T6SS效应蛋白Tae4对金黄色葡萄球菌和产单核细胞李氏杆菌的抑菌作用

doi:10.11843/j.issn.0366-6964.2024.10.037

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4660-4669.doi: 10.11843/j.issn.0366-6964.2024.10.037

T6SS效应蛋白Tae4对金黄色葡萄球菌和产单核细胞李氏杆菌的抑菌作用

占乐杨(), 苟婧萱(), 张曼琪, 傅唯轩, 兰守信, 涂健, 王振宇, 邵颖, 宋祥军*()

安徽农业大学动物科技学院兽医病理生物学与疫病防控安徽省重点实验室, 安徽合肥 230000

收稿日期:2023-11-24 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 宋祥军 E-mail:13705593713@163.com;1492648151@qq.com;sxj@ahau.edu.cn
作者简介:占乐杨(2003-), 女, 安徽屯溪人, 本科生, 主要从事禽致病性大肠杆菌研究, E-mail: 13705593713@163.com
苟婧萱(2003-), 女, 青海城中人, 本科生, 主要从事禽致病性大肠杆菌研究, E-mail: 1492648151@qq.com
第一联系人：
占乐杨和苟婧萱是同等贡献作者
基金资助:
2022年国家级大学生创新创业训练计划项目(S202210364039);2022年国家级大学生创新创业训练计划项目(S202210364026);2023年安徽农业大学省级大学生创新创业训练计划项目(X202310364040)

Study on the Antibacterial Effect of T6SS Effector Protein Tae4 on S. aureus and L. monocytogenes

Leyang ZHAN(), Jingxuan GOU(), Manqi ZHANG, Weixuan FU, Shouxin LAN, Jian TU, Zhenyu WANG, Ying SHAO, Xiangjun SONG*()

College of Animal Science and Technoogy, Anhui Agricultural University, Hefei 230000, China

Received:2023-11-24 Online:2024-10-23 Published:2024-11-04
Contact: Xiangjun SONG E-mail:13705593713@163.com;1492648151@qq.com;sxj@ahau.edu.cn

摘要/Abstract

摘要：

旨在探究Ⅵ型分泌系统(Type Ⅵ secretion systems，T6SS)的毒力效应蛋白Tae4酰胺酶对金黄色葡萄球菌(Staphylococcus aureus，S. aureus)及产单核细胞李氏杆菌(Listeria monocytogenes，L. monocytogenes)的抑菌作用。将pET-28a-Tae4重组质粒转化至大肠杆菌表达菌株BL21(Escherichia coli BL21，E. coli BL21)进行目的蛋白诱导表达，优化IPTG的诱导时间、浓度、温度，最后纯化后的Tae4蛋白通过琼脂孔扩散试验、最小抑菌浓度测定、抗生素联用抑菌试验分别作用于S. aureus和L. monocytogenes。SDS-PAGE电泳结果显示，本试验中重组蛋白诱导表达最佳条件为：IPTG终浓度为0.5 mmol·L^-1，16 ℃诱导16 h。重组蛋白大小约为21 ku，可溶性表达于培养上清中。琼脂孔扩散试验结果显示，Tae4蛋白浓度500 μg·mL^-1时S. aureus菌株均出现13mm以上抑菌圈，效果与菌株血清型有关，L. monocytogenes菌株均出现18mm以上抑菌圈；最小抑菌浓度试验结果显示，Tae4蛋白对S. aureus的MIC约为250 μg·mL^-1，对L. monocytogenes的MIC约为125 μg·mL^-1，点板法和微量肉汤稀释法结果一致；抗生素联用抑菌试验结果显示，Tae4蛋白与青霉素(penicillin，PG)联合使用均比单独使用的抑菌效果要好，且对L. monocytogenes的抑菌效果优于S. aureus。综上，Tae4蛋白对金黄色葡萄球菌和产单核细胞李氏杆菌具有抑菌效果，与青霉素联用抑菌效果得到了增强，为后续抗生素替代提供理论依据。

关键词: Tae4, Ⅵ型分泌系统, 金黄色葡萄球菌, 产单核细胞李氏杆菌, 抗生素替代

Abstract:

