Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target.
Topoisomerase I is essential in Cryptococcus neoformans: role In pathobiology and as an antifungal target.
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拓扑异构酶 I 在新型隐球菌中至关重要：在病理学中发挥作用并作为抗真菌靶点。
DOI：
10.1093/genetics/152.1.167
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发表时间：
1999
期刊：
影响因子：
3.3
通讯作者：
Perfect,JR
中科院分区：
文献类型：
--
作者：
DelPoeta,M;Toffaletti,DL;Rude,TH;Dykstra,CC;Heitman,J;Perfect,JR
拓扑异构酶 I 是多种毒素和化疗药物的靶标，该酶对于某些生物体（包括小鼠和果蝇）的生存至关重要。我们从机会性真菌病原体新型隐球菌中克隆了编码拓扑异构酶 I 的 TOP1 基因。新型隐球菌拓扑异构酶 I 含有真菌插入片段，该插入片段也存在于来自白色念珠菌和酿酒酵母的拓扑异构酶 I 中，而哺乳动物酶中不存在该插入片段。我们无法通过在分析的 8000 多个转化体中进行同源重组来破坏这种单倍体生物中的拓扑异构酶 I 基因。当 TOP1 基因的第二个功能性拷贝被引入基因组时，拓扑异构酶 I 基因可以很容易地通过同源重组被破坏（效率为 7%）。因此，拓扑异构酶 I 在新型隐球菌中是必需的。这种针对新型隐球菌的新分子策略也可能有助于鉴定其他病原真菌中的必需基因。为了解决该酶的生理和病理生物学功能，将 TOP1 基因与 GAL7 基因启动子融合。由此产生的 GAL7::TOP1 融合基因在新型隐球菌 A 血清型菌株中受到碳源的适度调节。拓扑异构酶 I 的适度过度表达赋予对热休克、γ 射线和喜树碱的敏感性。相比之下，拓扑异构酶 I 水平的改变对一类新型抗真菌剂——双芳香族化合物 (DAC) 的毒性没有影响，表明拓扑异构酶 I 不是 DAC 的靶标。在隐球菌脑膜炎的动物模型中，拓扑异构酶 I 的调节对于已建立的感染并不至关重要，但可能会影响对感染的初始应激反应。总之，我们的研究表明拓扑异构酶 I 在人类病原体新型隐球菌中至关重要，并且代表了抗真菌药物的新靶标。
Topisomerase I is the target of several toxins and chemotherapy agents, and the enzyme is essential for viability in some organisms, including mice and drosophila. We have cloned the TOP1 gene encoding topoisomerase I from the opportunistic fungal pathogen Cryptococcus neoformans. The C. neoformans topoisomerase I contains a fungal insert also found in topoisomerase I from Candida albicans and Saccharomyces cerevisiae that is not present in the mammalian enzyme. We were unable to disrupt the topoisomerase I gene in this haploid organism by homologous recombination in over 8000 transformants analyzed. When a second functional copy of the TOP1 gene was introduced into the genome, the topoisomerase I gene could be readily disrupted by homologous recombination (at 7% efficiency). Thus, topoisomerase I is essential in C. neoformans. This new molecular strategy with C. neoformans may also be useful in identifying essential genes in other pathogenic fungi. To address the physiological and pathobiological functions of the enzyme, the TOP1 gene was fused to the GAL7 gene promoter. The resulting GAL7::TOP1 fusion gene was modestly regulated by carbon source in a serotype A strain of C. neoformans. Modest overexpression of topoisomerase I conferred sensitivity to heat shock, γ-rays, and camptothecin. In contrast, alterations in topoisomerase I levels had no effect on the toxicity of a novel class of antifungal agents, the dicationic aromatic compounds (DACs), indicating that topoisomerase I is not the target of DACs. In an animal model of cryptococcal meningitis, topoisomerase I regulation was not critically important to established infection, but may impact on the initial stress response to infection. In summary, our studies reveal that topoisomerase I is essential in the human pathogen C. neoformans and represents a novel target for antifungal agents.