构效优化的新型抗菌肽MSI-1抗耐甲氧西林金黄色葡萄球菌的机制研究

The spread of methicillin-resistant staphylococcus aureus (MRSA) resulted from the abuse of broad-spectrum antibiotics aggravates the treatment difficulty of clinical MRSA infection. It is urgent to develop anti-MRSA drugs with novel mechanisms of action. Antibacterial peptides have become a research hotspot of great interest in more than two decades due to their novel mechanisms of action and rare drug resistance. In this study, a series of Magainin II-derived peptides were designed accounting for the balance between hydrophobicity and alpha-helix, which is closely related to the antibacterial activity and cytotoxicity of peptides. Importantly, a structure-optimized peptide, MSI-1, which has potent anti-MRSA activity, good selectivity to bacteria and desirable stability to pH and pepsin, was obtained through the mutation of amino acids. .We further explore the underlying mechanisms of action of MSI-1 against MRSA based on the peptide-membrane mode of action (the changes of bacterial membrane permeability and membrane integrity), the peptide-DNA mode of action (the inhibition of DNA replication, mecA and/or crtN transcription and translation) and the peptide-protein mode of action (the interaction between MSI-1 and CrtN protein to interfering the synthesis of S. aureus staphyloxanthin). Additionally, to further confirm the anti-MRSA activity of MSI-1 in vivo, the bacteria that the crtN gene was knocked out (recombinant COL-△crtN mutated bacteria), the mouse peritonitis model (systemic infection) and burn/scald model (local infection) were used. It can provide a theoretical foundation for the clinical application of peptides against refractory MRSA infection by illuminating the relationships between peptide structure and anti-MRSA activity, toxicity and/or stability, and explaining the anti-MRSA targets and molecular mechanisms of action of peptide in the levels of gene and protein.

广谱抗生素滥用导致耐甲氧西林金黄色葡萄球菌（MRSA）感染的临床用药困难，寻求新机制抗MRSA药物迫在眉睫。抗菌肽作用机制新颖且不易耐药，成为研究热点。项目以Magainin家族抗菌肽为研究对象，考虑疏水性和α螺旋度的平衡关系，经构效优化，获得对MRSA选择性强，耐强酸、胃蛋白酶的新型抗菌肽MSI-1。拟进一步基于细菌细胞膜探讨多肽改变膜通透性、破坏膜完整性的肽-膜机制；基于DNA研究多肽干扰DNA复制、抑制mecA和crtN基因及其编码蛋白的肽-DNA机制；基于CrtN蛋白分析多肽干扰CrtN蛋白功能，抑制金黄色素合成的肽-蛋白机制；借助成功构建的COL-△crtN重组菌和细菌感染的小鼠败血症/烫伤模型，明确多肽的体内抗MRSA活性。通过明确抗MRSA多肽设计中结构与活性、毒性和稳定性的关系，阐明多肽在基因和蛋白水平的抗菌作用靶点和分子机制，为多肽抗难治性MRSA感染的临床研究奠定基础。

近年来，广谱抗生素的滥用催生了包括耐甲氧西林金黄色葡萄球菌（MRSA）在内的“超级细菌”，寻求具有新作用机制的抗耐药细菌药物，尤其是抗MRSA药物迫在眉睫。本项目以具有广谱抗菌活性的多肽MSI-1为研究对象，深入阐述多肽MSI-1对多种革兰氏阳性菌、阴性菌、真菌的抗菌活性、机制及其安全性。通过稳定性和毒性研究，我们发现多肽MSI-1经强酸、强碱、高热和高盐处理后，其抗菌活性没有明显变化；细胞毒性实验结果表明，在多肽MSI-1的体外有效抑菌浓度下，其对哺乳动物细胞基本无毒性作用及溶血活性。体外抗菌活性筛选结果显示：多肽MSI-1对革兰氏阳性菌、阴性菌和真菌均具有良好的抗菌活性，包括MRSA、耐青霉素E. coli和耐氟康唑新型隐球菌等）。机制研究结果表明：多肽MSI-1可与青霉素耐药E. coli的脂多糖或MRSA细胞壁上的磷壁酸结合导致细菌细胞壁塌陷，改变细胞膜的通透性，最终导致核酸和蛋白质等重要物质泄露，使细菌快速死亡；或与新型隐球菌荚膜的主要成分荚膜多糖结合，增加新型隐球菌细胞膜流动性和渗透性，进而破坏细胞结构，引发真菌死亡。此外，MSI-1在亚抑菌浓度下可与E. coli的基因组DNA结合最终导致细菌总蛋白的合成量减少或特异性地抑制S. aureus中CrtN蛋白的活性以抑制S. aureus毒力因子—金黄色类胡萝卜素的产生，进而有效地增敏宿主免疫系统对耐药S. aureus的清除作用。综上所述，多肽MSI-1抗菌谱广、抗菌活性好，有望成为一种治疗耐药细菌/真菌感染性疾病的新制剂。