拟态弧菌OmpU蛋白的黏附功能及所介导的致病作用

Adhesion function of OmpU protein from Vibrio mimicus and its role in virulence

为了探明拟态弧菌OmpU蛋白的黏附功能,利用同源重组技术敲除基因组中OmpU基因,并构建其互补株,再经组合PCR方法和序列测定,证实了OmpU基因的缺失和互补。对野生株、缺失株和互补株进行了遗传稳定性、生长特性、生化特性、细胞黏附性、致病性等方面比较研究。结果显示,缺失株具有遗传稳定性;在相同的培养条件下,与野生株相比,突变株的培养特性和生化特性没有明显变化,生长速率略减慢,对实验草鱼的毒力降低了4倍,对鲤上皮瘤细胞(EPC)的黏附能力显著降低,下降了66.6%,而互补株的黏附能力和毒力又得到恢复,与野生株无明显差异。研究首次确证了拟态弧菌OmpU蛋白具有黏附功能,OmpU蛋白通过黏附参与致病作用。

Vibrio mimicus is the causative agent of ascites disease in aquatic animals. It was found that there was an adhesion motif at amino-terminal of OmpU protein,and OmpU-specific antibody exhibited significant inhibition effects on the adhesion of V. mimicus to cells in our previous studies,but adhesion function of OmpU protein is still unclear. To investigate the role of OmpU protein from V. mimicus in cell adhesion and virulence,the OmpU gene deletion mutant of V. mimicus 04-14 strain was constructed by the homologous recombination. A recovery mutant was obtained by introducing a low-copy plasmid strain. PCR and sequence analysis confirmed that OmpU gene was successfully deleted in the mutant and restored in the complementary strain. We compared the differences of genetic stability,growth and biochemical characteristics,adhesion ability and virulence between the gene deletion mutant and wild strains,as well as the complemented strain. The results showed that the mutant strain stabilized genetically and grewslower than the wild strain. There was no significant difference between them in morphology,culture and biochemical characteristics. Compared with wild strain,deletion of OmpU gene significantly reduced bacterial adhesion to EPC cells monolayers by 66. 6%,but the adhesion ability was restored in the complementary strain. Furthemore,the 50% lethal doses（ LD50） of the mutant strain was four times lower than those of the wild strain and complementary strain. These data indicate that the OmpU protein is involved in both adhesion and virulence in V. mimicus. The adhesion function of OmpU protein from V. mimicus and its role in virulence were comfirmed for the first time.