Development of a Tightly Regulatable Copper-Mediated Gene Switch System in Dermatophytes
Development of a Tightly Regulatable Copper-Mediated Gene Switch System in Dermatophytes
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DOI：
10.1128/aem.00464-12
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发表时间：
2012-08-01
影响因子：
4.4
通讯作者：
Yamada, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Iwata, Atsushi;Alshahni, Mohamed Mahdi;Yamada, Tsuyoshi
目前，皮肤癣菌的分子遗传学研究主要采用靶基因缺失的方法，一些基因的生理功能已被阐明。然而，这种方法不能应用于必需基因，这可能是潜在的药物靶点。为了克服这一限制，我们已经开发了一个条件基因敲除系统使用铜响应启动子。研究了红色毛癣菌铜转运蛋白基因CTR 4（P-CTR 4）和铜外排泵基因CRP 1（P-CRP 1）的启动子序列在范氏节皮霉中对铜的反应。P-CTR 4被证明在铜的存在下抑制报告基因的表达，而P-CRP 1的活性被铜诱导。重要的是，P-CTR 4更紧密地调控基因表达。此外，当P-CTR 4被用于调节内源基因ERG 1和TRP 5的表达时，它们的条件突变体在抑制条件下表现出降低的生长活性。这些结果表明，Pc，P-CTR 4为基础的基因调控系统代表了一个强大的工具，用于识别和表征的广泛的基因，包括必需基因，在皮肤癣菌。
Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P-CTR4) and that of the copper efflux pump gene CRP1 (P-CRP1) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P-CTR4 was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P-CRP1 was induced by addition of copper. Importantly, P-CTR4 regulated the gene expression more tightly. Furthermore, when P-CTR4 was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the Pc,P-CTR4-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.