嗜水气单胞菌Ⅵ型分泌系统假定效应蛋白基因tle1^AH的生物学功能  

Biological functions of the type Ⅵ secretion system putative effector gene tle1^AH in Aeromonas hydrophila

作　　者：马水燕 王楠楠[1] 董雨豪 刘锦 陆承平[1] 刘永杰[1] MA Shuiyan;WANG Nannan;DONG Yuhao;LIU Jin;LU Chengping;LIU Yongjie(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学动物医学院,江苏南京210095

出　　处：《南京农业大学学报》2020年第5期896-902,共7页Journal of Nanjing Agricultural University

基　　金：国家自然科学基金项目(31372454);江苏省水产三新工程项目(D2017-3-1)。

摘　　要：[目的]本文旨在鉴定和研究嗜水气单胞菌中国流行株NJ-35的Ⅵ型分泌系统(T6SS)新型效应蛋白及其功能,进一步阐明其致病机制。[方法]基于已报道的保守结构域DUF4123,对NJ-35株全基因组序列进行潜在的T6SS效应蛋白基因预测,采用同源重组技术构建基因缺失株和相应互补株。对野生株、缺失株和互补株分别进行细菌竞争试验,并测定细菌生长曲线、运动性、生物被膜形成能力、抗巨噬细胞吞噬能力及其对斑马鱼的半数致死量(LD50)等。[结果]成功预测到假定的效应蛋白基因tle1^AH(U876-17550),并构建tle1^AH基因缺失株(Δtle1^AH)和相应互补株(CΔtle1^AH)。特性分析发现,与野生株比较,缺失株抑制大肠杆菌生长和抗小鼠巨噬细胞吞噬的能力明显降低,生物被膜形成能力增强,对斑马鱼的LD50增加10余倍(P<0.01),互补株各项指标均恢复到野生株水平。[结论]鉴定到嗜水气单胞菌T6SS的一个假定效应蛋白基因(tle1^AH),并对其功能进行初步研究,为进一步探讨T6SS在该菌致病过程中的作用奠定基础。[Objectives]The aim of the study was to identify the novel effectors of typeⅥsecretion system(T6SS)and explore its function in Aeromonas hydrophila Chinese epidemic strain NJ-35,which was beneficial to further investigating the pathogenesis of pathogens.[Methods]The putative effector was screened in the whole genome of A.hydrophila NJ-35 based on the reported conservative domain DUF4123.Furthermore,the mutant and complementation strains were constructed via homologous recombination,and the competitiveness,growth,motility,biofilm formation,anti-phagocytosis and virulence in zebrafish were measured.[Results]A putative gene was obtained and named tle1^AH(U876-17550),and its mutant(Δtle1^AH)and complemented(CΔtle1^AH)strains were constructed.TheΔtle1^AH showed a significant decrease in anti-bacterial efficacy against Escherichia coli and anti-phagocytosis activity.In addition,the biofilm formation ability ofΔtle1^AH increased significantly,and its virulence decreased in zebrafish,more than a 10-fold increase in LD50 over the wild-type strain(P<0.01).Meanwhile,the above characteristics of CΔtle1^AH were restored to the same level of the wild type strain.[Conclusions]In this study,a functional analysis was conducted on a putative effector protein of T6SS in A.hydrophila,which provides a basis for further research on the role of T6SS in the pathogenic mechanism of this bacteria.

关 键 词：嗜水气单胞菌 Ⅵ型分泌系统 效应蛋白 tle1^AH基因 生物学功能 

分 类 号：S855.12[农业科学—临床兽医学]