DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes
DNA methylation from a Type I restriction modification system influences gene expression and virulence in Streptococcus pyogenes
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DOI：
10.1371/journal.ppat.1007841
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发表时间：
2019-06-01
期刊：
影响因子：
6.7
通讯作者：
Watson, Michael E., Jr.
中科院分区：
文献类型：
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作者：
Nye, Taylor M.;Jacob, Kristin M.;Watson, Michael E., Jr.
DNA甲基化在生命的各个领域都很普遍。在细菌中，N6-甲基腺苷（m6 A）的存在已被检测到在不同的物种，但m6 A的基因表达调控的贡献是不清楚的许多生物体。在这里，我们研究了人类病原体化脓性链球菌（或A组链球菌）中DNA甲基化对基因表达和毒力的影响。单分子实时测序和随后的甲基化分析在整个1.8 Mb基因组中鉴定了412个推定的m6 A位点。推定的I型限制性修饰系统的限制性、特异性和甲基化基因亚基（Delta RSM菌株）的缺失丢失了识别位点处的所有可检测的m6 A，并且未能阻止外源甲基化DNA的转化。RNA测序确定了1,895个预测编码区中的20个基因，它们具有显著不同的基因表达。所有差异表达基因在Delta RSM菌株中相对于亲本菌株均下调。重要的是，我们发现m6 A DNA修饰的存在影响了Mga的表达，Mga是S.化脓使用鼠皮下感染模型，与感染亲本或补充突变株的小鼠相比，感染Delta RSM株的小鼠表现出增强的宿主免疫应答，皮肤病变更大，促炎细胞因子水平增加，表明m6 A甲基化的改变影响毒力。此外，我们发现Delta RSM菌株在人嗜中性粒细胞中的存活率很低，并且与人上皮细胞的粘附性降低。这些结果表明，除了限制外源DNA，革兰氏阳性菌还使用限制性修饰系统来调节基因网络的表达，这对毒力很重要。作者总结DNA甲基化在许多细菌物种中很常见，但在有限数量的革兰氏阴性菌之外，DNA甲基化对基因表达调节的贡献尚不清楚。我们的特点是整个基因组的革兰氏阳性病原体化脓性链球菌或A组链球菌的DNA甲基化位点。我们确定了功能性限制性修饰系统的基因产物负责全基因组m6 A。与亲本菌株相比，缺乏DNA甲基化的突变菌株显示出改变的基因表达，其中几个对引起人类疾病重要的基因下调。此外，我们发现，缺乏DNA甲基化的突变株表现出改变的毒力特性相比，使用各种模型的发病机制的亲本菌株。该突变株在人中性粒细胞内的存活和对人上皮细胞的粘附均被减毒，并且在小鼠皮下感染模型中不能抑制宿主免疫应答。总之，这些结果表明，细菌m6 A有助于差异基因表达，并影响A组链球菌引起疾病的能力。DNA甲基化是细菌中的一个保守特征，可能是一个潜在的干预目标，以干扰细菌引起人类疾病的能力。
DNA methylation is pervasive across all domains of life. In bacteria, the presence of N6-methyladenosine (m6A) has been detected among diverse species, yet the contribution of m6A to the regulation of gene expression is unclear in many organisms. Here we investigated the impact of DNA methylation on gene expression and virulence within the human pathogen Streptococcus pyogenes, or Group A Streptococcus. Single Molecule Real-Time sequencing and subsequent methylation analysis identified 412 putative m6A sites throughout the 1.8 Mb genome. Deletion of the Restriction, Specificity, and Methylation gene subunits (Delta RSM strain) of a putative Type I restriction modification system lost all detectable m6A at the recognition sites and failed to prevent transformation with foreign-methylated DNA. RNA-sequencing identified 20 genes out of 1,895 predicted coding regions with significantly different gene expression. All of the differentially expressed genes were down regulated in the Delta RSM strain relative to the parent strain. Importantly, we found that the presence of m6A DNA modifications affected expression of Mga, a master transcriptional regulator for multiple virulence genes, surface adhesins, and immune-evasion factors in S. pyogenes. Using a murine subcutaneous infection model, mice infected with the Delta RSM strain exhibited an enhanced host immune response with larger skin lesions and increased levels of pro-inflammatory cytokines compared to mice infected with the parent or complemented mutant strains, suggesting alterations in m6A methylation influence virulence. Further, we found that the Delta RSM strain showed poor survival within human neutrophils and reduced adherence to human epithelial cells. These results demonstrate that, in addition to restriction of foreign DNA, gram-positive bacteria also use restriction modification systems to regulate the expression of gene networks important for virulence.Author summary DNA methylation is common among many bacterial species, yet the contribution of DNA methylation to the regulation of gene expression is unclear outside of a limited number of gram-negative species. We characterized sites of DNA methylation throughout the genome of the gram-positive pathogen Streptococcus pyogenes or Group A Streptococcus. We determined that the gene products of a functional restriction modification system are responsible for genome-wide m6A. The mutant strain lacking DNA methylation showed altered gene expression compared to the parent strain, with several genes important for causing human disease down regulated. Furthermore, we showed that the mutant strain lacking DNA methylation exhibited altered virulence properties compared to the parent strain using various models of pathogenesis. The mutant strain was attenuated for both survival within human neutrophils and adherence to human epithelial cells, and was unable to suppress the host immune response in a murine subcutaneous infection model. Together, these results show that bacterial m6A contributes to differential gene expression and influences the ability of Group A Streptococcus to cause disease. DNA methylation is a conserved feature among bacteria and may represent a potential target for intervention in effort to interfere with the ability of bacteria to cause human disease.