高效抗耐药鲍曼不动杆菌人防御素5简化肽的设计、筛选及抗菌机制研究

Drug-resistant Acinetobacter baumannii seriously threatens human health, which necessitates the development of novel antibiotics. Human defensin 5 (HD5) is a potent antibacterial peptide. Because bacteria are difficult to resist HD5, it is a promising candidate to traditional antibiotics. The complex process in peptide preparation, as well as the limited efficiency in saline solution，are two major problems slowing down the development of HD5. Because it is unnecessary for disulfide bonds to protect peptide from degradation when HD5 is locally used, disulfide removal is an instrumental method to simplify synthesis. Accordingly, this project plans to screen the right intermolecular disulfide bond and electropositive residue to enhance the bacterial killing of simplified HD5 against drug-resistant Acinetobacter baumannii in the context of high concentrations of saline solution by a structure-activity relationship study, where biolayer interferometry, bacterial colony count assay, and cytotoxicity evaluation are used. Furthermore, a Radiation-Wound-Infection model is employed to detect the antibacterial efficiency of simplified HD5 against drug-resistant Acinetobacter baumannii in vivo. TEM, laser scanning confocal, and bacterial adhesion-invasion assay are used as well to uncover the underlying antibacterial mechanism. This project is set up to design a novel antibiotic against drug-resistant Acinetobacter baumannii and promote the application of HD5 in clinic.

耐药鲍曼不动杆菌严重威胁人类健康，开发新型抗生素迫在眉睫。人防御素5（HD5）具有抗菌活性强、不易被细菌耐受等特点，是传统抗生素的理想替代物。制备复杂和活性易受盐离子干扰是制约HD5药用开发的两个主要问题。由于防御素局部应用抗菌时无需二硫键规避酶解，本项目前期通过构效关系研究明确了以去除二硫键简化HD5合成的方案。在此基础上，拟使用生物膜干涉、菌落计数和细胞毒性实验，筛选利于简化肽在高浓度盐溶液中抗耐药鲍曼不动杆菌的分子间二硫键和正电荷氨基酸。体外实验筛选出高效简化肽后，拟使用放创-感染模型评估多肽的体内抗菌效果，用透射电镜、激光共聚焦以及细菌黏附侵袭实验探讨高效简化肽抗耐药鲍曼不动杆菌的机制。项目旨在设计出新型抗耐药鲍曼不动杆菌抗生素，推进HD5在临床感染防治方面的应用。

耐药鲍曼不动杆菌严重威胁人类健康，开发新型抗生素迫在眉睫。人防御素5（HD5）具有抗菌活性强、不易被细菌耐受等特点，是传统抗生素的理想替代物。制备复杂和活性易受盐离子干扰是制约HD5药用开发的两个主要问题。由于防御素局部应用抗菌时无需二硫键规避酶解，本项目采用减少二硫键数量的方式简化HD5的制备。结构效应关系研究揭示，Cys2-4二硫键对HD5高效抗菌尤为重要。将仅含Cys2-4二硫键突变肽的非正电荷、非疏水性氨基酸替换为Arg后，衍生肽HD5d5抗多重耐药鲍曼不动杆菌（MDRAb）的活性显著增强。HD5d5生物安全性好，在高浓度盐离子环境中可保持高效抗菌作用。局部涂抹HD5d5可显著改善放射-创面复合伤MDRAb感染小鼠的存活率，降低创面痂下菌量。抗菌机制研究发现，HD5d5在盐溶液中结合细菌外膜脂质A和外膜蛋白A、机械损伤菌膜、诱导菌膜去极化的能力均强于HD5；此外，HD5d5结构弹性强，可穿膜进入细菌胞浆，结合并减弱过氧化氢酶活性，上调活性氧水平，干扰细菌正常代谢。鉴于增加多肽净电荷能补偿构象缺陷导致的功能不足，后续研究设计并制备了富含Arg的HD5线性衍生肽AR32。AR32通过机械损伤菌膜致死MDRAb，最低抑菌浓度为2.5 μg/mL。经鼻给予AR32可显著改善放射-肺部MDRAb感染小鼠的存活率，减少肺部细菌定植。HD5还原成线性肽后抗菌作用减弱、结构弹性和中和细菌内毒素能力增强。结构效应关系研究发现，线性还原型HD5破坏菌膜、致死细菌的能力高度依赖于保守的半胱氨酸和精氨酸，多肽中和细菌内毒素的作用则依赖于包括半胱氨酸、精氨酸、盐键、Gly18等在内的所有进化保守元素。根据以上结果，设计并制备多能抗菌肽AcmCys-E21R-HD5；该多肽在高效抗菌的同时可有效抑制内毒素释放引起的促炎反应。项目基于人体内源性多肽HD5研发出系列高效抗菌肽，为临床耐药鲍曼不动杆菌感染防治提供了新方案。