An in vitro gram-negative envelope mimetic: a new way to study membrane biology

Gram-negative bacteria account for 9 out of the 12 priority pathogens identified by the WHO, with new antibiotics for these pathogens being desperately needed to combat the threat of antimicrobial resistance. The bacterial cell envelope has been and remains one of the best targets for antimicrobials development. In gram-negative bacteria, this envelope is a complex, essential structure formed by two membranes sandwiching a peptidoglycan layer and periplasm that together maintain the integrity and shape of the cell. Targeting the outer of these two membranes remains massively underexploited due to our lack of understanding of its biology.

Currently it is impossible to accurately emulate the complex architecture of the gram-negative membrane in vitro, making understanding of its formation and regulation extremely difficult.

We believe we have discovered a route to recreating the gram-negative envelope atop a planar surface, opening up the ability to modify any surface-based detection technology (E.g. surface plasmon resonance, quartz crystal microbalance, neutron reflectometry) to become a gram-negative envelope biosensor, providing a novel ground-breaking platform for studying envelope biology. BBSRC pioneer award funding is being sought to test proof-of-principle of this early stage but high reward proposal.

We have identified a bacterial multi-protein complex, termed PqiABC, that spans across both the inner and outer membranes, and which can be tethered to a sensor surface. We believe these properties offer the potential for stepwise construction of the double membrane architecture on a surface, allowing absolute control over its formation and the addition of any components that warrant study.

PqiABC will form the initial focus of our study. To date, it remains unknown how phospholipids get to the outermost membrane in gram-negative bacteria, we believe PqiABC is responsible. Furthermore, we hypothesise it is driven by the proton motive force (PMF) based on its homology to other PMF driven transporters. We will investigate this by measuring phospholipid population changes across the envelope following induction of transmembrane electrochemical proton gradients.

Next, by introducing a known non-functional variant, PqiABC will move from subject to scaffold and our focus will move to establishing the system as an enabler for probing other membrane biology. We will focus on further proving the capabilities of our double bilayer mimetic by incorporating the complete outer membrane protein biogenesis pathway. If successful it will allow real time monitoring of all stages of outer membrane protein biogenesis from protein transport across the inner membrane, periplasmic shuttling via chaperones, to folding of integral outer membrane proteins at the outer membrane.

We have chosen this pathway because of the need to understand outer membrane protein biogenesis. The outer membrane is responsible for a vast range of biological functions including cellular homeostasis, pathogenesis and virulence. Central to these processes are outer membrane proteins, yet we still do not fully understand how they are transported, folded and inserted into the outer membrane. An in vitro OMP biogenesis platform would provide a unique resource to probe and perturb OMP biogenesis, providing novel insights into how the myriad OMPs are targeted and folded.

Moving forward this platform technology could also be used to understand how antibiotics interact with and penetrate the envelope or provide insight into host/pathogen interactions and how membranes remodel to facilitate infection.

Membranes are essential to all life, they are the means to encapsulate the cell, separating inside from out. Over 60% of all drugs target membrane proteins, yet we remain largely in the dark regarding many aspects of their function. The technology platform proposed here, if successful, could revolutionise their study, making the most complex system amenable to study.