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

项目摘要 最近的研究表明，难辨梭状芽胞杆菌对体内感染具有保护作用。抗生素- 诱导的生物失调会导致胆汁酸谱的改变，这种改变被认为是艰难梭菌生长和引起 感染。先前的研究表明，宿主胆汁酸如牛磺胆酸可以诱导艰难梭菌的孢子。 在胃肠道中萌发；而宿主胆汁酸转化为次生胆汁酸，如脱氧胆酸 辛辛德氏菌产生的酸(DCA)可阻止艰难梭菌孢子萌发和营养细胞生长。然而，一种基因 目前缺乏C.scindens的系统，这是证明DCA因果关系的障碍 艰难梭菌生长和毒力的形成和抑制。我们收集了十几个品系的C. Scindens基因组序列未知的菌株。在目标1中，我们建议对完整的 这些菌株的基因组。我们已经完成了华支睾吸虫ATCC 35704T的基因组/甲基组测序。 确定丝状念珠菌的‘泛基因组’将允许确定菌株变异，并可能允许 鉴定更易受基因操纵影响的菌株。我们还将进行转录分析 在一种新定义的培养基中与两株辛氏假丝酵母菌进行比较，并鉴定差异表达基因 胆汁酸的存在。在目标2中，我们检验了这样一个假设，即遗传操作的障碍 是限制修饰(FM)系统。甲基组数据在以下几个方面发现了广泛的m6A修饰 基因组中的保守序列。我们将利用一种质粒人工修饰(PAM)的方法 开发一种PYRE反选择标记。这些研究将在以下方面取得实质性进展： 对梭状芽胞杆菌生物学、胆汁酸代谢和遗传学研究的重大进展 对华支睾吸虫的操纵。这将使未来旨在测试DCA形成的假设的提案成为可能 辛辛德杆菌可在体内保护艰难梭菌免受感染。

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.