Cell wall synthetic lipid microdomains: composition and mechanism of formation

Lead Research Organisation: Newcastle University
Department Name: Biosciences Institute

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Title Resist NOW! 
Description A sci-fi comic anthology about antibiotic resistant bacteria to raise awareness of the AMR crisis to which we contributed. 
Type Of Art Creative Writing 
Year Produced 2022 
Impact Size print run: 500 copies Sold copies: 260 Science communication goals: - Feature AMR as a current problem - Highlight scientists currently working on AMR - Feature different approaches to combat AMR - Highlight female scientists as part of this project Kickstarter backers: April 2022 Run: 186 £7,294 (reached goal) November 2021 run: 252 - £13,674 (did not reach goal) Impressions via the AMR_Comics account on twitter: 144.7K impressions over the run of Resist NOW - Volume 1 (April-May) 415.2K impressions over the run of Resist Now (Nov-Dec) Impressions via LizahvdAart on twitter: 315K impressions over the run of Resist NOW - Volume 1 (April-May) 251K impressions over the run of Resist Now (Nov-Dec) Invited talks/events: - Resist NOW was part of the AMR event at the University of Plymouth (Poster and panel talk) - Resist NOW was featured at the Microbiology society meeting in Birmingham (Talk by Eliza Wolfson) - Invited talk Charlotte Roughton (PhD student with the project) Blogs: Nicola - Leiden University website: https://www.universiteitleiden.nl/en/news/2021/11/what-if-superbugs-were-as-tall-as-buildings Jacob/Katie- John Innnes Center website: https://www.jic.ac.uk/blog/comic-book-science/ Lifeology blog: https://lifeology.io/blog/2021/11/30/scicomm-via-kickstarter/ Microbiology Today article: https://microbiologysociety.org/publication/past-issues/engaging-microbiology.html https://microbiologysociety.org/publication/past-issues/engaging-microbiology/article/science-art-it-s-all-about-connecting-the-dots.html 
URL https://www.kickstarter.com/projects/lizah/resist-now-volume-1
 
Description The bacterial lipid domains studied in the framework of this research project are the cellular target of the last resort antibiotic daptomycin, which is used to treat life-threatening multidrug resistant bacterial infections. Perhaps surprisingly, the mechanism though which this antibiotic kills bacteria, and indeed the nature of its cellular target are only poorly understood. Through the research carried out in this project, we have now a much better understanding of the molecular composition of this type of lipid domains, and why they are targeted by daptomycin. This knowledge will be valuable to identify and characterise other novel antibiotics that target this vulnerable cellular structure. Furthermore, our research has allowed us to gain deeper insight into the antibacterial mechanism of daptomycin and indeed demonstrated that is exhibits two independent antibacterial activities. This dual activity very likely contributes to the remarkably slow resistance development towards daptomycin, and provides a valuable example of the type of a novel antibiotics that can overcome the rapid resistance development that drives the current AMR crisis.

Bacterial cell wall synthesis, which is associated with the lipids domains studied in this project, is also the target for some our most successful antibiotics classes such as penicillins, cephalosporins, and for the last resort antibiotic vancomycin used to tread life-threatening multidrug resistant bacterial infections. These classes on antibiotics are generally assumed to kill bacteria by inducing cell disintegration (cell lysis). However, our work on protein-membrane interactions of the cell wall synthetic machinery, which is funded by this grant, has allowed identify another, cell lysis independent mechanism though which these effective antibiotics are able to kill bacterial cells. This finding provides an explanation for several long-standing questions regarding how antibiotics kill bacteria and also how some bacterial cell types evade antibiotic activity, thereby leading the treatment failures and re-occurring infections. These findings not only allow us to better understand the biological processes underpinning antibacterial activity, but also to guide the development of novel antibiotics towards ones with higher likelihood of good clinical performance.
Exploitation Route Our findings regarding a novel mechanism though which existing, clinically successful antibiotic classes kill bacteria has great potential in increasing the predictive value of novel antibiotic development. One of the largest challenges of antibiotic discovery and development pipeline is that we do not have a good understanding of which type of antibiotic targets are intrinsically less vulnerable to rapid antibiotic resistance development. Our findings on how cell wall and membrane targeting antibiotics kill bacteria though a multi-pronged mechanisms has allowed us to identify the key cellular processes than link immediate inhibitory activity of an antibiotic to severe and pleiotropic disturbances overwhelming the cellular defense mechanisms, which ultimately lead to bacterial cell death. This knowledge can be utilised to steer target-based drug development, already in the early stages, towards targets associated with robust, bactericidal consequences and, thus, reduced risk or rapid antibiotic resistance development.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description EVALUATING ELONGASOME TUG-OF-WAR AS A KEY REGULATOR OF BACTERIAL CELL WALL SYNTHESIS
Amount £388,118 (GBP)
Funding ID BB/X001512/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 03/2023 
End 09/2026
 
