Department of Biotechnology, Chung-Ang University, Anseong 456-756, Republic of Korea.
Fungal Genet Biol. 2012 Nov;49(11):955-66. doi: 10.1016/j.fgb.2012.08.006. Epub 2012 Sep 4.
The high-affinity reductive iron uptake system that includes a ferroxidase (Cfo1) and an iron permease (Cft1) is critical for the pathogenesis of Cryptococcus neoformans. In addition, a mutant lacking CFO1 or CFT1 not only has reduced iron uptake but also displays a markedly increased susceptibility to azole antifungal drugs. Altered antifungal susceptibility of the mutants was of particular interest because the iron uptake system has been proposed as an alternative target for antifungal treatment. In this study, we used transcriptome analysis to begin exploring the molecular mechanisms of altered antifungal susceptibility in a cfo1 mutant. The wild-type strain and the cfo1 mutant were cultured with or without the azole antifungal drug fluconazole and their transcriptomes were compared following sequencing with Illumina Genome Analyzer IIx (GAIIx) technology. As expected, treatment of both strains with fluconazole caused elevated expression of genes in the ergosterol biosynthetic pathway that includes the target enzyme Erg11. Additionally, genes differentially expressed in the cfo1 mutant were involved in iron uptake and homeostasis, mitochondrial functions and respiration. The cfo1 mutant also displayed phenotypes consistent with these changes including a reduced ratio of NAD(+)/NADH and down-regulation of Fe-S cluster synthesis. Moreover, combination treatment of the wild-type strain with fluconazole and the respiration inhibitor diphenyleneiodonium dramatically increased susceptibility to fluconazole. This result supports the hypothesis that down-regulation of genes required for respiration contributed to the altered fluconazole susceptibility of the cfo1 mutant. Overall, our data suggest that iron uptake and homeostasis play a key role in antifungal susceptibility and could be used as novel targets for combination treatment of cryptococcosis. Indeed, we found that iron chelation in combination with fluconazole treatment synergistically inhibited the growth of C. neoformans.
高亲和力还原铁摄取系统包括一个亚铁氧化酶（Cfo1）和一个铁渗透酶（Cft1），对于新型隐球菌的发病机制至关重要。此外，缺乏 CFO1 或 CFT1 的突变体不仅铁摄取减少，而且对唑类抗真菌药物的敏感性显著增加。突变体改变抗真菌敏感性特别有趣，因为铁摄取系统已被提议作为抗真菌治疗的替代靶标。在这项研究中，我们使用转录组分析开始探索 cfo1 突变体改变抗真菌敏感性的分子机制。将野生型菌株和 cfo1 突变体在含有或不含有唑类抗真菌药物氟康唑的情况下培养，并在使用 Illumina Genome Analyzer IIx (GAIIx) 技术进行测序后比较它们的转录组。正如预期的那样，两种菌株用氟康唑处理都会导致包括靶酶 Erg11 在内的麦角固醇生物合成途径中的基因表达上调。此外，在 cfo1 突变体中差异表达的基因参与铁摄取和稳态、线粒体功能和呼吸。cfo1 突变体还表现出与这些变化一致的表型，包括 NAD(+) / NADH 比例降低和铁硫簇合成下调。此外，野生型菌株与氟康唑和呼吸抑制剂二苯基碘鎓联合处理显著增加了对氟康唑的敏感性。这一结果支持了这样一种假设，即呼吸所需基因的下调导致了 cfo1 突变体改变氟康唑敏感性。总的来说，我们的数据表明铁摄取和稳态在抗真菌敏感性中起着关键作用，并可作为新型隐球菌联合治疗的新靶标。事实上，我们发现铁螯合与氟康唑联合治疗协同抑制新型隐球菌的生长。
