微生物学报

Primer names	Primer sequences (5′→3′)	Note
pRE112 F	ACGCCATTCATGGCCATATCA	pRE112 validation primer
pRE112 R	GTTATTGGTGCCCTTAAACGC
pRE112-anti-F	TCTAGAAGAAGCTTGGGATCG	Primers for pRE112 vector linearization
pRE112-anti-R	GAGCTCTCCCGGGAATTCATG
vasH F0	GGATGCTGGAGTCCGTCATT	Validation of vasH gene knockout
vasH R0	CTCCTTGTTGCCTGATTGCGC
vasH F1	AATTCCCGGGAGAGCTCATCATCGACTGCAAGAACGTG	Amplification of the upstream homologous sequence of vasH
vasH R1	ATGATGCTCAGGTCAGCCCTCCACCTCG
vasH F2	GCTGACCTGAGCATCATGGGTTTGTTGC	Amplification of the downstream homologous sequence of vasH
vasH R2	CCAAGCTTCTTCTAGAATCCCTGATCCCGGATAAACC
vasH F3	GCTTGATATCGAATTCATGGAGCAAGCCCTCGCATT	vasH complementation plasmid primers
vasH R3	TAGAACTAGTGGATCCTCAGTTCACCTCCAGTTTCTG
pBBR-anti-F	GAATTCGATATCAAGCTTATCG	Primers for pBBR vector linearization
pBBR-anti-R	GGATCCACTAGTTCTAGAGC
pBBR-F	AGCGGCTATTTAACGACCCTGCC	Primers for pBBR plasmid verification
pBBR-R	GTGCTGATGCCGCTGGCGATTCAGGT
gyrB-qF	GCTCACCATTCGTCGTAACGG	Primers for RT-qPCR
gyrB-qR	GCCACCCTCGTAGCAGAAAT
hcp-qF	GGTCAGCGTGTCCACAAACC
hcp-qR	GGCATCTGGCAGTCGATGTC
vgrG(cluster2)-qF	AAGCCCGATGAAATCCTC
vgrG(cluster2)-qR	CCTTGCACTTCTCACACTCCTCA
vgrG(cluster134)-qF	ACACAGAAGCCTGACGAA
vgrG(cluster134)-qR	GTGTTTGCCCTGCTCGAA
tse1-qF	CACTGGCACCAGCAAAACCG
tse1-qR	GACATGCTCGCGCTTTACATCG
tse2-qF	AGGGACGAGGTTTGACCCAT
tse2-qR	ATTCGTGGGCACACCAAACA
tse3-qF	GCCAACAACACCACCAAGCA
tse3-qR	TCTTCCTTGTTGCGGCAACT

Primer names	Primer sequences (5′→3′)	Note
pRE112 F	ACGCCATTCATGGCCATATCA	pRE112 validation primer
pRE112 R	GTTATTGGTGCCCTTAAACGC
pRE112-anti-F	TCTAGAAGAAGCTTGGGATCG	Primers for pRE112 vector linearization
pRE112-anti-R	GAGCTCTCCCGGGAATTCATG
vasH F0	GGATGCTGGAGTCCGTCATT	Validation of vasH gene knockout
vasH R0	CTCCTTGTTGCCTGATTGCGC
vasH F1	AATTCCCGGGAGAGCTCATCATCGACTGCAAGAACGTG	Amplification of the upstream homologous sequence of vasH
vasH R1	ATGATGCTCAGGTCAGCCCTCCACCTCG
vasH F2	GCTGACCTGAGCATCATGGGTTTGTTGC	Amplification of the downstream homologous sequence of vasH
vasH R2	CCAAGCTTCTTCTAGAATCCCTGATCCCGGATAAACC
vasH F3	GCTTGATATCGAATTCATGGAGCAAGCCCTCGCATT	vasH complementation plasmid primers
vasH R3	TAGAACTAGTGGATCCTCAGTTCACCTCCAGTTTCTG
pBBR-anti-F	GAATTCGATATCAAGCTTATCG	Primers for pBBR vector linearization
pBBR-anti-R	GGATCCACTAGTTCTAGAGC
pBBR-F	AGCGGCTATTTAACGACCCTGCC	Primers for pBBR plasmid verification
pBBR-R	GTGCTGATGCCGCTGGCGATTCAGGT
gyrB-qF	GCTCACCATTCGTCGTAACGG	Primers for RT-qPCR
gyrB-qR	GCCACCCTCGTAGCAGAAAT
hcp-qF	GGTCAGCGTGTCCACAAACC
hcp-qR	GGCATCTGGCAGTCGATGTC
vgrG(cluster2)-qF	AAGCCCGATGAAATCCTC
vgrG(cluster2)-qR	CCTTGCACTTCTCACACTCCTCA
vgrG(cluster134)-qF	ACACAGAAGCCTGACGAA
vgrG(cluster134)-qR	GTGTTTGCCCTGCTCGAA
tse1-qF	CACTGGCACCAGCAAAACCG
tse1-qR	GACATGCTCGCGCTTTACATCG
tse2-qF	AGGGACGAGGTTTGACCCAT
tse2-qR	ATTCGTGGGCACACCAAACA
tse3-qF	GCCAACAACACCACCAAGCA
tse3-qR	TCTTCCTTGTTGCGGCAACT

Strain or plasmid	Relevant genotype or characteristic(s)	Source
Aeromonas veronii C4	Wild type, ampicillin resistance, virulent to fish	Lab stock
Aeromonas veronii C4 ΔrsmA	Ampicillin resistant, rsmA gene deleted from WT, T6SS-activated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔtssB	Ampicillin resistant, tssB gene deleted from WT, T6SS-inactivated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔvasH	Ampicillin resistant, vasH gene deleted from WT	This study
Aeromonas veronii C4 ΔvasH/P-vasH	Ampicillin and kanamycin resistant, vasH gene deleted from WT and complemented with functional vasH gene on pBBR plasmid	This study
pRE112	Suicide plasmid for homologous recombination to construct knockout strains, chloramphenicol (Cam) resistant	Laboratory stock
Escherichia coli WM3064	Encodes a relaxase, a mating pair formation (MPF) complex and a type Ⅳ coupling protein on the chromosome, diaminopimelic acid (DAP) auxotroph	Laboratory stock
pBBR-vasH	Derived from pBBR-MCS-2, contains the ORF region of vasH; kanamycin resistance	This study
pBBR-eGFP	Derived from pBBR-MCS-2, contains the ORF region of eGFP; gentamicin resistance	Previously constructed in our laboratory^[12]
Escherichia coli BL21(DE3)	Used for plasmid cloning and protein expression; served as the target strain for competition assay in this study	Laboratory stock

Strain or plasmid	Relevant genotype or characteristic(s)	Source
Aeromonas veronii C4	Wild type, ampicillin resistance, virulent to fish	Lab stock
Aeromonas veronii C4 ΔrsmA	Ampicillin resistant, rsmA gene deleted from WT, T6SS-activated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔtssB	Ampicillin resistant, tssB gene deleted from WT, T6SS-inactivated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔvasH	Ampicillin resistant, vasH gene deleted from WT	This study
Aeromonas veronii C4 ΔvasH/P-vasH	Ampicillin and kanamycin resistant, vasH gene deleted from WT and complemented with functional vasH gene on pBBR plasmid	This study
pRE112	Suicide plasmid for homologous recombination to construct knockout strains, chloramphenicol (Cam) resistant	Laboratory stock
Escherichia coli WM3064	Encodes a relaxase, a mating pair formation (MPF) complex and a type Ⅳ coupling protein on the chromosome, diaminopimelic acid (DAP) auxotroph	Laboratory stock
pBBR-vasH	Derived from pBBR-MCS-2, contains the ORF region of vasH; kanamycin resistance	This study
pBBR-eGFP	Derived from pBBR-MCS-2, contains the ORF region of eGFP; gentamicin resistance	Previously constructed in our laboratory^[12]
Escherichia coli BL21(DE3)	Used for plasmid cloning and protein expression; served as the target strain for competition assay in this study	Laboratory stock

