含铁肠杆菌素受体调节蛋白VPA0148对副溶血弧菌毒力的影响

doi:10.11978/2020146

摘要/Abstract

摘要：

副溶血弧菌(Vibrio parahaemolyticus)是一种嗜盐性革兰氏阴性菌, 广泛分布于海洋环境中, 能够引起肠胃炎、伤口感染以及败血症, 是人类重要的病原菌之一。宿主体内被认为是一种低铁环境, 能够激发致细菌的毒力。副溶血弧菌能够分泌弧菌素或利用外源铁载体来获取铁。在低铁条件下, 编码双组份调控系统的基因簇VPA0148-VPA0149转录上调, 使含铁肠杆菌素受体PeuA转录出有活性的蛋白。VPA0148编码产物为一个响应调节因子, 具有一个磷酸基团接收结构域和一个DNA结合结构域。本研究发现, VPA0148的缺失增强了菌株的生物膜形成能力和对斑马鱼的致死能力, 同时能够促进菌株在低铁环境中的生长, 并增强菌株的群集运动。研究结果表明含铁肠杆菌素受体调节蛋白VPA0148对副溶血弧菌致病力具有调控作用, 增进了对铁调节副溶血弧菌毒力机制的认识。

关键词: 副溶血弧菌, 含铁肠杆菌素受体调节蛋白, 毒力, 运动能力, 生物膜

Abstract:

Vibrio parahaemolyticus is a halophilic Gram-negative bacterium originally inhabiting marine environment. It is one of the primary human pathogenic bacteria, which causes gastroenteritis, wound infections and septicemia. The host environment is low-iron conditions, thus capable of inducing the virulence of pathogens. V. parahaemolyticus utilizes vibriocin or heterogenous siderophore to obtain iron. The transcription of gene cluster VPA0148-VPA0149 (encoding a two-component regulatory system) is upregulated under iron-limiting conditions, which increases the active transcriptional form of ferric enterobactin receptor gene peuA. VPA0148 encodes a response regulator containing a phosphoacceptor receiver (REC) domain and a DNA-binding domain. In this study, the deletion of VPA0148 enhanced the lethality to zebrafish as well as biofilm formation in V. parahaemolyticus. Moreover, under iron-limiting conditions, the growth and swarming motility were improved in VPA0148 deletion mutant. The results indicate that VPA0148 contributes to regulating the pathogenesis of V. parahaemolyticus. This study provides an important insight for further understanding the mechanism of how iron regulates virulence of V. parahaemolyticus.

Key words: Vibrio parahaemolyticus, VPA0148, virulence, swarming, biofilm

中图分类号:

S941.42

朱馨媛, 刘敏, 黄颖, 赵哲. 含铁肠杆菌素受体调节蛋白VPA0148对副溶血弧菌毒力的影响[J]. 热带海洋学报, 2021, 40(6): 93-101.

ZHU Xinyuan, LIU Min, HUANG Ying, ZHAO Zhe. Effects of the ferric enterobactin receptor regulator VPA0148 on virulence of Vibrio parahaemolyticus[J]. Journal of Tropical Oceanography, 2021, 40(6): 93-101.

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	名称	描述	来源或参考文献
菌株	RIMD2210633 (WT)	副溶血弧菌血清型O3:K6临床分离株; 野生型菌株	(Makino et al, 2003)
	ΔVPA0148	副溶血弧菌VPA0148基因缺失株	本研究
	ΔVPA0148::pVPA0148	副溶血弧菌VPA0148基因回补株	本研究
	WT::pVPA0148	含有VPA0148-pBBR1-MCS-1质粒的副溶血弧菌野生型菌株	本研究
	S17-1λpir	Thi pro hsdR hsdM+ recA RP4-2-Tc::Mu-Km::Tn7λpir接合供体菌, 大肠杆菌	(Milton et al, 1992)
质粒	pDM4	Cm^r, 含依赖π蛋白oriP6K复制子和sacBR基因的自杀质粒,	(Milton et al, 1996)
	pBBR1-MCS-1	Cm^r, 广宿主蛋白表达或克隆载体质粒, 高拷贝	(Kovach et al, 1994)
	VPA0148-pBBR1-MCS-1	Cm^r, 插入VPA0148基因及其启动子片段的pBBR1-MCS-1质粒	本研究
	ΔVPA0148-pDM4	Cm^r, 插入VPA0148基因缺失片段的pDM4质粒	本研究

名称	序列(5′—3′)	用途
d0148-1F	CCCTCGAGGTTGCCAGTTGGCACGATG	用于扩增VPA0148侧翼区域进行基因敲除
d0148-1R	CCACAGATTCTAAGCCAAATTACCACTTAAATAAAAATGG
d0148-2F	TATTTAAGTGGTAATTTGGCTTAGAATCTGTGGGCGCGTG
d0148-2R	CGAGCTCTCATCAATGCTGCGTTTGAATTG
p0148-F	GTCGACGGTATCGATAAGCTTGTTGCCAGTTGGCACGATG	扩增VPA0148启动子及基因片段进行回补质粒构建
p0148-R	GGGCGAATTGGAGCTTCACTTGTCATCGTCATCCTTGTAATCCGCGCCCACAGATTCTA	扩增VPA0148启动子及基因片段进行回补质粒构建
RT0148F	CATTTGTAATACCGCCCAGAG	荧光定量
RT0148R	GATCACCACGCCTTGATTCTT	荧光定量