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

Tripartite efflux pumps enable Gram-negative bacteria to extrude diverse toxins, contributing tobacterial multidrug resistance and the emerging threat of untreatable bacterial infections. These effluxpumps 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 recentlydiscovered that CusCBA, an RND-family metal efflux pump, undergoes dynamic assembly in responseto cellular demands for metal efflux. Understanding such mechanisms of these efflux pumps andexploring novel methods to compromise their functions are crucial for developing new and effectiveantibacterial 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 stressesmay affect the assembly and function of tripartite efflux pumps such as CusCBA, even though mechanicalforces are experienced in bacterial growth environments. The long-term goal of this research is tounderstand how bacterial efflux can be manipulated for preventive and therapeutic purposes. Theobjective here is to understand how mechanical stress can alter the assembly of CusCBA in live E. colicells and thus cells? resistance to metal stress. The central hypothesis here, supported by preliminarystudies, is that mechanical stress, by inducing cell deformations, can compromise the assembly ofCusCBA in cells and thus their efflux function, making cells less resistant to metal stress. This hypothesiswill be tested using combined approaches of single-molecule tracking, nanofluidics-based mechanicalmanipulations, chemical/genetic manipulations, and bulk biophysical/biochemical/cellular assays. Theapplicant will be advised by a mentoring team that includes a chemist with expertise in single-moleculeimaging 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 devisemechanical strategies to impair the assembly of CusCBA and related tripartite efflux pumps and thusbacterial efflux to increase the efficacy of antibiotic treatments. The proposed research has two specificaims: 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 researchis significant because it will advance the mechanobiology of bacterial efflux, the development ofmechanical strategies to intervene in bacterial efflux for antibacterial therapy, and new technologies formechanically manipulating single bacterial cells. It is innovative because it introduces the novel conceptof mechano-efflux coupling and it uses the novel techniques of single-molecule tracking via time-lapsestroboscopic imaging and nanofluidic manipulation of individual bacterial cells.