":"Wagner Luiz","family":"Batista","sequence":"additional","affiliation":[{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Diadema, SP, Brazil, 09913-030"]},{"name":"Federal University of Sa\u0303o Paulo , , , ,","place":["Sa\u0303o Paulo, SP, Brazil, 04023-062"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,7]]},"reference":[{"key":"2026090409142597500_cit1","doi-asserted-by":"publisher","first-page":"e428","DOI":"10.1016\/S1473-3099(23)00692-8","article-title":"Global\nIncidence and Mortality of Severe Fungal Disease","volume":"24","author":"Denning","year":"2024","journal-title":"Lancet Infect. 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Rep."},{"key":"2026090409142597500_cit4","doi-asserted-by":"publisher","first-page":"646","DOI":"10.1007\/s00103-019-02931-z","article-title":"Climate Change\nand Systemic Fungal Infections","volume":"62","author":"Rickerts","year":"2019","journal-title":"Bundesgesundheitsblatt\nGesundheitsforschung Gesundheitsschutz"},{"key":"2026090409142597500_cit5","doi-asserted-by":"publisher","DOI":"10.3390\/jof7020116","article-title":"Updates in Paracoccidioides Biology\nand Genetic Advances in Fungus Manipulation","volume":"7","author":"Chaves","year":"2021","journal-title":"J. Fungi"},{"key":"2026090409142597500_cit6","doi-asserted-by":"publisher","DOI":"10.1093\/ofid\/ofac559","article-title":"Fungal Nomenclature: Managing Change\nIs the Name of the Game","volume":"10","author":"Kidd","year":"2023","journal-title":"Open Forum Infect.\nDis."},{"key":"2026090409142597500_cit7","doi-asserted-by":"publisher","first-page":"1177","DOI":"10.2217\/fmb.13.68","article-title":"Paracoccidioidomycosis: Eco-Epidemiology,\nTaxonomy\nand Clinical and Therapeutic Issues","volume":"8","author":"Bocca","year":"2013","journal-title":"Future\nMicrobiol."},{"key":"2026090409142597500_cit8","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1093\/molbev\/msj008","article-title":"Cryptic Speciation and Recombination\nin the Fungus Paracoccidioides brasiliensis as Revealed by Gene Genealogies","volume":"23","author":"Matute","year":"2006","journal-title":"Mol. 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Trop."},{"key":"2026090409142597500_cit22","doi-asserted-by":"publisher","first-page":"182","DOI":"10.1055\/s-2008-1063857","article-title":"Pulmonary\nParacoccidioidomycosis","volume":"29","author":"Restrepo","year":"2008","journal-title":"Semin. Respir. Crit.\nCare Med."},{"key":"2026090409142597500_cit23","doi-asserted-by":"publisher","first-page":"S87","DOI":"10.1080\/13693780400029486","article-title":"Thermotolerance and Virulence of Aspergillus fumigatus: Role of the Fungal Nucleolus","volume":"43","author":"Bhabhra","year":"2005","journal-title":"Med. Mycol."},{"key":"2026090409142597500_cit24","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1007\/978-1-59745-204-5_13","article-title":"Real-Time\nand Semiquantitative RT-PCR\nMethods to Analyze Gene Expression Patterns during Aspergillus\u2013Host Interactions","volume":"470","author":"Bok","year":"2009","journal-title":"Methods Mol. Biol."},{"key":"2026090409142597500_cit25","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0006806","article-title":"Secreted\nAspartyl Proteinase PbSap Contributes to the Virulence of Paracoccidioides brasiliensis Infection","volume":"12","author":"Castilho","year":"2018","journal-title":"PLoS Neglected Trop. Dis."