An efficient gene targeting system using Δku80 and functional analysis of Cyp51A in Trichophyton rubrum.

IF 3.5 3区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY AMB Express Pub Date : 2024-08-31 DOI:10.1186/s13568-024-01755-8
Masaki Ishii, Tsuyoshi Yamada, Shinya Ohata
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Abstract

Trichophyton rubrum is one of the most frequently isolated fungi in patients with dermatophytosis. Despite its clinical significance, the molecular mechanisms of drug resistance and pathogenicity of T. rubrum remain to be elucidated because of the lack of genetic tools, such as efficient gene targeting systems. In this study, we generated a T. rubrum strain that lacks the nonhomologous end-joining-related gene ku80 (Δku80) and then developed a highly efficient genetic recombination system with gene targeting efficiency that was 46 times higher than that using the wild-type strain. Cyp51A and Cyp51B are 14-α-lanosterol demethylase isozymes in T. rubrum that promote ergosterol biosynthesis and are the targets of azole antifungal drugs. The expression of cyp51A mRNA was induced by the addition of the azole antifungal drug efinaconazole, whereas no such induction was detected for cyp51B, suggesting that Cyp51A functions as an azole-responsive Cyp51 isozyme. To explore the contribution of Cyp51A to susceptibility to azole drugs, the neomycin phosphotransferase (nptII) gene cassette was inserted into the cyp51A 3'-untranslated region of Δku80 to destabilize the mRNA of cyp51A. In this mutant, the induction of cyp51A mRNA expression by efinaconazole was diminished. The minimum inhibitory concentration for several azole drugs of this strain was reduced, suggesting that dermatophyte Cyp51A contributes to the tolerance for azole drugs. These findings suggest that an efficient gene targeting system using Δku80 in T. rubrum is applicable for analyzing genes encoding drug targets.

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使用 Δku80 的高效基因打靶系统和红毛癣菌中 Cyp51A 的功能分析。
红色毛癣菌是皮肤癣菌病患者最常分离到的真菌之一。尽管它具有重要的临床意义,但由于缺乏基因工具,如高效的基因打靶系统,它的耐药性和致病性的分子机制仍有待阐明。在这项研究中,我们生成了缺乏非同源末端连接相关基因 ku80(Δku80)的红念珠菌菌株,然后开发了一种高效基因重组系统,其基因靶向效率是野生型菌株的 46 倍。Cyp51A和Cyp51B是红念珠菌中的14-α-甘露醇脱甲基酶同工酶,能促进麦角甾醇的生物合成,是唑类抗真菌药物的靶标。添加唑类抗真菌药物依芬康唑会诱导 Cyp51A mRNA 的表达,而 Cyp51B 则没有这种诱导作用,这表明 Cyp51A 作为唑类反应型 Cyp51 同工酶发挥作用。为了探索 Cyp51A 对唑类药物敏感性的贡献,将新霉素磷酸转移酶(nptII)基因盒插入到 Δku80 的 Cyp51A 3'- 非翻译区,以破坏 Cyp51A mRNA 的稳定性。在该突变体中,依芬康唑对 cyp51A mRNA 表达的诱导作用减弱。该菌株对几种唑类药物的最小抑菌浓度降低,表明皮癣菌 Cyp51A 对唑类药物有耐受性。这些研究结果表明,在红皮病菌中使用 Δku80 的高效基因靶向系统适用于分析编码药物靶标的基因。
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来源期刊
AMB Express
AMB Express BIOTECHNOLOGY & APPLIED MICROBIOLOGY-
CiteScore
7.20
自引率
2.70%
发文量
141
审稿时长
13 weeks
期刊介绍: AMB Express is a high quality journal that brings together research in the area of Applied and Industrial Microbiology with a particular interest in ''White Biotechnology'' and ''Red Biotechnology''. The emphasis is on processes employing microorganisms, eukaryotic cell cultures or enzymes for the biosynthesis, transformation and degradation of compounds. This includes fine and bulk chemicals, polymeric compounds and enzymes or other proteins. Downstream processes are also considered. Integrated processes combining biochemical and chemical processes are also published.
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