SreE介导的铁调节对皮炎外瓶霉形态发生、药物敏感性及致病性的影响

Central nerve system infections of Exophiala dermatitidis are always fatal and occur mostly in immunocompetent individuals. Iron homeostasis is not only essential for fungal metabolism, but also important for fungal virulence. GATA-binding protein regulates iron homeostasis via repressing the expression of siderophore under iron-replete condition, and also has a crucial role in virulence factor expression in some fungal species. SreA deficiency in Aspergillus fumigatus results in intra-cellular iron accumulation and upregulation of antioxidative pathways. Cir1 controls the known major virulence factors of Cryptococcus neoformans including the capsule, the formation of the anti-oxidant melanin in the cell wall, and the ability to grow at host body temperature. Inspection of the annotated genomic sequence of E. dermatitidis revealed the presence of a gene (GenBank: EHY52687.1) displaying significant identity to SreA coding gene. Analysis of the cDNA sequence of E. dermatitidis GATA-binding protein which we named SreE revealed identity to the A. fumigatus orthologue and all typical features common to this class of fungal iron-regulatory GATA-transcription factors identified so far in the ascomycetes. By knocking-down SreE in E. dermatitidis, the role of SreE in the morphogenesis, antifungal sensitivity and virulence under different conditions of E. dermatitidis will be investigated. By establishing animal model of invasive E. dermatitidis infection, the role of SreE in the pathogenicity of E. dermatitidis will be investigated. This study aims to demonstrate the role of SreE in the morphogenesis, antifungal sensitivity and virulence of E. dermatitidis.

皮炎外瓶霉可引起致死性的中枢神经系统感染，且多发生于免疫正常的健康人群。铁平衡不仅对真菌的新陈代谢至关重要，也是影响真菌侵袭性的重要因素。GATA结合蛋白通过抑制真菌铁载体合成调节铁平衡，同时也参与调节真菌毒力因素的表达。研究证明烟曲霉SreA缺乏导致细胞内铁聚集及抗氧化应激途径转录增加；新生隐球菌中Cir1不仅调节铁代谢，同时也是毒力决定因素表达所必需。同源性比较发现皮炎外瓶霉具有与SreA高度同源的基因，该基因编码蛋白（我们命名为SreE）与其他子囊菌纲真菌GATA转录因子结构相似：具有两个GATA型锌指结构和中间富含半胱氨酸区。本项目拟通过构建皮炎外瓶霉SreE缺陷株，观察在不同铁离子浓度培养基和不同温度下菌丝形态发生、细胞内铁离子浓度及药物敏感性变化；通过构建小鼠感染模型，观察缺陷株致病性变化，深入研究SreE介导的铁代谢对皮炎外瓶霉形态发生、药物敏感性及致病性的影响。

皮炎外瓶霉可引起致死性的中枢神经系统感染，且多发生于免疫正常的健康人群。铁平衡不仅对真菌的新陈代谢至关重要，也是影响真菌侵袭性的重要因素。首先，在不同铁离子条件下（缺铁/高铁）观察皮炎外瓶霉生长和抗真菌药物敏感性的变化，显示低铁环境下皮炎外瓶霉生长减缓。其次利用基因敲除技术，试图明确GATA结合蛋白SreE对皮炎外瓶霉铁代谢和毒力的影响，但结果显示SreE蛋白对皮炎外瓶霉生长及毒力并没有显著的影响。此外，我们进一步探索了新型抗真菌联合方案。研究显示基于亚甲蓝和LED的光动力疗法能够有效杀伤皮炎外瓶霉游离态及生物膜，而传统药物扑蛲灵单用即具有抗皮炎外瓶霉效应。联合方案中扑蛲灵、钙调磷酸酶抑制剂他克莫司、Hsp90抑制剂17AAG、法尼基转移酶抑制剂Lonafarnib及凋亡抑制蛋白抑制因子AT406等与唑类联合均有协同抗皮炎外瓶霉效应，其中他克莫司、AT406及光动力与唑类联合具有协同抗皮炎外瓶霉生物膜效应。我们的研究极大丰富了联合抗真菌治疗方案，为研发临床难治性皮炎外瓶霉感染的治疗策略奠定了重要的基础，具有重要的应用价值。