Engineering bacterial traps to understand and inspire next-generation antibiotics

Session

Poster Session Two

Authors

Vincent Fideli (1, 2)

Affiliations

1. Imperial College
2. London Centre for Nanotechnology, University College London

Keywords

Atomic force microscopy (AFM), Bacterial physical traps, Micro-/Nano-fabrication, Antimicrobial peptides (AMP)

Abstract text

Antimicrobial peptides (AMPs) are a promising class of potent antibiotics that act by disrupting bacterial cell membranes. Atomic force microscopy (AFM) has been effective for studying the impacts of AMPs on model membranes, but the complexity of bacterial cell membranes poses difficulties in imaging the effects of AMPs on live bacterial surfaces at nanometre resolutions. Building upon physical methods of immobilising live bacteria, we aim to develop a microfluidics-based trap that facilitates microscopy whilst the bacteria are exposed to varying doses of antibiotics. This research can enable the rational design of antibiotics that are more potent and bacteria-selective to tackle antimicrobial resistance.

AFM has been used to show the membrane of live, growing E. coli at nm resolutions^1,2. AFM has also been used to study the impact of AMPs on bacterial cell membrane³. However, many studies have so far investigated the effects of AMPs on model cell membranes instead of live bacteria since it is difficult to capture nanometre-resolution images of growing and dividing bacteria. These difficulties arise from both the complex nature of the bacterial cell membrane and because it is difficult to immobilise dividing cells for AFM. 

It is therefore important to develop immobilisation protocols for bacterial AFM to increase spatial resolution, and to facilitate imaging of growing and dividing bacteria. Bacterial immobilisation strategies can be divided into two: chemical substrate modification and physical traps. Physical trapping is more advantageous because it is not reliant on the chemical properties of the buffer, so should be more versatile, and because chemical substrate modification can alter cell viability³. This project aims to improve PDMS-based micro-channels, previously established by Chen et al (2014)⁴, which were made using a combination of micro-fabrication and soft lithography. These traps would allow bacteria to grow and divide whilst still sufficiently immobilising them. Once these traps have been developed and optimised for bacterial scanning with AFM, we can add antibiotics to track changes on the cell surface over time⁵.

Here, I will present first attempts of alternative trapping methods for live, growing bacteria. I will compare the effectiveness of physical trapping with chemical substrate modification in terms of resolution and ability to immobilise growing bacteria. Using these micro-channels, I will then show the effects on the cell membrane of AMPs such as polymyxins, bacteriocins, and epidermicins. By understanding the organisation of the bacterial surface and the mechanisms of action of AMPs on the outer membrane of bacteria, we can further develop more potent antibiotics. For example, targeting bacteria-specific essential features on the outer membrane will increase selectivity for bacteria than eukaryotes, also making it more difficult for bacteria to evolve resistance mechanisms, tackling AMR.

References

1. Benn, G. et al. Phase separation in the outer membrane of Escherichia coli. Proc Natl Acad Sci USA 118, e2112237118 (2021).

2. Beaussart, A. & El-Kirat-Chatel, S. Microbial adhesion and ultrastructure from the single-molecule to the single-cell levels by Atomic Force Microscopy. The Cell Surface 5, 100031 (2019).

3. Louise Meyer, R. et al. Immobilisation of living bacteria for AFM imaging under physiological conditions. Ultramicroscopy 110, 1349–1357 (2010).

4. Chen, P. et al. Nanoscale Probing the Kinetics of Oriented Bacterial Cell Growth Using Atomic Force Microscopy. Small 10, 3018–3025 (2014).

5. Hammond, K., Ryadnov, M. G. & Hoogenboom, B. W. Atomic force microscopy to elucidate how peptides disrupt membranes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1863, 183447 (2021).