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Studies towards a pan-genome and genetic manipulation of Clostridium scindens

梭菌的全基因组和遗传操作研究

基本信息

批准号：
10113519
负责人：
Jason Michael Ridlon
金额：
$ 7.54万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-24 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10113519
关键词：
Adenine Adoptive Transfer Anaerobic Bacteria Animal Model Anion Exchange Resins Antibiotics Bacteria Bile Acids Binding Biology Cecum Cholestyramine Cholic Acids Clostridium Clostridium difficile Collection Conserved Sequence DNA DNA Restriction-Modification Enzymes Data Deoxycholic Acid Development Diarrhea Digestion Disease Etiology Feces Firmicutes Future Gastrointestinal tract structure Generations Genes Genetic Genetic Recombination Genome Germination Growth Health Human Infection Intestinal Content Malignant neoplasm of gastrointestinal tract Mediating Medical Methods Methylation Modification Molecular Biology Mus Operon Patients Peptide Antibiotics Personal Communication Phylogeny Physiology Plasmids Probability Rapid screening Regulon Reporting Reproduction spores Research Research Personnel Resistance Role Secondary to System Taurocholic Acid Testing Variant Virulence Work bile acid metabolism cell growth comparative genomics differential expression dysbiosis experimental study functional genomics genetic manipulation genome sequencing gut bacteria host-microbe interactions in vivo methylome mouse model mutant nanopore pan-genome prevent response transcriptome transcriptome sequencing transcriptomics whole genome

项目摘要

Project Summary Recent studies suggest that Clostridium scindens is protective against C. difficile infection in vivo. Antibiotic- induced dysbiosis results in altered bile acid profile which is thought to allow C. difficile to grow and cause infection. Prior studies have shown that host bile acids such as taurocholic acid induce C. difficile spore germination in the GI tract; whereas conversion of host bile acids to secondary bile acids such as deoxycholic acid (DCA) by C. scindens prevents C. difficile spore germination and vegetative cell growth. However, a genetic system is currently lacking in C. scindens, representing a barrier to demonstrating causation with respect to DCA formation and inhibition of C. difficile growth and virulence. We have a collection of over a dozen strains of C. scindens strains whose genome sequences are not known. In Aim 1, we propose to sequence the complete genomes of these strains. We have already completed the genome/methylome of C. scindens ATCC 35704T. Determining the ‘pan-genome’ of C. scindens will allow determination of strain variation, and may allow identification of strains more susceptible to genetic manipulation. We will also perform transcriptomic analysis with two C. scindens strains in a newly developed defined medium and identify genes differentially expressed in the presence of bile acids. In Aim 2 we test the hypothesis that the barrier to genetic manipulation of C. scindens is restriction modification (FM) systems. Methylome data has identified extensive m6A modification among conserved sequences in the genome. We will utilize a plasmid artificial modification (PAM) approach to developing a pyrE counter-selectable marker. These studies will provide substantial progress in the understanding of Clostridium scindens biology, bile acid metabolism, and significant progress towards genetic manipulation of C. scindens. This will allow future proposals aimed at testing the hypothesis that DCA formation by C. scindens protects against C. difficile infection in vivo.

项目概要最近的研究表明，Clostridium scindens 对体内艰难梭菌感染具有保护作用。抗生素- 诱导的生态失调导致胆汁酸谱改变，这被认为允许艰难梭菌生长并导致感染。先前的研究表明，牛磺胆酸等宿主胆汁酸会诱导艰难梭菌孢子在胃肠道中发芽；而宿主胆汁酸转化为次级胆汁酸，例如脱氧胆酸 C. scindens 产生的酸 (DCA) 可阻止艰难梭菌孢子萌发和营养细胞生长。然而，一种遗传目前 C. scindens 中缺乏该系统，这对于证明 DCA 的因果关系构成了障碍艰难梭菌生长和毒力的形成和抑制。我们收集了十多个 C. scindens 菌株的基因组序列未知。在目标 1 中，我们建议对完整的这些菌株的基因组。我们已经完成了 C. scindens ATCC 35704T 的基因组/甲基化组。确定 C. scindens 的“泛基因组”将允许确定菌株变异，并可能允许鉴定对基因操作更敏感的菌株。我们还将进行转录组分析在新开发的特定培养基中使用两种 C. scindens 菌株，并鉴定差异表达的基因胆汁酸的存在。在目标 2 中，我们检验了 C. scindens 遗传操作障碍的假设是限制修改（FM）系统。甲基化组数据已鉴定出广泛的 m6A 修饰基因组中的保守序列。我们将利用质粒人工修饰（PAM）方法开发pyrE反选择标记。这些研究将在以下方面取得实质性进展：了解梭菌生物学、胆汁酸代谢以及遗传学方面的重大进展 C. scincens 的操纵。这将允许未来的提案旨在测试 DCA 形成的假设 C. scindens 可以防止体内艰难梭菌感染。