Dose of challenge/(CFU/mL)	Number of death/Total		Death rate/%
5×10⁶	0	0	0	0.00	0.00	0.00
5×10⁷	0	0	1	0.00	0.00	3.33
5×10⁸	10	0	1	33.33	0.00	3.33
5×10⁹	30	30	30	100.00	100.00	100.00
LD₅₀	7.34×10¹⁰	1.58×10¹¹	1.36×10¹¹

Dose of challenge/(CFU/mL)	Number of death/Total		Death rate/%
5×10⁶	0	0	0	0.00	0.00	0.00
5×10⁷	0	0	1	0.00	0.00	3.33
5×10⁸	10	0	1	33.33	0.00	3.33
5×10⁹	30	30	30	100.00	100.00	100.00
LD₅₀	7.34×10¹⁰	1.58×10¹¹	1.36×10¹¹

维氏气单胞菌VasH正调控Ⅵ型分泌系统表达并影响细菌竞争力和毒力

PDF下载

高士杰 , 马香

微生物学报 | 研究报告 2026,66(8): 4135-4149

收起

微生物学报 |研究报告 2026 , 66 (8) : 4135 -4149

维氏气单胞菌VasH正调控Ⅵ型分泌系统表达并影响细菌竞争力和毒力

全屏

[Author(id=1297571068416390019, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1297571068496081797, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, authorId=1297571068416390019, language=EN, stringName=Shijie GAO, firstName=Shijie, middleName=null, lastName=GAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=School of Life and Health Sciences, Hainan University, Haikou, Hainan, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1297571068571579270, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, authorId=1297571068416390019, language=CN, stringName=高士杰, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=海南大学生命健康学院，海南海口, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1297571068324115327, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, xref=null, ext=[AuthorCompanyExt(id=1297571068332503936, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, companyId=1297571068324115327, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Life and Health Sciences, Hainan University, Haikou, Hainan, China), AuthorCompanyExt(id=1297571068340892545, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, companyId=1297571068324115327, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=海南大学生命健康学院，海南海口)])]), Author(id=1297571068655465352, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, orderNo=1, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=993034@hainanu.edu.cn, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1297571068730962826, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, authorId=1297571068655465352, language=EN, stringName=Xiang MA, firstName=Xiang, middleName=null, lastName=MA, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=School of Life and Health Sciences, Hainan University, Haikou, Hainan, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1297571068819043211, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, authorId=1297571068655465352, language=CN, stringName=马香, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=海南大学生命健康学院，海南海口, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1297571068324115327, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, xref=null, ext=[AuthorCompanyExt(id=1297571068332503936, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, companyId=1297571068324115327, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Life and Health Sciences, Hainan University, Haikou, Hainan, China), AuthorCompanyExt(id=1297571068340892545, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571065065141102, companyId=1297571068324115327, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=海南大学生命健康学院，海南海口)])])]

高士杰, 马香

作者信息

海南大学生命健康学院，海南海口

通讯作者:

马香

作者简介:

作者贡献声明

高士杰：实验具体实施人，完成实验操作、数据分析及论文初稿撰写；马香：获取资金资助并对实验进行总体设计及指导，论文撰写和修改。

VasH positively regulates type Ⅵ secretion system expression and influences competition and virulence of Aeromonas veronii

Shijie GAO, Xiang MA

Affiliations

School of Life and Health Sciences, Hainan University, Haikou, Hainan, China

Corresponding Author:

E-mail: 993034@hainanu.edu.cn

出版时间: 2026-08-04 doi: 10.13343/j.cnki.wsxb.20260025

文章导航

摘要

收起

维氏气单胞菌(Aeromonas veronii)属革兰氏阴性致病菌，可引发水产动物及人类多种疾病，对水产养殖业和人类公共健康构成严重威胁。Ⅵ型分泌系统(type 6 secretion system, T6SS)是决定维氏气单胞菌致病性的关键毒力因子，已知细菌增强子结合蛋白(bacterial enhancer-binding protein, bEBP) VasH负责调控T6SS功能，但维氏气单胞菌中是否存在这一调控关系尚不明确。 【目的】 揭示VasH蛋白对维氏气单胞菌T6SS表达及功能的影响，为深入探究T6SS介导的维氏气单胞菌致病机制，以及后续防控维氏气单胞菌感染提供理论依据。 【方法】 以A. veronii C4为原始菌株，通过同源重组策略构建VasH缺失菌株ΔvasH和回补株ΔvasH/p-vasH。通过RT-qPCR测定各菌株中T6SS基因的相对表达量，明确VasH对T6SS基因表达水平的影响；通过测定生长曲线、透射电镜观察菌体形态，采用结晶紫法测定生物膜形成能力，确定VasH对病原菌致病相关特征的影响；通过体外细菌竞争实验及斑马鱼模型体内半数致死浓度(median lethal dose, LD₅₀)测定评估VasH对病原菌竞争力和致病性的综合效应。 【结果】 成功构建了vasH基因敲除和回补菌株。VasH缺失导致T6SS结构蛋白和效应蛋白编码基因的表达均显著降低(P<0.05)，说明VasH正向调控T6SS基因表达。vasH基因敲除未显著影响维氏气单胞菌的生长能力、表面形态及结构，但导致病原菌生物膜形成能力显著增强，且体外竞争能力显著下降。斑马鱼体内感染实验显示，ΔvasH菌株的LD₅₀为1.58×10¹¹，约为野生型菌株LD₅₀ (7.34×10¹⁰)的2.15倍；在感染浓度为5×10⁸ CFU/mL时，ΔvasH菌株感染导致斑马鱼的死亡率由野生型感染时的33.3%降至0，表明VasH缺失降低了维氏气单胞菌的致病性。 【结论】 维氏气单胞菌VasH蛋白对T6SS基因表达具有正调控活性，其可能不参与调控病原菌的生长状态及外部形态，但显著影响生物膜形成能力和种间竞争力，进而调控病原菌对斑马鱼宿主的综合毒力。本研究为进一步探究VasH通过调控维氏气单胞菌T6SS活性与功能介导病原菌致病性的分子机制提供了必要的研究工具，并奠定了初步基础。

