Institute of Biology Leiden, Leiden University, Molecular Microbiology and Biotechnology, Sylviusweg 72, 2333 BE Leiden, The Netherlands.
Fungal Genet Biol. 2012 Apr;49(4):284-93. doi: 10.1016/j.fgb.2011.12.008. Epub 2011 Dec 29.
The identification of suitable targets is crucial for the discovery and development of new antifungals. Since the fungal cell wall is an essential organelle, the identification of genes involved in cell wall biosynthesis is expected to help discover new antifungal targets. From our previously obtained collection of cell wall mutants with a constitutively active cell wall stress response pathway, we selected a thermosensitive, osmotic-remediable mutant with decreased resistance to SDS for complementation analysis. The phenotypes of this mutant were complemented by a gene encoding a protein with high sequence similarity to subunit d of the eukaryotic Vacuolar-H(+)-ATPase (VmaD). Genetic analysis of this thermosensitive mutant revealed that the conditional mutant allele encodes a protein that lacks 12 amino acids at the C-terminus due to a point mutation that introduces a stop codon. Deletion of the entire gene resulted in very poor growth. The conditional mutant displayed several phenotypes that are typical for V-ATPase mutants, including increased sensitivity to zinc ions and reduced acidification of the vacuole as observed by quinacrine staining. Treatment of Aspergillus niger with the V-ATPase inhibitor bafilomycinB(1) induced the expression of agsA and other cell wall related genes. Furthermore genes involved in cell wall reassembly like fksA, agsA and phiA were clearly up-regulated in the conditional mutant. Our results indicate that the ATP-driven transport of protons and acidification of the vacuole is crucial for the strength of the fungal cell wall and that reduced activity of the V-ATPase induces the cell wall stress response pathway.
合适靶点的鉴定对于新抗真菌药物的发现和开发至关重要。由于真菌细胞壁是一种必需的细胞器，因此鉴定参与细胞壁生物合成的基因有望有助于发现新的抗真菌靶点。从我们之前获得的具有组成性激活细胞壁应激反应途径的细胞壁突变体集合中，我们选择了一个对 SDS 抗性降低的热敏、渗透压修复突变体进行互补分析。该突变体的表型可以通过编码与真核液泡 H(+)-ATP 酶（VmaD）亚基 d 具有高度序列相似性的蛋白质的基因来互补。对这个热敏突变体的遗传分析表明，条件突变等位基因编码的蛋白质由于点突变而在 C 末端缺失 12 个氨基酸，从而引入了一个终止密码子。该基因的缺失导致生长非常不良。该条件突变体表现出几种典型的 V-ATP 酶突变体的表型，包括对锌离子的敏感性增加和通过吖啶橙染色观察到的液泡酸化减少。用 V-ATP 酶抑制剂巴弗洛霉素 B(1)处理黑曲霉诱导了 agsA 和其他细胞壁相关基因的表达。此外，在条件突变体中，细胞壁重排相关基因如 fksA、agsA 和 phiA 明显上调。我们的结果表明，质子的 ATP 驱动运输和液泡的酸化对于真菌细胞壁的强度至关重要，而 V-ATP 酶活性的降低会诱导细胞壁应激反应途径。