},{"key":"2026090409142597500_cit26","doi-asserted-by":"publisher","first-page":"511","DOI":"10.1016\/j.mib.2008.09.018","article-title":"Fungal Adaptation\nto the Mammalian Host: It Is a New\nWorld, after All","volume":"11","author":"Cooney","year":"2008","journal-title":"Curr. Opin. Microbiol."},{"key":"2026090409142597500_cit27","doi-asserted-by":"publisher","first-page":"575","DOI":"10.1016\/j.micinf.2015.03.012","article-title":"The Response of Paracoccidioides spp.\nto Nitrosative Stress","volume":"17","author":"Parente","year":"2015","journal-title":"Microbes Infect."},{"key":"2026090409142597500_cit28","doi-asserted-by":"publisher","first-page":"213","DOI":"10.1128\/MMBR.00045-10","article-title":"Regulatory\nCircuitry Governing Fungal\nDevelopment, Drug Resistance, and Disease","volume":"75","author":"Shapiro","year":"2011","journal-title":"Microbiol.\nMol. Biol. Rev."},{"key":"2026090409142597500_cit29","doi-asserted-by":"publisher","DOI":"10.1186\/s12964-024-01624-8","article-title":"Emerging Roles\nof Lactate in Acute and Chronic Inflammation","volume":"22","author":"Fang","year":"2024","journal-title":"Cell Commun. Signal."},{"key":"2026090409142597500_cit30","doi-asserted-by":"publisher","first-page":"605","DOI":"10.1046\/j.1365-2958.1996.5441070.x","article-title":"Internal\npH Crisis, Lysine Decarboxylase and the Acid Tolerance Response of Salmonella typhimurium","volume":"20","author":"Park","year":"1996","journal-title":"Mol.\nMicrobiol."},{"key":"2026090409142597500_cit31","doi-asserted-by":"publisher","DOI":"10.2741\/2364","article-title":"The role of the activated macrophage in clearing Listeria monocytogenes infection","volume":"12","author":"Shaughnessy","year":"2007","journal-title":"Front. Biosci."},{"key":"2026090409142597500_cit32","doi-asserted-by":"publisher","first-page":"849","DOI":"10.1038\/nm.1795","article-title":"A Membrane\nProtein Preserves Intrabacterial pH in Intraphagosomal Mycobacterium tuberculosis","volume":"14","author":"Vandal","year":"2008","journal-title":"Nat. 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Clin. Microbiol."},{"key":"2026090409142597500_cit37","doi-asserted-by":"publisher","first-page":"469","DOI":"10.1128\/CMR.9.4.469","article-title":"Virulence\nFactors of Medically Important\nFungi","volume":"9","author":"Hogan","year":"1996","journal-title":"Clin. Microbiol. Rev."},{"key":"2026090409142597500_cit38","doi-asserted-by":"publisher","first-page":"225","DOI":"10.1080\/mmy.40.3.225.242","article-title":"Paracoccidioides brasiliensis and paracoccidioidomycosis: Molecular approaches to morphogenesis,\ndiagnosis, epidemiology, taxonomy and genetics","volume":"40","author":"Blas","year":"2002","journal-title":"Med. Mycol."},{"key":"2026090409142597500_cit39","doi-asserted-by":"publisher","DOI":"10.3390\/jof7121014","article-title":"The Role of Dimorphism\nRegulating Histidine Kinase Drk1 in the Pathogenic Fungus Paracoccidioides brasiliensis Cell Wall","volume":"7","author":"Navarro","year":"2021","journal-title":"J. Fungi"},{"key":"2026090409142597500_cit40","doi-asserted-by":"publisher","first-page":"197","DOI":"10.1016\/j.micinf.2005.06.018","article-title":"Melanin in the Dimorphic\nFungal Pathogen Paracoccidioides brasiliensis: Effects on Phagocytosis, Intracellular Resistance and Drug Susceptibility","volume":"8","author":"Da Silva","year":"2006","journal-title":"Microbes Infect."