低温条件下灿烂弧菌JZ6致病性的初步研究

Metalloprotease is one of the most important virulence factors in pathogenic Vibrio splendidus, which has caused serious disease to many aquatic animals. In our previous studies, a cold-adapted pathogen, V. splendidus JZ6, was isolated and identified at low temperature. A kind of metalloprotease (named SPF1), which was secreted from strain JZ6, shared 94% similarity with the virulence factor Vsm of V. splendidus LGP32 in amino acid sequence. And the protein structure analysis showed that SPF1 possessed the same domains with the psychrotrophilic metalloprotease MCP-02 of Pseudoalteromonas sp. SM9913. Therefore, SPF1 might have both features of pathogenic factor and psychrotrophilic protease. Based on the previous studies, the metalloprotease SPF1 is used as the main object to study the primary reason for the pathogenicity of V. splendidus JZ6 at low temperature. And the theme of this research is to elucidate the regulatory mechanism of low temperature for the expression and secretion of SPF1. In this project, SPF1 will be cloned and expressed in Escherichia coli to identify the pathogenicity to the hemocyte of scallop, and test the influence for this pathogecity at different temperatures. Strain JZ6 will be cultured both at high and low temperatures to analyze the secretion of all extracellular proteins and SPF1. The key molecules, which involved in the transcription and secretion of SPF1 at low temperature, will be selected to clear the regulation for the functions of these critical molecules. Depending on above studies and experiments, we will exposit the regulatory mechanism for metalloprotease SPF1 of V. splendidus JZ6 at low temperature. And this project will be significant for in-depth studies to explore the cold-adapted and pathogenic mechanisms of pathogens in low temperature condition.

我们在前期研究中分离获得一株在低温条件下有致病性的适冷灿烂弧菌JZ6。该菌分泌的金属蛋白酶（SPF1）经序列和结构分析与灿烂弧菌LGP32毒力因子金属蛋白酶Vsm的相似度为94%；与交替假单孢菌低温金属蛋白酶MCP-02具有相同结构域。因此，SPF1兼具有毒力因子和低温酶的结构特性。本项目拟采用现代分子生物学和微生物学等手段，克隆和体外重组表达灿烂弧菌JZ6金属蛋白酶SPF1，鉴定金属蛋白酶SPF1的致病性以及低温对其致病性的影响；研究温度对灿烂弧菌JZ6胞外蛋白产物和SPF1分泌量的调节作用；筛选参与低温条件下SPF1转录、表达以及分泌过程的关键分子，明确低温对这些关键分子功能的调节作用；比较分析在低温和高温条件下SPF1的转录、表达和分泌情况，探讨低温对SPF1致病性的调节作用，进一步揭示灿烂弧菌在低温条件下的致病机制，丰富和发展水产动物病原学内容。

本项目以虾夷扇贝低温致病性灿烂弧菌V. splendidus JZ6为研究对象，旨在初步揭示灿烂弧菌JZ6的低温的适应性和致病性机制。主要成果概述如下：. （1）明确了灿烂弧菌JZ6中金属蛋白酶Vsm的毒性以及不同温度对其毒性的影响. 通过对灿烂弧菌V. splendidus JZ6进行全基因组测序分析，获得了其主要毒力因子金属蛋白酶基因全长；根据金属蛋白酶Vsm的结构特性，分别对VsmM中的不同结构域进行原核重组表达，获得了有活性的Vsm结构域重组蛋白；分析了不同温度条件下Vsm重组蛋白对扇贝血细胞的毒性作用，确定了温度对Vsm细胞毒性的影响。. （2）明确了温度对灿烂弧菌JZ6致病性的调节作用. 分别将灿烂弧菌JZ6和另一株非致病性灿烂弧菌TZ19在10℃和28℃的培养菌体进行转录组测序，构建了4个转录组文库并对它们的表达差异基因进行了比较，共获得2359个差异基因。发现共有358个基因与灿烂弧菌JZ6的低温适应性和致病性相关。. （3）明确了低温条件下参与灿烂弧菌JZ6致病性的关键分子. 利用转录组数据的分析和qRT-PCR方法的验证，在低温条件下上调表达的基因中获得了10个与灿烂弧菌JZ6致病性密切相关的基因，这些基因主要参与灿烂弧菌JZ6中菌毛形成、密度感应系统激活、毒力因子合成以及分泌过程。以上成果在转录水平上解释了灿烂弧菌JZ6在低温条件下致病性升高的原因，为进一步揭示灿烂弧菌JZ6在低温条件下的致病机制奠定了基础。. （4）明确了灿烂弧菌JZ6在低温条件下调控金属蛋白酶表达和致病性的机制. 利用基因组和转录组的共同分析，发现灿烂弧菌JZ6中存在的受温度影响的毒力因子表达调控机制与CsrA-RNA互作系统密切相关。发现了一个调控金属蛋白酶Vsm毒性的蛋白酶PepTL，该蛋白酶主要参与金属蛋白酶Vsm前体的切割，使无活性的Vsm转变成有活性的Vsm。该蛋白酶在低温条件下同样发生了高表达，说明了灿烂弧菌JZ6中存在较为完整的毒力因子表达和活性的调控机制。. 本研究获得了以上原创性的科学发现，研究成果在国内外相关领域发表SCI论文2篇。以上研究成果对揭示灿烂弧菌JZ6低温适应和致病性调控机制意义重大。