金黄色葡萄球菌噬菌体裂解酶Ply187的CHAP结构域的表达及抗菌活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.031

摘要/Abstract

摘要： 噬菌体裂解酶是一种高效的抗菌蛋白,系统地分析噬菌体裂解酶催化域的活性,有助于设计高效杂合裂解酶。合成噬菌体裂解酶Ply187的CHAP催化结构域（CHAP_Ply187）基因,并构建表达载体pET28a-CHAP_Ply187转化至大肠埃希菌BL21（DE3）,经IPTG诱导表达和两步纯化,获得高纯度的CHAP_Ply187,再与溶葡萄球菌酶催化域（CAT_Lysn）进行活性比较。比浊法检测显示CHAP_Ply187和CAT_Lysn对金黄色葡萄球菌都具有较强的抗菌活性;CHAP_Ply187具有更宽的抗菌谱,最适作用pH范围更大,对离子强度的耐受能力更好,易受EDTA的影响。低浓度的二价金属离子Ca²⁺、Mg²⁺、Mn²⁺对两种催化域都有明显的促进作用,低浓度的Zn²⁺、Cu²⁺、Fe²⁺对它们有很强的抑制作用。

关键词: 噬菌体裂解酶, 溶葡萄球菌酶, 催化结构域, CHAP, 抗菌活性

Abstract: Bacteriophage lysins are efficiently antibacterial proteins,thus systematically analysing the activity of catalytic domain of it is conducive to the design of efficient heterozygous lysins. The coding sequence of the CHAP catalytic domain of Ply187（CHAP_Ply187）was synthesized,and a recombinant expression plasmid pET28a-CHAP_Ply187was constructed,which then was transformed into Escherichia coli BL21（DE3）. The highly pure recombinant CHAP_Ply187 protein was produced by IPTG induction,further purified by a two-step method,reaching > 95% purity,and finally compared with the catalytic domain of lysostaphin（CAT_Lysn）. The results from turbidimetry showed that both CHAP_Ply187 and CAT_Lysn possessed the solid antibacterial activities to Staphylococcus aureus.CHAP_Ply187showed a much broader antibacterial spectrum and optimal activity in a wider range of the pH,tolerated higher ionic strength,and more easily affected by EDTA. Ca²⁺,Mg²⁺,and Mn²⁺in low concentration significantly promoted the catalytic domains of both proteins,while the Zn²⁺,Cu²⁺,and Fe²⁺in low concentration strongly inhibited the catalytic domains.

Key words: bacteriophage lysin, lysostaphin, catalytic domain, CHAP, antibacterial activity

吴蒙, 陆海荣, 黄青山. 金黄色葡萄球菌噬菌体裂解酶Ply187的CHAP结构域的表达及抗菌活性分析[J]. 生物技术通报, 2016, 32(9): 232-238.

WU Meng¹, LU Hai-rong^{1, 2}, HUANG Qing-shan^{1, 2}. Expression and Antibacterial Activity of CHAP Catalytic Domain of Staphylococcus aureus Phage Lysin Ply187[J]. Biotechnology Bulletin, 2016, 32(9): 232-238.