The aim of this study was to investigate the antibacterial effect of Tae4 amidase, a virulent effector protein of Type Ⅵ secretion systems, on Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes). The recombinant plasmid pET-28a-Tae4 was transformed into E. coli expression strain BL21 to induce expression, and the induction time, concentration and temperature of IPTG were optimized. Finally, the purified Tae4 protein was tested by Agar pore diffusion test, minimal inhibitory concentration test and antibacterial test combining with antibiotic on S. aureus and L. monocytogenes, respectively. SDS-PAGE results showed that the optimal expression conditions were as follows: the final concentration of IPTG was 0.5 mmol·L^-1 at 16 ℃ for 16 h, the size of the recombinant protein was about 21 ku, and existed in the form of soluble supernatant. The results of Agar pore diffusion test showed that when the concentration of Tae4 protein was 500 μg·mL^-1, S. aureus strains showed bacteriostatic zones above 13mm, and the effect was related to the serotype of strains, and L. monocytogenes strains showed bacteriostatic zones above 18 mm. Minimum inhibitory concentration test showed that MIC of Tae4 against S.aureus was about 250 μg·mL^-1, MIC of Tae4 against L. monocytogenes was about 125 μg·mL^-1, which were consistent with spot plate method and microbroth dilution method. The combination test showed that the bacteriostatic effect of Tae4 protein combined with penicillin was better than that of penicillin alone. Furthermore, the antibacterial effect of L. monocytogenes was better than that of S. aureus. In summary, Tae4 protein has bacteriostatic effect on S.aureus and L. monocytogenes, and the bacteriostatic effect combined with penicillin has been enhanced, which provides a theoretical basis for subsequent antibiotic substitution.

Key words: Tae4, type Ⅵ secretion system, S. aureus, Listeria monocytogenes, alternative to antibiotics

中图分类号:

S859.796

占乐杨, 苟婧萱, 张曼琪, 傅唯轩, 兰守信, 涂健, 王振宇, 邵颖, 宋祥军. T6SS效应蛋白Tae4对金黄色葡萄球菌和产单核细胞李氏杆菌的抑菌作用[J]. 畜牧兽医学报, 2024, 55(10): 4660-4669.

Leyang ZHAN, Jingxuan GOU, Manqi ZHANG, Weixuan FU, Shouxin LAN, Jian TU, Zhenyu WANG, Ying SHAO, Xiangjun SONG. Study on the Antibacterial Effect of T6SS Effector Protein Tae4 on S. aureus and L. monocytogenes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4660-4669.

