杀多菌广谱裂解酶的结构基础与作用机制研究

The increasing antimicrobial resistance is one of the most urgent threats to public health worldwide. Due to their many advantages, including evolution with the host bacteria and rare resistance, bacteriophage lysins, produced by phages in the late phase of infection cycle to digest bacterial cell wall for the release of progeny virions, have showed great potential as anti-infectives and attracted much attentions during the past decade. However, the clinical development and application of lysins was hampered by their narrow host ranges. Benefitting from the rapid improvement of the screening technologies for chimeric lysins and the great efforts from researchers worldwide, several lysins with broad host range have been developed. However, due to the less understanding between the structural characteristics as well as the binding substrates of lysins with broad host range and their broad killing capacity, the designing and engineering of lysins with broad host range was still blindness. In this study, the novel chimeric lysin, ClyR (showing broad lytic activity against most streptococci, including S. pneumoniae, S. pyogenes, S. suis, S. uberis, S. agalactiae, S. dysgalactiae, S. mutans, S. equi and susceptible staphylococci and enterococci, developed by high throughput screening method in our laboratory previously) was used as a model molecule to study the relationship between structure and function in lysins with broad host range. We will study the binding substrate of ClyR, resolution the structures of ClyR, ClyR/substrate complex to discover the structural characteristics and multiple-killing mechanisms of ClyR. This study will not only provide guidelines for developing new lysins with broad host-range, but also provide potent targets for new antibacterial drug development.

日益严重的细菌耐药已成为全球公共健康领域的一项重大挑战。噬菌体裂解酶因具有天然的进化优势和极低的耐药性，是近十年来备受关注的天然抗菌分子。然而裂解酶的临床开发和应用却受限于其较强的宿主特异性。得益于嵌合裂解酶高通量筛选技术的发展和多国科研人员的长期探索，广谱裂解酶近几年取得了一些进展。但由于缺乏对现有广谱裂解酶静态、动态结构与杀菌机理关系的精确认识，以及对其底物识别机制的深入研究，广谱裂解酶的设计仍存在很大的盲目性和偶然性。本项目拟以申请人实验室构建的目前最广谱的裂解酶ClyR（杀菌谱覆盖了几乎所有种类的链球菌、肠球菌和金黄色葡萄球菌）为模式分子，用生物化学、结构生物学和体外进化等方法鉴定ClyR的识别底物，解析ClyR及其与底物复合物的结构，揭示其杀多菌的结构基础和作用机制。本研究不仅能为发展新型广谱裂解酶提供理论指导，而且能为抗菌药物的筛选提供潜在的靶标，具有非常重要的理论和实际意义。

噬菌体裂解酶（简称为裂解酶）是双链DNA噬菌体感染宿主后晚期表达的一类肽聚糖水解酶，由于他们能在体外快速水解革兰氏阳性细菌的肽聚糖，引起细菌的裂解和死亡，因此是一类天然的新型抗菌分子。裂解酶通常包含一个N端的肽聚糖水解功能域以及一个C端的细胞壁结合功能域。裂解酶的种属特异性，相比于小分子抗生素而言，具有能保留有益共生菌、不破坏微环境的生态平衡等优点，但在实际应用中却面临适应症单一、治疗效果片面等挑战。因此，如何突破裂解酶的种属特异性，研究和发展具有一定广谱杀菌活性的裂解酶是目前急需解决的科学问题。本课题组经过筛选获得了一个具有广谱杀菌活性的广谱裂解酶ClyR。该酶能高效杀灭肺炎链球菌、化脓链球菌、猪链球菌、乳房链球菌、无乳链球菌、停乳链球菌、变形链球菌、马链球菌等以及多种肠球菌和金黄色葡萄球菌。与特异性的裂解酶相比，具有相对广谱杀菌活性的裂解酶能够满足临床上混合细菌感染的需要，然而，广谱杀菌的分子机理尚不清楚。.. 本项目研究了广谱裂解酶ClyR杀多菌的结构基础与分子机制。功能域解析发现ClyR的CHAP催化域能够水解多种细菌的肽聚糖，而其SH3-b细胞壁结合域亦能识别多种细菌，为ClyR杀多菌提供了生物学基础。通过解析ClyR细胞壁结合域以及全长蛋白的3D结构，明确了ClyR的结构特征以及底物结合口袋特点，为ClyR杀多菌提供了结构基础。结构生物学和生化实验进一步研究表明，ClyR的两个功能域之间通过分子内相互作用而维持两个功能域的生物学活性，为ClyR杀多菌提供了分子机制。基于对ClyR催化功能域的认识，我们通过功能域筛选与优化构建了专一性杀灭肺炎链球菌的嵌合裂解酶ClyJ及其突变体。此外，本项目还揭示了ClyR通过小窝蛋白介导的内吞途径进入上皮细胞的结构特征和分子机制，为裂解酶治疗的有效性和安全性提供了有力支撑。