关键词

维氏气单胞菌 / vasH / T6SS / 毒力

Abstract

收起

Aeromonas veronii is a Gram-negative pathogenic bacterium that causes various diseases in aquatic animals and humans, posing a serious threat to aquaculture and public health. The type Ⅵ secretion system (T6SS) is a key virulence factor determining the pathogenicity of A. veronii. It is known that the bacterial enhancer-binding protein (bEBP) VasH is responsible for regulating the T6SS function, while whether this regulatory relationship exists in A. veronii remains unknown. [Objective] To elucidate the impacts of VasH on T6SS expression and function in A. veronii, thereby providing a theoretical basis for deciphering the mechanism of T6SS-mediated pathogenicity of A. veronii and for the subsequent prevention and control of A. veronii infections. [Methods] With A. veronii C4 as the wild-type strain, the VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were constructed via a homologous recombination strategy. RT-qPCR was employed to measure the relative expression levels of T6SS genes in each strain to clarify the effect of VasH on T6SS gene expression. Growth curve establishment, transmission electron microscopy (TEM) for observing bacterial morphology, and the crystal violet assay for biofilm quantification were performed to determine the influences of VasH on pathogenic characteristics. An in vitro bacterial competition assay and a zebrafish model for determining the median lethal dose (LD₅₀) were employed to assess the comprehensive effects of VasH on bacterial competitiveness and pathogenicity. [Results] The VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were successfully constructed. The deletion of VasH resulted in a decrease (P<0.05) in the expression of genes encoding both T6SS structural and effector proteins, indicating that VasH was responsible for regulating T6SS gene expression. The deletion of vasH did not significantly affect the growth or surface morphology/structure of A. veronii. However, it led to a significant increase in the biofilm formation and a significant decrease in in vitro bacterial competitiveness. The zebrafish infection assay showed that the LD₅₀ of ΔvasH was 1.58×10¹¹, which was 2.15 times that (7.34×10¹⁰) of the wild type. Moreover, at an infection concentration of 5×10⁸ CFU/mL, the death rate of zebrafish decreased from 33.3% in the wild-type infection group to 0 following infection with ΔvasH, indicating that deletion of vasH attenuated the virulence of A. veronii. [Conclusion] In A. veronii, VasH positively regulates T6SS gene expression. It may not be involved in regulating the growth state or external morphology of the pathogen but significantly influences the biofilm formation and competitiveness of A. veronii, thereby modulating the overall virulence during infection of the zebrafish host. This study provides essential research tools and lays a preliminary foundation for further exploration of the molecular mechanisms by which VasH mediates pathogenicity through regulating T6SS activity and function in A. veronii.

Key words

Aeromonas veronii / vasH / T6SS / virulence

引用本文

高士杰, 马香. 维氏气单胞菌VasH正调控Ⅵ型分泌系统表达并影响细菌竞争力和毒力. 微生物学报, 2026 , 66 (8) : 4135 -4149 . DOI: 10.13343/j.cnki.wsxb.20260025

Shijie GAO, Xiang MA. VasH positively regulates type Ⅵ secretion system expression and influences competition and virulence of Aeromonas veronii[J]. Acta Microbiologica Sinica, 2026 , 66 (8) : 4135 -4149 . DOI: 10.13343/j.cnki.wsxb.20260025

正文

收起

维氏气单胞菌(Aeromonas veronii) C4属于气单胞菌科(Aeromonadaceae)、气单胞菌属(Aeromonas)，是一种具有运动能力的革兰氏阴性短杆菌，广泛分布于地下水、湖泊及经氯处理或未经处理的饮用水等多种水体中^[1-2]。维氏气单胞菌感染可引起鱼类全身性出血、烂尾病、败血症等^[3]，也可导致人类肠胃炎、脑膜炎等疾病^[4-6]。目前，维氏气单胞菌已被公认为水产养殖业和人类公共卫生安全的潜在威胁。

分泌系统是介导维氏气单胞菌毒力的关键结构，通过分泌气溶素、溶血素、肠毒素及蛋白酶等效应蛋白调控细菌致病性^[7]。其中，Ⅵ型分泌系统(type 6 secretion system, T6SS)在介导细菌竞争力和环境适应性方面发挥关键作用^[8]。T6SS是广泛存在于革兰氏阴性菌中的纳米级生物大分子机器，能够向外分泌具有脂酶、溶菌酶等功能的效应蛋白以杀伤邻近细胞，从而确保自身竞争优势^[9]。T6SS的活性通常受到严格调控，仅在特定条件下被激活。深入研究T6SS的调控机制，不仅有助于进一步解析维氏气单胞菌的致病机理，也可为预防和治疗其引起的动物与人类感染、减少经济损失提供重要理论依据。

维氏气单胞菌C4菌株基因组中存在一套编码T6SS的基因簇，包括1个主簇和3个副簇，主簇中含有编码细菌增强子结合蛋白VasH的基因。已知VasH能够与σ⁵⁴协同作用，在霍乱弧菌、嗜水气单胞菌等细菌中调控T6SS的转录活性及功能^[10-11]。为初步鉴定维氏气单胞菌中VasH的生物学功能，本研究采用同源重组基因敲除技术构建了vasH基因敲除菌株及其回补菌株；通过与野生型菌株比较，初步鉴定vasH缺失对T6SS基因表达水平的影响；通过测定病原菌生长能力和生物膜形成能力，明确vasH缺失对病原菌致病相关特征的影响；通过体外种间竞争实验及斑马鱼感染模型测定体内毒力和半数致死浓度(median lethal dose, LD₅₀)，阐明vasH缺失对病原菌综合毒力的影响。本研究以期为深入解析VasH蛋白调控维氏气单胞菌T6SS功能的分子机制奠定基础，同时也为制定维氏气单胞菌感染的防控策略提供新思路。

1 材料与方法

收起

1.1 材料

自杀型质粒pRE112、大肠杆菌(Escherichia coli) WM3064均为本实验室保存菌株。

细菌基因组快速提取试剂盒、一步克隆酶试剂盒、2×Phanta UniFi Master Mix、Clon Express Ultra One Step Cloning Kit、HiScript^® Ⅲ All-in-one RT SuperMix Kit、ChamQ SYBR Color qPCR Master Mix Kit，南京诺唯赞生物科技股份有限公司；蛋白胨、酵母膏，OXOID公司；抗生素、琼脂粉、三氯乙酸、无机盐，北京索莱宝科技有限公司；RNAprep Pure Cell/Bacteria Kit、PCR产物纯化试剂盒，天根生化科技(北京)有限公司。

透射电子显微镜，Hitachi公司；Roche LightCycle^® 96仪器，Roche Diagnostics公司。

实验所用引物合成及样品测序服务由生工生物工程(上海)股份有限公司提供。本研究所用引物如表1所示，所构建及使用的菌株和质粒如表2所示。

1.2 重组菌株构建

采用细菌基因组快速提取试剂盒从A. veronii C4野生型菌液中提取基因组DNA作为PCR模板，以vasH F1/R1和vasH F2/R2 (表1)为引物，分别PCR扩增vasH基因的上、下游同源臂片段。PCR反应体系(50 µL)：2×Phanta UniFi Master Mix 25 µL，上、下游引物(10 µmol/L)各2 µL，DNA模板1 µL，ddH₂O 20 µL。PCR反应条件：95 ℃预变性5 min；95 ℃变性30 s，60 ℃退火30 s，72 ℃延伸30 s，共30个循环；72 ℃终延伸5 min。使用PCR产物纯化试剂盒对PCR扩增产物进行纯化回收。以上、下游同源臂片段为模板，利用Overlap PCR获得同源臂连接产物；以pRE112-anti-F/R为引物通过反向PCR线性化pRE112载体(PCR反应体系同上，延伸时间为2.5 min)，再利用一步克隆酶试剂盒将线性化载体与vasH上、下游同源臂连接产物连接，构建重组敲除质粒pRE112-ΔvasH，并热激转化至E. coli WM3064感受态细胞。采用同源臂与载体的交叉引物验证阳性克隆，PCR产物送生工生物工程(上海)股份有限公司测序。将鉴定正确的pRE112-ΔvasH重组菌株转接培养至OD₆₀₀为0.4-0.6，与相同浓度的维氏气单胞菌C4 WT按1:1、3:1、1:3的比例混合，终体积为800 μL。分别取50 μL混合菌液滴加于含50 μg/mL二氨基庚二酸(diaminopimelic acid, DAP)的LB平板上，30 ℃倒置培养24 h。挑取平板上的菌落，涂布于含25 μg/mL氯霉素(chloramphenicol, Cam)和50 μg/mL氨苄青霉素(ampicillin, Amp)的双抗平板上，以pRE112 F/R为引物进行PCR验证接合子。挑取阳性接合子的单菌落接种于LB液体培养基中，30 ℃振荡培养过夜，取100 μL菌液涂布于含20%蔗糖的LB平板上，30 ℃倒置培养24 h。通过菌落PCR验证vasH敲除菌株，PCR产物送生工生物工程(上海)股份有限公司测序。

