副溶血弧菌新II型毒素-抗毒素系统的鉴定和功能研究

Vibrio parahaemolyticus is an important food-borne pathogen causing severe threats to public health. Toxin-antitoxin (TA) systems are widely distributed in the genomes of bacteria and archaea, and possess important biological functions, including regulation of biofilm formation, drug resistance and pathogenicity. However, the impact of these systems on biofilm formation, drug resistance and pathogenicity of V. parahaemolyticus remains to be explored. By bioinformatics analysis, several putative type II TA loci have been predicted in the genome of V. parahaemolyticus RIMD2210633. In this study, we target identifying novel type II TA systems based on the features of a typical type II TA system. In-frame deletion mutants of these TA systems and the complementation strains will be constructed, and differences in growth, motility, biofilm formation, drug resistance and pathogenicity among these strains will be assessed. To investigate the effect of the newly identified type II TA systems on protein expression and reveal the molecular mechnism of the regulation by these systems, iTRAQ-based quantitative proteomics analysis of the mutants and wild-type strain will be performed, and the differentially expressed proteins will be identified. This study will be helpful to understand the mechanism of drug resistance and pathogenesis of V. parahaemolyticus, thus providing theoretical basis for prevention of the infection.

副溶血弧菌是一种重要的食源性病原菌，给公众健康带来严重危害。毒素-抗毒素系统广泛存在于细菌和古细菌的基因组中，具有重要的生物学功能，包括调控生物被膜形成、耐药和致病性等。然而，毒素-抗毒素系统对副溶血弧菌生物被膜形成、耐药和致病性的影响仍有待探索。申请人前期基于生物信息学分析，预测了副溶血弧菌基因组中编码的假定II型毒素-抗毒素系统。本项目拟根据II型毒素-抗毒素系统的典型特征，从中鉴定新的II型毒素-抗毒素系统；构建新鉴定II型毒素-抗毒素系统的基因缺失突变株和互补株，比较野生株、突变株和互补株在生长、运动能力、生物被膜形成、耐药和致病性方面的差异；通过iTRAQ定量蛋白质组学，分析突变株和野生株的差异表达蛋白，明确新鉴定II型毒素-抗毒素系统对蛋白表达的影响，并探讨其调控作用的分子机制。本项目研究成果有助于副溶血弧菌耐药和致病机制的阐明，也为预防其感染提供理论依据。

副溶血弧菌是全球范围内重要的食源性病原菌，食用被该菌污染的食品可能会引起急性肠胃炎。副溶血弧菌还是对虾急性肝胰腺坏死病的主要病原。毒素-抗毒素系统是细菌和古生菌基因组中的小型遗传元件，具有重要的生物学功能。本项目主要从毒素-抗毒素系统的角度探讨副溶血弧菌生物被膜形成、耐药和致病的分子机制。取得的主要研究成果如下：（1）在副溶血弧菌基因组中预测了6个假定II型毒素-抗毒素系统，证实vp1821-vp1820编码YefM-YoeB毒素-抗毒素系统；抗毒素YefM正调控启动子，YefM-YoeB负调控启动子。（2）对YefM-YoeB毒素-抗毒素系统的功能进行了研究，发现缺失yefM-yoeB影响副溶血弧菌的热应激反应，但不影响其它应激反应；YefM-YoeB对副溶血弧菌的运动性、生物被膜形成、耐药和致病性也没有显著影响。（3）发现在猪链球菌中诱导表达副溶血弧菌YoeB毒素具有显著的抑菌效应；以新鉴定的YoeB毒素为负筛选标记，构建了spc-PcopA-yoeBVp基因盒，该基因盒可以极大地提高猪链球菌基因缺失的效率。（4）基于TASmania数据库，预测副溶血弧菌基因组中编码另外28个假定毒素基因。在大肠杆菌中诱导这些假定毒素基因的表达，发现其中5个基因的表达会影响大肠杆菌的生长，说明他们编码毒素。本项目研究成果有助于揭示副溶血弧菌环境适应性的分子机制，也为基于毒素-抗毒素系统设计新药靶标、开发基因编辑工具奠定了基础。