Characterization of Toll-like receptor 22 in striped catfish (Pangasianodon hypophthalmus): Recognition of Gram-positive and Gram-negative bacteria

Abstract

Toll-like receptor 22 (TLR22) in Pangasianodon hypophthalmus is a key component of the fish innate immune system, responsible for recognizing pathogen-associated molecular patterns (PAMPs). However, its bacterial recognition mechanisms remain largely unexplored, particularly in striped catfish. In this study, four recombinant subunits of TLR22 - TLR22 (LRR1-17), TLR22 (LRR1-13), TLR22 (LRR1-10), and TLR22 (LRR1-4) - were successfully produced in Escherichia coli SHuffle® T7 Express, and protein expression was confirmed via SDS-PAGE and Western blot analysis. Dot blot and ELISA assays were employed to evaluate bacterial-binding activity against Escherichia coli, Aeromonas hydrophila, Streptococcus agalactiae, and Lactiplantibacillus plantarum. Among the subunits, TLR22 (LRR1–4) exhibited the strongest binding affinity, particularly toward Gram-positive bacteria. Furthermore, pre-blocking bacterial peptidoglycan with mouse serum significantly reduced TLR22 (LRR1-4) binding, indicating a likely peptidoglycan-mediated interaction. These findings provide the first experimental evidence that striped catfish TLR22 can recognize both Gram-negative and Gram-positive bacteria, broadening current understanding of its ligand recognition spectrum. This study highlights the potential role of TLR22 in bacterial immune detection and establishes a foundation for further investigations into the molecular mechanisms of fish innate immunity.