Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG-ODN) can be specifically recognized by Toll-like receptor 9 (TLR9), provoking innate immune responses. Designed according to this structural feature, many synthetic phosphorothioate CpG-ODNs successfully activate macrophages. However, it is difficult to find potent stimulatory CpG-DNA fragments in microbial genomes. Therefore, whether microbial CpG-DNA substantially contributes to infectious and immune diseases remains controversial. In this study, high-throughput scanning was carried out for thousands of bacterial genomes with bioinformatics tools to comprehensively evaluate the distribution of CpG-DNA fragments. A random sampling test was then performed to verify their immunostimulatory properties by experiments in vitro and in vivo. Natural TLR9-dependent and potent stimulatory CpG-DNA fragments were found in microbial genomes. Interestingly, highly conserved stimulatory CpG-DNA fragments were found in 16S and 23S rDNA sequences with multiple copies, while others were species-specific. Additionally, we found that the reported active motifs were mostly non-stimulatory in natural CpG fragments. This evidence indicates that the previous structural descriptions of functional CpG-ODNs are incomplete. Our study has assessed the distribution of microbial CpG-DNA fragments, and identified natural stimulatory CpG-DNA fragments. These findings provide a deeper understanding of CpG-ODN structures and new evidence for microbial DNA inflammatory function and pathogenicity.

中文翻译：

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有效的免疫刺激性CpG-ODNs的发现广泛分布在细菌基因组中。

包含未甲基化的CpG二核苷酸（CpG-ODN）的寡脱氧核苷酸可以被Toll样受体9（TLR9）特异性识别，从而引发先天免疫应答。根据这种结构特征进行设计，许多合成的硫代磷酸酯CpG-ODN成功激活了巨噬细胞。但是，很难在微生物基因组中找到有效的刺激性CpG-DNA片段。因此，微生物CpG-DNA是否实质上有助于感染和免疫疾病仍存在争议。在这项研究中，利用生物信息学工具对数千种细菌基因组进行了高通量扫描，以全面评估CpG-DNA片段的分布。然后进行随机抽样测试，以通过体外和体外实验验证其免疫刺激特性。体内。在微生物基因组中发现了天然的依赖TLR9的有效刺激性CpG-DNA片段。有趣的是，在具有多个拷贝的16S和23S rDNA序列中发现了高度保守的刺激性CpG-DNA片段，而其他片段则具有物种特异性。此外，我们发现，报道的活性基序在天然CpG片段中主要是非刺激性的。该证据表明功能性CpG-ODN的先前结构描述不完整。我们的研究评估了微生物CpG-DNA片段的分布，并确定了自然刺激性CpG-DNA片段。这些发现为CpG-ODN结构提供了更深入的了解，并为微生物DNA的炎症功能和致病性提供了新的证据。