图/表 8

图 1

图 2

图 3

图 4

图 5

图 6

图 7

图 8

参考文献 22

1	韩塔拉, 王俊瑞. 金黄色葡萄球菌异质性耐药机制及实验室检测技术[J]. 中国感染控制杂志, 2022, 21 (12): 1249- 1256. doi: 10.12138/j.issn.1671-9638.20222087
	HAN T L , WANG J R . Mechanisms and laboratory detection technologies of heteroresistance in Staphylococcus aureus[J]. Chinese Journal of Infection Control, 2022, 21 (12): 1249- 1256. doi: 10.12138/j.issn.1671-9638.20222087
2	张旭. 单核细胞增生李斯特菌(Listeria monocytogenes)分型及检测新方法探究[D]. 广州: 华南理工大学, 2021.
	ZHANG X. Research on new methods for typing and detection of Listeria monocytogenes[D]. Guangzhou: South China University of Technology, 2021. (in Chinese)
3	VOLLMER W , BERTSCHE U . Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli[J]. Biochim Biophys Acta (BBA)-Biomembr, 2008, 1778 (9): 1714- 1734. doi: 10.1016/j.bbamem.2007.06.007
4	王增航, 董涛. 霍乱弧菌Ⅵ型分泌系统的效应蛋白对细菌细胞壁的降解机制[J]. 微生物学通报, 2021, 48 (1): 135- 144.
	WANG Z H , DONG T . Bacterial cell wall degradation by type Ⅵ secretion system effector proteins in Vibrio cholerae[J]. Microbiology China, 2021, 48 (1): 135- 144.
5	TONG G , PAN Y , DONG H , et al. Structure and dynamics of pentaglycyl bridges in the cell walls of Staphylococcus aureus by¹³C-¹⁵N REDOR NMR[J]. Biochemistry, 1997, 36 (32): 9859- 9866. doi: 10.1021/bi970495d
6	BOYER F , FICHANT G , BERTHOD J , et al. Dissecting the bacterial type Ⅵ secretion system by a genome wide in silico analysis: what can be learned from available microbial genomic resources?[J]. BMC Genomics, 2009, 10 (1): 104. doi: 10.1186/1471-2164-10-104
7	HOFER U . T6SS: shoot and scrub[J]. Nat Rev Microbiol, 2020, 18 (8): 412- 413.
8	SANA T G , BERNI B , BLEVES S . The T6SSs of Pseudomonas aeruginosa strain PAO1 and their effectors: beyond bacterial-cell targeting[J]. Front Cell Infect Microbiol, 2016, 6, 61.
9	RUSSELL A B , SINGH P , BRITTNACHER M , et al. A widespread bacterial type Ⅵ secretion effector superfamily identified using a heuristic approach[J]. Cell Host Microbe, 2012, 11 (5): 538- 549. doi: 10.1016/j.chom.2012.04.007
10	NAYLOR N R , SILVA S , KULASABANATHAN K , et al. Methods for estimating the burden of antimicrobial resistance: a systematic literature review protocol[J]. Syst Rev, 2016, 5 (1): 187. doi: 10.1186/s13643-016-0364-8
11	RUSSELL A B , LEROUX M , HATHAZI K , et al. Diverse type Ⅵ secretion phospholipases are functionally plastic antibacterial effectors[J]. Nature, 2013, 496 (7446): 508- 512. doi: 10.1038/nature12074
12	WHITNEY J C , CHOU S , RUSSELL A B , et al. Identification, structure, and function of a novel type vi secretion peptidoglycan glycoside hydrolase effector-immunity pair[J]. J Biol Chem, 2013, 288 (37): 26616- 26624. doi: 10.1074/jbc.M113.488320
13	LU D F , ZHENG Y S , LIAO N S , et al. The structural basis of the Tle4-Tli4 complex reveals the self-protection mechanism of H2-T6SS in Pseudomonas aeruginosa[J]. Acta Crystallogr D Biol Crystallogr, 2014, 70 (Pt 12): 3233- 3243.
14	MATSUMOTO T , TAKAHASHI S , UEDA M , et al. Preparation of high activity yeast whole cell bioctalysts by optimization of intracellular production of recombinant Rhizopus oryzae lipase[J]. J Mol Catal B Enzym, 2002, 17 (3/5): 143- 149.
15	柴政斌, 张更林, 王学政, 等. 融合蛋白GST-PADI4可溶性表达条件的优化及纯化[J]. 中国生物制品学杂志, 2014, 27 (3): 404-408, 411.
	CHAI Z B , ZHANG G L , WANG X Z , et al. Optimization of condition for soluble expression of GST-PADI4 fusion protein and purification of expressed product[J]. Chinese Journal of Biologicals, 2014, 27 (3): 404-408, 411.
16	RAMÍREZ O T , ZAMORA R , ESPINOSA G , et al. Kinetic study of penicillin acylase production by recombinant E. coli in batch cultures[J]. Process Biochem, 1994, 29 (3): 197- 206.
17	PENNA T C V , ISHII M , DE SOUZA L C , et al. Expression of green fluorescent protein (GFPuv) in Escherichia coli DH5-a, under different growth conditions[J]. Afr J Biotechnol, 2004, 3 (1): 105- 111.
18	狄志欣. 人胰岛素原基因的克隆以及在大肠杆菌中的高效表达、质谱鉴定[D]. 兰州: 兰州理工大学, 2015.
	DI Z X. Gene cloning, prokaryotic expression, purification and MS identification of human proinsulin[D]. Lanzhou: Lanzhou University of Technology, 2015. (in Chinese)
19	BENZ J , REINSTEIN J , MEINHART A . Structural Insights into the Effector-Immunity System Tae4/Tai4 from Salmonella typhimurium[J]. PLoS One, 2013, 8 (6): e67362.
20	张馨元. 抗菌肽LL-37与酰胺酶联合应用治疗粪肠球菌感染的研究[D]. 呼和浩特: 内蒙古农业大学, 2021.
	ZHANG X Y. The effect of the combined use of antimicrobial peptide LL-37 and amidase on Enterococcus faecalis[D]. Hohhot: Inner Mongolia Agricultural University, 2021. (in Chinese)
21	SPRATT B G . Distinct penicillin binding proteins involved in the division, elongation, and shape of Escherichia coli K12[J]. Proc Natl Acad Sci U S A, 1975, 72 (8): 2999- 3003.
22	WISE E M Jr , PARK J T . Penicillin: its basic site of action as an inhibitor of a peptide cross-linking reaction in cell wall mucopeptide synthesis[J]. Proc Natl Acad Sci U S A, 1965, 54 (1): 75- 81.

