Cell wall synthetic lipid microdomains: composition and mechanism of formation

The wide use of antibiotics in healthcare and agriculture has caused the appearance of bacterial strains that are resistant against most or even all antibiotics. As a result, bacterial infections have re-emerged as a serious health concern, and an increasing financial burden for the healthcare systems. To counteract this trend, research and development of new antibiotics is a top priority. We now need to identify new classes of antibiotics that are not readily compromised by existing resistance mechanisms, and to direct our long-term efforts towards antibiotics with an intrinsically lower risk of resistance development.

Historically, our most successful classes of antibiotics have been natural compounds produced by other organisms to counteract bacteria in their environment. These antibiotic classes were identified based on good antibacterial activity, and usually feature a complex antibacterial mode of action with several cellular systems inhibited at the same time. In recent decades, efforts to develop new antibiotics have been dominated by a target-driven approach, which first identifies a single, theoretically good antibiotic target, and then screens for specific inhibitors against it. This approach has largely failed due to the rapid rate of resistance development against single mode-of-action antibiotics. Consequently, we now need to re-focus on antibiotics that do not act by a single inhibitory mechanism.

Compounds that target bacterial cell membranes are widely used in nature as antimicrobials. These molecules are produced by other bacteria, fungi, plants and animals to combat undesired bacteria. This evolutionally highly successful strategy has two crucial advantages over our current antibiotics. Firstly, instead of inhibiting gene-encoded targets such as proteins or ribosomes, the cellular targets are membrane lipids. Consequently, mutations that modify the target and thereby prevent the binding do not easily emerge. Secondly, the disruption of the cell membrane simultaneously inhibits a large number of membrane-associated cellular processes, thus making it difficult for bacteria to evolve a meaningful defense. One crucial cellular process disrupted by membrane-targeting antibiotics is the synthesis of cell wall, a rigid structure that encloses the cell and provides it with physical stability. The interference with the cell wall synthesis provides membrane-targeting antibiotics the ability to cause irreversible disintegration of the cell, a process termed bacteriolysis. Consequently, membrane-targeting antimicrobials kill bacteria very rapidly, and the rate of resistance development is either remarkably low or even undetectable.

The reason why the bacterial cell wall synthesis machinery is sensitive to membrane-targeting antibiotics has remained elusive. Recently, we showed that proteins responsible for the cell wall synthesis induce a specific area in their membrane surrounding (lipid domain) that differs from the remaining membrane in its properties and composition. Such lipid domains are preferred targets for membrane-targeting antibiotics, hence providing the first plausible explanation why cell wall synthesis is efficiently inhibited. In this project, we will identify the mechanism through which the lipid domains associated with the cell wall synthesis machinery are induced, and characterise their detailed composition. This is important in order to understand how bacteria synthesise their protecting cell wall envelope, and how membrane-targeting antimicrobials kill bacteria by disrupting its synthesis. By providing direct insight into the mechanisms underpinning their potency, our research will guide the design and development of novel membrane-targeting antibiotics that exploit this cellular weak point.

In virtually all bacteria, the cell shape is determined by a peptidoglycan-based cell wall that provides the cell with rigidity. Upon growth, the cell wall needs to expand by incorporation of new peptidoglycan. In rod-shaped bacteria, this is regulated by cytoskeletal structures formed by the actin-homolog MreB, which position the cell wall synthesis machinery through direct protein-protein interactions.

Recently, we showed that the MreB cytoskeleton induces a novel type of lipid domain in its immediate membrane surrounding. The MreB cytoskeleton, thus, bears intriguing similarity with the eukaryotic cortical actin cytoskeleton that is also associated with lipid domains. The defining characteristic of these lipid domains is an increased membrane fluidity, thus giving rise to the term regions of increased fluidity (RIFs). Lipid domains are preferred binding sites for membrane-targeting antimicrobials. The tight association of cell wall synthesis proteins with lipid domains now provides a plausible explanation for the long-standing question why cell wall synthesis is efficiently inhibited by membrane-targeting compounds, as exemplified by our recent finding that RIFs are the molecular target for the last resort antibiotic daptomycin.

Despite their relevance for the cell wall synthesis and the mode of action of membrane targeting antibiotics, the mechanism through which RIFs are induced, and their composition is still unknown. In this project, we aim to close this gap by deciphering the fundamental mechanism underpinning the RIF formation, by characterising their detailed protein and lipid composition, and by verifying the mechanism through in vitro reconstruction.

These studies will reveal the biological function of this novel type of bacterial lipid domain involved in cell wall synthesis, and deliver the first characterisation of a structure that is frequently targeted by natural antimicrobial compounds, but that is currently only poorly understood.

Relevance to Human Health:
Antibiotic resistance is developing into a serious threat to human health. The need to focus our research efforts towards the development of new, resistance-braking antibiotics has been identified as top priority by the UK Government and the World Health Organization. In recent decades, antibiotic research has largely focussed on screening compound libraries against specific selected targets, mostly single proteins. This approach has dramatically failed to deliver novel antibiotics not rapidly compromised by resistance development. Consequently, we need a change in strategy, and focus on compounds that exhibit a more complex mode-of-action and are therefore less prone to resistance development. Membrane-targeting antimicrobials, which are able to kill multidrug resistant bacteria, are a promising class of antibacterial lead compounds. These antibacterial compounds feature a complex mode-of-action that includes inhibition of bacterial cell wall synthesis as a central component, thus leading to a remarkably low resistance development. The proposed research provides a detailed characterisation of lipids domains associated with the bacterial cell wall synthesis machinery; a recently discovered link explaining the high sensitivity of cell wall synthesis towards membrane targeting antibiotics. Consequently, the planned research analyses an antibiotic target that is commonly exploited by membrane targeting antimicrobials, but that is currently only poorly understood. Our research, thus, will guide the development of novel membrane-targeting antibiotics with an intrinsically reduced risk of resistance development.

Commercial Exploitation:
This study is directly relevant for the development of novel antibiotics exploiting the cell wall synthetic lipids domains as antibiotic targets. In parallel to this project, I have initiated a collaboration with Demuris, a company specialising in antibiotic discovery, to screen natural compound libraries for such substances. This line of research is currently shortlisted and advertised for an MRC funded iCASE (Industrial Cooperative Awards in Science & Technology) PhD studentship, which is intended to start in September 2018.

Recently, we showed that the last resort antibiotic daptomycin directly targets the lipid domains associated with the cell wall synthesis machinery. The planned in vitro reconstruction opens the door for the development of high throughput drug screen allowing the identification of novel compounds that disrupts the cell wall synthetic lipid domains in a manner comparable to daptomycin. As detailed in the 'Pathways to Impact' document, this line of research will be developed in close collaboration with the High Throughput Screening Facility (HTSF) based in the Faculty of Medical Sciences at Newcastle University.

Public Awareness:
I strongly believe that promoting public understanding of scientific research is critical for global scientific endeavour, and that all scientists should actively engage with the public as part of their routine work. This is especially important in the context of the antibiotics resistance crisis, which necessitates a high level of public awareness. The public engagement activities detailed in the 'Pathways to Impact' statements will help raise the awareness of this serious threat to public health, and to inform the public about ongoing active research to seek solutions.

Training:
The planned research programme integrates elements from cutting-edge cell biology such as super resolution microscopy, from membrane biology including work with liposomes and supported lipid bilayers, and from protein biochemistry. Consequently, the PDRA will enjoy an exceptionally broad training with a wide spectrum of complementary, state of the art techniques, thereby strongly enhancing future career prospects.