低铁环境诱导白念珠菌耐药性形成的细胞与分子机制研究

The incidence of candidiasis increased year by year, and it now ranks the fourth nosocomial infeciton in hospital, which get more and more attention. Drug resistance caused by fluconazole and other commonly used drugs in clinic is becoming more and more serious. Our previous studies have demonstrated that the combination of ginseng stem-leave saponins and fluconazole had the activity against resistant strains of Candida.The mechanism of ginseng stem-leave saponins against Candida was further verified through animal experiments. We used protein mass spectrometric analysis to find the difference of proteins in Candida albicans before and after using Fluconazole combined with Ginseng stem-leave saponins, and found that the expression of the transcription factor Hap43 related to iron metabolism in Candida albicans and other proteins which areregulated by Hap43 up and down obivously. It suggests that Hap43 may play an important role in the mechanism of drug resistance underthe iron-limiting environment. This project aims to further observe the important role of Hap43 in maintaining the homeostasis of intracellular iron ions and regulating the expression of genes related with iron metabolism by gene knockout, Real-Time PCR and animal experiment methods. To investigate the role of Hap43 in drug resistance of Candida albicans . It not only helps us to find new anti-fungal drug targets but also give theoretical basis and experimental base to improve the theory of drug resistance of fungi.

念珠菌病的发病率逐年上升，现已位居院内感染的第四位，临床上常用的氟康唑等药物引起的耐药现象日益严重。本课题组前期发现人参茎叶皂苷与氟康唑联用具有抗耐药念珠菌的活性，通过动物实验进一步得到验证，并且探讨了人参茎叶皂苷抗念珠菌的机制。我们利用蛋白质谱技术分析发现白念珠菌耐药株在人参茎叶皂苷作用后与铁代谢相关转录因子Hap43及其调控的相关蛋白发生了显著变化，提示我们Hap43可能参与了低铁环境诱导白念珠菌耐药性的形成过程。本项目拟进一步通过基因敲除、Real-Time PCR及动物实验等方法，观察Hap43在维持细胞内铁离子的动态平衡和调控铁代谢相关基因表达方面的重要作用；探讨Hap43在白念珠菌耐药形成中的作用，不仅为丰富念珠菌耐药理论提供理论依据，也为开发高效低毒的念珠菌药物提供靶位点。

念珠菌病的发病率逐年上升，现已位居院内感染的第四位，临床上常用的氟康唑等药物引起的耐药现象日益严重，目前已知白念珠菌有多种耐药机制，但仍缺乏精准、有效的对耐药菌治疗手段，因此进一步探索、丰富白念珠菌耐药机制以期开发出靶向治疗手段尤为重要。本课题组前期研究发现Hap43可能参与了铁环境诱导白念珠菌耐药性形成的过程。因此本项目旨在探讨Hap43是否参与了白念珠菌耐药及其与铁稳态的关系。本课题首先通过基因敲除，构建Hap43缺失菌hap43Δ/HAP43和hap43Δ/Δ;通过体外实验：药敏实验（MIC）、固体点板实验、液体生长曲线等，以明确Hap43蛋白在白念珠菌氟康唑耐药形成过程中起到一定的作用；进行体内实验：建立小鼠白念珠菌系统性真菌感染模型，以小鼠肾脏菌落形成单位(CFU)、肝脏菌落形成单位(CFU)作为毒力和侵袭能力的评判指标，来考察Hap43对白念珠菌毒力和侵染宿主能力的影响; 利用 Real-Time PCR 方法，检测白念珠菌亲本菌株SN152和耐药株103的Hap43表达水平的差异；利用细胞壁抑制剂刚果红(Congo red)和荧光白(Fluorescent white , CFW)作用于Hap43缺失菌，观察Hap43缺失对白念珠菌生长抑制的机制;通过活性氧检测实验（Ros Assay）、点板实验（Spot Assy）、生长曲线（Growth Curve）观察Hap43与氧化应激、铁稳态的关系。本实验成功构建Hap43缺失菌；Hap43很可能是在富铁环境下参与白念珠菌耐药的形成；Hap43的缺失使白念珠菌的毒力和侵染能力减弱；Hap43缺失对白念珠菌在富铁和低铁条件下菌丝形成能力均无明显影响；氧化应激和低铁环境在一定条件下不利于白念珠菌的生长，且两者具有协同抑菌作用。Hap43缺失不是通过抑制细胞壁α-1，6-甘露醇骨架的合成来抑制白念珠菌生长；Hap43有利于降低白念珠菌对氧化应激的敏感性，具有“抗氧化应激”作用，Hap43有可能是通过在高铁环境下发挥“抗氧化应激”作用而促进白念珠菌耐药。本课题的开展丰富了白念珠菌耐药理论依据，Hap43有可能成为白念珠菌耐药基因，这为开发高效低毒的抗念珠菌药物提供靶点，对未来念珠菌病的精准治疗提供了重要的理论依据。