Single-cell Imaging of Mechanically Coupled Assembly of Metal Efflux Complexes in Bacteria

细菌中金属流出复合物机械耦合组装的单细胞成像

• 批准号: 9883623
• 负责人: Lauren A Genova
• 金额: $ 2.7万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-25 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9883623
• 关键词: Adaptor Signaling Protein; Affect; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Bacteria; Bacterial Drug Resistance; Bacterial Infections; Bacterial Physiology; Biochemical; Biomechanics; Biomedical Engineering; Biophysics; Cells; Cellular Assay; Chemicals; Collaborations; Communicable Diseases; Complex; Consultations; Coupled; Coupling; Development; Environment; Escherichia coli; Family; Goals; Gram-Negative Bacteria; Growth; Habitats; Image; Impairment; Individual; Infection; Ion Channel; Knowledge; Link; Mechanical Stress; Mechanics; Mediating; Membrane; Mentors; Metals; Methods; Microbial Biofilms; Microfluidics; Mission; Modeling; Multi-Drug Resistance; National Institute of Allergy and Infectious Disease; Nodule; Outcome; Play; Poison; Preventive; Proteins; Pseudomonas aeruginosa; Public Health; Pump; Research; Resistance; Resolution; Role; Stress; Study Section; Supervision; Techniques; Testing; Therapeutic; Time; Toxin; Virulence; base; biological adaptation to stress; cell envelope; cellular imaging; chemical genetics; combat; efflux pump; experience; genetic manipulation; global health; innovation; mechanical force; molecular imaging; nanofluidic; new technology; novel; periplasm; prevent; protein complex; response; single molecule; toxic metal

Tripartite efflux pumps enable Gram-negative bacteria to extrude diverse toxins, contributing to bacterial multidrug resistance and the emerging threat of untreatable bacterial infections. These efflux pumps require the assembly of an inner-membrane pump, a periplasmic adaptor protein, and an outer- membrane channel into a protein complex to extrude chemicals. In a collaboration, the applicant recently discovered that CusCBA, an RND-family metal efflux pump, undergoes dynamic assembly in response to cellular demands for metal efflux. Understanding such mechanisms of these efflux pumps and exploring novel methods to compromise their functions are crucial for developing new and effective antibacterial treatments. On the other hand, while the effects of chemical stressers, such as antibiotics, on bacterial physiology are well described, nothing is known about whether or how mechanical stresses may affect the assembly and function of tripartite efflux pumps such as CusCBA, even though mechanical forces are experienced in bacterial growth environments. The long-term goal of this research is to understand how bacterial efflux can be manipulated for preventive and therapeutic purposes. The objective here is to understand how mechanical stress can alter the assembly of CusCBA in live E. coli cells and thus cells’ resistance to metal stress. The central hypothesis here, supported by preliminary studies, is that mechanical stress, by inducing cell deformations, can compromise the assembly of CusCBA in cells and thus their efflux function, making cells less resistant to metal stress. This hypothesis will be tested using combined approaches of single-molecule tracking, nanofluidics-based mechanical manipulations, chemical/genetic manipulations, and bulk biophysical/biochemical/cellular assays. The applicant will be advised by a mentoring team that includes a chemist with expertise in single-molecule imaging of bacterial metal efflux, a mechanical/biomedical engineer with expertise in mechanobiology, and a microbiologist. The rationale for this research is that, once it is accomplished, it will help devise mechanical strategies to impair the assembly of CusCBA and related tripartite efflux pumps and thus bacterial efflux to increase the efficacy of antibiotic treatments. The proposed research has two specific aims: 1) Define how mechanical stress alters CusCBA assembly and cells’ resistance to toxic metals. 2) Identify the role of cell stiffness in coupling mechanical stress to CusCBA assembly in cells. The research is significant because it will advance the mechanobiology of bacterial efflux, the development of mechanical strategies to intervene in bacterial efflux for antibacterial therapy, and new technologies for mechanically manipulating single bacterial cells. It is innovative because it introduces the novel concept of mechano-efflux coupling and it uses the novel techniques of single-molecule tracking via time-lapse stroboscopic imaging and nanofluidic manipulation of individual bacterial cells.

三方外排泵可以使革兰氏阴性细菌挤压潜水毒素，从而有助于 细菌多药的耐药性和不可治疗的细菌感染的新兴威胁。这些外排 泵需要内膜泵的组装，外围适配器蛋白和外部 - 膜通道进入蛋白质复合物以挤出化学物质。在合作中，最近适当的 发现RND金属外排泵库斯巴（Cuscba）经历了动态组件的响应 对金属外排的细胞需求。了解这些外排泵的这种机制和 探索损害其功能的新颖方法对于开发新有效的方法至关重要 抗菌治疗。另一方面，诸如抗生素等化学胁迫的作用，但 在细菌生理学上得到很好的描述，关于机械应力或如何进行机械应力尚无 即使机械 力在细菌生长环境中经历。这项研究的长期目标是 了解如何为预防和治疗目的操纵细菌外排。这 这里的目的是了解机械应力如何改变现场大肠杆菌中cuscba的组装 细胞以及细胞对金属应激的抗性。这里的中心假设，由初步支持 研究是，通过诱导细胞变形，机械应力会损害组装 细胞中的cuscba及其外排功能，使细胞对金属应激的抗性较小。这个假设 将使用基于纳米流体的机械方法的组合方法进行测试 操纵，化学/遗传操作以及批量生物物理/生化/细胞分析。这 申请人将由一个心理团队提供建议，其中包括一名具有单分子专家的化学家 细菌金属外排的成像，金属外排，机械/生物医学工程师，具有机制专业知识， 和一名微生物学家。这项研究的理由是，一旦完成，它将有助于设计 机械策略损害了cuscba和相关三方外排泵的组装，因此 细菌外排以提高抗生素处理的效率。拟议的研究有两个特定的 目的：1）定义机械应力如何改变CUSCBA组装和细胞对有毒金属的抗性。 2） 确定细胞刚度在耦合机械应力与细胞中cuscba组装的作用。研究 之所以重要，是因为它会推进细菌外排的机制， 介入细菌以进行抗菌治疗的机械策略和新技术 机械操纵单细菌细胞。它具有创新性，因为它介绍了新颖的概念 机械效率耦合，并通过延时使用单分子跟踪的新技术 频镜成像和单个细菌细胞的纳米流体操纵。