Collaborative Research: Single-molecule in vivo analysis of mechanosensitive channels in bacteria using force spectroscopy

The objective of this research project is to discover how the mechanical safety valves, so called “mechanosensitive channels”, embedded in the tough outer shells of bacteria function and how they help to protect bacteria against rupture due to excessive internal pressure during sudden changes in their environment. An understanding of this essential bacterial function could impact human health and the control of bacterial disease.

Multiple drug resistance is an immense health threat. A detailed understanding of bacterial protective functions may lead to new pharmacological approaches to overcoming these protections. Furthermore, bacteria play crucial roles in commercial agriculture, environmental remediation, and alternative energy production.

In all these situations, understanding of growth regulation and reactions to changing environmental conditions is critical. The work of this collaborative research program lies at the intersection of biology, experimental biophysics, and mechanical engineering, and graduate students and postdoctoral researchers in the team will be trained to work at this intersection.

Undergraduate students at Duke and UCLA will also participate in this research project. Undergraduates from underrepresented minority groups will participate in summer research experiences at UCLA that focus on computational modeling. Middle and high school students from diverse backgrounds will participate in afterschool and camp experiences at Duke that introduce participants to state-of-the-art cell imaging technologies.

Through the educational outreach, this work will increase and diversify the group of undergraduates interested in STEM-based careers, including those from community colleges and Minority Serving Institutions.

Bacteria are enclosed by a complex multi-layered cell envelope that enables them to maintain a high internal turgor pressure of one or more atmospheres. When external osmolarity drops significantly, excessive turgor can cause cells to burst. To prevent this, mechano-sensitive channels (MSCs) embedded in the inner lipid membrane act as safety valves and release solutes to decrease turgor.

It remains unknown if MSCs in the living cell open only when the lateral tension in the inner cell membrane increases, or if they also react to other mechanical stimuli transmitted through their complex mechanical microenvironments. It also remains unknown how biochemical regulation affects force transmission leading to channel gating. This project will utilize a new approach to observe the opening of single MSCs in live bacteria in response to mechanical compression, essentially between flat plates in an atomic force microscope (AFM).

This method can precisely quantify turgor pressure and, at the same time, resolve cell volume changes as small as 0.01 femtoliters, produced by the gating of individual MSCs. The project will study gram-negative E. coli and gram-positive B. subtilis bacteria and compare the behavior of wildtype strains with strains expressing only specific MSCs. Experiments will be combined with analytical and numerical coarse-grained modeling to understand force transmission to MSCs through the complex cell wall structures, including the lipid membrane(s), the proteoglycan layer, and the periplasmic polyelectrolyte layer, with a focus on the role of cell wall defects.

The new approach to in vivo characterization of MSCs will help to solve fundamental puzzles about MSC function in their native physiological environment.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.