Fisheries College, Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Jimei University, Xiamen, Fujian, 361021, China.
State Key Laboratory of Large Yellow Croaker Breeding, Ningde, Fujian, 352000, China.
Fish Shellfish Immunol. 2019 Oct;93:949-957. doi: 10.1016/j.fsi.2019.08.040. Epub 2019 Aug 18.
Pseudomonas plecoglossicida is a Gram-negative aerobic bacterium that causes high mortality and serious economic losses in some commercial marine fish. Expression of secY was found to be significantly upregulated at 18 °C compared to 28 °C by RNA-seq and qRT-PCR. All five tested recombinant vectors (pCM130/tac + shRNA) significantly reduced secY mRNA levels in P. plecoglossicida. The recombinant vector encoding shRNA-1165 exhibited the best gene-silencing efficiency, 82.4% and was used to create an RNAi strain for further studies. Compared with the wildtype strain, infections of Larimichthys crocea with the RNAi strain resulted in a 2-day delay in onset time and a 35% reduction in mortality, as well as the alleviation of spleen symptoms. The spleens of L. crocea infected by the wild type or RNAi strain of P. plecoglossicida were subjected to dual RNA-seq at 2 dpi. Compared with the wildtype strain, infection of P. plecoglossicida with the RNAi strain resulted in significant changes in the transcriptomes of both host and pathogen. KEGG analysis showed that the complement and coagulation cascade and the Toll-like receptor signalling pathway were the most enriched host pathways. In the pathogen, genes of the "Sec secretion system" were significantly downregulated. This downregulation of "Sec secretion system" genes hindered the secretion of bacterial proteins and reduced the virulence of P. plecoglossicida. Thus, it was easier for L. crocea to clear the RNAi strain of P. plecoglossicida, and the immune response was similarly reduced. The results indicated that secY was a virulence gene of P. plecoglossicida and played roles in the host-pathogen interactions of L. crocea and P. plecoglossicida.
鮰爱德华氏菌是一种革兰氏阴性需氧菌，可导致一些商业海水鱼的高死亡率和严重的经济损失。通过 RNA-seq 和 qRT-PCR 发现，与 28°C 相比，secY 在 18°C 时表达显著上调。所有五种测试的重组载体（pCM130/tac+shRNA）均显著降低了鮰爱德华氏菌中的 secY mRNA 水平。编码 shRNA-1165 的重组载体表现出最好的基因沉默效率，为 82.4%，并用于构建 RNAi 菌株进行进一步研究。与野生型菌株相比，感染 RNAi 菌株的大黄鱼的发病时间延迟了 2 天，死亡率降低了 35%，同时脾脏症状也得到了缓解。在 2dpi 时，用野生型或 RNAi 菌株感染大黄鱼的脾脏进行了双重 RNA-seq。与野生型菌株相比，感染 RNAi 菌株的鮰爱德华氏菌的宿主和病原体的转录组均发生了显著变化。KEGG 分析表明，补体和凝血级联以及 Toll 样受体信号通路是最丰富的宿主途径。在病原体中，“Sec 分泌系统”的基因显著下调。这种“Sec 分泌系统”基因的下调阻碍了细菌蛋白的分泌，降低了鮰爱德华氏菌的毒力。因此，大黄鱼更容易清除鮰爱德华氏菌的 RNAi 菌株，免疫反应也相应降低。结果表明，secY 是鮰爱德华氏菌的一个毒力基因，在大黄鱼和鮰爱德华氏菌的宿主-病原体相互作用中发挥作用。