参考文献

[1] 汪复, 朱德妹, 胡付品. 2012 年中国 CHINET 细菌耐药性监测[J]. 中国感染与化疗杂志, 2013, 13（5）：321-330.
[2] File TM, Srinivasan A, Bartlett JG. Antimicrobial stewardship：importance for patient and public health[J]. Clin Infect Dis, 2014, 59（suppl. 3）：S93-S96.
[3] Nelson DC, Schmelcher M, Rodriguez-Rubio L, et al. Endolysins as antimicrobials[J]. Adv Virus Res, 2012, 83：299-365.
[4] 方圆子, 王琰, 孙建和. 噬菌体裂解酶——现状与未来[J]. 微生物学通报, 2009, 36（12）：1888-1893.
[5] Jun SY, Jung GM, Yoon SJ, et al. Preclinical safety evaluation of intravenously administered SAL200 containing the recombinant phage endolysin SAL-1 as a pharmaceutical ingredient[J]. Antimicrob Agents Chemother, 2014, 58（4）：2084-2088.
[6] Schmelcher M, Donovan DM, Loessner MJ. Bacteriophage endolysins as novel antimicrobials[J]. Future Microbiol, 2012, 7（10）：1147-1171.
[7] Wu HY, Lu HR, Huang JJ, et al. EnzyBase：a novel database for enzybiotic studies[J]. BMC Microbiol, 2012, 12（1）：54.
[8] 王铮, 沈文彬, 等. 噬菌体裂解酶作为抗菌药物的研究进展 [J]. 上海交通大学学报：医学版, 2013, 33（3）：368-373.
[9] Kunde S, Cloney D, Conrad H, et al. Fecal microbial transplantation shows efficacy in children with refractory ulcerative colitis-early results of phase I clinical trial：P-128[J]. Inflamm Bowel Dis, 2012, 18：S66-S67.
[10] Saravanan SR, Paul VD, George S, et al. Properties and mutation studies of a bacteriophage-derived chimeric recombinant staphylolytic protein P128：Comparison to recombinant lysostaphin[J]. Bacteriophage, 2013, 3（3）：e26564.
[11] Young R, Bläsi U. Holins：form and function in bacteriophage lysis[J]. Fems microbiol Rev, 1995, 17（1-2）：195-205.
[12] Briers Y, Volckaert G, et al. Muralytic activity and modular struct-ure of the endolysins of Pseudomonas aeruginosa bacteriophages φKZ and EL[J]. Mol Microbiol, 2007, 65（5）：1334-1344.
[13] Lu HR, Gu MG, et al. Hydrogen/deuterium exchange mass spectro-metry and site-directed disulfide cross-linking suggest an important dynamic interface between the two lysostaphin domains[J]. Antimicrob Agents Chemother, 2013, 4：1872-1881.
[14] Lawrence R, Jeyakumar E. Antimicrobial resistance：a cause for global concern[J]. BMC Proceedings, 2013, 7（3）：1-14.
[15] 王琰, 陆承平. 噬菌体裂解酶的抗菌特性[J]. 微生物学报, 2009, 49（10）：1277-1277.
[16] 杨航, 余军平, 危宏平. 裂解酶治疗的研究进展与应用前景[J]. 微生物学通报, 2015, 42（1）：178-184.
[17] Dong Q, Wang J, Yang H, et al. Construction of a chimeric lysin Ply187N-V12C with extended lytic activity against staphylococci and streptococci[J]. Microb Biotechnol, 2015, 8（2）：210-220.
[18] Daniel A, Euler C, Collin M, et al. Synergism between a novel chi- meric lysin and oxacillin protects against infection by methicillin- resistant Staphylococcus aureus[J]. Antimicrob Agents Chemo-ther, 2010, 54（4）：1603-1612.
[19] Donovan DM, Dong S, Garrett W, et al. Peptidoglycan hydrolase fusions maintain their parental specificities[J]. Appl Environ Microb, 2006, 72（4）：2988-2996.
[20] Pastagia M, Euler C, Chahales P, et al. A novel chimeric lysin shows superiority to mupirocin for skin decolonization of methicillin-resi- stant and-sensitive Staphylococcus aureus strains[J]. Antimicro- b Agents Chemother, 2011, 55（2）：738-744.
[21] Schmelcher M, Tchang VS, et al. Domain shuffling and module engineering of Listeria phage endolysins for enhanced lytic activity and binding affinity[J]. Microb Biotechnol, 2011, 5：651-662.
[22] Layec S, Decaris B, Leblond-Bourget N. Characterization of proteins belonging to the CHAP-related superfamily within the Firmicutes[J]. J Mol Microb Biotech, 2008, 14（1-3）：31-40.
[23] Rigden DJ, Jedrzejas MJ, Galperin MY. Amidase domains from bacterial and phage autolysins define a family of γ-D, L-glutamate-specific amidohydrolases[J]. Trends Biochem Sci, 2003, 28（5）：230-234.
[24] Filatova LY, Becker SC, et al. LysK, the enzyme lysing Staphyloco-ccus aureus cells：specific kinetic features and approaches towards stabilization[J]. Biochimie, 2010, 92（5）：507-513.
[25] 顾敬敏. 金黄色葡萄球菌噬菌体 GH15及其裂解酶三维结构与分子作用机制研究[D]. 长春：吉林大学, 2014.
[26] 杨信怡, 游雪甫, 蒋建东. 溶葡萄球菌酶的抗菌活性研究进展[J]. 中国新药杂志, 2006, 14（9）：1113-1117.
[27] Mao J, Schmelcher M, Harty WJ, et al. Chimeric Ply187 endolysin kills Staphylococcus aureus more effectively than the parental enzyme[J]. Fems Microbiol Lett, 2013, 342（1）：30-36.
[28] Koehl JL, Muthaiyan A, Jayaswal RK, et al. Cell wall composition and decreased autolytic activity and lysostaphin susceptibility of glycopeptide-intermediate Staphylococcus aureus[J]. Antimicrob Agents Chemother, 2004, 48（10）：3749-3757.
[29] Yang H, Zhang Y, Yu J, et al. Novel chimeric lysin with high-level antimicrobial activity against methicillin-resistant Staphylococcus aureus in vitro and in vivo[J]. Antimicrob Agents Chemother, 2014, 58（1）：536-542.
[30] Filatova LY, Donovan DM, Becker SC, et al. An investigation of the structure and function of antistaphylococcal endolysins using kinetic methods[J]. Moscow Univ Chem Bull, 2014, 69（3）：107-111.