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.
Given the importance of gut bacteria in health and disease, we propose to explore the effect of the widespread modification of bacterial DNA with sulfur in the form of a phosphorothioate (PT), which is present in many human gut bacteria and which confers resistance to the stress of inflammation, such as occurs in inflammatory bowel disease and colitis. We will test the hypothesis that PT modifications confer a selective advantage to gut microbes during the oxidative and nitrosative stresses of inflammation and colitis, using a convergence of novel technologies, including mouse models of colitis, ultrasensitive analytical techniques, genome sequencing and bioinformatics, to define the spectrum of gut bacteria possessing PT modifications and changes in gut microbiota caused 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.
| Kellner, Stefanie; DeMott, Michael S; Cheng, Ching Pin et al. (2017) Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability. Nat Chem Biol 13:888-894 |
| Chen, Chao; Wang, Lianrong; Chen, Si et al. (2017) Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes. Proc Natl Acad Sci U S A 114:4501-4506 |
| Su, Dan; Chan, Clement T Y; Gu, Chen et al. (2014) Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry. Nat Protoc 9:828-41 |
| Cao, Bo; Cheng, Qiuxiang; Gu, Chen et al. (2014) Pathological phenotypes and in vivo?DNA cleavage by unrestrained activity of a phosphorothioate-based restriction system in Salmonella. Mol Microbiol 93:776-85 |
| Cao, Bo; Chen, Chao; DeMott, Michael S et al. (2014) Genomic mapping of phosphorothioates reveals partial modification of short consensus sequences. Nat Commun 5:3951 |
