Two tripartite motif-containing proteins regulate autophagy-related gene expression to mediate anti-Vibrio immunity in Takifugu obscurus

Tripartite motif (TRIM) proteins play crucial roles in innate immunity, but their functions in antibacterial defense in teleost fish remain poorly understood. In this study, two TRIM genes, designated ToTRIM16 and ToTRIM65, were identified in the obscure pufferfish Takifugu obscurus. The domain architectures are distinct, ToTRIM16 consists of three low-complexity, one B-box, two coiled-coil, and one PRY/SPRY domains, whereas ToTRIM65 comprises single RING, B-box, coiled-coil, and PRY/SPRY domains. Phylogenetic analysis confirmed their evolutionary conservation among pufferfish lineages. Subcellular localization studies showed pathogen-specific dynamic changes; Vibrio harveyi infection induced the aggregation of ToTRIM16 into cytoplasmic puncta and increased the foci density of ToTRIM65. Tissue-specific expression profiling indicated that ToTRIM16 was predominantly expressed in the intestine, while ToTRIM65 was highly expressed in the liver, with distinct induction patterns following V. harveyi challenge. Notably, overexpression of either TRIM protein significantly upregulated five autophagy-related genes (ULK1, P62, LC3B, ATG5, and ATG12) in FHM cells. Conversely, in vivo siRNA knockdown of these TRIMs not only substantially suppressed the expression of these autophagy-related genes but also led to significantly increased bacterial loads in immune tissues after V. harveyi infection. These findings demonstrate that ToTRIM16 and ToTRIM65 are pivotal regulators of autophagy-mediated antibacterial immunity, providing novel insights for the development of immunomodulatory strategies in aquaculture.