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

> **NIH NIH F31** · CORNELL UNIVERSITY · 2020 · $27,016

## Abstract

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...

## Key facts

- **NIH application ID:** 9883623
- **Project number:** 5F31AI143208-02
- **Recipient organization:** CORNELL UNIVERSITY
- **Principal Investigator:** Lauren A Genova
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $27,016
- **Award type:** 5
- **Project period:** 2019-01-25 → 2020-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9883623

## Citation

> US National Institutes of Health, RePORTER application 9883623, Single-cell Imaging of Mechanically Coupled Assembly of Metal Efflux Complexes in Bacteria (5F31AI143208-02). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/9883623. Licensed CC0.

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