铜绿假单胞菌噬菌体vB_PaeM-YQ78生物学与基因组特征及其裂解酶的改造

孙素越　于姣阳　王卫晓　董慧　卢晓亮　韩丽梅　王世伟　胡春梅　陈伟

摘要：目的 从医院废水中筛选并鉴定铜绿假单胞菌烈性噬菌体，表达和优化其裂解酶。方法 以收集的临床铜绿假单胞菌为宿主菌，分离纯化烈性噬菌体，并深入鉴定其中一株裂解能力强的广谱噬菌体的生物学特性，分析其基因组特征和进化特征，表达和改造噬菌体裂解酶，测定噬菌体与抗生素的协同杀菌作用以及不同穿膜辅助剂对于裂解酶杀菌活性的影响。结果噬菌体vB_PaeM-YQ78是一株新的铜绿假单胞菌肌尾噬菌体，潜伏期为10 min，裂解量为43；最佳感染复数为0.00001；在40℃以下和pH 5～10范围内保持稳定。噬菌体与环丙沙星具有较强的协同作用，在24 h内无突变菌株生长。不使用穿膜辅助剂时，裂解酶LysYQ78在1 h内能将多重耐药铜绿假单胞菌降低1个log单位，在添加5 mmol/L EDTA时，LysYQ78可以将细菌浓度降低6个log单位。将ApoE蛋白N端穿膜肽融合到LysYQ78的N端后，Lys1410-YQ78的杀菌活性得到显著提高，可以在1 h内将宿主菌浓度降低2个log单位。结论 本实验分离鉴定了一株新的铜绿假单胞菌烈性噬菌体，其具有较宽的宿主谱，与环丙沙星具有较强的协同作用。并且，其裂解酶本身就可以穿透细胞外膜，展现杀菌活性，而与穿膜肽融合后，杀菌活性得到显著增强。本实验的裂解酶改造为进一步优化提供了思路，为将来铜绿假单胞菌的噬菌体治疗提供新的选项。

关键词：铜绿假单胞菌；噬菌体；协同作用；裂解酶；穿膜肽

中图分类号：R978.1文献标志码：A

Characterization of biological and genomic features of Pseudomonas phage

vB_PaeM-YQ78 and optimization of its endolysin

Sun Su-yue1，2， Yu Jiao-yang3， Wang Wei-xiao1， Dong Hui1， Lu Xiao-liang3，

Han Li-mei1， Wang Shi-wei3， Hu Chun-mei1， and Chen Wei1，2

（1 Department of Tuberculosis， the Second Hospital of Nanjing， Nanjing University of Chinese Medicine， Nanjing 210003; 2 The Clinical Infectious Disease Center of Nanjing， Nanjing 210003; 3 Key Laboratory of Resources Biology and Biotechnology in Western China， Ministry of Education， College of Life Sciences， Northwest University， Xi'an 710069）

Abstract Objective To isolate and identify Pseudomonas aeruginosa phages and optimize their endolysins， in order to provide new therapeutic option for the P. aeruginosa infection. Methods The clinically collected MDR-P. aeruginosa strains were used as the host strains to isolate and identify lytic phages. One of the broad-spectrum phages， vB_PaeM-YQ78 was deeply characterized in terms of the biological and genomic features， as well as its interaction with ciprofloxacin. Its endolysin gene was cloned， expressed， purified and assessed in the presence or absence of membrane-penetrating adjuvants. Results 22 P. aeruginosa lytic phages were isolated from hospital waste water and their host spectrums were identified for a panel of multiple drug-resistant P. aeruginosa strains. vB_PaeM-YQ78 was a novel Myoviridae phage with linear double-strand DNA. Its activity was maintained below 40℃ and at pH 5～10. The optimal MOI when lysing its original host was 0.00001， with which the phage titer could reach 1011 PFU/mL. vB_PaeM-YQ78 displayed synergistic interaction with ciprofloxacin， which suppressed bacterial growth within 24 h. Its endolysin， LysYQ78 was able to reduce 1 log and 6 log units of MDR P. aeruginosa within 1 h in the absence and presence of 5 mmol/L EDTA， respectively. When fused with a membrane-penetrating peptide from ApoE， Lys1410-YQ78 had significantly higher bactericidal activity. Conclusions vB_PaeM-YQ78 was a novel P. aeruginosa lytic phage with broad host spectrum， strong lytic capability， and synergy with ciprofloxacin in vitro. Its endolysin exhibited bactericidal activity without adjuvants. On fusing with a membrane-penetrating peptide， the recombinant endolysin exhibited significantly stronger activity. Our study shed light on the further modification of endolysin and would provide new option for phage therapy of P. aeruginosa infection.

Key words Pseudomonas aeruginosa; Phage; Synergy; Endolysin; Membrane-penetrating peptide

收稿日期：2022-10-29

基金項目：国家自然科学基金面上项目（No. NSFC 82272340）；江苏省自然面上项目（No. BK20221722）

作者简介：孙素越，女，生于1997年，在读硕士研究生，研究方向为内科学，E-mail： 20201631@njucm.edu.cn

*通信作者：胡春梅，E-mail： njyy003@njucm.edu.cn；陈伟，E-mail： njyy039@njucm.edu.cn