Protein-protein interactions in the Gram-negative bacterial cell envelope
Bacteria envelope themselves in robust layers of membrane and cell wall that are vital to their survival. In my laboratory, we aim to understand how protein-protein interactions in bacteria help support the organisation, structure and stability of the outer membrane and how such interactions can also be exploited by protein antibiotics known as bacteriocins to kill bacterial cells.
We use a variety of biochemical, biophysical, structural, biophysical and cell-based approaches to address the areas below:
Bacteriocin translocation – Bacteriocins are species-specific protein antibiotics that parasitize a variety of outer membrane and periplasmic proteins in Gram-negative bacteria. Our work centres on the entry mechanisms of colicins, which target Escherichia coli, pyocins, which target Pseudomonas aeruginosa and klebicins, which target Klebsiella pneumoniae. These toxins serve as important agents of competition within microbial communities. We study both nuclease (DNases, rRNases and tRNases) and pore-forming bacteriocins, which use their network of protein-protein interactions within the cell envelope to establish a translocon complex that delivers a toxic domain into the cell. Hence, bacteriocin translocation represents a highly simplified model for understanding energised cellular protein import. We recently demonstrated that Tol- and Ton-dependent bacteriocins translocate across the outer membrane by the same basic mechanism in which the unfolded toxin is pulled through a specific translocator protein and powered by the proton-motive force of the inner membrane.
Developing bacteriocins as protein antibiotics – The rise of multidrug resistant bacteria coupled with the lack of new classes of antibiotics in over 40 years means there is a pressing and urgent need for new antibiotics especially for molecules that target pathogenic Gram-negative bacteria. In collaboration with colleagues in Glasgow, we are exploiting the species selectivity and potency of bacteriocins as next-generation protein antibiotics.
Supramolecular assembly of outer membrane proteins – The asymmetric bilayer of the outer membrane contains outer membrane proteins (OMPs) and lipoproteins. Using colicins as OMP-specific probes, we discovered that OMPs cluster to form ‘OMP islands’ in the membrane and that this clustering behaviour underpins their turnover. Through the creation of a variety of OMP-specific labels we are dissecting the organisation of OMP islands and investigating the ramifications of these micro-domains to bacterial physiology. For example, we demonstrated that the inherent organisation of outer membrane proteins into islands becomes imprinted on inner membrane proteins when they are connected by the energized protein bridge of the Tol-Pal system.
The Tol-Pal assembly – Tol-Pal is a little understood complex that is required for the stable maintenance of the Gram-negative outer membrane and which is recruited to the cell division apparatus late during division. We recently discovered a unique ‘mobilisation-and-capture’ mechanism underpins Tol-Pal function whereby force transduction across the cell envelope enables the accumulation of the peptidoglycan-binding protein Pal at division septa, stabilising the invaginating outer membrane. Ongoing work aims to understand the molecular basis of this unusual mechanism.