内生真菌Aspergillus sp. TJ23次生代谢产物及其抗MRSA活性研究

Methicillin-resistant Staphylococcus aureus (MRSA), a “superbug”, which could express a specific resistanceprotein-penicillin-binding protein 2a (PBP2a) that produces inducible resistance to many drugs, is one of the pathogens with the highest morlality rate in the world. Therefore, an endophytic fungi Aspergillus sp. TJ23 was isolated from the medicine plant Acorus tatarinowii using the bioassay-guided method, exhibiting the best antibacterial activity against MRSA. A series of novel meroterpenoids were isolated from the Aspergillus sp. TJ23, in which the new skeleton compound exhibited significant anti-MRSA acticity and inhibited the expression of PBP2a in MRSA. To search for more structurally novel and bioactive products from Aspergillus sp. TJ23，the applicant find the optimized fermentation by means of OSMAC. Molecular docking and evaluation of antibacterial activity in vitro will be performed to study the anti-MRSA activity and mechanism by reducing the expression of PBP2a. Finally, the compounds with significant activities will be evaluated in vivo. This project will greatly promote the development of anti-MRSA agent by inhibiting the expression of PBP2a.

耐甲氧西林金黄色葡萄球菌（MRSA），被认为是一种“超级细菌”，因其能大量表达一种特殊的耐药蛋白━青霉素结合蛋白2a (PBP2a)，使得其对多种抗生素均产生不同程度的耐药性，成为世界死亡率最高的病原菌。因此，申请人通过抗MRSA 活性为导向，从药用植物石菖蒲的内生真菌中寻找到了一株抗MRSA活性良好的真菌Aspergillus sp. TJ23。对其次生代谢产物进行提取分离，发现了一系列结构新颖的杂萜类化合物，其中新骨架化合物具有明显的抗MRSA活性，并能抑制PBP2a的表达。项目申请者拟通过OSMAC策略，发酵得到不同条件的代谢产物，寻找到更多类似结构新颖的杂萜类化合物，通过计算机模拟及体外抗菌活性评价，探讨其通过抑制PBP2a而发挥抗MRSA作用及机制。对活性显著的化合物进一步探讨其体内效果及机制。该项目的实施对开发通过抑制PBP2a作用而发挥抗MRSA活性的新药具有重要意义。

本项目对内生真菌Aspergillus sp.TJ23次生代谢产物进行提取分离，得到了46个单体化合物(6个新化合物和1个新骨架），并通过计算ECD，质谱分析，X单晶衍射等方法对绝对构型进行了确定。化合物2可能为PBP2a的潜在抑制剂，并且能够协同增强β-lactam类抗生素oxacillin和piperacillin的抗MRSA作用，MIC值为1 μg/mL，协同增效值∑FIC小于0.5；反向对接虚拟筛选，结合MST实验提示化合物2潜在靶点为PBP2a；透射电镜TEM观测结果表明化合物2直接影响MRSA细胞形态；化合物15体外显示出较好的降糖活性，其降糖作用强于阳性药二甲双胍；通过对差异基因KEGG（Kyoto Encyclopedia of Genes and Genomes）通路分析、GO（Gene Ontology）功能富集、蛋白互作网络分析以及qT-PCR验证，发现化合物15通过激活胰岛素通路发挥降糖作用，其靶基因可能为FGF21，并且化合物15能剂量依赖性地激活该基因的表达。