基于化学表观遗传调控的煤矿区真菌抗耐G-菌次生代谢物及其机制研究

Drug-resistant Gram-negative (G-) pathogens have been reported as current key pathogens which are gradually threatening global public health due to the depletion of antibiotics. The coal mine area is a complex environment area with extreme acidity, rich salt, high heavy metal content, etc., breeding a variety of special fungi. However, for the inability of restoring extreme environments for fungi culture condition in laboratory, it is particularly important to exogenously activate secondary metabolites silent synthesis gene clusters. In the preliminary experiment, two strains of fungi with anti-multi-drug resistant Escherichia coli were isolated from the soil of the Late Permian coal mine in Xingren, Guizhou Province. The chemical constituents from metabolites of one strain were studied, and a new isocoumarin revealed potential decreasing the amount of Cephalosporin sodium (1/4MIC) in the combination antibacterial test. And the strain can produce diverse metabolites under the induction of exogenous chemical molecule. Therefore, the project is to collect the fungi from this special environment with four drug-resistant G- bacteria as indicator, to obtain new compounds with anti-drug resistance G- pathogens under the chemical epigenetic regulation of strains, combining with bioautography and LC-MS analysis as well as modern separation and identification, and to explain the mechanism of 1 to 2 compounds by molecular biology techniques.

耐药性革兰氏阴性（G-）病原菌已列为当前重点病原体，抗生素的枯竭正使其逐步威胁全球公共健康。煤矿区是一个极酸、极盐、高重金属含量等多极端复杂环境区域，孕育了多种特殊的真菌，是新药发现的重要资源。但由于实验室培养条件无法还原极端环境，外源激活沉默型次生代谢产物合成基因簇显得尤为重要。前期从贵州省兴仁晚二叠纪煤矿区土壤中分离筛选获得2株具有抗多药耐药性大肠杆菌的真菌，经对其中一株代谢物的化学成分研究，发现其新型异香豆素类化合物在联合抗耐大肠杆菌实验中使头孢曲松钠的MIC从1024μg/mL降到256μg/mL，同时该菌株在外源化学诱导下能代谢合成结构多样性的化学分子。因此本项目拟在此基础上，广泛采集该特境下的真菌，以4种耐G-菌为指示菌，通过化学表观遗传调控，结合生物自显影及液质分析和现代分离、鉴定手段，以期获得具有抗耐G-病原菌的新型化合物，并利用分子生物学技术阐明1-2个化合物的作用机制。

耐药革兰氏阴性病源菌是目前全球公共健康最大威胁之一，抗生素的“失效”让众多疾病面临巨大的威胁，研发新型抗生素已迫在眉睫。煤矿区是一个多极端复杂环境区域，孕育了多种特殊的真菌，是新药发现的重要资源。本研究从煤矿区分离了222株真菌。以耐药革兰氏阴性大肠杆菌、肺炎克雷伯菌、铜绿假单胞菌、鲍曼不动杆菌以及嗜麦芽窄食单胞菌为受试病原菌进行抗菌活性筛选，发现33株真菌次生代谢产物对不同受试菌具有潜在的抗菌活性(20mg/mL)。完成了5株真菌次生代谢产物研究，共分离121个化合物，发现抗耐药大肠杆菌或抗耐药嗜麦芽窄食单胞菌活性分子11个。其中helvolinic acid抗耐药嗜麦芽窄食单胞菌活性最优，并进一步通过化学结构改造获得更优衍生物6a(MIC=2μg/mL，MIC头孢曲松钠=128μg/mL)。初步机制研究表明6a对嗜麦芽窄食单胞菌的抑菌作用与破坏细胞膜和抑制生物膜的形成有关；RNA-seq分析结果显示化合物6a影响嗜麦芽窄食单胞菌中多个基因显著性地差异表达；目前已通过RT-qPCR证实其抗菌作用与ABC转运蛋白中IoID，modA基因，双组分系统narH，narI，kdpB，kdpA基因，以及氧化磷酸化涉及cyoD2，catD，coxB基因的显著下调有关。本项目为开发抗耐革兰氏阴性病原菌药物奠定一定基础。