{"title":"利用烟曲霉的菌丝分散突变体实时监测液体培养物中的菌丝生长。","authors":"Ken Miyazawa, Takashi Umeyama, Shogo Takatsuka, Yasunori Muraosa, Yasutaka Hoshino, Shigekazu Yano, Keietsu Abe, Yoshitsugu Miyazaki","doi":"10.1093/mmy/myae011","DOIUrl":null,"url":null,"abstract":"<p><p>Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.</p>","PeriodicalId":18586,"journal":{"name":"Medical mycology","volume":null,"pages":null},"PeriodicalIF":2.7000,"publicationDate":"2024-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.\",\"authors\":\"Ken Miyazawa, Takashi Umeyama, Shogo Takatsuka, Yasunori Muraosa, Yasutaka Hoshino, Shigekazu Yano, Keietsu Abe, Yoshitsugu Miyazaki\",\"doi\":\"10.1093/mmy/myae011\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p>Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.</p>\",\"PeriodicalId\":18586,\"journal\":{\"name\":\"Medical mycology\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.7000,\"publicationDate\":\"2024-03-07\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Medical mycology\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1093/mmy/myae011\",\"RegionNum\":3,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"INFECTIOUS DISEASES\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Medical mycology","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/mmy/myae011","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"INFECTIOUS DISEASES","Score":null,"Total":0}
Real-time monitoring of mycelial growth in liquid culture using hyphal dispersion mutant of Aspergillus fumigatus.
Hyphal pellet formation by Aspergillus species in liquid cultures is one of the main obstacles to high-throughput anti-Aspergillus reagent screening. We previously constructed a hyphal dispersion mutant of Aspergillus fumigatus by disrupting the genes encoding the primary cell wall α-1,3-glucan synthase Ags1 and putative galactosaminogalactan synthase Gtb3 (Δags1Δgtb3). Mycelial growth of the mutant in liquid cultures monitored by optical density was reproducible, and the dose-response of hyphal growth to antifungal agents has been quantified by optical density. However, Δags1Δgtb3 still forms hyphal pellets in some rich growth media. Here, we constructed a disruptant lacking all three α-1,3-glucan synthases and galactosaminogalactan synthase (Δags1Δags2Δags3Δgtb3), and confirmed that its hyphae were dispersed in all the media tested. We established an automatic method to monitor hyphal growth of the mutant in a 24-well plate shaken with a real-time plate reader. Dose-dependent growth suppression and unique growth responses to antifungal agents (voriconazole, amphotericin B, and micafungin) were clearly observed. A 96-well plate was also found to be useful for the evaluation of mycelial growth by optical density. Our method is potentially applicable to high-throughput screening for anti-Aspergillus agents.
期刊介绍:
Medical Mycology is a peer-reviewed international journal that focuses on original and innovative basic and applied studies, as well as learned reviews on all aspects of medical, veterinary and environmental mycology as related to disease. The objective is to present the highest quality scientific reports from throughout the world on divergent topics. These topics include the phylogeny of fungal pathogens, epidemiology and public health mycology themes, new approaches in the diagnosis and treatment of mycoses including clinical trials and guidelines, pharmacology and antifungal susceptibilities, changes in taxonomy, description of new or unusual fungi associated with human or animal disease, immunology of fungal infections, vaccinology for prevention of fungal infections, pathogenesis and virulence, and the molecular biology of pathogenic fungi in vitro and in vivo, including genomics, transcriptomics, metabolomics, and proteomics. Case reports are no longer accepted. In addition, studies of natural products showing inhibitory activity against pathogenic fungi are not accepted without chemical characterization and identification of the compounds responsible for the inhibitory activity.