Cell Envelope Biogenesis in Clostridioides difficile

艰难梭菌的细胞包膜生物发生

• 批准号: 10295470
• 负责人: Craig D Ellermeier
• 金额: $ 52.59万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10295470
• 关键词: Address; Animal Model; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Attention; Bacteria; Biochemistry; Biogenesis; Biology; CRISPR interference; Carboxypeptidase; Categories; Cell Wall; Cell division; Cellular Morphology; Cellular biology; Centers for Disease Control and Prevention (U.S.); Cessation of life; Clostridium difficile; Colon; Complex; Enzymes; Essential Genes; Fluorescence Microscopy; Foundations; Gene Deletion; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Genome; Glucosamine; Goals; Growth; Health; Homeostasis; Hospital Nursing; Human; In Situ; Individual; Infection; Investigation; Knowledge; Label; Lead; Libraries; Maps; Masks; Microscopy; Morphology; Nursing Homes; Organism; PPBP gene; Pathway interactions; Penicillin-Binding Proteins; Peptides; Peptidoglycan; Peptidyltransferase; Process; Proteins; Public Health; Regulation; Reproduction spores; Role; Seeds; Site; System; Testing; United States; Vancomycin; base; beta-Lactams; cell assembly; cell envelope; combat; crosslink; experimental study; glycosyltransferase; gut bacteria; insight; microbiota; new therapeutic target; novel; novel strategies; pathogen; prevent; tool

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organism forms dormant spores that survive antibiotic therapy and seed recolonization of the gut when antibiotics are withdrawn.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

项目概要 美国每年有近 50 万人受到结肠艰难梭菌感染 州，导致近30,000人死亡。疾病预防控制中心已宣布这种生物体对公众健康构成“紧急”威胁， 最高威胁类别。艰难梭菌感染很难治疗，很大程度上是因为该生物体形成 休眠孢子在抗生素治疗后存活下来，并在抗生素撤除后在肠道内重新定殖。 由于用于对抗艰难梭菌的抗生素也会杀死许多健康人​​，这一事实加剧了这个问题。 肠道细菌，为孢子萌发时艰难梭菌重新定殖扫清道路。由此可见，存在着巨大的 需要针对艰难梭菌而不破坏健康微生物群的新药物。本提案提出的前提 更深入地了解细胞被膜生物发生可以为开发更好的方法铺平道路 治疗艰难梭菌感染。细胞包膜是经过充分验证的抗生素靶标，在艰难梭菌中 包膜具有一些不寻常的特征，表明其组装需要可以利用的新型蛋白质 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 了解交联肽聚糖细胞壁的酶的作用和调节。这些酶 引起我们注意的是，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”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 交联的形成以及它们是否起作用 在分裂、伸长或两者兼而有之时？ 3-3与4-3交联的比例如何调节？艰难梭菌如何 主要使用 3-3 个交联会受益吗？在目标 2 中，我们将利用一种强大的新型基因沉默工具，称为 CRISPR 干扰 (CRISPRi) 将一组约 50 个假定必需的包膜生物发生基因分配给更多 特定的功能途径。这些基因本质上很有趣，构成了潜在的新抗生素 目标。我们还将对在一项研究中发现的新型转录调控系统进行详细分析。 我们提议的屏幕的试点版本。总的来说，这里要进行的调查路线将极大地 通过鉴定参与细胞组装的新蛋白质，增进我们对艰难梭菌生物学的理解 信封并揭示他们的活动如何协调以完成复杂的成长过程和 分配。