Genome-Wide Identification, Characterization, and Expression Profiles of TLR Genes in Darkbarbel Catfish (Pelteobagrus vachelli) Following Aeromonas hydrophila Infection

Toll-like receptors (TLRs) are central to pathogen recognition in teleost innate immunity. In this study, we surveyed 41 genomes from four representative teleost orders (i.e., Cypriniformes, Siluriformes, Perciformes, and Pleuronectiformes) for 15 TLR genes (TLR1-9, 12, 13, 18, 20-22) revealed a conserved core (TLR2/3/7 in nearly all examined species) alongside lineage-specific losses (TLR4/9/18/20/21/22), indicating both strong conservation and dynamic diversification of the TLR repertoire. We further identified and characterized 12 TLR genes in economically important darkbarbel catfish (Pelteobagrus vachellii). Corresponding cDNAs span 2089-4456 bp and encode proteins of 789-1,087 aa, with canonical extracellular LRR arrays and C-terminal TIR domains but notable "non-classical" features (such as absence of signal peptides in TLR1/13; no transmembrane segment in TLR7; multiple transmembranes in TLR3/8/13/18/22), suggesting subcellular and functional heterogeneity of various TLR genes. Subsequent gene-structure comparisons uncovered gene-specific exon-intron organizations and variable UTR lengths, implicating differential post-transcriptional regulation. Predicted 3D structures retain the traditional hallmark LRR horseshoe fold with subtle variations potentially tuning ligand specificity. Genomic synteny with Pseudobagrus ussuriensi and Pangasianodon hypophthalmus reveals conserved chromosomal organization, and phylogeny construction resolves each TLR subtype into well-supported monophyletic clades, which underscore evolutionary stability. Functionally, exogenous Aeromonas hydrophila challenge triggered rapid, tissue-dependent TLR up-regulation in the kidney, liver, and especially gill (with some transcripts > 1000-fold), highlighting coordinated mucosal and systemic surveillance in darkbarbel catfish. Taken together, these valuable data provide a comprehensive framework for the structural, evolutionary, and inducible expression landscape of catfish TLRs and establish a foundation for in-depth studies on antibacterial immunity in diverse teleost species.