Sulfur DNA modifications in gut microbes confer resistance to oxidative stress

肠道微生物中的硫 DNA 修饰赋予其对氧化应激的抵抗力

• 批准号: 8751068
• 负责人: Peter C Dedon
• 金额: $ 19.5万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8751068
• 关键词: Affect; Affinity; Affinity Chromatography; Bacteria; Bacterial DNA; Bacterial Genome; Bioinformatics; Clinical; Colitis; Colon Carcinoma; DNA; DNA Modification Process; Data; Dinucleoside Phosphates; Disease; Eukaryota; Foundations; Gene Cluster; Genes; Genome; Goals; Growth; Health; Human; Human Microbiome; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Light; Microbe; Modification; Mus; Nucleotides; Oxidative Stress; Patients; Proteins; Publishing; Research Personnel; Resistance; Ribosomal RNA; Role; Sampling; Sodium Dextran Sulfate; Stress; Sulfur; System; Techniques; Testing; Translating; Widespread Disease; experience; genome sequencing; gut microbiota; member; microbiome; mouse model; new technology; next generation sequencing; nitrosative stress; novel; pathogen; phosphorothioate; public health relevance

DESCRIPTION (provided by applicant): The goals of these exploratory studies are to characterize gut microbiota that possess phosphorothioate (PT) modifications of their genomes and to assess the impact of PT modifications on the composition of the gut microbiome during inflammation. We recently discovered that bacteria possessing the 5-member dnd gene cluster (dndA-E) incorporate sulfur (S) into DNA as sequence- and stereo-specific PT modifications, with >200 different species of diverse bacteria known to possess PT and dnd genes, including normal human and mouse microbiota and >30 human pathogens. As the focus of our studies, we have found that PT modifications confer resistance to oxidative stress in bacteria, so we propose to test the hypothesis that PT modifications confer a selective advantage to gut microbes during the oxidative and nitrosative stresses of inflammation and colitis. In the first of two Aims, we propose to use IL10-/- mice (1) to analyze the quantity and sequence context of PT modifications in the gut microbiome; (2) to identify gut bacteria possessing PT; and (3) to quantify PT levels and speciate PT-containing bacteria in IL10-/- mice in which the native gut flora has been replaced with altered Schaedler flora (ASF). Our preliminary studies revealed seven PT sequence contexts in fecal DNA from wild- type C57BL/6J mice and the presence of dnd genes in at least one of the eight strains of ASF, so we will first use our bioanalytical platform to define the quantities of PT and the spectrum of their dinucleotide sequence contexts in native gut flora and ASF in the C57BL/6J IL10-/- mouse model of colitis used in Aim 2 to assess the effect of inflammation on PT-containing microbiota. We will also identify PT-containing bacteria using a novel affinity purification strategy to isolate PT-containing DNA for next generation sequencing and quantitative PCR. In Aim 2, we test the hypothesis that bacteria possessing PT modifications have a selective advantage in the inflamed gut. Here we will use the dextran sodium sulfate (DSS)-treated IL10-/- mouse model of colitis in conjunction with 16S rRNA sequencing of fecal DNA from inflamed and control mice to define the IL10-/- gut microbiome and changes caused by colitis. Second, using the information from Aim 1, we will assess the effect of colitis on the quantity and dinucleotide sequence context of PT modifications from fecal DNA obtained from control and inflamed mice. Third, we will compare the sequences of affinity-purified, PT-containing DNA from control and inflamed mice in an attempt to identify specific bacterial species affected by inflammation. With etiological implications for inflammatory bowel disease and colon cancer, the significance of these studies lies in the potential clinical impact of a horizontally-transferred DNA modification system that may confer resistance to inflammation and that is widespread in both the human microbiome and clinically important bacterial pathogens. The results lay the foundation for proceeding into other mouse models of inflammation-induced colitis and colon cancer (e.g., H. hepaticus-infected Rag2-/- mice) and for proceeding into human microbiome studies utilizing fecal samples from IBD patients.

描述（由申请人提供）：这些探索性研究的目标是表征具有其基因组磷酸座（PT）修饰的肠道菌群，并评估PT修饰对炎症过程中肠道微生物组组成的影响。我们最近发现，拥有5个成员DND基因簇（DNDA-E）的细菌将硫掺入DNA中，作为序列和立体特异性PT修饰，> 200种不同种类的多种细菌，这些细菌具有PT和DND基因，包括正常的人类和小鼠Microbobiota和小鼠Microbobiota和> 30个人类型阳性元素。作为我们研究的重点，我们发现PT修饰赋予细菌中氧化应激的耐药性，因此我们建议测试以下假设：PT修饰在炎症和结肠炎的氧化和亚硝化应激期间为肠道微生物提供选择性优势。在第一个 两个目的，我们建议使用IL10 - / - 小鼠（1）分析肠道微生物组中PT修饰的数量和序列上下文； （2）鉴定具有PT的肠道细菌； （3）在IL10 - / - 小鼠中量化PT水平并识别含PT的细菌，其中天然肠道菌群已被改变的Schaedler Flora（ASF）取代。 Our preliminary studies revealed seven PT sequence contexts in fecal DNA from wild- type C57BL/6J mice and the presence of dnd genes in at least one of the eight strains of ASF, so we will first use our bioanalytical platform to define the quantities of PT and the spectrum of their dinucleotide sequence contexts in native gut flora and ASF in the C57BL/6J IL10-/- mouse AIM 2中使用的结肠炎模型来评估炎症对含PT的微生物群的影响。我们还将使用新型的亲和力纯化策略来鉴定含PT的细菌，以分离含PT的DNA，以进行下一代测序和定量PCR。在AIM 2中，我们检验了具有PT修饰的细菌在发炎的肠道中具有选择性优势的假设。在这里，我们将使用经硫酸钠（DSS）处理的结肠炎的IL10 - / - 小鼠模型以及16S rRNA测序来自发炎和对照小鼠的粪便DNA的16S rRNA测序来定义IL10 - / - 肠道微生物组和由结肠炎引起的变化。其次，使用来自AIM 1的信息，我们将评估结肠炎对从对照和发炎小鼠获得的粪便DNA的数量和二核苷酸序列上下文的影响。第三，我们将比较来自对照和发炎小鼠的亲和纯化，含PT的DNA的序列，以尝试鉴定受炎症影响的特定细菌。这些研究对炎症性肠病和结肠癌的病因影响，这些研究的重要性在于水平转移的DNA修饰系统的潜在临床影响，该系统可能赋予对炎症的耐药性，并且在人类微生物组和临床上重要的细菌病原体中广泛存在。结果为进入其他小鼠炎症诱导的结肠炎和结肠癌的小鼠模型（例如H. H. H. H. H. H. H.肝病感染的RAG2 - / - 小鼠）奠定了基础。