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: 8898718
• 负责人: Peter C Dedon
• 金额: $ 23.4万
• 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/8898718
• 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

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（由申请方提供）：这些探索性研究的目的是表征其基因组具有硫代磷酸酯（PT）修饰的肠道微生物群，并评估PT修饰对炎症期间肠道微生物群组成的影响。我们最近发现，拥有5-member dnd基因簇（dndA-E）的细菌将硫（S）作为序列特异性和立体特异性PT修饰掺入DNA中，已知有超过200种不同的细菌拥有PT和dnd基因，包括正常的人类和小鼠微生物群和超过30种人类病原体。作为我们研究的重点，我们已经发现PT修饰赋予细菌对氧化应激的抗性，因此我们建议测试PT修饰在炎症和结肠炎的氧化和亚硝化应激期间赋予肠道微生物选择性优势的假设。中的第一 为了两个目的，我们提出使用IL 10-/-小鼠（1）分析肠道微生物组中PT修饰的数量和序列背景;（2）鉴定具有PT的肠道细菌;和（3）定量IL 10-/-小鼠中的PT水平并使含PT的细菌形成物种，其中天然肠道植物群已被改变的Schaedler植物群（ASF）取代。我们的初步研究揭示了野生型C57 BL/6 J小鼠粪便DNA中的七种PT序列背景和八种ASF菌株中至少一种中存在dnd基因，因此，我们将首先使用我们的生物分析平台来确定PT的量及其二核苷酸序列背景在天然肠道植物群和ASF中的C57 BL/6 J IL 10-/-目的2中使用的结肠炎小鼠模型，以评估炎症对含PT微生物群的影响。我们还将使用一种新的亲和纯化策略来鉴定含PT的细菌，以分离含PT的DNA用于下一代测序和定量PCR。在目标2中，我们检验了具有PT修饰的细菌在炎症肠道中具有选择性优势的假设。在这里，我们将使用葡聚糖硫酸钠（DSS）治疗的IL 10-/-小鼠结肠炎模型，结合炎症小鼠和对照小鼠粪便DNA的16 S rRNA测序，以确定IL 10-/-肠道微生物组和结肠炎引起的变化。其次，使用来自目标1的信息，我们将评估结肠炎对来自对照和发炎小鼠的粪便DNA的PT修饰的量和二核苷酸序列背景的影响。第三，我们将比较亲和纯化的，含有PT的DNA序列，从控制和发炎的小鼠，试图确定受炎症影响的特定细菌物种。由于炎症性肠病和结肠癌的病因学意义，这些研究的意义在于水平转移DNA修饰系统的潜在临床影响，该系统可能赋予对炎症的抵抗力，并且在人类微生物组和临床重要的细菌病原体中广泛存在。该结果为进一步研究炎症诱导的结肠炎和结肠癌的其他小鼠模型奠定了基础（例如，H.肝感染的Rag 2-/-小鼠），并利用来自IBD患者的粪便样品进行人类微生物组研究。