五种蕨类植物的内生真菌抗MRSA活性代谢产物的研究

Antibiotic resistance is the single biggest public health threat today, of which MRSA is a leading cause of bacterial infection-related mortality. It is an urgent need and a continuous task to discover new anti-MRSA agents in combating antibiotic resistance. Ferns (pteridophytes) are all observed to be highly resistant to diseases. This common characteristic of ferns allows to reasoning that their endophytes should be potential as a microbial resource for discovery of new antibiotic leads. In consequence, we have carried out isolation and antibacterial activity screening of endophytes in ferns. As a result, 476 fungal endophytes have been isolated from 5 ferns widely distributed in South China, of which 42 strains have been found to be antibacterial against MRSA. In this proposed project, the 42 anti-MRSA stains will be investigated for their metabolites responsible for the activity, which includes the follows. The active strains will be subjected to HPLC-DAD-MS/MS analysis associated with anti-MRSA activity-based growth curve to identify promising stains which have the potential for producing new active metabolites. The promising strains will be applied to large-scale fermentation and the extracts of fermented cultures will be subjected to fractionation and separation under the guidance of anti-MRSA activity and HPLC-MS detection, followed by structural elucidation of the isolated compounds. The new active compounds will be evaluated for the antibacterial activity against MRSA and other various bacteria as well as for cytotoxicity against mammal cells to obtain novel compounds with potent anti-MRSA activity and selectively acting on bacteria. It is predicted that this project can discover a number of novel anti-MRSA compounds which would be promising as leads for development of new anti-MRSA antibiotics.

抗生素耐药菌是当前最大的公众健康威胁，其中的MRSA是细菌感染所致死亡的主要诱因，寻找新型抗MRSA药物是应对耐药菌威胁的迫切需要和长期任务。蕨类植物所具有的超强抗病性预示着其内生菌在寻找抗菌素先导物上有潜在研究价值。在前期研究中，我们从5种蕨类植物分离获得了476株内生真菌，通过活性筛选从中发现了42个抗MRSA菌株。本项目将在此基础上，对这些活性菌株的抗MRSA代谢产物进行研究。通过基于抗菌活性生长曲线的UPLC-DAD-MS/MS分析，筛选有产生抗MRSA活性新化合物潜力的菌株；在抗MRSA活性跟踪和UPLC-MS检测下对潜力菌株的发酵物进行分离并鉴定活性代谢产物的结构，获得抗MRSA活性新化合物；通过测试活性新化合物的抗MRSA活性、抗菌谱和哺乳动物细胞毒作用，获得抗MRSA活性显著、选择性作用于细菌的新化合物，为新型抗MRSA药物的设计和合成提供先导物。

MRSA是细菌感染所致死亡的主要诱因之一，寻找抗MRSA药物先导物是新型抗菌素研发的迫切需要。在前期研究中，我们从476株分离自蕨类植物的内生真菌中筛选获得了42个抗MRSA活性菌株。本项目在基于抗菌活性生长曲线的UPLC-DAD-MS分析基础上对其中9个活性菌株的抗MRSA代谢产物进行了研究。在抗MRSA活性跟踪下从9个菌株的发酵物中分离、鉴定了230个化合物，包括94个新化合物，其中来自J551菌株的4个pestalone型二苯甲酮新化合物和来自J453菌株的5个萘醌螺缩酮类新化合物对MRSA具有高活性（MIC ≤ 6.3 ug/mL）。此外，对前期发现的土壤来源SC1169菌株的抗MRSA活性代谢产物进行了深入挖掘，获得了34个苯基四环聚酮新化合物，其中8个化合物对MRSA有高活性（MIC ≤ 5.0 ug/mL）。进一步抗菌活性和细胞毒活性测定发现得自J551菌株的3个C-14醛基还原为氧甲基的pestalone型二苯甲酮新化合物和得自SC1169菌株的7个fasamycin型苯基四环聚酮新化合物对四种供试革兰氏阳性菌包括VRE均有显著活性（MIC ≤ 10.0 ug/mL），其中对MRSA活性（MIC = 1.25–2.5 ug/mL）接近于万古霉素（MIC = 0.63 ug/mL），而其对4种供试人癌细胞和正常细胞Vero的毒性显著弱于抗MRSA活性（IC50 > 摩尔MIC），表明二类新化合物具有选择性抗MRSA活性。从二类选择性抗MRSA活性新化合物中分别选取了1个代表性化合物进行小鼠体内抗MRSA活性试验，结果显示2个化合物对小鼠皮肤MRSA感染有治疗作用。上述生物活性评价结果表明C-14为氧甲基的pestalone型二苯甲酮和fasamycin型苯基四环聚酮二类新化合物有成为抗MRSA药物先导物的潜力。