脂多糖诱导抗菌肽HAL-2形成淀粉样纤维结构的分子机制研究

Antimicrobial peptides (AMPs) are important components of the innate immune systems in all living organisms. AMPs possess unusually efficient antimicrobial effects with little drug-resistant against a broad range of microorganisms, which makes them attractive candidates for the development of therapeutic drugs in the fight against resistant pathogens. The interaction between AMPs and lipopolysaccharide (LPS) contributes to its antimicrobial activity against Gram-negative bacteria. However, the molecular mechanism of interaction between them and how it relates to the activity are not clear. Our present study showed that LPS is capable of inducing HAL-2 amyloid formation when against E. coli, which may play a vital role in its antimicrobial activity. But as yet unknown molecular basis regulate that specific amyloid aggregation and the antibacterial activity. Effect factors of HAL-2/LPS system including HAL-2 size, sequence changes, conformation, pH, ionic strength and divalent cations are investigated, by using various biophysical methods, such as nuclear magnetic resonance and surface plasmon resonance. Furthermore, the interaction between amyloid formation of HAL-2 and its activity will be discovered. In conclusion, this study will increase our understanding of the molecular basis of HAL-2 mediated by LPS, and it could provide information to design novel antimicrobial peptides for future therapeutic purposes.

抗菌肽广泛存在于各种生物体内，是宿主先天免疫防御系统的重要组成部分，具有抗菌谱广、不易产生耐药性等优点，有望成为治疗耐药性病原菌感染所引发疾病的理想药物。在抑杀革兰氏阴性菌过程中，抗菌肽与脂多糖的相互作用对其发挥抑菌活性至关重要，但其具体作用细节及与抑菌活性之间的内在联系仍不清楚。申请人发现抗菌肽HAL-2在抑杀大肠杆菌过程中，脂多糖可诱导其形成淀粉样纤维结构，此淀粉体结构的形成对其抑菌活性至关重要。但这种淀粉体结构形成的具体分子机制及其与HAL-2抑菌活性之间的内在联系还需深入研究。本项目拟运用核磁共振、表面等离子共振等方法，主要研究氨基酸序列长度与组成、分子构象及溶液酸碱性、离子强度、二价阳离子等对HAL-2/LPS作用体系的影响；结合体外抑菌实验，揭示HAL-2淀粉体结构发挥抑菌活性的分子机制，为阐明HAL-2作用机制提供重要理论依据，为发展有针对性的新型抗菌肽类药物奠定基础。

抗菌肽广泛存在于各种生物体内，能够抵抗病原微生物入侵,是宿主先天免疫防御系统的重要组成部分，它不易产生耐药性，是一类极具潜力的治疗耐药性病原菌感染所引发疾病的理想药物。在抑杀革兰氏阴性菌过程中，抗菌肽与脂多糖（LPS）的相互作用对其发挥抑菌活性至关重要，但其具体作用细节及与抑菌活性之间的内在联系仍不清楚。本项目运用核磁共振、圆二色谱、红外光谱、静态光散射等多种生物物理学方法，研究抗菌肽氨基酸序列组成、分子构象、离子强度及二价阳离子等对HAL-2/LPS作用体系的影响，并深入探究抗菌肽HAL-2与LPS之间的相互作用细节及其对细菌的抑制作用，揭示HAL-2淀粉体结构发挥抑菌活性的分子机制。.我们完成了抗菌肽HAL-2的克隆表达及纯化，并对其进行同位素标记，收集三维核磁共振波谱，归属其化学位移，确定了HAL-2与LPS发生作用的氨基酸位点；HAL-2与LPS相互作用过程中，HAL-2中的大部分脂肪族侧链与LPS距离接近，整个HAL-2抗菌肽与LPS发生了较强的相互作用，且HAL-2能够将LPS胶束聚集体解离。HAL-2与LPS作用过程中，HAL-2自身不会发生聚集，LPS可诱导其以β-sheet结构存在进而产生淀粉样聚集体，而在其他类膜环境如30% TFE，15mM DPC，100mM SDS中，HAL-2折叠为典型的α-螺旋结构。HAL-2与LPS之间的相互作用对其抑制革兰氏阴性菌至关重要，外界盐离子对HAL-2的抑菌活性具有较大影响。本项目为阐明HAL-2作用机制提供重要理论依据，为发展有针对性的新型抗菌肽类药物奠定了理论基础。项目执行过程中发表SCI论文两篇，申请专利3项。