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

三方外排泵使革兰氏阴性菌能够排出各种毒素，有助于 细菌多药耐药性和不可治愈的细菌感染的新威胁。这些外排 泵需要组装内膜泵、周质衔接蛋白和外膜泵， 膜通道进入蛋白质复合物以挤出化学物质。在一次合作中，申请人最近 发现CusCBA是一种RND系列金属外排泵， 对金属流出的细胞需求。了解这些外排泵的机制， 探索新的方法来折衷它们的功能对于开发新的有效的 抗菌治疗另一方面，虽然化学应激物，如抗生素， 关于细菌生理学的描述很好，关于机械应力是否或如何 可能会影响三方外排泵（如CusCBA）的组装和功能，即使是机械外排泵， 在细菌生长环境中受到力的作用。这项研究的长期目标是 了解如何操纵细菌外排以达到预防和治疗目的。的 目的是了解机械应力如何改变CusCBA在活大肠杆菌中的组装。杆菌 细胞和因此细胞对金属应力的抵抗力。这里的中心假设，由初步的 研究表明，机械应力通过诱导细胞变形，可以损害细胞的组装， CusCBA在细胞中，因此它们的外排功能，使细胞对金属胁迫的抵抗力降低。这一假设 将使用单分子跟踪、基于纳米流体的机械 操作、化学/遗传操作和批量生物物理/生化/细胞测定。的 申请人将由一个指导小组提供建议，该小组包括一名具有单分子专业知识的化学家 细菌金属流出的成像，机械/生物医学工程师，具有机械生物学专业知识， 和微生物学家这项研究的基本原理是，一旦完成，它将有助于设计 机械策略，以削弱组装的CusCBA和相关的三方外排泵， 细菌外流，以增加抗生素治疗的功效。该研究提出了两个具体的 目的：1）研究机械应力如何改变CusCBA的组装和细胞对有毒金属的耐受性。（二） 确定细胞刚度在将机械应力耦合到细胞中的CusCBA组装中的作用。研究 是重要的，因为它将推进细菌外排的机械生物学， 机械策略干预细菌外排抗菌治疗，以及新技术， 机械操纵单个细菌细胞。它是创新的，因为它引入了新的概念， 机械-外排耦合，它使用了单分子跟踪的新技术， 频闪成像和纳米流体操纵单个细菌细胞。

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.