Engineering Colicins to Control Antibiotic-Tolerant Bacteria

改造大肠杆菌素来控制抗生素耐受性细菌

• 批准号: 9516317
• 负责人: Seok Hoon Hong
• 金额: $ 43.71万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9516317
• 关键词: Address; Affect; Aftercare; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Antimicrobial Resistance; Area; Back; Bacteria; Bacterial Infections; Biotechnology; Cell Survival; Cells; Characteristics; Chronic; Chronic Disease; Clinical; Cloning; Communicable Diseases; Custom; DNA; Data; Deoxyribonucleases; Development; Directed Molecular Evolution; Drops; Engineering; Environment; Escherichia coli; Fluorescence; Genetic Transcription; Goals; Growth; Health; Human; Illinois; Infection; Institutes; Interdisciplinary Study; Knowledge; Membrane; Metabolic; Microbial Biofilms; Modification; Molecular Biology; Pathway interactions; Population; Problem Solving; Production; Property; Protein Biosynthesis; Protein Engineering; Proteins; RNA; Research; Site-Directed Mutagenesis; Specificity; Stress; Structure; Students; Technology; Tertiary Protein Structure; Therapeutic; Therapeutic Uses; Toxin; Variant; Work; antibiotic tolerance; bactericide; base; cell envelope; cell killing; chronic infection; colicin; combat; cytotoxicity; design; experience; fight against; graduate student; high throughput screening; innovation; insight; novel strategies; pathogen; premature; programs; receptor; receptor binding; resistance mechanism; skills; student training; therapeutic protein; undergraduate student

PROJECT SUMMARY Chronic bacterial infections are difficult to treat through standard antibiotics because a small population of the bacteria becomes tolerant to antibiotics by entering a dormant state. Such bacteria are known as persisters and pose a significant problem in clinical settings. As antibiotic levels drop post-treatment, persister cells revert back into actively growing cells resulting in recalcitrant chronic infections. Currently, there are very limited approaches available for eradicating persister cells. We propose leveraging antibacterial protein colicins as an anti-persister agent. Colicins kill cells physically by disrupting cell envelopes and degrading DNA and RNA. Colicins are excellent candidates for killing persisters because the mechanisms of colicin action are growth- independent. We will utilize cell-free protein synthesis (CFPS) to produce colicins, as CFPS offers rapid protein production and characterization of such toxic proteins without cell-viability constraints. The goal of this project is to investigate and engineer colicins to control non-growing persister cells for therapeutic applications. The proposed research will be advanced with three integrated research Aims. In Aim 1, we will characterize the effects of different types of colicins on persister cells to get insights on what kind of cell killing mechanisms enable colicins to eradicate persister cells. Colicins have great potential to be developed into target-specific therapeutics without affecting beneficial bacteria. In Aim 2, we will evolve colicins via directed evolution to have enhanced activity. The engineered colicins will be identified through a fluorescence-based high-throughput screening approach using CFPS. We anticipate developing highly active and target-specific colicins that can be applied for treating persistent infections. In Aim 3, we will investigate redirecting the activity of colicins toward other pathogens which are not recognized by native colicins. The chimeric colicins will be constructed by replacing domain proteins of colicins with those of colicin-like proteins from other bacteria. Our research will open the development of customized anti-persister agents that can selectively kill antibiotic-tolerant pathogens.

项目概要 慢性细菌感染很难通过标准抗生素治疗，因为人口很少 的细菌通过进入休眠状态而对抗生素产生耐受性。这种细菌被称为持久性细菌 并在临床环境中造成重大问题。随着治疗后抗生素水平下降，持久细胞会恢复 回到活跃生长的细胞，导致顽固性慢性感染。目前，数量非常有限 可用于根除持续细胞的方法。我们建议利用抗菌蛋白大肠菌素作为 抗持久剂。大肠杆菌素通过破坏细胞包膜并降解 DNA 和 RNA 来物理性杀死细胞。 大肠杆菌素是杀死持久性细菌的绝佳候选者，因为大肠杆菌素的作用机制是生长- 独立的。我们将利用无细胞蛋白质合成 (CFPS) 来生产大肠杆菌素，因为 CFPS 可以快速提供蛋白质 在没有细胞活力限制的情况下生产和表征此类有毒蛋白质。 该项目的目标是研究和改造大肠杆菌素来控制非生长的持久细胞 治疗应用。拟议的研究将通过三个综合研究目标来推进。瞄准 1，我们将表征不同类型的大肠菌素对持续细胞的影响，以深入了解哪种大肠菌素 细胞杀伤机制使大肠菌素能够消灭持久细胞。大肠杆菌素具有巨大的开发潜力 进入特定目标的治疗而不影响有益细菌。在目标 2 中，我们将通过定向进化大肠杆菌素 进化到具有增强的活性。工程化的大肠杆菌素将通过基于荧光的方法进行鉴定 使用 CFPS 的高通量筛选方法。我们预计开发高度活跃且针对特定目标的 大肠杆菌素可用于治疗持续性感染。在目标 3 中，我们将研究重定向活动 大肠杆菌素针对天然大肠杆菌素无法识别的其他病原体。嵌合大肠杆菌素将是 通过用其他细菌的大肠菌素样蛋白替换大肠菌素的结构域蛋白而构建。我们的 研究将开启定制抗持久剂的开发，可以选择性地杀死抗生素耐受者 病原体。