Investigating AsmA-like Proteins as Key Mediators of Lipid Transport in Gram-negative Bacteria University of Birmingham in United Kingdom

Project Overview

Antibiotic-resistant bacteria pose a significant and escalating global health challenge, particularly due to the rise of multidrug-resistant Gram-negative bacteria. These pathogens are difficult to treat because of their unique outer membrane, which acts as a formidable barrier against antibiotics. With limited new antibiotics in development, there is an urgent need for alternative strategies to combat these infections.

Research Focus

The Gram-negative outer membrane is a complex structure comprising lipopolysaccharides (LPS) on the outer surface and phospholipids on the inner layer. The arrangement forms a highly effective barrier that hinders the entry of hydrophilic molecules and slows down hydrophobic compound penetration, thus enhancing resistance to antibiotics and detergents. Outer membrane proteins (OMPs) also play a key role in bacterial virulence, pathogenesis, and nutrient uptake, making them crucial targets for antimicrobial development.

A critical yet poorly understood aspect of Gram-negative bacterial physiology is the transport of phospholipids from the inner membrane (IM) to the outer membrane (OM). Recent discoveries have identified a novel class of lipid transporter, known as the repeating ß-groove (RBG) protein superfamily, which are believed to facilitate bulk lipid transfer by forming bridge-like structures between the membranes containing hydrophobic grooves along which phospholipids move. These transporters, originally found in eukaryotes, have now been identified across didermic bacteria, including E. coli.

In E. coli, six members of the RBG superfamily have been identified, all belong to the AsmA-like protein family. These proteins are all anchored to the inner membrane via an N-terminal alpha helix and contain a large periplasmic RBG domain.

Key Objectives

This PhD project aims to uncover the molecular mechanisms and biological roles of AsmA-like proteins in Gram-negative bacteria, focusing on their involvement in phospholipid transport and outer membrane integrity. Specific objectives include:

• Characterizing the structure and transport mechanisms of AsmA-like proteins using advanced structural biology techniques.

• Developing biophysical assays and molecular dynamic simulations to understand lipid transport dynamics.

• Investigating the cellular roles of AsmA-like proteins using genetic and biochemical approaches to link their activity to bacterial physiology and pathogenesis.

Methodology

This interdisciplinary project will employ a combination of structural biology, biophysics, and cell biology techniques. The use of cryo-EM, X-ray crystallography, and neutron reflectometry will provide detailed insights into protein structures, while biophysical assays and molecular simulations will elucidate their transport mechanisms. Additionally, cell-based experiments will be conducted to assess the functional significance of these proteins in bacterial cells.

Ideal Candidate

We are seeking a motivated and curious candidate with a background in molecular biology, biochemistry, microbiology, or structural biology. Experience with techniques such as cryo-EM or protein biochemistry, or an interest in antimicrobial resistance, will be highly valued.

Why Join Us?

This project offers an exciting opportunity to contribute to a cutting-edge area of bacterial cell biology with implications for antibiotic development. You will gain hands-on experience with state-of-the-art technologies and work within a collaborative, multidisciplinary research environment, opening new avenues in the fight against bacterial infections.

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.

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.