回补菌株的构建流程与上述基本一致，仅将载体更换为pBBR质粒，线性化引物更换为pBBR-anti-F/R，目的基因片段扩增引物更换为vasH F3/R3。

1.3 生长曲线测定

挑取WT、ΔvasH和ΔvasH/p-vasH的单菌落，分别接种于LB培养基中，30 ℃、150 r/min培养过夜。按每毫升OD₆₀₀=0.02的浓度分别转接至LB和M9培养基；转接至M9培养基前，先用M9培养基清洗菌体1次。30 ℃下使用酶标仪每隔1 h测定OD₆₀₀值，记录数据并绘制生长曲线。每组设3个生物学重复，实验独立重复3次。使用GraphPad Prism 10.0软件进行统计分析并绘制生长曲线图。

1.4 生物膜形成能力测定

挑取WT、ΔvasH和ΔvasH/p-vasH单菌落，分别按每毫升OD₆₀₀=0.02的浓度转接至LB培养基中，并按照每孔300 μL菌液转移至48孔板，以含50 μg/mL Amp的LB培养基作为空白对照。30 ℃静置培养36 h后吸出培养液，用无菌PBS缓冲液冲洗残留的成膜菌体3次，加入200 μL 0.5%结晶紫染色液染色10 min，再用无菌水充分冲洗去除残留染色液。于37 ℃烘箱中彻底干燥后加入200 μL 33%醋酸溶液，37 ℃孵育30 min，使用酶标仪于570 nm波长下测定吸光度值。每组设3个生物学重复，实验重复4次。

1.5 透射电镜(transmission electron microscopy, TEM)观察

将培养过夜的菌体转接至新鲜LB培养基中，培养至OD₆₀₀为0.4-0.6。收集1.5 mL菌液至离心管中，6 000 r/min离心5 min去除上清，用1 mL无菌PBS清洗3次，加1 mL电镜固定液室温固定2 h。取涂有碳膜的铜网，碳膜面朝上，滴加10 μL菌液，静置15 min，去除多余液体，室温晾干。使用HT7800透射电子显微镜观察并拍照。每组设3个生物学重复，实验重复3次。

1.6 RT-qPCR检测

收集LB培养基中培养至稳定期的细菌，10 000 r/min离心1 min去除培养基，用DEPC水洗涤残余培养基，按照RNAprep Pure Cell/Bacteria Kit说明书提取细菌总RNA。以1 000 ng RNA为模板，去除gDNA和逆转录反应体系的配制和程序按照HiScript^® Ⅲ All-in-one RT SuperMix Kit说明书进行。每个菌株设3个生物学重复，每个生物学重复设3个技术重复。各检测基因所用引物如表1所示，内参基因为gyrB。实时荧光定量逆转录PCR (RT-qPCR)按照ChamQ SYBR Color qPCR Master Mix Kit的说明书进行，使用Roche LightCycle^® 96仪器测定循环阈值(C_q)。采用2^-∆∆^C^q计算各基因的相对表达量，并用单因素方差分析(one-way ANOVA)进行显著性检验。

1.7 细菌竞争实验

将维氏气单胞菌重组菌株作为攻击菌，携带pBBR-eGFP质粒的大肠杆菌BL21作为目标菌株，分别取培养过夜菌液调整至10⁸ CFU/mL。按攻击菌与靶标菌数量比1:1或10:1混合，将混合液滴加于无抗生素LB平板上，37 ℃培养24 h。随后刮取平板上的混合菌落，采用抗生素选择性平板或荧光标记法分离2株菌，通过滴板实验、涂板计数及流式细胞术评估竞争后靶标菌的存活情况。每组设3个生物学重复，实验重复3次。

1.8 斑马鱼LD₅₀ 测定

采用LD₅₀评价ΔvasH菌株相对于野生型(WT)菌株的毒力变化。选取健康的5 dpf AB系斑马鱼幼鱼，随机分为3组，每组设4个平行亚组，每亚组30尾，于28 ℃ E3培养基中培养，光照周期为14 h光照/10 h黑暗。在更换新鲜E3培养基后，将各组斑马鱼幼鱼分别浸泡于WT、ΔvasH及ΔvasH/p-vasH菌液的10倍梯度稀释液(浓度范围5×10⁶-5×10⁹ CFU/mL)中，对照组以PBS处理。每4 h记录死亡情况，持续48 h，根据简易寇氏法^[13]计算LD₅₀值。本研究所有动物实验均获得海南大学动物伦理委员会批准，编号为HNUAUCC-2023-00141。

1.9 数据处理

实验数据使用Excel整理，采用SPSS 24.0进行统计分析，GraphPad Prism 10.0进行数据可视化。统计方法包括采用双尾t检验、单因素方差分析(one-way ANOVA)、Dunnett检验及Log-rank (Mantel-Cox)检验。

2 结果与分析

收起

2.1 vasH 敲除菌株ΔvasH 和回补菌株ΔvasH/p-vasH 的构建

分别扩增vasH基因上、下游同源臂(图1A、1B)并线性化pRE112载体(图1C)，利用一步克隆酶构建敲除质粒pRE112-ΔvasH。经菌落PCR验证阳性克隆，ΔvasH敲除株的扩增产物为309 bp，而A. veronii C4野生型对照的扩增产物为1 848 bp (图1D)，条带大小符合预期，表明目的基因vasH已成功敲除。进一步对PCR产物测序，确认vasH基因已完全缺失。同时，利用pBBR质粒验证引物对回补菌株进行PCR验证，结果显示扩增产物条带大小与理论值2 440 bp一致，表明回补菌株构建成功(图1E)。

通过RT-qPCR检测所构建敲除及回补菌株中vasH基因的转录水平，结果如图1F所示。与野生型相比，ΔvasH菌株中vasH基因表达水平显著降低；而回补菌株ΔvasH/p-vasH中vasH基因表达水平显著高于野生型，达到了过表达的效果，这可能归因于回补质粒pBBR所携带的组成型高表达启动子。

2.2 VasH蛋白对维氏气单胞菌T6SS基因表达具有正调控活性

在其他病原菌中VasH是T6SS发挥功能所必需的蛋白，其缺失可导致T6SS失活^[10]。为明确VasH蛋白对维氏气单胞菌T6SS基因表达水平的影响，以维氏气单胞菌野生型、vasH敲除菌株ΔvasH和回补菌株ΔvasH/p-vasH为研究对象，通过RT-qPCR比较T6SS结构标志性蛋白Hcp、VgrG及效应蛋白Tse1、Tse2、Tse3的基因表达水平。结果显示，ΔvasH菌株中T6SS代表性结构基因和效应蛋白基因的表达水平均显著降低，而回补菌株中相应基因的表达水平恢复至接近野生型的水平(图2)。上述结果表明，VasH对维氏气单胞菌T6SS基因的表达具有正调控活性。