Description Membrane targeting antimicrobials as promising resistance-breaking antibiotic candidates.
Amount £85,670 (GBP)
Funding ID 2117896 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 03/2022
 
Description Membrane-adaptive processes in bacterial resistance against membrane targeting antibiotics and host immunity
Amount £97,000 (GBP)
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 08/2020 
End 09/2023
 
Description Novel mode of action of cell wall-targeting antibiotics
Amount £97,000 (GBP)
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 09/2018 
End 09/2021
 
Description Optimising protein production from high density Bacillus subtilis cultures by supressing autolysis
Amount £97,109 (GBP)
Funding ID 2306748 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2019 
End 09/2023
 
Description Uncovering the killing-mechanism of bactericidal antibiotics
Amount £97,109 (GBP)
Funding ID 2601737 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2021 
End 09/2025
 
Description Understanding an ancient universal membrane effector system
Amount £4,431,990 (GBP)
Funding ID BB/X003035/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 11/2022 
End 08/2027
 
Title Cell based screen for rapid identification of membrane depolarising, membrane pore forming, and lysis-inducing antibacterial compounds. 
Description The new established screen allows rapid identification of the antibacterial mode of action of known antibacterial compounds, or screen for novel antibacterial compounds with bacterial cell-envelope targeting activities. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact The tool was used to screen for novel cell envelope -targeting antibiotics (in collaboration with Demuris Ltd). This resulted in identification of 20 actinobacterial species that produce likely novel bacterial cell envelope-targeting antimicrobial compounds. 
 
Description Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics 
Organisation Durham University
Department Department of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Lead-supervision of a shared BBSRC DTP PhD student.
Collaborator Contribution Co-supervision of a shared BBSRC DTP PhD student including hosting the student during research stays.
Impact No outputs yet beyond funding for a BBSRC DTP PhD studensthip.
Start Year 2021
 
Description Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics 
Organisation Polish Academy of Sciences
Country Poland 
Sector Public 
PI Contribution Lead-supervision of a shared BBSRC DTP PhD student.
Collaborator Contribution Co-supervision of a shared BBSRC DTP PhD student including hosting the student during research stays.
Impact No outputs yet beyond funding for a BBSRC DTP PhD studensthip.
Start Year 2021
 
Description Cellular processes underpinning bactericidal and bacteriolytic activities of envelope-targeting antibiotics 
Organisation University of Sheffield
Country United Kingdom 
Sector Academic/University 
PI Contribution Lead-supervision of a shared BBSRC DTP PhD student.
Collaborator Contribution Co-supervision of a shared BBSRC DTP PhD student including hosting the student during research stays.
Impact No outputs yet beyond funding for a BBSRC DTP PhD studensthip.
Start Year 2021
 
Description Evaluating elongasome tug-of-war as a key regulator of bacterial cell wall synthesis 
Organisation University of Warwick
Department School of Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research due to commence in April 2023.
Collaborator Contribution Collaborative research due to commence in April 2023.
Impact No outputs yet
Start Year 2023
 
Description Membrane-targeting antimicrobials as promising resistance-breaking antibiotic candidates 
Organisation Demuris Limited
Country United Kingdom 
Sector Private 
PI Contribution This is an MRC iCASE studentship with Demuris Ltd as the industrial partner. While the start of the project coincided with the time period between the grant approval and grant start, the project was explicitly mentioned in the grant proposal as one of the translational research activities linked to the project. The MreB membrane interactions that lie in the core of the grant are involved in bacteriolysis induced by cell envelope targeting antibiotics. Furthermore, disruption of MreB membrane interactions are critically involved in the mode of action membrane-targeting last resort antibiotics such as Daptomycin, Polymyxin B and Colistin. In this collaborative project, we aim to use the fundamental understanding of the role of MreB in antibiotic mode of action to screen for novel, natural product antibiotics that target bacterial cell envelopes. For this aim, we have developed a novel assay that allows such antibiotics to be rapidly screened and identified.
Collaborator Contribution Building on the established assays, novel antibiotic screens were carried out at the premised of Demuris, using their unique library of actinobacterial species. This was carried out in the framework of an industrial placement of the PhD student.
Impact We have identified 20 so far uncharacterised actinobacterial species that produce bacterial envelope-targeting compounds. These include both those targeting bacterial membranes, novel compounds that exhibit characteristics similar to the last resort antibiotic Daptomycin, and compounds that target bacterial cell wall synthesis machinery. We are currently negotiating agreements with Odyssey Therapeutics (which purchased Demurs Ltd during the course of this project) to continue work on these promising and likely novel antibacterial lead compounds.
Start Year 2018
 
