Characteristics comparison and functional analysis of TLR5S in two mandarin fish species (Siniperca scherzeri and...

Toll-like receptor 5 (TLR5) is a key pattern-recognition receptor that senses bacterial flagellin, and teleosts typically possess both membrane-bound and soluble isoforms. To explore species-specific TLR5-mediated antibacterial responses in mandarin fish, we cloned and characterized soluble TLR5 (TLR5S) from Siniperca chuatsi (ScTLR5S) and Siniperca scherzeri (SsTLR5S), and evaluated their functional roles during Aeromonas veronii challenge.ScTLR5S encoded a 646-aa protein that lacks the classical TIR/transmembrane regions and contains 14 LRR domains, whereas SsTLR5S encoded a 646-aa protein lacking the classical TIR/transmembrane regions and containing 13 LRR domains; the two proteins shared high sequence similarity and clustered together phylogenetically. Upon A. veronii infection, both genes were significantly induced, peaking at 48 h post-infection with 2.85-fold (ScTLR5) and 5.61-fold (SsTLR5) increases. In MFF-1 cells, ScTLR5-GFP and SsTLR5-GFP signals were predominantly cytoplasmic. In vivo RNAi-mediated TLR5 knockdown reduced survival following SJ4 challenge in both species and broadly suppressed cytokine transcription. At the protein level, both TLR5S proteins co-localized with flagellin (FlaB) at the plasma membrane region, while SsTLR5S showed a higher co-localization coefficient and stronger FlaB co-immunoprecipitation, indicating tighter binding. Consistently, co-expression of TLR5S with FlaB amplified inflammatory cytokine induction, with SsTLR5S producing a stronger enhancement than ScTLR5S. Collectively, these results indicate that TLR5S contributes to flagellin recognition and downstream immune activation in mandarin fish, and the stronger SsTLR5S-FlaB interaction may help explain interspecific differences in antibacterial responsiveness.