金黄色葡萄球菌噬菌体vB_Sau_P68的基因组分析和裂解酶

Expression and sequence analysis of Staphylococcus aureus phage lysin

摘要:

【背景】金黄色葡萄球菌是常见的人畜共患条件致病菌，随着多耐药菌株分离率的增长，研发与抗生素作用模式不同的抗菌剂迫在眉睫。【目的】分离高效且特异性强的金黄色葡萄球菌噬菌体，对其进行功能注释，并对其编码的裂解酶进行功能验证。【方法】通过对噬菌体全基因组序列进行分析找到裂解酶基因，利用原核表达系统对其编码的2个裂解酶蛋白进行克隆，用SDS-PAGE与蛋白免疫印迹法(Western blotting)鉴定目的蛋白是否表达，并采用单斑法验证其裂解活性。【结果】本研究的噬菌体为一株新的金黄色葡萄球菌噬菌体，命名为vB_Sau_P68，该基因组全长为139 409 bp，GC含量为31.0%，编码220个开放阅读框(open reading frame，ORF)，透射电镜观察具有正二十面体头部和收缩性尾部，形态学分类属于肌尾噬菌体。该噬菌体编码2个裂解酶基因，分别具有CHAP催化结构域与SH3_5结合结构域，SDS-PAGE与Western blotting表明Lys161能够表达且有裂解活性，Lys163则无法外源表达。对Lys161序列进行分析，该裂解酶无信号肽，无跨膜区域，以无规则卷曲为主。【结论】本研究对一株新的金黄色葡萄球菌噬菌体编码的2个裂解酶基因进行了克隆表达，结果提示CHAP催化结构域具有裂解活性，而SH3_5结合结构域则不表达。这一结果可为裂解酶的机制探索及应用提供理论基础。

关键词:全基因组分析;CHAP结构域;SH3_5结构域;蛋白结构

Abstract:

[Background] Staphylococcus aureus is a common zoonotic opportunistic pathogen. With the emergence of multidrug resistant strains, it is urgent to develop antimicrobial agents with different modes of action from antibiotics. [Objective] To isolate S. aureus phage and identify its functional lysin components as an effective specific antimicrobial agent. [Methods] The whole genome sequence of the phage was assembled and annotated for the mining of putative lysin encoding genes. Two putative lysin genes were respectively cloned into a prokaryotic expression vector. SDS-PAGE and Western blotting were employed to confirm the expression of the target proteins. We then verified the lytic activity by spotting the expression product on the host bacterial lawn. [Results] The isolated phage in this study could lyse its host bacterium and was named vB_Sau_P68. The phage genome was 139 409 bp with the GC content of 31.0% and encoded 220 open reading frames (ORFs). Under the transmission electron microscope, the phage appeared as an icosahedron with a contractile tail, which belonged to Myoviridae. Two putative lysin genes were annotated in the phage genome. ORF161 was predicted to encode lysin with a CHAP catalytic domain and ORF163 with a SH3_5 binding domain. The results of SDS-PAGE and Western blotting showed that Lys161 was expressed successfully and had lytic activity, while the expression of Lys163 was not be detected. The Lys161 sequence had no signal peptide or transmembrane region, and random coil was its major secondary structural element. [Conclusion] In this study, two lysin genes were cloned from a Staphylococcus aureus phage genome and expressed. The results suggested that the CHAP catalytic domain had lytic activity, while the SH3_5 binding domain was not expressed. The findings provide a theoretical basis for the exploration of the acting mechanism and application of lysin.

Key words:whole genome analysis;CHAP domain;SH3_5 domain;protein structure