组蛋白去甲基化酶基因JHD1调控白念珠菌对唑类药物耐药性的作用及机制研究

The drug-resistance of azoles results in fungal infections difficult to cure and relapse. It is an effective approach to overcome the fungal infections problem by further clarifying the mechanisms of antifungal drug resistance and developing novel antifungal agents. It has been reported that epigenetic was implicated in azoles drug-resistance, and histone deacetylation modification could regulate fungal drug-resistance, and it has become a new target of antifungal drugs. Histone demethylation could affect the anticancer drug-resistance and stress-resistance in Saccharomyces cerevisiae. However, there are rarely researches on the histone demethylase (HDMs) function in pathogenic fungus. It has important sense to explore the effect of HDMs on the azoles drug-resistance of pathogenic fungus. Our pretest results showed that Jhd1 could demethylate the histone 3 of C. albicans, and the expression of JHD1 could affect the antifungal activity and drug-resistance of azoles in vitro. This project will determine the relationship between HDMs gene JHD1 and azoles drug-resistance in vivo using Caenorhabditis elegans-C. albicans infection model, then establish the relation of Jhd1 expression and sensitivity or resistance of azoles by detecting the expression of Jhd1 in clinical susceptible or resistant azoles Candida isolates, so as to expand its clinical value, finally explore the mechanisms of HDMs gene JHD1 regulating azoles drug-resistance in C. albicans by testing the change of known azoles antifungal and drug-resistant pathways and transcriptome bioinformatic analysis. This project will extend the mechanisms of azoles drug-resistance from the perspective of epigenetic, which providing new clues to development of novel antifungal agents and new fungal infections diagnostic or treatment methods.

唑类耐药导致真菌感染反复难愈，是临床亟待解决的难题。深入探究唑类耐药机制，研发新型抗真菌药物是克服真菌感染的有效途径。研究表明，唑类耐药与表观遗传相关，组蛋白乙酰化修饰可调控唑类耐药，是抗真菌药物研发的新靶点；组蛋白去甲基化修饰与抗肿瘤药物耐药性及酿酒酵母抗逆性相关，而在病原真菌中的生物学功能尚不明确。因此，探究组蛋白去甲基化修饰调控病原真菌耐药性的作用具有重要意义。预实验显示，白念珠菌中Jhd1蛋白具有组蛋白去甲基化功能，且可影响体外唑类的耐药性产生。本项目拟通过秀丽隐杆线虫感染模型和临床唑类敏感及耐药念珠菌检测，明确JHD1基因调控唑类耐药性的作用，建立其表达水平与临床唑类敏感、耐药性的联系，拓展其临床应用价值；通过检测已知唑类耐药相关靶点、通路，结合转录组分析，构建JHD1参与的唑类耐药基因调控网络，从表观遗传角度拓展唑类耐药机制，为抗真菌药物新靶点发掘及新型诊疗方法开展提供新思路。

真菌感染是临床常见感染性疾病之一，侵袭性真菌感染在免疫低下患者中具有较高的发病率和死亡率。目前，唑类、多烯类和棘白霉素类是一线的真菌感染治疗药物，唑类药物虽然口服利用度好、价格经济、毒副作用小，但容易在长期使用过程中产生耐药性，威胁患者生命。深入探究唑类药物的耐药机制，研发新的真菌感染治疗药物或治疗策略是解决真菌感染治疗现状的有效手段。研究表明，表观遗传在真菌耐药性形成以及检测中发挥重要作用，组蛋白修饰可调控唑类耐药，是唑类耐药机制研究的新方向。本项目通过数据库检索、比对以及体外蛋白表达，酶活反应、质谱鉴定等手段，确认白念珠菌中的组蛋白去甲基化酶（HDMs）基因JHD1具有组蛋白去甲基化功能。利用白念珠菌的JHD1敲除菌株，通过药敏试验、诱导耐药、动物感染及治疗等实验，发现白念珠菌HDMs基因JHD1能够影响其对唑类药物敏感性以及诱导耐药性。通过进一步机制研究结合转录组测序分析发现，白念珠菌HDMs基因JHD1通过麦角甾醇、乙醇代谢等途径，调控唑类抗菌及耐药性。通过收集临床唑类耐药及敏感菌株，检测其JHD1基因表达量，以确定其是否可作为临床耐药念珠菌的检测指标。结果显示，唑类敏感度与JHD1表达量间不存在显著的相关性，其不能作为临床念珠菌唑类耐药性的检测指标。本项目明确了HDMs 基因JHD1对唑类药物的敏感性及耐药性的调控作用及机制，为表观遗传调控真菌耐药提供了新的证据。本项目的完成为以JHD1及相关基因为靶点的抗真菌药物、增效剂及逆转耐药剂的研发提供了理论依据和实验基础。