Site-specific methylation of a TLR1 promoter SNP impedes CREB binding to impair antibacterial immunity in grass carp

As the most abundantly farmed freshwater fish species, grass carp (Ctenopharyngodon idella) faces a serious threat from Aeromonas hydrophila infection, underscoring the critical need to understand the genetic basis of disease resistance for selective breeding. This study deciphered the functional association and mechanism between polymorphisms in the CiTLR1 gene and resistance to A. hydrophila. CiTLR1 shows a broad expression profile, predominantly in immune organs and is significantly upregulated following bacterial infection. Among 13 single nucleotide polymorphisms (SNPs) identified in CiTLR1, 2 (-2575C/T and -1717A/T) are significantly associated with host resistance to A. hydrophila. Notably, only the -2575C/T SNP shows a correlation with CiTLR1 expression. Furthermore, the -2575C/T SNP falls within a CpG island, whose methylation is significantly higher in susceptible (-2575CC) individuals compared to resistant (-2575TT) ones. Multiple transcription factor and methylation experiments confirmed that CREB is a key transcription factor activating the TLR1 promoter and -2575CC methylation impedes its binding. Re-challenge experiments revealed a significant survival advantage in -2575TT homozygous individuals (44%) over -2575CC homozygotes (16%), definitively establishing the CiTLR1-2575C/T locus as a key genetic marker conferring resistance. Furthermore, resistant grass carp (-2575TT) mounted a robust immune response post-infection, as evidenced by significantly reduced bacterial loads and concomitant tissue damage/apoptosis, a markedly enhanced induction of key immune genes, and elevated serum-mediated innate immune activity. Collectively, this study bridges genetic variation, epigenetic regulation, and phenotypic resistance in grass carp. By pinpointing a causal SNP and the methylation mechanism that disrupts transcription factor binding, providing a theoretical foundation and molecular resources for disease-resistant breeding.