{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:51:56Z","timestamp":1786117916369,"version":"build-2736575974"},"reference-count":34,"publisher":"American Society for Microbiology","issue":"8","license":[{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,4]],"date-time":"2026-08-04T00:00:00Z","timestamp":1785801600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/journals.asm.org\/non-commercial-tdm-license"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2023YFE0198700"],"award-info":[{"award-number":["2023YFE0198700"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004826","name":"Natural Science Foundation of Beijing Municipality","doi-asserted-by":"publisher","award":["7232178"],"award-info":[{"award-number":["7232178"]}],"id":[{"id":"10.13039\/501100004826","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Investigator-Initiated Studies Program of Merck Sharp & Dohme LLC","award":["2024Res301-001"],"award-info":[{"award-number":["2024Res301-001"]}]}],"content-domain":{"domain":["journals.asm.org"],"crossmark-restriction":true},"short-container-title":["Microbiol Spectr"],"published-print":{"date-parts":[[2026,8,4]]},"abstract":"ABSTRACT<\/jats:title>\n \n \n \n The clinical management of invasive aspergillosis (IA) is limited by the scarcity of antifungal agents and the emergence and spread of drug resistance, which impairs therapeutic efficacy and poses a significant public health threat. Olorofim, a first-in-class clinical antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapeutic option for IA. It shows activity against several filamentous fungi, including\n Aspergillus<\/jats:italic>\n spp., both\n in vitro<\/jats:italic>\n and\n in vivo<\/jats:italic>\n . Olorofim-resistant\n Aspergillus fumigatus<\/jats:italic>\n isolates, characterized by various amino acid substitutions (with a hotspot at G119), have been generated under laboratory conditions through olorofim exposure. However, the potential olorofim resistance mechanisms in\n Aspergillus flavus<\/jats:italic>\n , the second-most common pathogen causing IA globally, remain entirely unexplored. In this study, we revealed that the residue G118 in\n A. flavus<\/jats:italic>\n , conserved in many filamentous fungi, is homologous to G119 in\n A. fumigatus<\/jats:italic>\n by DHODH amino acid sequence alignment. Using a CRISPR-based point mutation approach, we further confirmed that substitutions at the DHODH G118 residue confer acquired olorofim resistance in\n A. flavus<\/jats:italic>\n . Additionally, we established a\n Galleria mellonella<\/jats:italic>\n infection model and observed that these resistant isolates exhibited no obvious fitness cost, and olorofim treatment was ineffective against infections caused by DHODH G118 mutants. Molecular docking models further indicated that substitutions at the G118 locus may introduce steric hindrance, reducing the binding affinity between olorofim and the DHODH active site and thereby driving resistance.\n <\/jats:p>\n <\/jats:sec>\n \n IMPORTANCE<\/jats:title>\n \n Invasive aspergillosis, a life-threatening infection in immunocompromised patients, is increasingly difficult to treat due to limited antifungal options and rising resistance. Olorofim, a novel antifungal targeting dihydroorotate dehydrogenase (DHODH), represents a promising therapy. While resistance in\n Aspergillus fumigatus<\/jats:italic>\n has been linked to DHODH G119 mutations, the mechanisms in the globally prevalent pathogen\n Aspergillus flavus<\/jats:italic>\n remained unknown. Using CRISPR-based mutagenesis, we demonstrate that amino acid substitutions at G118 confer olorofim resistance, supported by molecular docking showing reduced drug binding. Importantly, olorofim failed to protect larvae infected with resistant mutants in an infection model. These findings provide essential insights for proactively monitoring and managing olorofim resistance in\n Aspergillus flavus<\/jats:italic>\n , aiding clinical and public health responses.\n <\/jats:p>\n \n This study is registered with ClinicalTrials.gov as\n NCT05101187<\/jats:related-object>\n and\n NCT06969703<\/jats:related-object>\n .\n <\/jats:p>\n <\/jats:sec>","DOI":"10.1128\/spectrum.00251-26","type":"journal-article","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T13:06:30Z","timestamp":1784034390000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Amino acid substitutions at the G118 hotspot in dihydroorotate dehydrogenase confer olorofim resistance in\n Aspergillus 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