Cell Envelope Biogenesis in Clostridioides difficile

项目摘要 在美国，每年有近50万人因艰难梭状芽胞杆菌结肠罢工而感染 造成近30,000人死亡。美国疾病控制与预防中心已宣布这种微生物对公共健康构成“紧急”威胁， 最高威胁级别。艰难梭菌感染很难治疗，很大程度上是因为 休眠的孢子在抗生素治疗后存活下来，当抗生素被取出时，种子在肠道中重新繁殖。 对艰难梭菌使用的抗生素也杀死了许多健康的人，这一事实加剧了这个问题。 肠道细菌，为艰难梭菌在孢子萌发时重新克隆扫清了道路。因此，有一个巨大的 需要针对艰难梭菌的新药，而不破坏健康的微生物区系。这项建议的前提是 对细胞膜生物发生的更深入的了解可以为开发更好的方法铺平道路 治疗艰难梭菌感染。细胞被膜是抗生素的有效靶标，在艰难梭菌中 包膜有一些不同寻常的特征，这表明它的组装需要可以开发的新蛋白质 作为艰难梭菌选择性抗生素的靶标。在目标1中，我们将使用遗传学、生物化学和显微镜来 了解交联肽多糖细胞壁的酶的作用和调节。这些酶 引起了我们的注意，因为在艰难梭菌中，细胞壁含有异常高比例的“3-3” 与大多数细菌中占主导地位的“4-3”型交联物相比。我们的实验将解决 以下问题：哪些酶负责3-3和4-3交联物的形成？它们是否起作用？ 在分裂期间，伸长还是两者兼而有之？3-3和4-3交联剂的比例是如何调节的？艰难梭菌是如何 从主要使用3-3个交联链中获益？在目标2中，我们将利用一种强大的新基因沉默工具，称为 CRISPR干扰(CRISPRi)将一组约50个假定必不可少的包膜生物发生基因分配给更多 特定的功能通路。这些基因在本质上很有趣，并构成了潜在的新抗生素 目标。我们还将对一种新的转录调控系统进行详细分析，该系统在 我们提议的屏幕的试运行版本。总的来说，这里要进行的调查将极大地 通过识别参与细胞组装的新蛋白来促进我们对艰难梭菌生物学的理解 包装并揭示他们的活动是如何协调的，以完成复杂的增长和 组织。

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organism forms dormant spores that survive antibiotic therapy and seed recolonization of the gut when antibiotics are withdrawn.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.