Abstract
Chinese Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes, leading to the production of excessive reactive oxygen species (ROS) and irreversible apoptotic cell death. Emerging evidence suggests that mitochondrial autophagy (mitophagy) is the most effective method for eliminating damaged mitochondria and ROS, with choline dehydrogenase (CHDH) identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3 (LC3) in vertebrates. However, the functional role of CHDH in invertebrates is largely unknown. In this study, we observed a significant increase in the mRNA and protein expression levels of A. japonicus CHDH (AjCHDH) in response to V. splendidus infection and lipopolysaccharide (LPS) challenge, consistent with changes in mitophagy under the same conditions. Notably, AjCHDH was localized to the mitochondria rather than the cytosol following V. splendidus infection. Moreover, AjCHDH knockdown using siRNA transfection significantly reduced mitophagy levels, as observed through transmission electron microscopy and confocal microscopy. Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region (LIR) for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1. The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis. Furthermore, laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells. In contrast, AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria, a critical step in damaged mitochondrial degradation. Thus, AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS, followed by increased apoptosis and decreased coelomocyte survival. Collectively, these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A. japonicus following V. splendidus infection. 先前的研究表明灿烂弧菌感染会引起刺参（ Apostichopus japonicus）体腔细胞线粒体损伤，从而产生过多的活性氧（reactive oxygen species, ROS），导致不可逆的凋亡性细胞死亡。越来越多的证据表明，线粒体自噬是消除受损线粒体和ROS最有效的方法，而胆碱脱氢酶（CHDH）作为一种新的线粒体自噬受体，能同时识别脊椎动物非泛素化损伤信号和微管相关蛋白1轻链3（LC3）。但在无脊椎动物中，CHDH的功能作用在很大程度上是未知的。在该研究中，我们观察到灿烂弧菌和LPS胁迫后AjCHDH的基因和蛋白表达水平显著升高，这与相同条件下线粒体自噬水平的变化一致。值得注意的是，灿烂弧菌感染后，AjCHDH被发现定位于线粒体而不是细胞质。紧接着，我们利用透射电镜和共聚焦显微镜观察到，siRNA转染敲除AjCHDH后线粒体自噬水平显著降低。为进一步探究CHDH调控线粒体自噬的分子机制，我们克隆并分析刺参CHDH的结构域，发现AjCHDH缺乏直接结合LC3的LC3相互作用区，但它具有FB1结构域，因此推测其可与SQSTM1结合。免疫沉淀结果进一步证实了AjCHDH与SQSTM1的相互作用。此外，我们在激光共聚焦显微镜下观察到AjCHDH在体腔细胞和HEK293T细胞中均可募集SQSTM1和LC3。相比之下，干扰AjCHDH阻碍了SQSTM1和LC3向线粒体的募集，而这一步是清除受损线粒体的关键步骤。最后，我们发现干扰AjCHDH会引起胞内ROS水平显著增加，进而导致细胞凋亡增加，体腔细胞存活率降低。综上所述，这些结果表明AjCHDH介导的线粒体自噬在灿烂弧菌感染后刺参体腔细胞存活中起着至关重要的作用。.