基于多靶点的多肽抗耐药性细菌感染的机制研究

In view of the serious infection induced by drug-resistant bacteria, the structure of peptides derived from natural protein was improved. To get new peptide molecules with high activity and stability and low toxicity, the structure-activity relationship of these optimized peptides were evaluated based on their physicochemical properties and anti-microbial activity in vitro/in vivo. . To clarify the multiple targets mechanism of peptides against drug-resistant bacteria, the NDM-1 carrying bacteria (recombinant E. coli BL21 (DE3)-NDM-1,” superbugs”) infected cell/animal models and the molecular targeted drug delivery system mediated by peptide-oligonucleotide conjugates were used. The involved targets include bacterial membrane (or LPS), drug-resistant gene (encoded by plasmid), regulator gene (ropD which regulate the DNA-dependent RNA polymerase (RNAP) primary sigma factor 70 (δ70)) and mRNA (which regulate the translation of drug resistant-related proteins). . Meanwhile, for bacterial inflammation, the cell and animal inflammation models were stimulated by lipopolysaccharide (LPS) and lipoteichoic acid (LTA) to reveal the molecular mechanisms of interaction between peptides and NAPDH oxidase complex and/or the IKK-IκB-NF-κB signaling pathway. The involved targets include NADPH oxidase p47-Phox (C-terminal SH3 domain) and IκBa subunits predominated by ubiquitin-proteasome pathway.. It can provide a theoretical foundation for the clinical application of peptides against drug-resistant bacterial infection by illuminating the structure-activity relationship, antibacterial and anti-inflammatory molecular mechanisms and multiple targets effect of peptides.

针对严重的耐药细菌感染问题，进行天然蛋白（Cathelicidin 家族）来源抗菌肽结构优化，根据理化参数和体内/外活性，评价抗菌肽构效关系，获得高效、低毒、稳定的新型多肽分子。基于细菌细胞膜（或膜LPS）、耐药基因（质粒编码）、调节基因（调控RNAP关键因子δ70的ropD基因）和耐药相关蛋白的mRNA等靶点，借助本室构建的重组E. coli BL21 (DE3)-NDM-1（超级细菌）细胞/小鼠感染模型及多肽-寡核苷酸缀合物分子靶向递药体系等关键技术，阐明多肽对耐药细菌作用的多靶点分子机制。针对细菌感染性炎症，基于NADPH氧化酶p47-Phox的SH3结构域及泛素-蛋白酶通路IκBa亚基靶点，通过LTA/LPS诱导的细胞/动物炎症模型，揭示多肽对NADPH氧化酶复合体及NF-κB信号通路的影响。通过阐明多肽的构效关系、感染与炎症、多靶点，为多肽抗耐药细菌感染的临床应用奠定基础。

耐药细菌感染严重威胁人类健康，如何克服细菌耐药，是亟待解决的问题。抗菌肽（Antimicrobial Peptides）是一类由生物体产生的具有抗菌活性的小分子多肽，因其广谱的抗病原微生物活性，近年来成为抗耐药细菌感染研究的重点。.在本课题中，我们从天然来源的动物毒素出发，经活性氨基酸序列分析、截取、氨基酸位点替换等手段，通过构效关系研究，应用多种临床分离耐药菌株和携带ndm-1、mcr-1、cagA等耐药基因的重组菌，针对细菌耐药基因、毒力蛋白和基因组DNA等靶点，综合考虑细菌和宿主免疫防御之间的关系，经结构-活性筛选、不断构效优化，获得具有作用新靶点/新机制、低毒性、且不易产生诱导耐药性的新型抗菌肽Cbf-14、MSI-1和HF-18。构效研究发现，多肽的疏水性、电荷性和两亲性是影响其抗菌活性的关键因素。.体内外抗菌活性研究表明，多肽Cbf-14、MSI-1和HF-18对多种临床耐药细菌均具有良好的抑菌和杀菌效果。同时，多肽对青霉素耐药E. coli或MRSA感染的小鼠烧伤皮肤具有很好的保护作用。在小鼠急性败血症模型中，多肽能减少细菌向重要组织脏器的扩散，快速杀灭体内细菌，减轻细菌感染诱发的炎症反应，从而提高小鼠的存活率。.在抗菌作用机制研究中，我们发现，高浓度下（4×MIC）多肽能结合脂多糖（LPS）、磷壁酸（LTA）和荚膜多糖（GXM），中和细菌/真菌细胞表面的负电荷，逐渐在菌体表面聚集，当达到一定浓度后，多肽使得细菌/真菌细胞膜通透性增强，且亚抑菌浓度的多肽能在细菌细胞内共定位。琼脂糖凝胶电泳、紫外光谱测定、EB竞争性实验和CD光谱分析，表明多肽能够插入细菌DNA，与之结合，这种结合使得耐药细菌的耐药基因（ndm-1、mcr-1、cagA、tem-1、shv-1等）和DNA合成相关基因（如DnaA，gyrB和DNA polymerase III subunit beta）的表达减少，从而干扰细菌总蛋白的合成。同时，我们验证了在细胞和小鼠感染模型中，多肽通过由泛素-蛋白酶信号通路介导的IκBa亚基降解过程，来调节IKK-IκB-NF-κB信号通路或抑制NLRP3炎症小体的激活，阻止炎症因子的过度释放。此研究为临床治疗耐药菌感染提供思路，为多肽临床应用奠定基础。