CiTLR1 deficiency impairs intestinal immunity and gut microbiota homeostasis in grass carp after bacterial infection

In teleost fishes, TLRs are widely expressed in mucosal tissues and play crucial roles in host defense against pathogenic microbes, although the specific function of TLR1 in fish intestinal immunity remains poorly understood. In the present study, we generated TLR1 mutant Ctenopharyngodon idella (grass carp) using CRISPR/Cas9 technology and investigated the role of CiTLR1 in maintaining intestinal immune homeostasis and microbiota balance following Aeromonas hydrophila infection. We successfully generated F0 CiTLR1 mutant grass carp (CiTLR1-deficient) using CRISPR/Cas9. Following infection with A . hydrophila , CiTLR1-deficient fish exhibited exacerbated intestinal damage, reduced goblet cell abundance, and decreased IgT distribution and content in the intestinal mucosa compared to wild-type and negative control fish. qRT-PCR revealed that CiTLR1-deficient fish significantly downregulated key immune genes, including MyD88, IKKα, NF-κB1, TNF-α, IL6, IL22, MUC2, and Lysozyme, at 72 hpi. 16S rRNA sequencing revealed that CiTLR1 deficiency altered gut microbiota diversity, reduced beneficial bacteria ( Cetobacterium and Barnesiellaceae ), and enriched opportunistic pathogens ( Vibrio , Pseudomonas and Crenobacter ), with a marked increase in A. hydrophila abundance. These results indicate that CiTLR1 plays a critical role in maintaining intestinal immune homeostasis and microbiota balance, and its mutation compromises host defense against bacterial infection.