副溶血弧菌T3SS1效应蛋白的鉴定及分子致病机制研究

Vibrio parahaemolyticus belongs to Gram-negative halophilic bacterium, is an important foodborne pathogens, the relevant departments survey showed that the incidence of foodborne diseases increased year by year. The type III secretion system is a necessary factor in the pathogenesis of Gram-negative bacterial infections by which pathogenic bacteria release effector proteins (virulence factors) into host cells. Studies have shown that these effector proteins can interfere with autophagy, apoptosis and Immune response signal pathway and so on. At present, there are few reports on the type Ⅲ secretion system of Vibrio parahaemolyticus (T3SS), and the spectrum of effector proteins has not been elucidated. Therefore, bioinformatics software was used to predict T3SS effector protein of Vibrio parahaemolyticus in combination with proteomics technology. A series of technical methods were used to identify T3SS effector mediated Vibrio parahaemolyticus infection and to study the role of effector protein as a virulence factor in host cell autophagy, apoptosis and signal transduction pathway. Through GST pull-down and yeast two-hybrid system screening and identification of key effect protein interaction with the receptor of host, revealing the molecular interaction mechanism of T3SS effector protein as an important virulence factor of Vibrio parahaemolyticus, thus providing a valuable reference for identifying new drug targets of Vibrio parahaemolyticus and developing potential subunit vaccine tags.

副溶血弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌，该菌引发的食源性疾病逐年增多。革兰氏阴性菌可通过Ⅲ型分泌系统向宿主细胞释放效应蛋白来干扰细胞自噬、凋亡和免疫应答信号通路等，发挥毒性作用使宿主致病。目前，对副溶血弧菌的Ⅲ型分泌系统(T3SS)研究较少，其效应蛋白谱也没被阐明。因此，本研究以筛选副溶血弧菌T3SS效应蛋白为切入点，运用生物信息学软件预测并结合蛋白质组学技术筛选该菌T3SS效应蛋白，通过系列技术方法鉴定T3SS效应因子介导该菌感染的致病作用，研究效应蛋白作为毒力因子在干扰宿主细胞相关生理活动所发挥的作用；通过GST pull-down和酵母双杂交系统等技术筛选鉴定关键效应蛋白的宿主互作受体，揭示T3SS效应蛋白作为该菌重要毒力因子的分子互作机制，从而为副溶血弧菌新药物靶标筛选和潜在的亚单位疫苗标签研发提供参考。

副溶血弧菌(Vibrio parahaemolyticus)是一种重要的食源性致病菌，该菌引发的食源性疾病逐年增多。革兰氏阴性菌可通过Ⅲ型分泌系统向宿主细胞释放效应蛋白来干扰细胞自噬、凋亡和免疫应答等，从而使宿主致病。而副溶血弧菌的Ⅲ型分泌系统具体作用机制仍待探索，其效应蛋白谱尚未完全阐明。.本项目通过对副溶血弧菌的T3SS1效应蛋白进行生物信息学预测，应用T3_MM和Bastion3对 GenBank中公布的副溶血弧菌 RIMD2210633 菌株全基因组序列进行筛选，得到123个候选假定蛋白，随后构建过表达载体，转入POR-1和POR-2，在T3SS1诱导条件下，检测各菌株上清中的假定蛋白，筛选出能够向菌体外分泌的假定蛋白，并将相关菌株感染HeLa细胞以鉴定可向HeLa细胞易位的效应蛋白。最终研究确定了VPA0226是副溶血弧菌T3SS1新发现的效应蛋白，筛选出51个能够分泌到菌体外的细菌蛋白，其中14个能够向Hela细胞易位，是潜在的T3SS1效应蛋白；此外，本次研究还探索了T3SS1编码的针状蛋白基因vscF及其编码的内杆蛋白基因vscI对VPA0226发挥其功能的影响，发现VscF发挥分泌功能时需要潜在的伴侣蛋白VopD、VscP和VscG；除此外，研究还对针状蛋白基因vscF在副溶血弧菌感染Hela细胞期间的转录组显著调控基因进行了分析，发现了120个显著差异表达基因，其中70个基因显著上调和 50个基因显著下调，而其中差异表达基因在群体感应系统、细菌趋化性、分泌系统、脂肪酸代谢和生物合成以及牛磺酸和低牛磺酸代谢中得到了显著富集，此外T3SS1的下调对T3SS2的表达有积极影响。.本项目筛选了潜在的T3SS1效应蛋白，完善了副溶血弧菌的效应蛋白谱；并且通过项目开展进一步为副溶血弧菌感染过程中T3SS1发挥致病作用的具体机制进行了探索，为更好的阐明T3SS1致病机制奠定了基础。