高效筛选抗菌嵌合裂解酶的平台及其应用

Antimicrobial resistance is one of the biggest threats to human health. There are urgent needs for developing new antimicrobial molecular. Due to their many advantages, such as evolution with the host and wide resources, bacteriophage lysins, produced by phages in the late phase of infection cycle to digest bacterial cell wall for the release of progeny virions, has showed great potential as anti-infectives and attracted much attentions during the past decade. Chimeric lysins, created by domain shuffling of different catalytic domains and cell wall binding domains from various natural lysins, are the trend to develop new lysins with improved proprieties, such as enhanced lytic activity, extended lytic spectrum, and improved solubility, and especially to develop new lysins against Gram-negative bacteria. However, the current methods for developing chimeric lysins are largely trial-and-error and based on the lucky of the researchers. Therefore, the efficiency for discovering good chimeric lysins is very low. In this study, we will establish a novel highly efficient platform for screening bactericidal chimeric lysins based on the techniques of random cloning, induced releasing and bactericidal activity determination on plates. The platform is expected to have the capability to screen thousands of different chimeric lysins within 2-3 weeks. Multi-drug resistant Streptococcus pyogenes and Acinetobacter baumannii will be used as the example targets to test the efficiency of the platform and discover novel chimeric lysins against these two bacteria. This study will provide a new solution to solve the critical obstacles in the current chimeric lysins development, significantly improve the efficiency to discover novel bactericidal chimeric lysins, and discover a few new chimeric lysins with potentials to treat the infections caused by the multi-drug resistant Streptococcus pyogenes and Acinetobacter baumannii.

近年因细菌耐药性而导致抗生素失去有效性的问题，已成为人类面临的最大健康威胁之一，急需发展新的抗菌分子。噬菌体裂解酶因具有天然进化和来源广等特点，是一类近十年广受关注和极具开发潜力的抗菌分子。将不同来源裂解酶的功能域模块组合构建嵌合裂解酶是目前的研究趋势，特别是发展革兰氏阴性细菌裂解酶。但目前嵌合裂解酶研究存在着盲目组合和效率低等技术瓶颈。本项目拟建立一种基因片段随机组合克隆，诱导裂解释放E. coli胞内所表达的嵌合裂解酶以及平板抗菌活性鉴定的嵌合裂解酶高效筛选平台，实现2-3周内对上千种不同组合嵌合裂解酶的筛选。并以临床上耐药严重的化脓链球菌和鲍曼不动杆菌为靶标筛选新的嵌合裂解酶，验证平台的筛选能力和效率，评估新裂解酶抗感染潜力。本研究将解决目前嵌合裂解酶发展的关键瓶颈，为新型、高效嵌合裂解酶分子的发现提供新工具，为治疗耐药细菌感染提供几种新嵌合裂解酶药物候选分子，具有重要的研究意义。

由于抗生素广泛使用，近年来包括MRSA在内的超级耐药细菌日益增多，严重威胁人类健康。加之研制全新抗生素面临较大困难, 因此，研究和发展能对抗耐药细菌感染的新药物刻不容缓。噬菌体裂解酶是近十年研究比较多的一类天然抗菌分子，具有耐药性低、来源广泛等优点。在本项目中，我们利用噬菌体裂解酶模块化的特点结构，发展构建了嵌合裂解酶高效筛选平台，将不同来源和不同种类的天然裂解酶的催化域和结合域进行组合，或者将催化域与其它功能域组合所得到的重组裂解酶。发展嵌合裂解酶可以使全酶具有更好的性质，甚至获得一些全新的性质，如具有更广谱的裂解谱，具有更好的水溶性和具有杀灭阴性细菌的活性等。在阴性细菌裂解酶方面，通过在天然裂解酶的N端融合一个具有膜破坏功能的短肽有可能得到直接裂解阴性细菌的重组裂解酶。基于这种设想，我们建立和发展了一种在基因水平上随机组合克隆（random cloning），诱导释放（induced releasing）E. coli胞内所表达的嵌合裂解酶以及平板抗菌活性鉴定的筛选平台，并对平台性能和可靠性进行了测试。进一步依托该平台体系以金黄色葡萄球菌、肺炎链球菌、无乳链球菌、鲍曼不动杆菌等典型细菌为靶标实现了高活性嵌合裂解酶的快速发现，表达和研究了8种嵌合酶, 部分结果发表研究论文6篇，申请发明专利3项，为感染的预防与控制提供了新的候选分子。其中首次发现一个裂解酶的结合域V12CBD蛋白在小鼠模型上同时具有治疗（作用于细菌本身）和免疫保护（激活宿主免疫反应）效果，为研究治疗包括MRSA在内的超级耐药细菌感染药物提供了新的思路和潜在靶标，具有重要意义。