2.3 VasH蛋白不参与调控维氏气单胞菌富营养条件下的生长和外部形态

生长曲线结果显示，在寡营养培养基(M9培养基)中各菌株的生物量均低于富营养培养基(LB培养基)。培养至25 h时，寡营养条件下WT、ΔvasH和ΔvasH/p-vasH菌株的OD₆₀₀均值分别为0.7、0.8、0.8，而富营养条件下三者OD₆₀₀均值均为1.6。在富营养培养条件下，ΔvasH和ΔvasH/p-vasH菌株的生长曲线与野生型相比无显著差异(图3A)；而在寡营养培养条件下，ΔvasH和ΔvasH/p-vasH菌株的初期生长速度略有降低，野生型于14 h进入稳定期，重组菌株则延至约18 h (图3B)。上述结果表明，营养限制显著抑制了维氏气单胞菌的生长，VasH蛋白可能参与调控寡营养条件下的细菌生长，但在富营养条件下对细菌生长无显著影响。

透射电镜观察结果显示，ΔvasH和ΔvasH/p-vasH菌株的形态及外部结构与野生型相比未见显著差异，3株菌的鞭毛和菌毛均完整，未发现分裂异常或菌体长度异常等现象(图4)。该结果表明，VasH蛋白可能不参与细菌细胞壁或表面结构的组装，也不影响细菌的增殖与分裂过程。

2.4 VasH蛋白抑制菌群生物膜形成

采用结晶紫染色法检测WT、ΔvasH和ΔvasH/p-vasH菌株的生物膜形成能力，结果如图5所示。与野生型相比，ΔvasH菌株的生物膜形成能力显著增强，而ΔvasH/p-vasH菌株的生物膜形成能力恢复至接近野生型的水平，表明VasH可能参与抑制生物膜的形成^[14-17]。

2.5 VasH蛋白正调控细菌的种间竞争力

分别以维氏气单胞菌C4的WT、ΔvasH和ΔvasH/p-vasH为攻击菌，以携带pBBR-eGFP质粒的大肠杆菌BL21为目标菌株，通过体外竞争实验检测vasH基因敲除对细菌种间竞争能力的影响。本实验室前期构建的T6SS激活菌株ΔrsmA和T6SS结构组装缺陷菌株ΔtssB分别作为阳性对照和阴性对照^[12]。采用滴板法(图6A、6D)、CFU计数法(图6B、6E)和流式细胞术(图6C、6F)分别测定竞争处理后目标菌株的存活菌落数，结果表明，无论在攻击菌株与目标菌株数量比为10:1 (图6A-6C)还是1:1 (图6D-6F)的情况下，均获得一致的结果：野生型及ΔrsmA菌株的T6SS功能正常，能够有效抑制大肠杆菌BL21的生长；而与ΔvasH或ΔtssB菌株共培养的大肠杆菌BL21生长未受到显著抑制，经10⁵倍稀释后仍可存活(图6A、6D)；CFU计数结果进一步显示，与ΔvasH或ΔtssB共培养组中大肠杆菌BL21的存活数量显著高于WT或ΔrsmA共培养组(图6B、6E)；流式细胞术检测也观察到相应的荧光信号峰值变化(图6C、6F)。上述结果表明，VasH是调控维氏气单胞菌种间竞争能力所必需的蛋白。

2.6 VasH蛋白介导维氏气单胞菌毒力

以斑马鱼幼鱼为感染模型，探究vasH基因敲除对维氏气单胞菌毒力的影响。采用改良寇氏法^[13]计算WT、ΔvasH和ΔvasH/p-vasH菌株的半数致死浓度LD₅₀，结果显示ΔvasH菌株的LD₅₀约为WT的2.15倍(表3)，表明vasH基因敲除显著降低了维氏气单胞菌对斑马鱼幼鱼的致死能力。

进一步以5×10⁸ CFU/mL浓度分别感染斑马鱼幼鱼，绘制生存曲线并采用Log-rank (Mantel-Cox)法进行统计分析。与野生型相比，ΔvasH菌株的毒力显著减弱(P<0.001)，而ΔvasH/p-vasH回补菌株感染组的斑马鱼死亡时间早于野生型组(图7)，推测可能与回补菌株中vasH基因过表达(图1F)导致T6SS功能过度激活有关，但该差异未达显著水平(P>0.05)。

3 讨论

收起

本课题组前期研究表明，维氏气单胞菌基因组中存在一套完整的T6SS基因簇，T6SS可介导维氏气单胞菌种间竞争并调节其生态位适应性，从而协同促进其致病性^[12]。在病原菌感染宿主的过程中，T6SS的激活与功能表达受到精密调控^[18]，在不同细菌中存在环境信号激活^[18-22]、转录调控^[23-26]、转录后调控^[27-30]和翻译后调控^[31-32]等多种调控方式。然而，维氏气单胞菌在何种条件下激活T6SS组装目前仍不清楚，已知在实验室富营养培养条件下维氏气单胞菌T6SS处于非激活状态。维氏气单胞菌中存在rpoN基因编码的转录起始因子σ⁵⁴，且σ⁵⁴增强子结合蛋白VasH的编码基因位于T6SS基因簇中。结合现有研究^[10,33-35]，推测T6SS基因簇中的细菌增强子结合蛋白VasH可能参与维氏气单胞菌对T6SS的精密调控。

研究报道VasH蛋白是霍乱弧菌等细菌中T6SS表达和发挥功能所必需的元件，能够调控细菌毒力^[10,34]。本研究从基因表达水平、体外竞争能力及宿主体内毒力3个层面证实，在维氏气单胞菌中敲除vasH基因后T6SS活性及毒力显著下降。RT-qPCR结果表明，vasH缺失导致T6SS核心基因——包括重要结构蛋白Hcp和VgrG的编码基因，以及本实验室前期鉴定的3个效应蛋白Tse1、Tse2和Tse3的编码基因^[12]，其相对表达量均较野生型显著下调(图2)，表明VasH对维氏气单胞菌T6SS基因表达具有正调控活性。后续将进一步探究VasH是否通过直接结合T6SS启动子行使功能、其具体结合位点以及是否存在转录后调控等机制。在种间竞争方面，体外竞争实验结果显示，VasH缺失导致细菌的种间竞争能力显著降低至与T6SS结构组装缺陷菌株ΔtssB相近的水平(图6)。尽管VasH本身并非T6SS结构蛋白，但其缺失可能通过直接下调T6SS结构基因的表达而间接影响T6SS的组装及T6SS介导的细菌竞争能力。为进一步明确VasH蛋白缺失对维氏气单胞菌感染鱼类宿主毒力的影响，本研究以斑马鱼幼鱼为感染模型，比较了WT、ΔvasH和ΔvasH/p-vasH菌株的LD₅₀及其对斑马鱼存活率的影响。结果显示，ΔvasH菌株的LD₅₀为WT的2.15倍；在感染浓度为5×10⁸ CFU/mL时，ΔvasH菌株感染导致斑马鱼的死亡率由WT感染时的33.3%降至0，表明vasH基因敲除显著降低了维氏气单胞菌的毒力(表3、图7)。生存曲线显示回补株导致斑马鱼早期死亡，这可能是由于构建回补株时采用了组成型启动子，导致VasH过量表达而过度激活T6SS所致。后续将采用vasH基因自身启动子驱动或低拷贝表达载体进行回补以进一步验证其功能。T6SS通常通过分泌不同效应蛋白杀伤邻近细胞、维持自身竞争优势^[36-40]。后续将进一步鉴定VasH缺失菌株分泌至胞外的效应蛋白种类及浓度变化，以阐明其调控T6SS竞争能力及病原菌致病性的分子机制。

