Molecular basis of outer membrane stabilisation by the energised Tol-Pal system in Gram-negative bacteria

Bacteria are important for the health and well-being of most living organisms on earth. Examples in humans include their importance in our gut microbiomes for the digestion of food. Bacteria can also be pathogenic, meaning they can infect tissues and organs which, if left untreated, can be lethal. Much of modern medicine relies on the effective treatment of bacterial infections, such as pneumonia and sepsis, through the use of antibiotics. The rise of antibiotic resistance in bacteria is increasingly rendering many of our frontline antibiotics ineffective.

The current proposal focuses on the outer membrane, one of the main factors contributing to antibiotic resistance in Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The outer membrane is a highly impermeable barrier that excludes many types of antibiotics that are otherwise effective against Gram-positive bacteria that lack an outer membrane. During the process of division in Gram-negative bacteria newly replicated daughter cells must have their individual outer membranes pinned to the underlying cell wall. This process requires the input of energy. Because the outer membrane is an 'energy-less' environment the requisite energy is provided by the electrochemical gradient across the inner membrane of the bacterium. A complex, multiprotein protein nanomachine known as Tol-Pal is tasked with this transfer of energy. Tol-Pal taps into this energy source to stabilise the outer membrane by an unknown mechanism.

We discovered recently how Tol-Pal achieves this complex function, in the process identifying a biological mechanism we call 'mobilisation-and-capture'. The current proposal seeks to capitalise on these advances. We will investigate all the discrete interactions of the five Tol-Pal components and how they move in the membranes of the bacterium in order to understand how the cell's energy is exploited to pin the outer membrane to the cell wall. As part of this work, we will also exploit a new technology we have developed whereby energised complexes in Gram-negative bacteria can be trapped in their activated states for subsequent structural dissection using antibacterial proteins known as colicins.

Through this work we will obtain the most detailed view yet of how the outer membrane is stabilised in Gram-negative bacteria and how nanomachines like Tol-Pal work to achieve this, knowledge that can be exploited in the future fight against bacterial infections.

We recently discovered a mobilisation-and-capture (M&C) mechanism is the basis for Tol-Pal stabilisation of the outer membrane (OM). This involves the complex interplay of five proteins, both membranes of the cell envelope, the peptidoglycan and the PMF. Our multidisciplinary proposal aims to dissect the molecular basis for the M&C mechanism and has five objectives:

1. Determining the in vivo interaction partnerships of E. coli Tol-Pal proteins by photoactivatable crosslinking and LC-MS/MS; specifically, how the TolQ-TolR-TolA complex is recruited to the divisome, how the PMF activates peptidoglycan binding by TolR and how TolA reaches the OM.

2. Tracking the mobilities and associations of Tol-Pal complexes in the two membranes of live E. coli cells and the influence of the PMF. Mobilities of complexes will be followed using newly-developed FRET-based assays.

3. Following changes in the periplasmic localisation of TolB, the mediator of Pal diffusion in the OM using protease protection assays in permeabilised cells and fluorescence microscopy. The localisation of TolB relative to the peptidoglycan layer is predicted to change depending on the division state of the cell.

4. Defining the structural basis of PMF-induced activation of peptidoglycan-binding by the stator protein TolR. We will obtain structural information on the TolQ-TolR stator complex prior to its binding of the peptidoglycan using an inactivating disulphide and obtain structural information for the TolR-peptidoglycan complex.

5. Visualising by cryo-electron tomography the entire Tol-Pal nanomachine within minicells trapped in its PMF-activated state using a bacteirocin. We have already put this strategy in place for a Ton-dependent bacteriocin and will now extend the approach to a Tol-dependent bacteriocin. This approach promises to furnish structural data for these nanomachines in the context of the cell envelope and in their PMF-activated states.