Breaking down barriers against antimicrobials: elucidating a cross-kingdom novel lipid transport mechanism

Introduction

Antimicrobial resistance is on target to become the leading cause of death by 2050. A major determinant of resistance in Gram-negative bacteria is the tripartite cell envelope, which forms a physical barrier to drugs. Understanding how this structure is made, therefore, is of critical importance. Despite this, how the lipids that make the cell envelope are delivered remains poorly understood.

DedA Family of Proteins

Recently, a family of proteins has emerged that likely holds the key to understanding these lipid transport mechanisms. The DedA family of membrane proteins is conserved across all domains of life and early evidence suggests they are central to lipid transport, antimicrobial sensitivity, and virulence in bacteria.

We hypothesise that DedA proteins have fundamental lipid transport roles essential for biogenesis of the cell envelope, making them ideal targets for new approaches to combat antimicrobial resistance. However, the true functions of DedA proteins in Gram-negative bacteria remain unknown, and not a single DedA protein across life has been mechanistically characterised.

Research Goals

Our goal is to reveal the first structure of a DedA family protein, using emerging methods including the use of nanobodies with cryo electron microscopy, potentially unveiling an entirely novel cross-kingdom transport mechanism.

We will:

1. Functionally characterise DedA proteins to identify their substrates using cutting-edge native mass spectrometry methods.
2. Determine the route of lipid transport using a new in vivo cross-linking technique that I developed.
3. Study the cell envelope compositions of loss-of-function mutations in DedA proteins.
4. Develop a technique to assess alterations in membrane lipid distribution.

Expected Outcomes

This research will generate detailed mechanistic insight into how this elusive protein family functions across life, while characterising how bacteria build their bacterial envelope barrier, which is central to their ability to tolerate and resist antimicrobials.