孫素越　于姣陽　王衛(wèi)曉　董慧　盧曉亮　韓麗梅　王世偉　胡春梅　陳偉

摘要：目的 從醫(yī)院廢水中篩選并鑒定銅綠假單胞菌烈性噬菌體，表達和優(yōu)化其裂解酶。方法 以收集的臨床銅綠假單胞菌為宿主菌，分離純化烈性噬菌體，并深入鑒定其中一株裂解能力強的廣譜噬菌體的生物學特性，分析其基因組特征和進化特征，表達和改造噬菌體裂解酶，測定噬菌體與抗生素的協(xié)同殺菌作用以及不同穿膜輔助劑對于裂解酶殺菌活性的影響。結(jié)果噬菌體vB_PaeM-YQ78是一株新的銅綠假單胞菌肌尾噬菌體，潛伏期為10 min，裂解量為43；最佳感染復數(shù)為0.00001；在40℃以下和pH 5～10范圍內(nèi)保持穩(wěn)定。噬菌體與環(huán)丙沙星具有較強的協(xié)同作用，在24 h內(nèi)無突變菌株生長。不使用穿膜輔助劑時，裂解酶LysYQ78在1 h內(nèi)能將多重耐藥銅綠假單胞菌降低1個log單位，在添加5 mmol/L EDTA時，LysYQ78可以將細菌濃度降低6個log單位。將ApoE蛋白N端穿膜肽融合到LysYQ78的N端后，Lys1410-YQ78的殺菌活性得到顯著提高，可以在1 h內(nèi)將宿主菌濃度降低2個log單位。結(jié)論 本實驗分離鑒定了一株新的銅綠假單胞菌烈性噬菌體，其具有較寬的宿主譜，與環(huán)丙沙星具有較強的協(xié)同作用。并且，其裂解酶本身就可以穿透細胞外膜，展現(xiàn)殺菌活性，而與穿膜肽融合后，殺菌活性得到顯著增強。本實驗的裂解酶改造為進一步優(yōu)化提供了思路，為將來銅綠假單胞菌的噬菌體治療提供新的選項。

關(guān)鍵詞：銅綠假單胞菌；噬菌體；協(xié)同作用；裂解酶；穿膜肽

中圖分類號：R978.1文獻標志碼：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：國家自然科學基金面上項目（No. NSFC 82272340）；江蘇省自然面上項目（No. BK20221722）

作者簡介：孫素越，女，生于1997年，在讀碩士研究生，研究方向為內(nèi)科學，E-mail： 20201631@njucm.edu.cn

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