Both NLRC3 and NOD1 belong to regulatory NLR subfamily based on their best-characterized function. In mammals, NLRC3 was reported to function by attenuating signaling cascades initiated by other families of PRRs. In teleosts, multiple NLRC3-like genes were identified through transcriptome sequencing. However, the functions of many NLRC3-like genes, especially the fish-specific NLRC3-like genes, remain unclear. In the present study, we report the functional characterization of a novel category of NLRC3-like proteins (named as NLRC3-like 1) from the zebrafish, which consists of a fish-specific FISNA, a conserved NACHT and five C-terminal LRRs domains. The expression of zebrafish NLRC3-like 1 was inducible in response to Edwardsiella piscicida infection. During bacterial infection, the in vitro and in vivo studies revealed that zebrafish NLRC3-like 1 overexpression facilitated bacterial growth and dissemination, together with the decreased survival rate of zebrafish larvae infected with E. piscicida. The attenuated response by zebrafish NLRC3-like 1 in response to bacterial infection were characterized by the impaired expression of antibacterial genes, proinflammatory cytokines and Nox genes. Furthermore, zebrafish NLRC3-like 1 interacted with the adaptor protein RIPK2 of NODs signaling via the FISNA (Fish-specific NACHT associated domain) and NACHT domains. However, the interaction between zebrafish NLRC3-like 1 and RIPK2 inhibited the assembly of the NOD1-RIPK2 complex. Importantly, zebrafish NLRC3-like 1 inhibited NOD1-mediated antibacterial activity, NF-κB and MAPK pathways and proinflammatory cytokine production. All together, these results firstly demonstrate that zebrafish NLRC3-like 1 inhibits NOD1-RIPK2 antibacterial pathway via targeting the adaptor protein RIPK2.

NLRC3 和 NOD1 都属于调节性 NLR 亚家族，基于它们的最佳表征功能。在哺乳动物中，据报道 NLRC3 通过减弱由其他 PRR 家族发起的信号级联来发挥作用。在硬骨鱼中，通过转录组测序鉴定了多个 NLRC3 样基因。然而，许多 NLRC3 样基因的功能，尤其是鱼类特异性 NLRC3 样基因，仍不清楚。在本研究中，我们报告了来自斑马鱼的一类新型 NLRC3 样蛋白（命名为 NLRC3 样 1）的功能表征，该蛋白由鱼特异性 FISNA、保守的 NACHT 和五个 C 端 LRRs 域组成. 斑马鱼 NLRC3 样 1 的表达是响应于爱德华氏菌感染诱导的。在细菌感染过程中，体外和体内的研究显示，斑马鱼NLRC3样1倍的过表达促进了细菌的生长和传播，连同减少感染斑马鱼的存活率E. piscicida. 斑马鱼 NLRC3-like 1 对细菌感染的反应减弱，其特征是抗菌基因、促炎细胞因子和 Nox 基因的表达受损。此外，斑马鱼 NLRC3 样 1 通过 FISNA（鱼特异性 NACHT 相关域）和 NACHT 域与 NOD 信号转导的接头蛋白 RIPK2 相互作用。然而，斑马鱼 NLRC3-like 1 和 RIPK2 之间的相互作用抑制了 NOD1-RIPK2 复合物的组装。重要的是，斑马鱼 NLRC3-like 1 抑制了 NOD1 介导的抗菌活性、NF-κB 和 MAPK 通路以及促炎细胞因子的产生。总之，这些结果首先证明斑马鱼 NLRC3-like 1 通过靶向衔接蛋白 RIPK2 抑制 NOD1-RIPK2 抗菌途径。