特异性靶向抗菌肽对水产动物致病菌的抑菌活性和作用机制

The problems of bacterial resistance, drug residues and water pollution caused by the abuse of antibiotics in aquaculture have attracted great attention from the international community. Development of safe and reliable AMPs with enhanced antibacterial activities and decreased toxicity are the proposed research. Our previous study reports the identification of multiple uncharacterized AMPs. We utilized the AMPs as templates and modified their hydrophobicity and cationicity to design and synthesize a series of analogs. The AMP-derived peptides, have enhanced antimicrobial activities against several bacterial strains including five MDR strains, the minimum inhibitory concentration (MIC) was lower than that of many antibiotics and other antimicrobial peptides. These AMP-derived peptides are promising template for the design of novel peptide antibiotics against MDR microbes. Based on the previous research, we project to design new specifically targeted antimicrobial peptides (STAMPs) by bioinformatics and computer-aided drug design technology and then test their antibacterial activities and the involved mechanisms of action against pathogenic bacteria of aquatic animals. We project to screen out effective, stable and non-toxic antimicrobial peptides, and to explore the method of constructing targeted antimicrobial peptides against specific bacteria. The effect of synthetic antimicrobial peptides on gut microbiota was studied as a test index to evaluate the effect of antimicrobial peptides. This project provides solutions for the production of new antibiotic substitues for aquatic products and the control of the large-scale diffusion of multi-drug resistant bacteria and drug resistance genes caused by the abuse of antibiotics. The project is conducive to the realization of "green, healthy and safe" aquaculture development model.

水产养殖过程中抗生素滥用所导致的细菌耐药性、药物残留和水环境污染等问题已引起了国际社会的高度关注。开发安全可靠、无毒无害的新型抗菌肽是解决这一问题的理想方案。课题组以前期鉴定的多种抗菌肽为模板，合成了多条抗菌肽，其抗菌尤其是抗耐药性细菌活性显著提高，最小抑菌浓度（MIC）低于多种抗生素和其他抗菌肽。基于以上前期工作，本项目旨在通过生物信息学技术设计和改造抗菌肽分子，连接靶向片段构建针对水产动物致病菌的特异性靶向抗菌肽，研究其对不同种类水产致病菌的抗菌活性和作用机制，以筛选出高效稳定无毒性的抗菌肽，探索构建针对特定细菌的靶向抗菌肽的方法；研究合成的抗菌肽对肠道菌群的影响，作为检验指标评估抗菌肽的作用效果；本项目为新型水产用抗生素替代品的创制和控制抗生素滥用引起的细菌耐药、耐药基因的大规模扩散提供解决方案，有利于实现“绿色、健康、安全”的水产养殖业发展模式。

为了应对全球广泛关注的微生物耐药问题，迫切需要开发不易产生耐药性的新型抗菌剂。多数抗菌肽(Antimicrobial peptides, AMPs)与抗生素的抗菌机制不同，它不是通过作用于微生物的固定作用靶点发挥抑菌功能，因此不易引起微生物耐药性，被认为是一种有潜力的抗生素替代品。然而天然抗菌肽存在着抗菌活性弱、细胞毒性高等缺点，因此本论文聚焦于设计新型抗菌肽，旨在提高抗菌肽的抗菌功能，改善细胞毒性，为抗菌肽的广泛应用提供数据支撑。.天然抗菌肽但其抗菌效果不够理想，并且天然多肽不易被提取。因此本研究以mBjAMP1为模板，设计出八条新型抗菌肽，分析它们的理化性质，并对其生物活性和抑菌机制进行研究。主要研究内容如下：.1.新型抗菌肽的设计、合成与结构表征。通过用色氨酸和赖氨酸替换mBjAMP1中的一些氨基酸，并在此基础上添加细菌信息素，设计出八条新型抗菌肽。.2.新型抗菌肽的生物活性研究。首先，本研究验证了八条抗菌肽的抑菌及杀菌活性，与mBjAMP1相比，新型抗菌肽对多种水产致病菌的抑菌活性显著增强，在特定浓度下对细菌的杀菌率可达99%甚至100%。其次，以小鼠巨噬细胞RAW264.7为实验对象，验证了这些抗菌肽在抑菌浓度下不会对哺乳动物细胞产生细胞毒性。.3.新型抗菌肽的作用机制研究。抗菌肽能引起细菌细胞膜通透性增加，发生去极化现象，抗菌肽能显著提高细菌细胞内活性氧水平，引发细菌坏死或凋亡。.本项目通过氨基酸替换和细菌信息素修饰，对天然抗菌肽进行改造，成功设计出八条新型抗菌肽，具有良好的抑菌和杀菌活性，且在有效抑菌浓度范围内无细胞毒性，能够通过改变细菌细胞内外膜通透性、促进细胞膜发生去极化、增加细胞内活性氧水平等机制杀死细菌，不易产生耐药性，是良好的新型抗菌剂。本论文为新型抗菌肽的设计提供了模板和思路，为解决细菌耐药问题提供了方案。