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

项目摘要 最近的研究表明，Clostridium dendens对C.艰难梭菌体内感染。抗生素- 诱导的生态失调导致胆汁酸谱改变，这被认为允许C.难以生长和导致 感染先前的研究表明，宿主胆汁酸如牛磺胆酸诱导C。艰难梭菌抱子 在胃肠道中萌发;而宿主胆汁酸转化为二级胆汁酸，如脱氧胆酸， 酸（DCA），C. C.艰难孢子萌发和营养细胞生长。然而，一个基因 系统目前缺乏C.证据，代表证明DCA因果关系的障碍 C.艰难的生长和毒力。我们收集了一打以上的C. 其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C.白藓ATCC 35704 T。 确定了C.生物传感器将允许确定应变变化，并且可以允许 鉴定更易受遗传操作影响的菌株。我们还将进行转录组学分析 两个C。在新开发的确定成分培养基中培养菌株，并鉴定在 胆汁酸的存在。在目标2中，我们检验了C. scindens 是限制性修改系统。甲基化组数据已经确定了广泛的m6 A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反向选择标记。这些研究将在以下方面取得重大进展： 了解梭菌生物学，胆汁酸代谢，以及遗传学的重大进展， 操纵C。你好这将使未来的建议，旨在测试的假设，DCA的形成 由C.该菌对C.艰难梭菌体内感染。

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.