效应蛋白Afp17在哈维氏弧菌T3SS致鱼类细胞死亡中的作用及分子机制

Vibrio harveyi, a Gram-negative marine bacterium, is one of the major bacterial pathogens in aquaculture; however, its pathogenic mechanisms remain to be further elucidated. Type III secretion system (T3SS) is a highly-conserved apparatus among many gram-negative pathogenic bacteria, which delivers bacterial virulence proteins, called effectors, into host cells and is directly involved into toxic effect on host cells. In our early works, we found that a unique protein, named Afp17, was present in the effector-encoded region of V. harveyi T3SS, and can be secreted into culture supernatant in a T3SS-dependent pattern in the induced condition. Importantly, Afp17 contains a conserved ADP-ribosyltransferase domain (ADPRT) in its C-terminus, implying that the protein could be a virulence factor of V. harveyi. Based on the above analysis, we firstly confirm that Afp17 is an effector of V. harveyi T3SS by observing the translocation of Afp17 into fish cells using the fluorescence resonance energy transfer (FRET) translocation assay in this study. Then, we determine the role of effector Afp17 in V. harveyi T3SS-caused death of fish cells and the functional domain(s) and critical amino acid residue(s) that are essential for the cytotoxicity role using gene knockout and site-directed mutagenesis techniques. Secondly, we also identify protein substrate(s) in fish cells that is modified by the ADPRT activity of Afp17, and elucidate how the effector alter gene expression of host cells in protein level using quantitative proteomics based approaches and post-translational modification analysis, and accordingly, we figure out the cellular signaling pathways that are manipulated by effector Afp17, and verify that these pathways are important for effector Afp17-induced cell death in fish cells using RNA interference(RNAi). Finally we will cast light on the role of effector Afp17 in V. harveyi T3SS-caused death of fish cells at a molecular level. All data obtained from the study will contribute to illustrate pathogenic mechanisms of V. harveyi, and also provides valuable reference for finding novel molecular drug target to prevent and control the Vibriosis caused by the bacterium.

哈维氏弧菌是海水养殖动物的重要病原菌之一，但其致病机理尚不明确。III型分泌系统(T3SS)是许多病原菌中保守存在的毒力装置，通过分泌效应蛋白对宿主细胞产生毒性。前期工作发现哈维氏弧菌存在一个依赖T3SS而分泌的独特蛋白Afp17，其含有ADP核糖转移酶结构域。基于此，本项目拟先利用FRET技术观察Afp17被分泌至鱼类细胞内部，以证实其是T3SS的效应蛋白；再运用基因敲除和定点突变等技术明确Afp17在哈维氏弧菌T3SS致鱼类细胞死亡中的作用及发挥作用所必须的功能结构域和关键氨基酸；然后利用翻译后修饰蛋白质组学技术查找Afp17核糖基化修饰的细胞靶蛋白，并结合定量蛋白质组学和RNA干扰等手段查明并验证Afp17所操控的细胞死亡相关信号通路，最终在分子水平上阐明效应蛋白Afp17发挥致细胞死亡作用的机制。研究结果将有助于揭示哈维氏弧菌的致病机理，也为寻找防控弧菌疾病的分子靶标提供理论参考。

哈维氏弧菌是海水养殖动物的重要病原菌之一，其致病机理备受关注。III型分泌系统(type III secretion system, T3SS)是许多病原菌中保守存在的毒力装置，通过分泌效应蛋白对宿主细胞产生毒性，因此鉴定效应蛋白对于揭示弧菌致病机理具有重要意义。前期研究发现Afp17基因存在哈维氏弧菌T3SS基因簇，可能是其编码的新效应蛋白。为此，在本项目中首先系统分析了Afp17蛋白的分布及进化情况，发现其主要存在于Harveyi分支成员中，如：哈维氏弧菌、坎氏弧菌、欧文氏弧菌和塔氏弧菌，且在分化之前Afp17就已经存在；随后利用基于荧光共振能量转移（FRET）技术显示Afp17蛋白可依赖于哈维氏弧菌T3SS分泌，且在线EffectiveDB软件预测和截短实验显示在其N端存在一个强的T3SS信号肽。基因敲除和回补等实验揭示Afp17蛋白在哈维氏弧菌T3SS诱导的宿主细胞（鱼类细胞FHM和哺乳动物HeLa）变圆中的作用，在鱼类细胞中负责激活细胞凋亡途径，但不参与T3SS导致的LDH释放；定点突变、回补实验和体外转染表达等实验显示Afp17蛋白的N端和C端对其发挥细胞毒性作用都必不可少，其保守ADPRT结构域及Glu832和Glu834对Afp17发挥致细胞毒性功能都是必须的；免疫印迹实验证实Afp17蛋白可能对宿主细胞中细胞骨架蛋白Ras进行修饰，进而导致细胞变圆的表型；基于iTRAQ/TMT标记定量蛋白质组学技术分析显示哈维氏弧菌野生型菌株和Afp17缺失突变株感染的鱼类细胞组中分别鉴定到920个和849个表达上调的差异表达蛋白，475个和309个表达下调的差异表达蛋白；KEGG通路富集和聚类分析显示差异表达蛋白主要富集到4个与细胞死亡相关的信号通路，分别是凋亡信号通路、MAPK信号通路、肌动蛋白介导的细胞骨架信号通路和细胞紧密连接信号通路等。本项目还从副溶血弧菌和溶藻弧菌中鉴定出调控T3SS的双组分系统，其中副溶血弧菌为LuxQ-ArcB-LuxO，溶藻弧菌为ArcB-LuxU-LuxO，它们依此行使组氨酸激酶、磷酸转移（Hpt）蛋白和反应调节蛋白条件蛋白。以上研究结果阐明了效应蛋白Afp17的功能，有助于揭示哈维氏弧菌T3SS致宿主细胞死亡的机制，而且还拓展了弧菌T3SS基因表达的调控新机制，对于针对弧菌T3SS效应蛋白或调控途径寻找防控弧菌疾病的提供了靶标