Single-cell Super-resolution Imaging of Bacterial Metal Efflux Mechanobiology
Abstract
Tripartite efflux pumps (TEPs) enable Gram-negative bacteria to extrude toxic chemicals such as antibiotics and heavy metals, contributing to the development of bacterial multidrug resistance and the emerging threat of untreatable bacterial infections. Understanding the mechanisms of TEPs and identifying novel methods to compromise their function is crucial to the development of new and effective antibacterial treatments. To extrude toxic chemicals, all TEPs require the assembly of a protein complex consisting of an inner-membrane pump, a periplasmic adaptor protein, and an outer-membrane channel. Disrupting the assembly of the TEPs in the cell is thus a potential approach to impair their efflux functions and compromise bacterial resistance to antibiotics. Recently, mechanical forces have been shown to have a profound effect on bacterial cell physiology, yet virtually nothing is known about their effect on efflux pumps. The long-term goal here is to understand how bacterial efflux can be manipulated for preventive and therapeutic purposes. The overall objective here is to define how mechanical stress can alter the assembly and efflux functions of CusCBA, a Cu+ and Ag+ efflux complex of the resistance-nodulation-division (RND) family, which provides clinically relevant antibiotics resistance to Gram-negative bacteria. To attain this objective, the proposed research will apply controllable mechanical stress on E. coli, a model Gram-negative bacterium, and study the effects on the assembly and function of CusCBA. The research will test a hypothesis, formulated on the basis of our preliminary studies, that mechanical stress, by inducing cell deformation, can disrupt the assembly of CusCBA in cells and its efflux function, making the cell less resistant to toxic metals. The proposed research will combine the approaches of single-molecule super-resolution tracking via stroboscopic imaging, chemical/genetic manipulations, nanofluidics-based mechanical manipulations, and bulk biochemical/cellular assays. The proposed research involves close collaborations between two investigators: the PI Chen, a chemist with expertise in single-molecule super-resolution imaging of bacterial proteins, and the co-PI Hernandez, a mechanical engineer with expertise in biomechanics and mechanobiology. The rationale for this research is that, upon completion, the obtained knowledge has the potential to help devise mechanical strategies to compromise bacterial efflux and resistance to toxins such as toxic metals. Our proposed research has three specific aims: 1) Define how mechanical stress from extrusion loading alters CusCBA assembly in cells. 2) Determine the form and magnitude of mechanical stress generated in the bacterial cell envelope within the nanofluidic device using mechanical modeling. 3) Define how mechanical stress alters cellÕs resistance to toxic metals. This research is significant because: 1) it will elucidate how mechanical stresses can alter the assembly of CusCBA and its efflux-enabled resistance to copper/silver in Gram-negative bacteria, and 2) it will contribute fundamental knowledge to bacterial mechanobiology and for helping devise strategies to use mechanical stress to impair efflux pump assembly for antibiotic treatments and/or for the development of diagnostic technologies, and 3) The strategies can help preventing/treating microbial growth/infection, relevant for protecting soldiers in hostile environments and from bio-agent attacks, in line with AROÕs mission. This research is also innovative because it introduces the novel concepts of the substrate-responsive efflux pump assembly and of coupling between mechanical stress and efflux pump assembly, and because it uses the novel techniques of single-molecule super-resolution tracking and nanofluidic manipulation of individual bacterial cells.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 21, 2019
- Source ID
- W911NF1910121
Entities
People
- Peng Chen
Organizations
- Army Contracting Command
- Cornell University
- United States Army