本研究还发现，ΔvasH菌株在富营养培养基(LB培养基)中的生长能力及外部形态与野生型相比无显著差异(图3)，推测其原因可能是VasH调控的T6SS通路不参与细胞壁合成、营养物质转运等与生长及形态相关的基础代谢过程。现有研究也表明，多数细菌的T6SS并不直接参与基础生长代谢或细胞形态的维持，其核心功能集中于种内竞争、宿主互作等过程^[7-8,12]，本研究结果与之一致。

T6SS作为接触依赖性竞争武器，在生物膜这一密集的微生物群落中可能发挥关键作用，在铜绿假单胞菌、肺炎克雷伯菌等细菌中T6SS某些结构蛋白或效应蛋白的缺失可导致生物膜形成能力发生改变^[14-17]。本研究在维氏气单胞菌中也观察到类似现象：ΔvasH菌株的生物膜形成能力较野生型显著增强(图5)，提示VasH可能负调控维氏气单胞菌的生物膜形成。其具体机制尚不明确，有待后续实验进一步阐明VasH缺失是否通过影响T6SS效应蛋白的分泌，进而干扰效应蛋白参与的碳代谢等生物膜形成相关通路，最终促进细菌聚集与生物膜基质沉积。

4 结论

收起

本研究成功构建了维氏气单胞菌ΔvasH菌株及其回补菌株，从T6SS基因表达、细菌竞争能力及宿主感染能力3个层面证实VasH正调控维氏气单胞菌T6SS的表达与功能；通过检测ΔvasH及其回补菌株的生长能力、生物膜形成能力及细胞形态，初步明确VasH的核心功能集中于调控T6SS活性。本研究为后续深入解析维氏气单胞菌T6SS介导的致病机制奠定了基础。

基金

收起

海南省自然科学基金(324MS017)
国家自然科学基金(32360047)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

Fernández-Bravo

, Figueras

. An update on the genus Aeromonas: taxonomy, epidemiology, and pathogenicity[J]. Microorganisms, 2020, 8(1): 129.

[2]

Egorov

, Best

JMB

, Frebis

, Karapondo

. Occurrence of Aeromonas spp. in a random sample of drinking water distribution systems in the USA[J]. Journal of Water and Health, 2011, 9(4): 785-798.

[3]

Chen

, Sun

, Han

, Yang

, Xian

, Lv

, Hu

, Shi

. Isolation, identification and characteristics of Aeromonas veronii from diseased crucian carp (Carassius auratus Gibelio)[J]. Frontiers in Microbiology, 2019, 10: 2742.

[4]

Chen

, Tsai

, Chen

, Lu

, Chen

, Lee

, Li

, Wu

, Ko

. Aeromonas stool isolates from individuals with or without diarrhea in southern Taiwan of China: predominance of Aeromonas veronii [J]. Journal of Microbiology, Immunology and Infection, 2015, 48(6): 618-624.

[5]

Beaz-Hidalgo

, Figueras

. Aeromonas spp. whole genomes and virulence factors implicated in fish disease[J]. Journal of Fish Diseases, 2013, 36(4): 371-388.

[6]

Lin

, Zhong

, Chen

, Cui

, Xu

, Tang

. Aeromonas veronii-associated endogenous endophthalmitis: a case report[J]. Journal of Medical Case Reports, 2024, 18: 171.

[7]

Costa

TRD

, Felisberto-Rodrigues

, Meir

, Prevost

, Redzej

, Trokter

, Waksman

. Secretion systems in Gram-negative bacteria: structural and mechanistic insights[J]. Nature Reviews Microbiology, 2015, 13(6): 343-359.

[8]

Robitaille

, Trus

, Ross

. Bacterial defense against the type Ⅵ secretion system[J]. Trends in Microbiology, 2021, 29(3): 187-190.

[9]

Yadav

, Magotra

, Ghosh

, Krishnan

, Pradhan

, Kumar

, Das

, Sharma

, Jha

. Immunity proteins of dual nuclease T6SS effectors function as transcriptional repressors[J]. EMBO Reports, 2021, 22(6): e51857.

[10]

, Wu

, Chen

, Zhou

, Zhang

. VasH contributes to virulence of Aeromonas hydrophila and is necessary to the T6SS-mediated bactericidal effect[J]. Frontiers in Veterinary Science, 2021, 8: 793458.

[11]

Manera

, Caro

, Li

, Pei

, Hersch

, Mekalanos

, Dong

. Sensing of intracellular Hcp levels controls T6SS expression inVibrio cholerae [J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(25): e2104813118.

[12]

Jiang

, Li

, Ma

, Li

, Ma

, Tang

, Li

, Chi

, Deng

, Zeng

, Liu

. Role of Type VI secretion system in pathogenic remodeling of host gut microbiota during Aeromonas veronii infection[J]. The ISME Journal, 2024, 18: wrae053.

[13]

杨世洪. 半数致死量LD₅₀速算方法[J]. 卫生研究, 1974, 3(5): 73-78.

[14]

Yang

, Pan

, Tang

, Li

, Zhu

, Yu

, Zhu

, Wang

, Chen

, Li

. H3-T6SS of Pseudomonas aeruginosa PA14 contributes to environmental adaptation via secretion of a biofilm-promoting effector[J]. Stress Biology, 2022, 2: 55.

[15]

Fei

, Ji

, Yang

, Yu

, Qiao

, Yan

, Guan

, Yang

, Zhao

. Hcp of the type VI secretion system (T6SS) in Acidovorax citrulli group Ⅱ strain Aac5 has a dual role as a core structural protein and an effector protein in colonization, growth ability, competition, biofilm formation, and ferric iron absorption[J]. International Journal of Molecular Sciences, 2022, 23(17): 9632.

[16]

Mohamed

, Alrawy

, Makbol

, Mohamed

, Hemdan

, Shafik

. Type VI secretion system (T6SS) in Klebsiella pneumoniae, relation to antibiotic resistance and biofilm formation[J]. Iranian Journal of Microbiology, 2023: 601-608.

[17]

Zhang

, Huang

, Ding

, Ma

, Tong

, Zhang

, Tao

, Wang

. A σE-mediated temperature gauge orchestrates type VI secretion system, biofilm formation and cell invasion in pathogen Pseudomonas plecoglossicida [J]. Microbiological Research, 2023, 266: 127220.

[18]

Hespanhol

, Nóbrega-Silva

, Bayer-Santos

. Regulation of type VI secretion systems at the transcriptional, posttranscriptional and posttranslational level[J]. Microbiology, 2023, 169(8): 001376.

[19]

Ben-Yaakov

, Salomon

. The regulatory network ofVibrio parahaemolyticustype VI secretion system 1[J]. Environmental Microbiology, 2019, 21(7): 2248-2260.

[20]

Dos Passos Lima

, de Santana

, Lorenzetti

APR

, Lourenço

, de Moraes Ceseti

, Riva

, Bayer-Santos

, Boechat

, Baldini

, Farah

, Koide

, Alvarez-Martinez

. The AraC-type transcription factor TagK is a new player in the signaling cascade that induces the anti-eukaryotic T6SS of Xanthomonas citri [J]. Molecular Microbiology, 2022, 118(5): 552-569.

[21]

, Wu

, Hou

, Zhang

, Zhou

. Fur functions as an activator of T6SS-mediated bacterial dominance and virulence in Aeromonas hydrophila [J]. Frontiers in Microbiology, 2023, 13: 1099611.