Description Optimising protein production from high density Bacillus subtilis cultures by supressing autolysis 
Organisation DSM
Country Netherlands 
Sector Private 
PI Contribution This is a BBSRC CASE studentship with Royal DSM as the industrial partner. The MreB-membrane interactions that lie in the core of the grant are also involved in Bacillus subtilis cell lysis process. B. subtilis is a major production host in biotechnological production of heterologous proteins with high commercial value. In this collaborative project, we aim to translate the knowledge on MreB-membrane interactions towards biotechnological applications.
Collaborator Contribution Although the project has been significantly delayed by COVID-19, we have maintained an active collaboration through remote meetings. The student is scheduled to carry out the first collaborative placement at the industrial partner in April-June 2022.
Impact We have established novel assays that allow cell lysis to be monitored in a fermentation setup. This has allowed us to identify gene deletions that supress cell lysis. This is very exciting since cell lysis under these conditions leads to both lower product yields and contamination of the product with GMO-DNA, which necessitates costly additional purification steps. We have started initial conversations about patenting some of the strains.
Start Year 2019
 
Description Understanding an ancient universal membrane effector (sLOLA) 
Organisation Johannes Gutenberg University of Mainz
Country Germany 
Sector Academic/University 
PI Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Collaborator Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Impact No outputs yet.
Start Year 2022
 
Description Understanding an ancient universal membrane effector (sLOLA) 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Collaborator Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Impact No outputs yet.
Start Year 2022
 
Description Understanding an ancient universal membrane effector (sLOLA) 
Organisation University of Nottingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Collaborator Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Impact No outputs yet.
Start Year 2022
 
Description Understanding an ancient universal membrane effector (sLOLA) 
Organisation University of York
Department Department of Biology
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Collaborator Contribution Collaborative research in the framework of a BBSRC sLOLA consortium.
Impact No outputs yet.
Start Year 2022
 
Description Resist NOW! 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact A sci-fi comic anthology about antibiotic resistant bacteria to raise awareness of the AMR crisis to which we contributed.

Size print run: 500 copies
Sold copies: 260

Science communication goals:
- Feature AMR as a current problem
- Highlight scientists currently working on AMR
- Feature different approaches to combat AMR
- Highlight female scientists as part of this project

Kickstarter backers:
April 2022 Run: 186 £7,294 (reached goal)
November 2021 run: 252 - £13,674 (did not reach goal)

Impressions via the AMR_Comics account on twitter:
144.7K impressions over the run of Resist NOW - Volume 1 (April-May)
415.2K impressions over the run of Resist Now (Nov-Dec)

Impressions via LizahvdAart on twitter:
315K impressions over the run of Resist NOW - Volume 1 (April-May)
251K impressions over the run of Resist Now (Nov-Dec)

Invited talks/events:
- Resist NOW was part of the AMR event at the University of Plymouth (Poster and panel talk)
- Resist NOW was featured at the Microbiology society meeting in Birmingham (Talk by Eliza Wolfson)
- Invited talk Charlotte Roughton (PhD student with the project)

Blogs:
Nicola - Leiden University website:
https://www.universiteitleiden.nl/en/news/2021/11/what-if-superbugs-were-as-tall-as-buildings

Jacob/Katie- John Innnes Center website:
https://www.jic.ac.uk/blog/comic-book-science/

Lifeology blog:
https://lifeology.io/blog/2021/11/30/scicomm-via-kickstarter/

Microbiology Today article:
https://microbiologysociety.org/publication/past-issues/engaging-microbiology.html
https://microbiologysociety.org/publication/past-issues/engaging-microbiology/article/science-art-it-s-all-about-connecting-the-dots.html
Year(s) Of Engagement Activity 2021,2022
URL https://www.kickstarter.com/projects/lizah/resist-now-volume-1