},{"key":"2026090409142597500_cit41","doi-asserted-by":"publisher","first-page":"331","DOI":"10.1007\/s11046-007-9061-4","article-title":"Melanin\nas a Virulence Factor of Paracoccidioides brasiliensis and Other Dimorphic Pathogenic Fungi: A Minireview","volume":"165","author":"Taborda","year":"2008","journal-title":"Mycopathologia"},{"key":"2026090409142597500_cit42","doi-asserted-by":"publisher","first-page":"400","DOI":"10.1128\/MMBR.67.3.400-428.2003","article-title":"Candida albicans Secreted Aspartyl Proteinases in Virulence\nand Pathogenesis","volume":"67","author":"Naglik","year":"2003","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"2026090409142597500_cit43","doi-asserted-by":"publisher","first-page":"829","DOI":"10.1515\/hsz-2012-0157","article-title":"Extracellular\nAspartic Protease SAP2\nof Candida albicans Cleaves Human Kininogens\nand Releases Proinflammatory Peptides, Met-Lys-bradykinin and des-Arg9-Met-Lys-bradykinin","volume":"393","author":"Bras","year":"2012","journal-title":"Biol. Chem."},{"issue":"1","key":"2026090409142597500_cit44","doi-asserted-by":"publisher","DOI":"10.1186\/s12866-015-0394-8","article-title":"Kinin Release from Human Kininogen\nby 10 Aspartic Proteases Produced by Pathogenic Yeast Candida albicans","volume":"15","author":"Kozik","year":"2015","journal-title":"BMC Microbiol."},{"issue":"10","key":"2026090409142597500_cit45","doi-asserted-by":"publisher","first-page":"4259","DOI":"10.1021\/pr5002274","article-title":"Exploring Potential\nVirulence Regulators in Paracoccidioides brasiliensis Isolates of Varying Virulence through Quantitative Proteomics","volume":"13","author":"Castilho","year":"2014","journal-title":"J. Proteome Res."},{"key":"2026090409142597500_cit46","doi-asserted-by":"publisher","DOI":"10.3390\/jof9030375","article-title":"Characterization\nof Aspartic Proteases from Paracoccidioides brasiliensis and Their Role in Fungal Thermo-Dimorphism","volume":"9","author":"Silva","year":"2023","journal-title":"J. Fungi"},{"key":"2026090409142597500_cit47","doi-asserted-by":"publisher","DOI":"10.3390\/jof11070504","article-title":"Acidic pH Modulates Cell Wall and Melanization in Paracoccidioides brasiliensis, Affecting Macrophage\nInteraction","volume":"11","author":"de Souza Silva","year":"2025","journal-title":"J. Fungi"},{"key":"2026090409142597500_cit48","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1016\/B978-0-12-394318-7.00001-2","article-title":"The Molecular\nBasis of pH Sensing, Signaling, and Homeostasis in Fungi","volume":"79","author":"Bignell","year":"2012","journal-title":"Adv. Appl. Microbiol."},{"issue":"1","key":"2026090409142597500_cit49","doi-asserted-by":"publisher","first-page":"68","DOI":"10.1038\/s41596-018-0082-x","article-title":"Single-Pot,\nSolid-Phase-Enhanced Sample Preparation\nfor Proteomics Experiments","volume":"14","author":"Hughes","year":"2019","journal-title":"Nat. Protoc."},{"key":"2026090409142597500_cit50","doi-asserted-by":"publisher","DOI":"10.1016\/j.micpath.2024.106773","article-title":"Extracellular\nBSA-Degrading SAPs in the Rare Pathogen Meyerozyma guilliermondii Strain SO as Potential\nVirulence Factors in Candidiasis","volume":"193","author":"Lim","year":"2024","journal-title":"Microb. Pathog."},{"key":"2026090409142597500_cit51","doi-asserted-by":"publisher","first-page":"3332","DOI":"10.1128\/JCM.37.10.3332-3337.1999","article-title":"Quantitation of\nErgosterol Content: Novel Method for Determination of Fluconazole\nSusceptibility of Candida albicans","volume":"37","author":"Arthington-Skaggs","year":"1999","journal-title":"J. Clin. 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