[22]

, Lu

, Huo

, Wang

, Zhang

, Zong

, Wang

, Dong

, Li

, Chen

, Tan

. EvfG is a multi-function protein located in the Type VI secretion system for ExPEC[J]. Microbiological Research, 2024, 283: 127647.

[23]

Han

, Wang

, Chen

, Pu

, Liu

, Zhang

, Xu

, Wu

, Liang

. A Pseudomonas aeruginosa type VI secretion system regulated by CueR facilitates copper acquisition[J]. PLoS Pathogens, 2019, 15(12): e1008198.

[24]

, Wei

, He

, Liu

, Zuo

, Xiao

, Wang

, Shen

, Zhu

. OxyR-regulated T6SS functions in coordination with siderophore to resist oxidative stress[J]. Microbiology Spectrum, 2024, 12(2): e03231-23.

[25]

Bervoets

, Charlier

. Diversity, versatility and complexity of bacterial gene regulation mechanisms: opportunities and drawbacks for applications in synthetic biology[J]. FEMS Microbiology Reviews, 2019, 43(3): 304-339.

[26]

Zhang

, Yin

, Liu

, Wang

, Xu

, Pan

, Bai

, Cheng

, Wu

, Jin

. NrtR mediated regulation of H1-T6SS in Pseudomonas aeruginosa [J]. Microbiology Spectrum, 2022, 10: e01858-21.

[27]

Van Assche

, van Puyvelde

, Vanderleyden

, Steenackers

. RNA-binding proteins involved in post-transcriptional regulation in bacteria[J]. Frontiers in Microbiology, 2015, 6: 141.

[28]

Jørgensen

, Pettersen

, Kallipolitis

. sRNA-mediated control in bacteria: an increasing diversity of regulatory mechanisms[J]. Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms, 2020, 1863(5): 194504.

[29]

Bhowmik

, Pathak

, Pandey

, Devnath

, Sett

, Jyoti

, Bhando

, Akhter

, Chugh

, Singh

, Sharma

, Pathania

. Acinetobacter baumanniirepresses type VI secretion system through a manganese-dependent small RNA-mediated regulation[J]. mBio, 2025, 16(2): e03025-24.

[30]

Zammit

, Sy

, Shchepachev

, Mediati

, Poudyal

, Norris

, Bang

, Alquethamy

, Wu

, Ullah

, McAteer

, Rowell

, Michie

, Gally

, Tollervey

, Tree

. The type 6 secretion system immunity protein RhsFI has been repurposed for small RNA regulation in pathogenic Escherichia coli [J]. Nucleic Acids Research, 2025, 53(14): gkaf749.

[31]

Wang

, Brodmann

, Basler

. Assembly and subcellular localization of bacterial type VI secretion systems[J]. Annual Review of Microbiology, 2019, 73: 621-638.

[32]

Liu

, Zhou

, Bu

, Qin

, Zhang

, Shao

, Wang

. Lysine acetylation regulates the AT-rich DNA possession ability of H-NS[J]. Nucleic Acids Research, 2024, 52(4): 1645-1660.

[33]

Bernard

, Brunet

, Gavioli

, Lloubès

, Cascales

. Regulation of type VI secretion gene clusters by σ⁵⁴ and cognate enhancer binding proteins[J]. Journal of Bacteriology, 2011, 193(9): 2158-2167.

[34]

Kitaoka

, Miyata

, Brooks

, Unterweger

, Pukatzki

. VasH is a transcriptional regulator of the type VI secretion system functional in endemic and pandemic Vibrio cholerae [J]. Journal of Bacteriology, 2011, 193(23): 6471-6482.

[35]

Guckes

, Cecere

, Williams

, McNeil

, Miyashiro

. The bacterial enhancer binding protein VasH promotes expression of a type VI secretion system in Vibrio fischeri during symbiosis[J]. Journal of Bacteriology, 2020, 202(7): e00777-19.

[36]

Ren

, Jia

, Liu

, Zhou

, Wu

, Dong

, Cai

, Qu

, Liu

, Yang

, Zhang

. Acquisition of T6SS effector TseL contributes to the emerging of novel epidemic strains of Pseudomonas aeruginosa [J]. Microbiology Spectrum, 2023, 11: e03308-22.

[37]

Sá-Pessoa

, López-Montesino

, Przybyszewska

, Rodríguez-Escudero

, Marshall

, Ova

, Schroeder

, Barabas

, Molina

, Curtis

, Cid

, Bengoechea

. A trans-Kingdom T6SS effector induces the fragmentation of the mitochondrial network and activates innate immune receptor NLRX1 to promote infection[J]. Nature Communications, 2023, 14: 871.

[38]

Ahmad

, Wang

, Walker

, Tran

, Stogios

, Savchenko

, Grant

, McArthur

, Laub

, Whitney

. An interbacterial toxin inhibits target cell growth by synthesizing (p)ppApp[J]. Nature, 2019, 575(7784): 674-678.

[39]

, Pinedo

, Lopez

, Cava

, Feldman

. Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector[J]. Proceedings of the National Academy of Sciences of the United States of America, 2021, 118(40): e2106555118.

[40]

Hespanhol

, Sanchez-Limache

, Nicastro

, Mead

, Llontop

, Chagas-Santos

, Farah

, de Souza

, da Silva Galhardo

, Lovering

, Bayer-Santos

. Antibacterial T6SS effectors with a VRR-Nuc domain are structure-specific nucleases[J]. eLife, 2022, 11: e82437.

2026年第66卷第8期

PDF下载

238

引用本文

BibTeX

文章信息

doi: 10.13343/j.cnki.wsxb.20260025

接收时间：2026-01-10
首发时间：2026-08-21
出版时间：2026-08-04

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2026-01-10
录用日期：2026-03-31

基金

Hainan Province Natural Science Foundation(324MS017)

海南省自然科学基金(324MS017)

Natural Science Foundation of China(32360047)

国家自然科学基金(32360047)

作者信息

海南大学生命健康学院，海南海口

通讯作者:

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/wswxb/CN/10.13343/j.cnki.wsxb.20260025

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Primer names	Primer sequences (5′→3′)	Note
pRE112 F	ACGCCATTCATGGCCATATCA	pRE112 validation primer
pRE112 R	GTTATTGGTGCCCTTAAACGC
pRE112-anti-F	TCTAGAAGAAGCTTGGGATCG	Primers for pRE112 vector linearization
pRE112-anti-R	GAGCTCTCCCGGGAATTCATG
vasH F0	GGATGCTGGAGTCCGTCATT	Validation of vasH gene knockout
vasH R0	CTCCTTGTTGCCTGATTGCGC
vasH F1	AATTCCCGGGAGAGCTCATCATCGACTGCAAGAACGTG	Amplification of the upstream homologous sequence of vasH
vasH R1	ATGATGCTCAGGTCAGCCCTCCACCTCG
vasH F2	GCTGACCTGAGCATCATGGGTTTGTTGC	Amplification of the downstream homologous sequence of vasH
vasH R2	CCAAGCTTCTTCTAGAATCCCTGATCCCGGATAAACC
vasH F3	GCTTGATATCGAATTCATGGAGCAAGCCCTCGCATT	vasH complementation plasmid primers
vasH R3	TAGAACTAGTGGATCCTCAGTTCACCTCCAGTTTCTG
pBBR-anti-F	GAATTCGATATCAAGCTTATCG	Primers for pBBR vector linearization
pBBR-anti-R	GGATCCACTAGTTCTAGAGC
pBBR-F	AGCGGCTATTTAACGACCCTGCC	Primers for pBBR plasmid verification
pBBR-R	GTGCTGATGCCGCTGGCGATTCAGGT
gyrB-qF	GCTCACCATTCGTCGTAACGG	Primers for RT-qPCR
gyrB-qR	GCCACCCTCGTAGCAGAAAT
hcp-qF	GGTCAGCGTGTCCACAAACC
hcp-qR	GGCATCTGGCAGTCGATGTC
vgrG(cluster2)-qF	AAGCCCGATGAAATCCTC
vgrG(cluster2)-qR	CCTTGCACTTCTCACACTCCTCA
vgrG(cluster134)-qF	ACACAGAAGCCTGACGAA
vgrG(cluster134)-qR	GTGTTTGCCCTGCTCGAA
tse1-qF	CACTGGCACCAGCAAAACCG
tse1-qR	GACATGCTCGCGCTTTACATCG
tse2-qF	AGGGACGAGGTTTGACCCAT
tse2-qR	ATTCGTGGGCACACCAAACA
tse3-qF	GCCAACAACACCACCAAGCA
tse3-qR	TCTTCCTTGTTGCGGCAACT

