Developing a Surface-tethered Double Membrane Model to Study Bacterial Envelope Biogenesis University of Birmingham in United Kingdom

Project Overview

The rise of antimicrobial resistance, particularly among Gram-negative bacteria, is a pressing global health concern. Gram-negative pathogens account for 9 out of the 12 priority pathogens identified by the WHO, underscoring the urgent need for new therapeutic strategies. One of the most promising targets for novel antibiotics is the bacterial cell envelope. In Gram-negative bacteria, this envelope is a complex structure consisting of two membranes surrounding a peptidoglycan layer, essential for maintaining cell integrity and shape. Key biological processes within this envelope, such as multidrug efflux, secretion, nutrient import, and membrane biogenesis, often span both membranes, making them challenging to study using traditional methods.

Research Focus

This PhD project aims to develop an innovative in vitro system that mimics the Gram-negative bacterial envelope, enabling unprecedented insights into membrane structure, transport processes, host-pathogen interactions, and drug screening. The goal is to create a surface-based double bilayer model that replicates the native envelope environment, providing a novel platform to study envelope biology in a controlled setting. Central to this project is the reconstitution of the Escherichia coli PqiABC multiprotein complex, which naturally forms a trans-envelope tunnel connecting the inner and outer membranes. This complex consists of: PqiA: An integral inner membrane protein PqiB: An inner membrane-anchored periplasmic protein PqiC: An outer membrane lipoprotein By assembling the PqiABC complex on a planar surface, we aim to reconstruct a double membrane architecture that can serve as a scaffold for studying trans-envelope processes. This system will allow for stepwise assembly and precise control over membrane formation, enabling the study of complex envelope systems in a way that has not been possible before.

Key Objectives

The project will explore the following objectives:

1. Development of a Double Bilayer Membrane System: Construct a surface-tethered double membrane model using the PqiABC complex as a scaffold.

2. Incorporation of Trans-envelope Pathways: Integrate other essential membrane biogenesis systems, such as the Mla pathway, which is involved in retrograde phospholipid transport. This pathway includes key components like MlaA, MlaC, and the inner membrane ATPase complex MlaFEDB.

3. Real-time Monitoring and Functional Analysis: Use advanced techniques like quartz crystal microbalance and neutron reflectometry to assess the incorporation and activity of these systems in real-time.

4. Expansion to Outer Membrane Protein Biogenesis: Investigate the potential to incorporate more complex intermembrane machineries, such as the complete outer membrane protein (OMP) biogenesis pathway.

This could enable monitoring of OMP transport, periplasmic chaperone-mediated shuttling, and protein folding at the outer membrane.

Significance and Impact

Understanding outer membrane biogenesis is critical for bacterial homeostasis, virulence, and pathogenesis. Yet, the transport, folding, and insertion of outer membrane proteins remain poorly understood. This project aims to create a versatile in vitro platform to investigate these processes in detail, providing new insights into bacterial physiology and potential antibiotic targets. In the long term, this research could pave the way for novel antimicrobial development strategies.

Ideal Candidate

We are looking for a highly motivated candidate with a background in microbiology, biochemistry, molecular biology, or biophysics. Experience in membrane protein studies, structural biology, or biophysical assay development would be advantageous.

Why Join Us?

This project offers an exciting opportunity to work at the forefront of bacterial cell biology and antibiotic research. By joining this project, you will gain hands-on experience with state-of-the-art technologies and contribute to addressing a critical global health challenge. This research will be conducted in a collaborative, interdisciplinary environment, offering ample opportunities for skill development and innovation.

Funding notes:

1. Competition based funding available through the Midlands Integrative Biosciences Training Partnership - https://warwick.ac.uk/fac/cross_fac/mibtp/ https://www.birmingham.ac.uk/research/activity/mibtp 2. Competition based funding available through the Darwin Trust of Edinburgh 3. Self-funded Please contact [email protected] for more information regarding funding available

References:

Cooper, B.F., Ratkeviciute, G., Clifton, L.A., Johnston, H., Holyfield, R., Hardy, D.J., Caulton, S.G., Chatterton, W., Sridhar, P., Wotherspoon, P., Hughes, G.W., Hall, S.C., Lovering, A.L. and Knowles, T.J. (2024). "An octameric PqiC toroid stabilises the outer-membrane interaction of the PqiABC transport system." EMBO Rep 25(1): 82-101.

Hall, S.C.L., Hardy, D.J., Bragginton, E.C., Johnston, H., Onose, T., Holyfield, R., Sridhar, P., Knowles, T.J. and Clifton, L.A. (2024). "Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly." Nanoscale 16(28): 13503-13515.

Wotherspoon, P., Johnston, H., Hardy, D.J., Holyfield, R., Bui, S., Ratkeviciute, G., Sridhar, P., Colburn, J., Wilson, C.B., Colyer, A., Cooper, B.F., Bryant, J.A., Hughes, G.W., Stansfeld, P.J., Bergeron, J.R.C. and Knowles, T.J. (2024). "Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport." Nat Commun 15(1): 6394.