溶血相关基因缺失降低鳗弧菌感染花鲈的致病性

Deletion of hemolysis-related genes attenuates the pathogenicity of Vibrio anguillarum to Lateolabrax maculatus

为阐明毒力因子溶血素对鳗弧菌感染花鲈过程中发挥的作用,实验以花鲈为研究对象,通过对花鲈幼鱼分别注射5.0×10^(5)CFU鳗弧菌野生株和缺失株△vah1-vah4-rtxA,评估了溶血相关基因缺失对花鲈的感染能力、花鲈各组织的病理变化情况以及免疫响应的影响。结果显示,花鲈感染野生株后的LD_(50)为2.103×10^(5)CFU/mL,感染缺失株△vah1-vah4-rtxA后LD_(50)为1.837×10^(6)CFU/mL,溶血相关基因缺失使鳗弧菌毒性降到原来的11.44%;鳗弧菌主要定植在花鲈的肠道和鳃中,溶血相关基因缺失降低鳗弧菌在花鲈体内的定植能力,同时降低对鳃和肠道的损伤程度;转录组分析表明vah1、vah4和rtxA缺失后,头肾组织的差异表达基因显著富集到造血细胞谱系、抗原处理和呈递、细胞黏附分子、肠道免疫网络的IgA生产等免疫通路,并用实时荧光定量PCR(qRT-PCR)对表达水平进行了验证。研究表明,溶血相关基因缺失降低了鳗弧菌感染花鲈的致病能力。研究结果有助于进一步了解鳗弧菌的致病机制,并为开发花鲈抗弧菌免疫增强剂或减毒疫苗提供依据。

Lateolabrax maculatus is an important mariculture fish in China.In recent years,fishery losses caused by diseases have become more and more serious.Vibrio anguillarum is a gram-negative pathogenic bacterium widely distributed in seawater and marine organisms,and it is one of the important sources of bacterial diseases in mariculture and can cause serious infections in fish.Hemolysin is an essential component of virulence factor of V.anguillarum,which causes hemorrhagic septicemia in the host.However,very little information is available about hemolysin in L.maculatus.To explore the role of hemolysin in V.anguillarum infection of the fish,5.0×10^(5)CFU V.anguillarum wild strain or△vah1-vah4-rtxA was immunized by intraperitoneal injection,and the colonization ability of bacteria in L.maculatus,histopathological changes and immune response of L.maculatus after deletion of hemolysis-related genes were evaluated.The results showed that the virulence of V.anguillarum decreased by 8.74 times after the deletion of hemolysis-related genes,and the LD_(50)of wild strain or△vah1-vah4-rtxA was 2.103×10^(5)CFU/mL and 1.837×10^(6)CFU/mL,respectively.The intestine and gill were the main colonization sites of V.anguillarum,which were seriously damaged after infection.After the deletion of hemolysis-related genes,the colonizing ability of V.anguillarum and the damage degree of intestinal mucosa vacuolation and gill necrosis was reduced.The KEGG pathway enrichment analysis showed that the differentially expressed genes were significantly enriched into hematopoietic cell lineage,antigen processing and presentation,cell adhesion molecules and intestinal immune network for IgA production signal pathways related to immune response.The above results showed that deletion of hemolysis-related genes attenuated the pathogenicity of V.anguillarum to L.maculatus.The results of this study will help to further understand the pathogenic mechanism of V.anguillarum and provide a basis for the development of anti-Vibrio immune enhancer or attenuated vaccine for L.maculatus.