Primer names

Primer sequences (5′→3′)

Note

pRE112 F

ACGCCATTCATGGCCATATCA

pRE112 validation primer

pRE112 R

GTTATTGGTGCCCTTAAACGC

pRE112-anti-F

TCTAGAAGAAGCTTGGGATCG

Primers for pRE112 vector linearization

pRE112-anti-R

GAGCTCTCCCGGGAATTCATG

vasH F0

GGATGCTGGAGTCCGTCATT

Validation of vasH gene knockout

vasH R0

CTCCTTGTTGCCTGATTGCGC

vasH F1

AATTCCCGGGAGAGCTCATCATCGACTGCAAGAACGTG

Amplification of the upstream homologous sequence of vasH

vasH R1

ATGATGCTCAGGTCAGCCCTCCACCTCG

vasH F2

GCTGACCTGAGCATCATGGGTTTGTTGC

Amplification of the downstream homologous sequence of vasH

vasH R2

CCAAGCTTCTTCTAGAATCCCTGATCCCGGATAAACC

vasH F3

GCTTGATATCGAATTCATGGAGCAAGCCCTCGCATT

vasH complementation plasmid primers

vasH R3

TAGAACTAGTGGATCCTCAGTTCACCTCCAGTTTCTG

pBBR-anti-F

GAATTCGATATCAAGCTTATCG

Primers for pBBR vector linearization

pBBR-anti-R

GGATCCACTAGTTCTAGAGC

pBBR-F

AGCGGCTATTTAACGACCCTGCC

Primers for pBBR plasmid verification

pBBR-R

GTGCTGATGCCGCTGGCGATTCAGGT

gyrB-qF

GCTCACCATTCGTCGTAACGG

Primers for RT-qPCR

gyrB-qR

GCCACCCTCGTAGCAGAAAT

hcp-qF

GGTCAGCGTGTCCACAAACC

hcp-qR

GGCATCTGGCAGTCGATGTC

vgrG(cluster2)-qF

AAGCCCGATGAAATCCTC

vgrG(cluster2)-qR

CCTTGCACTTCTCACACTCCTCA

vgrG(cluster134)-qF

ACACAGAAGCCTGACGAA

vgrG(cluster134)-qR

GTGTTTGCCCTGCTCGAA

tse1-qF

CACTGGCACCAGCAAAACCG

tse1-qR

GACATGCTCGCGCTTTACATCG

tse2-qF

AGGGACGAGGTTTGACCCAT

tse2-qR

ATTCGTGGGCACACCAAACA

tse3-qF

GCCAACAACACCACCAAGCA

tse3-qR

TCTTCCTTGTTGCGGCAACT

Strain or plasmid	Relevant genotype or characteristic(s)	Source
Aeromonas veronii C4	Wild type, ampicillin resistance, virulent to fish	Lab stock
Aeromonas veronii C4 ΔrsmA	Ampicillin resistant, rsmA gene deleted from WT, T6SS-activated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔtssB	Ampicillin resistant, tssB gene deleted from WT, T6SS-inactivated strain	Previously constructed in our laboratory^[12]
Aeromonas veronii C4 ΔvasH	Ampicillin resistant, vasH gene deleted from WT	This study
Aeromonas veronii C4 ΔvasH/P-vasH	Ampicillin and kanamycin resistant, vasH gene deleted from WT and complemented with functional vasH gene on pBBR plasmid	This study
pRE112	Suicide plasmid for homologous recombination to construct knockout strains, chloramphenicol (Cam) resistant	Laboratory stock
Escherichia coli WM3064	Encodes a relaxase, a mating pair formation (MPF) complex and a type Ⅳ coupling protein on the chromosome, diaminopimelic acid (DAP) auxotroph	Laboratory stock
pBBR-vasH	Derived from pBBR-MCS-2, contains the ORF region of vasH; kanamycin resistance	This study
pBBR-eGFP	Derived from pBBR-MCS-2, contains the ORF region of eGFP; gentamicin resistance	Previously constructed in our laboratory^[12]
Escherichia coli BL21(DE3)	Used for plasmid cloning and protein expression; served as the target strain for competition assay in this study	Laboratory stock

Strain or plasmid

Relevant genotype or characteristic(s)

Source

Aeromonas veronii C4

Wild type, ampicillin resistance, virulent to fish

Lab stock

Aeromonas veronii C4 ΔrsmA

Ampicillin resistant, rsmA gene deleted from WT, T6SS-activated strain

Previously constructed in our laboratory^[12]

Aeromonas veronii C4 ΔtssB

Ampicillin resistant, tssB gene deleted from WT, T6SS-inactivated strain

Previously constructed in our laboratory^[12]

Aeromonas veronii C4 ΔvasH

Ampicillin resistant, vasH gene deleted from WT

This study

Aeromonas veronii C4 ΔvasH/P-vasH

Ampicillin and kanamycin resistant, vasH gene deleted from WT and complemented with functional vasH gene on pBBR plasmid

This study

pRE112

Suicide plasmid for homologous recombination to construct knockout strains, chloramphenicol (Cam) resistant

Laboratory stock

Escherichia coli WM3064

Encodes a relaxase, a mating pair formation (MPF) complex and a type Ⅳ coupling protein on the chromosome, diaminopimelic acid (DAP) auxotroph

Laboratory stock

pBBR-vasH

Derived from pBBR-MCS-2, contains the ORF region of vasH; kanamycin resistance

This study

pBBR-eGFP

Derived from pBBR-MCS-2, contains the ORF region of eGFP; gentamicin resistance

Previously constructed in our laboratory^[12]

Escherichia coli BL21(DE3)

Used for plasmid cloning and protein expression; served as the target strain for competition assay in this study

Laboratory stock

Dose of challenge/(CFU/mL)	Number of death/Total		Death rate/%
5×10⁶	0	0	0	0.00	0.00	0.00
5×10⁷	0	0	1	0.00	0.00	3.33
5×10⁸	10	0	1	33.33	0.00	3.33
5×10⁹	30	30	30	100.00	100.00	100.00
LD₅₀	7.34×10¹⁰	1.58×10¹¹	1.36×10¹¹

Dose of challenge/(CFU/mL)

Number of death/Total

Death rate/%

ΔvasH

ΔvasH/p-vasH

ΔvasH

ΔvasH/p-vasH

5×10⁶

0.00

5×10⁷

0.00

3.33

5×10⁸

33.33

0.00

3.33

5×10⁹

100.00

LD₅₀

7.34×10¹⁰

1.58×10¹¹

1.36×10¹¹