Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters
Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters
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DOI：
10.1016/j.fgb.2009.10.010
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发表时间：
2010-01-01
影响因子：
3
通讯作者：
Ramon De Lucas, J.
中科院分区：
文献类型：
--
作者：
Candida Monteiro, M.;Ramon De Lucas, J.
必需基因的鉴定是发现烟曲霉新型治疗靶点的关键一步。酿酒酵母RNA三磷酸酶的结构分析指出，这种酶作为一个有吸引力的治疗真菌感染的目标。此外，还论证了S.酿酒酵母RNA三磷酸酶编码基因增强了这一潜在治疗靶点的价值。然而，考虑真菌RNA三磷酸酶作为理想的治疗靶标需要确认真菌病原体中相应基因的必要性。本文分析了A.烟曲霉triA基因，编码RNA三磷酸酶，通过条件基因表达和异核体删除。使用由alcA启动子（alcA（P））驱动的条件基因表达，我们发现TriA缺失导致形态异常，从而导致非常强烈的生长抑制。然而，由于没有观察到严格的末端表型，因此不能确保triA基因的必要性。据此，利用异核体拯救技术分析了该基因的重要性。所得结果明确地证明了A.表明RNA三磷酸酶是治疗烟曲霉的理想靶点。烟曲霉感染此外，第二个条件基因表达系统，基于niA启动子（niA（P）），在这项工作中使用。尽管niiA（P）介导的triA抑制不如alcA（P）驱动的严重，但在niiA（P）-triA菌株中也发现了强烈的生长抑制。最后，为了确定triA下调的细胞是否对抗真菌药变得更加敏感而进行的E-试验表明，阿替霉素B和另一种抑制烟曲霉RNA三磷酸酶活性的抗真菌药之间存在协同作用。(C)2009 Elsevier Inc. All rights reserved.
The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter (alcA(P)), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter (niiA(P)), was utilized in this work. Although the niiA(P)-mediated repression of triA was less severe than that driven by the alcA(P), a strong growth inhibition was also found in niiA(P)-triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A fumigatus RNA triphosphatase activity. (C) 2009 Elsevier Inc. All rights reserved.