Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans
Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans
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DOI：
10.1128/iai.68.12.6712-6719.2000
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发表时间：
2000-12-01
影响因子：
3.1
通讯作者：
Aoki, Y
中科院分区：
文献类型：
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作者：
Nakayama, H;Mio, T;Aoki, Y
用于阐明基因功能的常规工具在白色念珠菌中相对稀缺，白色念珠菌是最普遍的人类真菌病原体。为此，我们开发了一个方便的系统来控制基因表达在C。通过四环素可调节（TR）启动子将白念珠菌转化为大肠杆菌。当将海肾荧光素酶基因（RLUC 1）置于该系统的控制下时，强力霉素（DOX）几乎完全抑制荧光素酶活性。在不存在DOX的情况下，诱导RLUC 1基因以比存在DOX的情况高400- 1，000倍的水平表达荧光素酶。在菌丝形成细胞中获得了相同的结果。用TR启动子替换N-肉豆蔻酰转移酶或翻译延伸因子3启动子可导致培养基中的DOX依赖性生长缺陷。此外，所有感染这些突变体的小鼠在DOX给药后存活，这些突变体仍然是有毒的。一致地，我们观察到从小鼠肾脏回收的这些突变细胞的数量在DOX施用后显著减少。因此，该系统对于研究基因功能很有用，因为该系统能够在体外和体内环境中发挥作用。
Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.