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具有磷酸化（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成员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的PT修饰结合到DNA中，已知拥有PT和dnd基因的不同细菌有>200种，包括正常的人类和小鼠微生物群和>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抵抗力，因此我们提出验证这一假设，即PT修饰在炎症和结肠炎的氧化和亚硝化应激中赋予肠道微生物选择性优势。在第一个

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.