基于三维结构和分子机制构建跨肺炎克雷伯菌K1和K2荚膜型解聚酶的研究

产K1和K2型荚膜的肺炎克雷伯菌（Kpn）占强毒力菌株的70%以上，加之耐药性非常严重，目前缺乏有效的防控药物。噬菌体解聚酶可特异性降解细菌荚膜，能帮助机体有效清除细菌，不影响正常菌群，是精准型抗菌物质。目前已发现的解聚酶三维结构和作用机制均不清楚，且降解谱过窄，限制了应用。本课题组前期已获得首个特异降解K2型Kpn荚膜的解聚酶Depo32，其体内治疗效果良好，已成功解析其三维结构；合作者台湾大学Wang Jin-Town教授也报道了目前唯一能特异降解K1型Kpn荚膜的解聚酶S2-4和K1-ORF34。在此基础上，本研究重点通过冷冻电镜技术等率先解析上述解聚酶及其与底物、中间体和产物分子复合物的三维结构、揭示相互作用的分子机制，据此对解聚酶进行改造，以获得对K1和K2型Kpn荚膜均具有降解活性的解聚酶改造体。本项目将为产荚膜多重耐药细菌多价解聚酶新型抗菌药物的研发奠定坚实的理论与实验基础。

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a zoonotic pathogen causing life-threatening infections in human and animals. More than 70% of hvKp strains produce the K1 or K2 serotypes capsular. In addition, the multidrug-resistant hvKp strains have been emerging worldwide. Therefore, there is an urgent need for novel therapeutic agents directed against this formidable pathogen. Phage-encoded capsular depolymerases can specifically destroy the capsule and favor the removing of bacteria by immune system. So far as we know, the three-dimensional structures and molecular mechanisms of all reported depolymerases have not been studied. And the narrow spectrum of phage depolymerases due to the specificity is a major constraint on affecting its therapeutic application. In our previous studies, the first depolymerase (named Depo32) specific for K2 capsule of K. pneumoniae has been obtained. Depo32 shows effective treatment on K2 K. pneumoniae infections. The three-dimensional structure of Depo32 has been revealed by Cryo-SEM. Otherwise, our collaborator Professor Wang Jin-Town of Taiwan University also has reported the only depolymerases S2-4 and K1-ORF34 that specifically degrade K1 capsule of K. pneumoniae. Based on these studies, this research will further focus on revealing the 3D structures of these three depolymerases and the complexes of depolymerases with their substrates, intermediates and products by Cryo-SEM, and revealing the molecular mechanisms of capsule degradation of depolymerases. Based on the 3D structures and molecular mechanisms, the smaller depolymerase which can degrade both K. pneumoniae K1 and K2 capsules will be constructed by truncation and modification. This project will provide a solid theoretical and experimental basis for the development of new antibacterial drugs of depolymerases against the multidrug-resistant and capsule producing bacteria.