| 描述信息 |
The balance between ammonia nitrogen metabolism and immune responses represents a conserved molecular mechanism for aquatic animals to adapt to environmental stress and resist pathogen infection. However, the molecular basis of how the host balances the activation of these two pathways to conserve energy remains unclear. This study began with an investigation of Litopenaeus vannameiaquaculture ponds, which revealed that shrimp from ponds experiencing outbreaks of White Spot Syndrome Virus (WSSV) or Decapod Iridescent Virus 1 (DIV1) had significantly elevated hemolymph ammonia levels compared to those from outbreak-free ponds. The STING-IRF-mediated interferon pathway was identified as the primary pathway responding to WSSV and DIV1 infection in shrimp. RNA sequencing analysis indicated that knockdown of STING led to an increase in glutamine synthetase (GS) expression. Shrimp GS shares high homology with human GS. Knocking down GS resulted in decreased GS activity in the gills, increased hemolymph ammonia concentration, reduced glutamine levels in both hemolymph and gills, and exacerbated ammonia toxicity in the gill filaments, demonstrating that GS in shrimp can convert ammonia into glutamine. To facilitate the detection of interferon pathway activation, phosphorylation activation sites of shrimp IRF were identified via liquid chromatography-mass spectrometry. Dual-luciferase reporter assays and co-immunoprecipitation confirmed that phosphorylation at site S341 is crucial for IRF activation, leading to the production of a phosphospecific antibody against this site. Further experiments involving RNAi, Western blotting, and qPCR showed that knockdown of STING or IRF reduced IRF phosphorylation activation, increased GS expression and activity in the gills, decreased hemolymph ammonia levels, elevated glutamine concentrations in hemolymph and gills, and alleviated ammonia toxicity in gill filaments. This suggests that silencing the STING-IRF-mediated interferon pathway enhances GS-dependent ammonia detoxification. Conversely, activating the STING-IRF pathway with poly(I:C) suppressed GS expression and activity in the gills, increased hemolymph ammonia concentration, decreased glutamine levels, and worsened ammonia toxicity, indicating that activation of the interferon pathway suppresses ammonia detoxification and promotes ammonia poisoning in shrimp. An IRF binding site was predicted on the GS promoter, and dual-luciferase assays demonstrated that IRF can transcriptionally repress GS. Electrophoretic mobility shift assays confirmed direct binding of IRF to the GS promoter sequence. This study elucidates the adaptive mechanisms by which crustaceans balance key signaling pathways under the threats of environmental stress and pathogen infection, providing valuable target gene resources for breeding L. vannameivarieties with enhanced disease resistance and environmental stress tolerance. |
