T6SS效应蛋白Tse1的原核表达及其对金黄色葡萄球菌的抑菌作用

doi:10.11843/j.issn.0366-6964.2025.06.034

摘要/Abstract

摘要：

Ⅵ型分泌系统(type Ⅵ secretion systems，T6SS)广泛存在于革兰阴性菌中，其效应蛋白Tse1在细菌种间竞争中发挥着重要的作用。本研究旨在探究Tse1对金黄色葡萄球菌(Staphylococcu saureus，S.aureus)的抑菌作用，探索其在耐药性细菌中的表现和影响。利用含有重组质粒pET-28a-Tse1的大肠杆菌原核表达系统诱导Tse1蛋白表达并纯化。通过荧光显微镜及扫描电镜观察重组蛋白Tse1对金黄色葡萄球菌细胞壁的破坏作用。最后，采用琼脂孔扩散抑菌试验、微量肉汤稀释法测定重组蛋白Tse1对金黄色葡萄球菌的抗菌活性及其与抗生素联合作用的效果。结果表明，在16℃，IPTG浓度为0.5 mmol·L^-1诱导16 h时，Tse1蛋白的表达量达到最高并以可溶性形式存在于上清液中，蛋白相对分子质量约为22 ku。重组蛋白Tse1与S.aureus共培后可发现蛋白结合在菌体表面并能破坏其细胞壁，破坏程度表现为剂量依赖形式。琼脂孔扩散抑菌试验表明重组蛋白Tse1对S.aureus不同菌株均有明显抑菌作用，并且与青霉素(PG)联合使用能明显提升Tse1蛋白的抑菌效果。本试验建立了Tse1蛋白的原核表达及纯化方法，证明了Tse1蛋白对金黄色葡萄球菌具有抑菌作用，与青霉素联合应用能增强其抑菌效果，为挖掘新型抗生素替代品提供了相关理论基础。

关键词: 抗生素替代, Ⅵ型分泌系统, Tse1, 金黄色葡萄球菌

Abstract:

Type Ⅵ secretion systems (T6SS) are widely found in Gram-negative bacteria, and its effector protein Tse1 plays an important role in bacterial interspecies competition. The aim of this study was to investigate the inhibitory effect of Tse1 on Staphylococcus aureus (S. aureus), and to explore its expression and effect in drug-resistant bacteria. Tse1 protein expression was induced and purified using an E.coli prokaryotic expression system containing the recombinant plasmid pET-28a-Tse1. The disruptive effect of the recombinant protein Tse1 on the cell wall of S. aureus was observed by fluorescence microscopy and scanning electron microscopy. Finally, the antibacterial activity of recombinant protein Tse1 against S. aureus and its combined effect with antibiotics were determined by agar pore diffusion inhibition test and micro broth dilution method. The results showed that the expression of Tse1 protein reached the highest level and existed in soluble form in the supernatant at 16 ℃ and IPTG concentration of 0.5 mmol·L^-1 for 16 h. The relative molecular mass of the protein was about 22 ku. The co-culture of recombinant protein Tse1 with S. aureus revealed that the protein was bound to the surface of the bacterium and was able to disrupt the cell wall of the bacterium, and the extent of the disruption was shown in a dose. The degree of disruption was dose-dependent. The agar pore diffusion inhibition assay showed that the recombinant protein Tse1 had significant inhibitory effects on different strains of S. aureus, and the inhibitory effect of Tse1 protein could be significantly enhanced by the combination of penicillin (PG). In this experiment, the prokaryotic expression and purification method of Tse1 protein were established, and it was proved that Tse1 protein had an inhibitory effect on S. aureus, and its inhibitory effect could be enhanced by combining with penicillin, which provided the theoretical basis for the excavation of new antibiotic substitutes.

Key words: antibiotic substitution, T6SS, Tse1, Staphylococcus aureus

中图分类号:

S859.799.1

张曼琪, 赵冰雨, 温如如, 张静雯, 孙孟冉, 占乐杨, 苟婧萱, 宋祥军. T6SS效应蛋白Tse1的原核表达及其对金黄色葡萄球菌的抑菌作用[J]. 畜牧兽医学报, 2025, 56(6): 2917-2926.

ZHANG Manqi, ZHAO Bingyu, WEN Ruru, ZHANG Jingwen, SUN Mengran, ZHAN Leyang, GOU Jingxuan, SONG Xiangjun. Prokaryotic Expression of the T6SS Effector Protein Tse1 and Its inhibitory Effect on Staphylococcus aureus[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(6): 2917-2926.

