Sulfur DNA modifications in gut microbes confer resistance to oxidative stress

描述(由申请人提供)：这些探索性研究的目标是表征拥有其基因组的硫代(PT)修饰的肠道微生物区系，并评估PT修饰对炎症期间肠道微生物组组成的影响。我们最近发现，拥有5个成员的DnD基因簇(Dnda-E)的细菌将硫(S)作为序列和立体特异性的PT修饰结合到dna中，已知的200种不同种类的细菌具有PT和DND基因，包括正常的人和鼠微生物群以及&gt；30种人类病原体。作为我们研究的重点，我们发现PT修饰可以增强细菌对氧化应激的抵抗力，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过程中对肠道微生物具有选择性优势的假设。在…的第一个 有两个目的，我们建议使用IL10-/-小鼠(1)分析肠道微生物组中PT修饰的数量和序列背景；(2)鉴定具有PT的肠道细菌；(3)在IL10-/-小鼠中量化PT水平并确定含有PT的细菌的种类，在IL10-/-小鼠中，天然肠道菌群已被改变的舍德勒菌群(ASF)所取代。我们的初步研究揭示了野生型C57BL/6J小鼠粪便DNA中的七种PT序列背景，以及在八种ASF菌株中至少一株ASF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定义天然肠道菌群和ASF中PT的数量及其二核苷酸序列背景的谱，以评估炎症对含有PT的微生物区系的影响。我们还将使用一种新的亲和纯化策略来分离含PT的DNA，以用于下一代测序和定量PCR，从而鉴定含PT的细菌。在目标2中，我们测试了这样的假设，即拥有PT修饰的细菌在发炎的肠道中具有选择性优势。在这里，我们将使用葡聚糖硫酸钠(DSS)治疗的IL10-/-结肠炎小鼠模型，结合发炎和对照小鼠粪便DNA的16S rRNA测序，以定义IL10-/-肠道微生物群和结肠炎引起的变化。其次，利用来自目标1的信息，我们将评估结肠炎对对照和炎症小鼠粪便DNA中PT修饰的数量和二核苷酸序列背景的影响。第三，我们将比较来自对照组和炎症小鼠的亲和纯化的、含有PT的DNA的序列，试图识别受炎症影响的特定细菌种类。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的DNA修饰系统可能对炎症产生潜在的临床影响，这种修饰系统在人类微生物群和临床重要的细菌病原体中都广泛存在。这些结果为进一步建立其他炎症诱导的结肠炎和结肠癌的小鼠模型(例如，感染H型肝炎病毒的Rag2-/-小鼠)以及利用IBD患者的粪便样本进行人类微生物组研究奠定了基础。

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.