Cell Envelope Biogenesis in Clostridioides difficile

项目摘要 在美国，每年有近50万人感染结肠艰难梭菌。 导致近3万人死亡。疾病预防控制中心宣布这种微生物对公共卫生构成“紧急”威胁， 最高的威胁类别。C.难治性感染很难治疗， 休眠孢子，在抗生素治疗和停止使用抗生素后肠道的种子繁殖中存活下来。 这一问题因针对C.艰难梭菌也会杀死许多健康的 肠道细菌，为C.当孢子萌发时很难形成孢子。因此， 需要针对C的新药。而不会破坏健康的微生物群。这项提议的前提是 更深入地了解细胞包膜生物发生可以为开发更好的方法铺平道路， 治疗C.艰难感染。细胞被膜是抗生素的有效靶点，在C。艰难的 包膜有一些不寻常的特征，这表明它的组装需要新的蛋白质， 作为C.难选抗生素在目标1中，我们将使用遗传学，生物化学和显微镜， 了解交联肽聚糖细胞壁的酶的作用和调节。这些酶 吸引了我们的注意力，因为在C。difficile细胞壁含有异常高的“3-3”百分比， 与在大多数细菌中占主导地位的“4-3”交联相比，我们的实验将解决 以下问题：哪些酶负责3-3和4-3交联的形成，它们是否起作用 在分裂、伸长或两者的过程中？3-3与4-3交联的比例如何调节？C.艰难 主要使用3-3个交叉链接的好处？在目标2中，我们将利用一种强大的新基因沉默工具， CRISPR干扰（CRISPRi）将一组约50个puerectin必需的包膜生物发生基因分配给更多 特定的功能路径。这些基因本质上是有趣的，并构成潜在的新抗生素 目标的我们还将对一个新的转录调控系统进行详细的分析，该系统是在一个 我们提议的屏幕的试点版本。总的来说，在这里进行的调查将大大 加深了对C.通过识别参与细胞组装的新蛋白质， 信封和揭示他们的活动是如何协调，以完成复杂的增长过程， 师.

Project Summary Clostridioides (Clostridium) difficile infections of the colon strike close to 500,000 people a year in the United States, leading to nearly 30,000 deaths. The CDC has declared this organism an “urgent” threat to public health, the highest threat category. C. difficile infections are difficult to treat in large part because the organism forms dormant spores that survive antibiotic therapy and seed recolonization of the gut when antibiotics are withdrawn. This problem is exacerbated by the fact that the antibiotics used against C. difficile also kill many of the healthy gut bacteria, clearing the way for C. difficile to recolonize when spores germinate. Thus, there is a tremendous need for new drugs that target C. difficile without disrupting the healthy microbiota. The premise of this proposal is that a deeper understanding of cell envelope biogenesis can pave the way towards developing better ways to treat C. difficile infections. The cell envelope is a well-validated target for antibiotics, and in C. difficile the envelope has some unusual features that suggest its assembly requires novel proteins that could be exploited as targets of C. difficile-selective antibiotics. In Aim 1 we will use genetics, biochemistry and microscopy to understand the roles and regulation of enzymes that crosslink the peptidoglycan cell wall. These enzymes captured our attention because in C. difficile the cell wall contains an unusually high percentage of “3-3” crosslinks as compared to the “4-3” crosslinks that predominate in most bacteria. Our experiments will address the following questions: Which enzymes are responsible for 3-3 and 4-3 crosslink formation and do they operate during division, elongation or both? How is the ratio of 3-3 to 4-3 crosslinking regulated? How does C. difficile benefit from using primarily 3-3 crosslinks? In Aim 2 we will leverage a powerful new gene-silencing tool called CRISPR interference (CRISPRi) to assign a set of ~50 putatively essential envelope biogenesis genes to more specific functional pathways. These genes are intrinsically interesting and constitute potential new antibiotic targets. We will also undertake a detailed analysis of a novel transcriptional regulatory system uncovered in a pilot version of our proposed screen. Collectively, the lines of investigation to be pursued here will greatly advance our understanding of C. difficile biology by identifying new proteins involved in assembly of the cell envelope and revealing how their activities are coordinated to accomplish the complex processes of growth and division.