Toll-like receptors (TLRs) are major pattern recognition receptors (PRRs) that recognize multiple pathogen-associated molecular patterns (PAMPs) through the leucine-rich repeat (LRR) domain and mount effective immune responses. Vibrio parahaemolyticus is the main pathogen that causes vibriosis in aquatic animals, yet the mechanisms of its recognition by innate immune system in teleost fish remain unknown. Here, the results reveal that TLR13 in orange-spotted grouper (Epinephelus coioides) (EcTLR13) recognizes a conserved 23S ribosomal RNA (23S rRNA) sequence in V. parahaemolyticus, and the 13-nucleotide motif near the 23S rRNA ribozyme activation site (VP13) acts as a PAMP. After challenge with RNA and 23S rRNA from V. parahaemolyticus and with the synthetic oligoribonucleotide VP13, the expression of EcTLR13 in grouper spleen cells (GS cells) was significantly increased. EcTLR13-knockdowned GS cells were stimulated with the same stimulants as listed above, the expression of IL-6, IL-12, IL-1β and TNFα was significantly reduced. RNA-protein immunoprecipitation revealed that VP13 could directly bind to EcTLR13. The dual-luciferase reporter assay also showed that EcTLR13 enhanced the fluorescence activity of IFNβ rather than that of NF-κB when the cells were challenged with RNA from V. parahaemolyticus or with synthetic VP13. Our study established the mechanism of fish TLR13-mediated recognition of microbial products during V. parahaemolyticus infection.

Toll 样受体 (TLR) 是主要的模式识别受体 (PRR)，可通过富含亮氨酸的重复 (LRR) 结构域识别多种病原体相关分子模式 (PAMP) 并产生有效的免疫反应。副溶血性弧菌是导致水生动物弧菌病的主要病原体，但硬骨鱼先天免疫系统对其的识别机制仍不清楚。在这里，结果表明橙斑石斑鱼 ( Epinephelus coioides ) ( Ec TLR13) 中的 TLR13 识别副溶血弧菌中保守的 23S 核糖体 RNA (23S rRNA) 序列，以及 23S rRNA 核酶激活位点附近的 13 个核苷酸基序。 VP13) 充当 PAMP。用来自的 RNA 和 23S rRNA 攻击后V. parahaemolyticus和合成寡核糖核苷酸 VP13 后，Ec TLR13 在石斑鱼脾细胞（GS 细胞）中的表达显着增加。用与上述相同的刺激物刺激Ec TLR13 敲低的 GS 细胞，IL-6、IL-12、IL-1β 和 TNFα 的表达显着降低。RNA-蛋白质免疫沉淀显示VP13 可以直接与Ec TLR13 结合。双荧光素酶报告基因分析还表明，当细胞受到副溶血性弧菌的 RNA 攻击时， Ec TLR13 增强了 IFNβ 的荧光活性，而不是 NF-κB 的荧光活性。或使用合成 VP13。我们的研究建立了副溶血性弧菌感染期间鱼类 TLR13 介导的微生物产物识别机制。