Selectivity of Human TLR9 for Double CpG Motifs and Implications for the Recognition of Genomic DNA

TLR9 acts as a first-line host defense against pathogens recognizing DNA comprising unmethylated CpG motifs present in bacteria and viruses. Species- and sequence-specific recognition differences were demonstrated for TLR9 receptors. Activation of human (h)TLR9 requires a pair of closely positioned CpG motifs within oligodeoxyribonucleotides (ODNs), whereas mouse TLR9 is effectively activated by an ODN with a single CpG motif. Molecular model-directed mutagenesis identified two regions, site A and site B, as important for receptor activation. Amino acid residues Gln³⁴⁶ and Arg³⁴⁸ within site A contribute to the sequence-specific recognition by hTLR9 in determining the bias for two appropriately spaced CpG motifs within immunostimulatory ODNs. Mutation of Gln⁵⁶² at site B, in combination with Gln³⁴⁶ and Arg³⁴⁸ mutations of mouse counterparts, increased activation of hTLR9 by mouse-specific ODN, mammalian genomic DNA, and bacterial DNA. We propose that the double CpG motif sequence-specificity of hTLR9 results in decreased activation by ODNs with a lower frequency of CpG motifs, such as from mammalian genomic DNA, which increases hTLR9 selectivity for pathogen versus host DNA.