图/表 8

图 1

图 2

图 3

图 4

图 5

表 1

图 6

图 7

参考文献 31

1	VESTERGAARD M , FREES D , INGMER H . Antibiotic resistance and the MRSA problem[J]. Microbiol Spectr, 2019, 7 (2): GPP3-0057-2018.
2	DE JONG N W M , VAN KESSEL K P M , VAN STRIJP J A G . Immune evasion by Staphylococcus aureus[J]. Microbiol Spectr, 2019, 7 (2): GPP3-0061-2019.
3	DEO S , TURTON K L , KAINTH T , et al. Strategies for improving antimicrobial peptide production[J]. Biotechnol Adv, 2022, 59, 107968. doi: 10.1016/j.biotechadv.2022.107968
4	BARDAN A , NIZET V , GALLO R L . Antimicrobial peptides and the skin[J]. Expert Opin Biol Ther, 2004, 4 (4): 543- 549. doi: 10.1517/14712598.4.4.543
5	YOKOO H , HIRANO M , MISAWA T , et al. Helical antimicrobial peptide foldamers containing non-proteinogenic amino acids[J]. ChemMedChem, 2021, 16 (8): 1226- 1233. doi: 10.1002/cmdc.202000940
6	LUO Y , SONG Y Z . Mechanism of antimicrobial peptides: antimicrobial, anti-inflammatory and antibiofilm activities[J]. Int J Mol Sci, 2021, 22 (21): 11401. doi: 10.3390/ijms222111401
7	KUMAR P , KIZHAKKEDATHU J N , STRAUS S K . Antimicrobial peptides: diversity, mechanism of action and strategies to improve the activity and biocompatibility in vivo[J]. Biomolecules, 2018, 8 (1): 4. doi: 10.3390/biom8010004
8	ZHANG Q Y , YAN Z B , MENG Y M , et al. Antimicrobial peptides: mechanism of action, activity and clinical potential[J]. Mil Med Res, 2021, 8 (1): 48.
9	LI X , ZUO S Y , WANG B , et al. Antimicrobial mechanisms and clinical application prospects of antimicrobial peptides[J]. Molecules, 2022, 27 (9): 2675. doi: 10.3390/molecules27092675
10	MIYACHIRO M M, CONTRERAS-MARTEL C, DESSEN A. Penicillin-binding proteins (PBPs) and bacterial cell wall elongation complexes[M]//HARRIS J R, MARLES-WRIGHT J. Macromolecular Protein Complexes Ⅱ: Structure and Function. Cham: Springer, 2019: 273-89.
11	CHERRAK Y , FLAUGNATTI N , DURAND E , et al. Structure and activity of the type Ⅵ secretion system[J]. Microbiol Spectr, 2019, 7 (4): PSIB-0031-2019.
12	HAYES B K , HARPER M , VENUGOPAL H , et al. Structure of a Rhs effector clade domain provides mechanistic insights into type Ⅵ secretion system toxin delivery[J]. Nat Commun, 2024, 15 (1): 8709. doi: 10.1038/s41467-024-52950-x
13	DING J J , WANG W , FENG H , et al. Structural insights into the Pseudomonas aeruginosa type Ⅵ virulence effector Tse1 bacteriolysis and self-protection mechanisms[J]. J Biol Chem, 2012, 287 (32): 26911- 26920. doi: 10.1074/jbc.M112.368043
14	ALLSOPP L P , WOOD T E , HOWARD S A , et al. RsmA and AmrZ orchestrate the assembly of all three type Ⅵ secretion systems in Pseudomonas aeruginosa[J]. Proc Natl Acad Sci U S A, 2017, 114 (29): 7707- 7712. doi: 10.1073/pnas.1700286114
15	岳舒. 鳗弧菌MHK3株T6SS的功能性表达及抗菌特性[D]. 青岛: 中国海洋大学, 2015.
	YUE S. Functional expression of T6SS of Vibrio anguillarum MHK3 and its antibacterial properties[D]. Qingdao: Ocean University of China, 2015. (in Chinese)
16	宋莉. 假结核耶尔森氏菌双功能效应蛋白Tce2参与细菌间竞争新机制研究[D]. 杨凌: 西北农林科技大学, 2022.
	SONG L. Study on the new mechanism of bifunctional effector Tce2 in Yersinia pseudotuberculosis participating in bacterial competition[D]. Yangling: Northwest A&F University, 2022. (in Chinese)
17	傅唯轩. Tae4的原核表达及对两种革兰氏阳性菌抑菌作用研究[D]. 合肥: 安徽农业大学, 2023.
	FU W X. Prokaryotic expression of Tae4 and its inhibitory effect on two Gram-positive bacteria[D]. Hefei: Anhui Agricultural University, 2023. (in Chinese)
18	DVORAK P , CHRAST L , NIKEL P I , et al. Exacerbation of substrate toxicity by IPTG in Escherichia coli BL21(DE3) carrying a synthetic metabolic pathway[J]. Microb Cell Fact, 2015, 14, 201. doi: 10.1186/s12934-015-0393-3
19	SINGH R P , KUMARI K . Bacterial type Ⅵ secretion system (T6SS): an evolved molecular weapon with diverse functionality[J]. Biotechnol Lett, 2023, 45 (3): 309- 331. doi: 10.1007/s10529-023-03354-2
20	CHEN Z D , MAO Y K , SONG Y Z , et al. Refined egoist: the toxin-antitoxin immune system of T6SS[J]. Microb Pathog, 2024, 196, 106991. doi: 10.1016/j.micpath.2024.106991
21	BENZ J , SENDLMEIER C , BARENDS T R M , et al. Structural insights into the effector-immunity system Tse1/Tsi1 from Pseudomonas aeruginosa[J]. PLoS One, 2012, 7 (7): e40453. doi: 10.1371/journal.pone.0040453
22	JIANG X L , LI H Z , MA J Y , et al. Role of Type Ⅵ secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection[J]. ISME J, 2024, 18 (1): wrae053. doi: 10.1093/ismejo/wrae053
23	PÉREZ-LORENTE A I , MOLINA-SANTIAGO C , DE VICENTE A , et al. Sporulation activated via σ^W protects Bacillus from a tse1 peptidoglycan hydrolase type Ⅵ secretion system effector[J]. Microbiol Spectr, 2023, 11 (2): e0504522. doi: 10.1128/spectrum.05045-22
24	SIMAS R G , PESSOA JUNIOR A , LONG P F . Mechanistic aspects of IPTG (isopropylthio-β-galactoside) transport across the cytoplasmic membrane of Escherichia coli-a rate limiting step in the induction of recombinant protein expression[J]. J Ind Microbiol Biotechnol, 2023, 50 (1): kuad034. doi: 10.1093/jimb/kuad034
25	TOLIA N H , JOSHUA-TOR L . Strategies for protein coexpression in Escherichia coli[J]. Nat Methods, 2006, 3 (1): 55- 64. doi: 10.1038/nmeth0106-55
26	BANEYX F , MUJACIC M . Recombinant protein folding and misfolding in Escherichia coli[J]. Nat Biotechnol, 2004, 22 (11): 1399- 1408. doi: 10.1038/nbt1029
27	ALVAREZ L , HERNANDEZ S B , TORRENS G , et al. Control of bacterial cell wall autolysins by peptidoglycan crosslinking mode[J]. Nat Commun, 2024, 15 (1): 7937. doi: 10.1038/s41467-024-52325-2
28	SHANG G J , LIU X H , LU D F , et al. Structural insight into how Pseudomonas aeruginosa peptidoglycanhydrolase Tse1 and its immunity protein Tsi1 function[J]. Biochem J, 2012, 448 (2): 201- 211. doi: 10.1042/BJ20120668
29	RUSSELL A B , HOOD R D , BUI N K , et al. Type Ⅵ secretion delivers bacteriolytic effectors to target cells[J]. Nature, 2011, 475 (7356): 343- 347. doi: 10.1038/nature10244
30	李翠翠, 马万鹏, 张毅, 等. 新疆某奶牛场奶源金黄色葡萄球菌分离鉴定、毒力基因检测和耐药性分析[J]. 动物医学进展, 2023, 44 (11): 40- 46.
	LI C C , MA W P , ZHANG Y , et al. Isolation, identification, virulence gene detection and drug resistance analysis of Staphylococcus aureus from milk in a dairy farm in Xinjiang[J]. Progress In Veterinary Medicine, 2023, 44 (11): 40- 46.
31	甘卫泽, 李益涛, 曹梦园, 等. 某规模化牧场致奶牛乳房炎金黄色葡萄球菌的鉴定及耐药性分析[J]. 中国奶牛, 2020 (11): 45- 48.
	GAN W Z , LI Y T , CAO M Y , et al. Identification and drug resistance analysis of Staphylococcus aureus mastitis in dairy cows from a large-scale pasture[J]. China Dairy Cattle, 2020 (11): 45- 48.

