acfA基因在溶藻弧菌定植过程中的调控机制

The intestinal colonization is an important step during the host infected by Vibrio. In previous study, we found the expression of acfA gene could promote the formation of the polar flagella and inhibit the synthesis of biofilm formation, thereby regulating their ability of colonization in host intestines. Based on the results, we intends to further study the mechanism for acfA regulating Vibrio alginolyticus colonization in in host intestines. The proposal is as following: (1) Analyzing the transcripts and whole cell proteins of the mutant strain and wild strain under the culture conditions of the simulative marine environment and infected fish using KEGG pathway enrichment Analysis of gene expression with RNA-Seq and 2D-DIGE technology; (2)Searching the target genes related with polar flagella and biofilm regulated by acfA regulation; (3)Constructing the mutant strain deleting a different target gene, then measuring the phenotype and colonization ability to grouper intestines; (4) verifying the specific regulatory relationships between acfA regulator and target genes with the experiments of qRT-PCR, lacZ reporter gene fusion and primer extension in vivo; (5) identifying the interaction between acfA regulator and target genes with the techniques of Western blotting, EMSA and Dnase I footprint in vitro. The results will reveal the intrinsic link and network control mechanism between acfA regulator and target genes, and ultimately clarify the mechanism for acfA Regulating Vibrio alginolyticus colonization.

菌体在水生动物肠道定植是弧菌侵染宿主的重要环节。申请人在前期工作中发现溶藻弧菌acfA基因的表达可以促进其极生鞭毛形成及抑制生物被膜合成，从而调控该菌对宿主定植能力。本项目拟通过构建溶藻弧菌acfA缺失株，RNA-Seq及2D-DIGE技术分析模拟海水环境和感染鱼体环境条件下培养的缺失株及野生株转录本和全菌蛋白，利用KEGG通路富集分析高表达基因，搜索acfA调控极生鞭毛和生物被膜的靶基因；构建不同靶基因缺失株，测定其表型及对斜带石斑鱼的肠道定植能力；qRT-PCR、lacZ报告基因融合及引物延伸等体内实验技术验证acfA调控子对靶基因的具体调控关系；Western blotting、EMSA及Dnase I足迹等体外实验技术鉴定acfA调控子与靶基因的互作关系；以期揭示acfA基因调控下不同靶基因之间的内在联系及网络调控机制，最终阐明溶藻弧菌acfA在其定植过程中的调控机制。

溶藻弧菌(Vibrio alginolyticus)是一种全球性分布的条件致病细菌，能致使鱼、虾、贝等各种海洋生物出现不同程度的发病，尤其近年来养殖水质环境频繁出现污染、水体富营养化的现象，由溶藻弧菌引起的弧菌病呈现流行范围广，暴发频繁的趋势，给水产养殖业造成巨大的损失并影响其健康发展。在宿主肠道上成功定植并进行繁殖是弧菌感染宿主和引起疾病的关键第一步。其中附属定植因子AcfA是弧菌重要毒力蛋白之一，AcfA可以使致病菌有效地与宿主黏膜粘附，而关于溶藻弧菌AcfA相关功能的研究较少。本研究以溶藻弧菌HY9901为野生株，利用同源重组技术构建溶藻弧菌acfA基因缺失株∆acfA。利用16S rDNA高通量测序技术研究溶藻弧菌野生株WT和缺失株∆acfA在石斑鱼肠道的定植情况。通过转录组测序和蛋白质组学研究AcfA对鞭毛组装、能量代谢等相关基因的调控。根据转录组测序和荧光定量PCR分析，敲除溶藻弧菌sodB基因，研究sodB基因的缺失是否影响溶藻弧菌的生物学特征和毒力。采用β-半乳糖苷酶报告基因和细菌单杂交实验来检测溶藻弧菌AcfA与sodB、flgF、flgG、flgI和dctQ基因启动子区的结合作用。结果发现，AcfA对sodB、flgG和dctQ具有正调控关系，对flgF和flgI具有负调控关系。AcfA可直接与flgF和flgI基因启动子区结合，但未与sodB、flgG和dctQ基因启动子区相结合，表明AcfA对其调控是间接的。