[1]	马金锐, 史文静, 田常青, 董志杰, 赵学慧, 芝吉, 曹青, 魏衍全, 宋维丽, 薛惠文, 苟惠天. 产单核细胞李氏杆菌内化素G单克隆抗体的制备及双抗体夹心ELISA方法的建立[J]. 畜牧兽医学报, 2024, 55(9): 4069-4076.
[2]	崔恒洁, 覃金珑, 朱志豪, 鲍雪, 栗绍文, 孟宪荣. 金黄色葡萄球菌对苯扎溴铵敏感性与生物被膜形成能力相关性分析[J]. 畜牧兽医学报, 2024, 55(8): 3669-3677.
[3]	任慧杰, 马勋, 王静, 刘彩霞, 曾东东, 寇丽君, 史唯地, 吕双飞, 钱瑞宣, 高盛杰. 产单核细胞李氏杆菌Lm4b_02325/26双基因缺失株构建及部分生物学特性试验[J]. 畜牧兽医学报, 2024, 55(6): 2578-2587.
[4]	和晓兰, 赵艳坤, 孟璐, 刘慧敏, 高姣姣, 郑楠. 金黄色葡萄球菌异质性耐药研究进展[J]. 畜牧兽医学报, 2024, 55(4): 1432-1445.
[5]	秦祎, 胡文洁, 方小伟, 郭骞, 田篮鑫, 刘芳, 方春. 脂磷壁酸合成酶ltaS基因缺失对产单核细胞李氏杆菌致病性的影响[J]. 畜牧兽医学报, 2024, 55(2): 670-679.
[6]	吴自豪, 蔡依龙, 陀海欣, 陈伟. 1株马乳源PVL⁺ST22型金黄色葡萄球菌致病性分析[J]. 畜牧兽医学报, 2024, 55(2): 718-726.
[7]	苑庆欣, 刘阔, 包旭华, 高东阳, 李鹤, 宋军, 周志新. 白屈菜红碱抗耐甲氧西林金黄色葡萄球菌作用机制研究[J]. 畜牧兽医学报, 2024, 55(10): 4670-4678.
[8]	孙盼盼, 曹志刚, 凌小雅, 孙娜, 孙耀贵, 李宏全. 苦参碱联合不同抗生素抗炎作用比较研究[J]. 畜牧兽医学报, 2023, 54(10): 4411-4421.
[9]	江南松, 吉星, 王亚新, 孙城涛, 汪洋, 陈红梅, 程龙飞, 黄瑜, 吴聪明. 猪源ST9型耐甲氧西林金黄色葡萄球菌中前噬菌体的流行状况与转导分析[J]. 畜牧兽医学报, 2023, 54(1): 338-350.
[10]	卢婉青, 赵莎莎, 蒋松宏, 童智子, 黄丹妮, 郭建华, 吴俊伟, 周洋. 金黄色葡萄球菌对BV2细胞IFN-α生成的影响[J]. 畜牧兽医学报, 2022, 53(8): 2633-2641.
[11]	毛彦妮, 常佳伟, 李娜, 王鑫, 康馨匀, 马强, 马靓, 王桂琴. 金黄色葡萄球菌在生物被膜态与浮游态的转录组差异表达分析[J]. 畜牧兽医学报, 2022, 53(8): 2697-2707.
[12]	江玲丽, 高有领, 胡兴娟, 章先, 杨云凯, 魏华, 方维焕. 检测产单核细胞李氏杆菌溶血素的胶体金试纸条制备及初步应用[J]. 畜牧兽医学报, 2022, 53(6): 1841-1848.
[13]	李天昊, 赵学慧, 宋晨, 委慧玲, 齐玉梅, 田常青, 祁国军, 史文静, 苟惠天, 薛慧文. 1株跨种裂解李氏杆菌噬菌体的分离鉴定[J]. 畜牧兽医学报, 2022, 53(5): 1544-1552.
[14]	王华健, 张宁, 杨威, 赵志强, 李茜, 陆安, 田勇, 何欣, 赵兴华, 李杰峰. 3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2022, 53(4): 1201-1209.
[15]	王迪, 俞英. 奶牛金葡菌乳房炎抗性的转录组及表观遗传学研究进展[J]. 畜牧兽医学报, 2022, 53(2): 329-338.

T6SS效应蛋白Tae4对金黄色葡萄球菌和产单核细胞李氏杆菌的抑菌作用

Study on the Antibacterial Effect of T6SS Effector Protein Tae4 on S. aureus and L. monocytogenes

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 8

参考文献 22

相关文章 15

编辑推荐

Metrics

本文评价