菌株编号 Strain No.	抑菌直径Inhibitory diameter
菌株编号 Strain No.	A	B	C	D	${\bar x}$±s
ATCC25923	1.0	1.60	1.20	1.70	1.38±0.33
ATCC6538	1.0	1.60	1.20	1.75	1.39±0.35
CMCC26003	1.1	1.3	1.1	1.4	1.23±0.15

[1]	吉星, 李俊, 王冉, 何涛. 金黄色葡萄球菌毒力调控及减毒药物研究进展[J]. 畜牧兽医学报, 2025, 56(4): 1594-1607.
[2]	和晓兰, 赵艳坤, 孟璐, 刘慧敏, 高姣姣, 郑楠. 生牛乳中金黄色葡萄球菌异质性耐药及机制研究[J]. 畜牧兽医学报, 2025, 56(4): 1934-1946.
[3]	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.
[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]	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.
[6]	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.
[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]	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.
[13]	张金柠, 钱梦樱, 唐永杰, 米思远, 师科荣, 俞英. 金黄色葡萄球菌表面蛋白A对奶牛乳腺上皮细胞的黏附作用[J]. 畜牧兽医学报, 2021, 52(5): 1369-1377.
[14]	吴香云, 刘亚娜, 周喆麒, 潘源虎, 郝海红, 程古月, 王玉莲. 多糖类化合物的抗菌作用及其机制研究进展[J]. 畜牧兽医学报, 2020, 51(6): 1167-1176.
[15]	马强, 杨蕊, 万佳宏, 常佳伟, 魏彦琴, 王桂琴. 宁夏地区牛源金黄色葡萄球菌β-内酰胺酶的分析及作用方式的研究[J]. 畜牧兽医学报, 2020, 51(5): 1138-1148.