Disruption in bacterial cell envelope following polymyxin challenge and adaptations in tolerant strains

Bacterial infections remain a major challenge to healthcare and the problem is exacerbated by the development or acquisition of resistance to antibiotics. The group of Gram-negative bacteria are particularly refractive to management, as the structure of their cell envelope includes an outer membrane that confers natural protection from antibiotics and antimicrobials. For example, the molecular targets for the most widely used antibiotics from the penicillin group, are protected within the bacterial outer membrane. Also protected is the inner bacterial membrane, which plays a defining role in maintaining the living state of cells. Both membranes have a continuous bilayer structure with distinctly different architecture and composition.

Polymyxins are one class of antimicrobial compounds with activity against Gram-negative bacteria that have found restricted clinical use as last line therapeutics. They show comparatively high toxicity and despite a wealth of research and history of clinical use, their mechanism of action remains poorly understood. During their action, polymyxins utilise and subvert a bacterial surface receptor, lipopolysaccharide, to cross the protective outer membrane and access the inner membrane, where they exercise their killing action.

In this proposal, we aim to decipher the molecular details of bacterial killing by polymyxins, describe the stages of polymyxin-induced bacterial death, and investigate the detailed structural and molecular changes in bacterial outer and inner membranes that result from adaptive changes in resistant bacteria. We seek a molecular template from the interaction between polymyxin and its target, lipopolysaccharide, from resistant bacteria, which will inform engineering of polymyxin antimicrobials with enhanced activity against resistant Gram-negative pathogens.

To achieve this, we combine advanced force microscopy that can map bacterial surfaces at molecular resolution, with magnetic resonance spectroscopy providing molecular and structural information at atomic level of detail, to investigate the interaction of polymyxins with bacterial outer and inner membranes, the changes in their structure, organisation and stability. we hypothesise initial outer membrane breach by polymyxin disrupts the normal exchange of molecular material between the two membranes and dysregulates organisation of both outer and inner membranes. To investigate the lethal consequences of this dysregulation, we will monitor bacterial viability and will use electron microscopy to monitor the sequential collapse of important structural elements in the bacterial envelope. Finally, we will employ this methodology to reveal what changes in bacterial membrane structure and molecular architecture confer protection against polymyxins in resistant bacteria.

Polymyxins are last-resort antibiotics for the treatment of infections caused by multi-drug-resistant pathogens such as carbapenemase-resistant E. coli. Polymyxins are known to target bacterial membranes, but how they kill Gram-negative bacteria remains poorly understood. The clinical use of polymyxins and other membrane-targeting antimicrobials is still restricted, due to associated host toxicity at near therapeutic concentrations. This highlights a significant gap in our biological understanding of the Gram-negative bacterial cell envelope and its barrier function, as well as the need for engineering polymyxins with improved activity and reduced host toxicity. We propose a research programme combining state-of-the-art biophysical, spectroscopic and microscopy techniques to determine how polymyxin antibiotics affect the outer and inner bacterial membranes and how this translates into cell envelope restructuring, disruption and bacterial cell death. We also investigate the molecular and structural impact of polymyxin on the bacterial envelope of adapted cells to understand the mechanism of self-protection and polymyxin resistance.

In this project, we investigate the perturbation of bacterial outer membranes by polymyxins. We look at outer membrane organisation, as well as molecular interactions that underpin outer membrane stability. We also look at the impact of polymyxins on the architecture, stability and integrity of inner bacterial membranes, challenged by polymyxins that have breached the outer membrane. We hypothesise that polymyxin may affect the exchange of lipopolysaccharide and lipid between the two membranes that is essential to maintaining the distinct morphology and essential physiological functions of the envelope membranes. Aiming to develop polymyxins with enhanced activity against resistant bacteria, we will characterise the molecular interface between polymyxin and its target, bacterial lipopolysaccharide from resistant strains.