MICA: Mechanistic understanding of cell wall biosynthesis to combat antimicrobial resistance

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

The discovery of the antibiotic penicillin opened the door to the treatment of a wide range of infections. It works by stopping bacteria from making the polymer in the cell wall (peptidoglycan, PG) that holds them together. This is assembled by specialised proteins (called penicillin-binding-proteins or PBPs, which are present in all bacteria) that either have the ability to stitch together both the sugar backbone and the peptides (these are known as bi-functional enzymes), or either just the sugar back bone or just the peptide-crosslinks (mono-functional enzymes). We know little about how the polymerization and cross linking activities are controlled or co-ordinated, or how they truly interact with their natural substrates. Furthermore, the construction of peptide cross-links by PBPs is famously the target inhibited by penicillin which stops cell wall construction and kills the bacterium.

Penicillin has been an excellent antibiotic, not least because it targets multiple PBPs simultaneously within a bacterium and resistance rarely develops by altering the PBP target (with the notable exception of bacteria that can acquire altered PBP genes from other species that are poor targets for the antibiotic). Unfortunately, many bacteria have acquired resistance to penicillin by other mechanisms. Primarily this has been due to the acquisition of enzymes that degrade the antibiotic (beta-lactamases), or reduce penetration (influx) of antibiotic into the bacterium or increasing the rate of efflux out of the bacterium. We urgently need to fight back and the strategy of exploring PBPs to make better versions of current antibiotics that are more active, can evade beta-lactamases or resistance due to changing influx or efflux. Global pharmaceutical companies have a real interest in progressing such developments, however, they need better mechanistic insight into how PBPs work. We attend to address these fundamental gaps in our understanding.

Why can we succeed where others have failed?

1. Progress in achieving this mechanistic insight has been hampered by past inability to routinely synthesise the key chemical components or precursors that make this polymer. From past MRC and BBSRC funding we can now make key chemical components at Warwick, and have developed an exceptional track record of providing reagents to study peptidoglycan biosynthesis to academia worldwide.

2. Having studied how to synthesise all of the chemical precursors used by different PBPs we have developed completely new continuous assays that will now help us to understand how PBPs polymerise precursors or how they crosslink these. We have one assay to finalise, which would bring together our ability to study polymerization and crosslinking in one reaction. Alongside a continuous crosslinking assay, these new technologies represent a 70 year long breakthrough and world first.

3. Super high resolution imaging is now available so that we can see how PBPs work inside bacteria and in the test tube, how they interact with each other and other proteins or lipids within bacterial cells. We can also study PBP structure at ultra-high resolution to understand how PBPs interact at the molecular level with their natural substrates and different well known antibiotics. We also have access to new chemical approaches, which along with our assays and structural biology will help direct us to new ways to stop these enzymes.

4. Finally, we have brought together international academic experts from across the UK with skills in microbiology, chemistry and physics to work in synchrony and closely with many industry experts and a wider scientific advisory panel. This concentration of effort across a wide skill base with new technology will help ensure rapid progress and results with broad application that will be valuable for future programs of antibiotic discovery and development.

Technical Summary

Penicillin and the wider family of beta-lactams are the single most important family of antibiotics. They target the final stage of bacterial cell wall biosynthesis, specifically cross-linking the structural polymer peptidoglycan (PG) by a family of enzymes called penicillin-binding-proteins (PBPs). Given this pivotal importance of PBPs we know little about how they interact with their natural substrates, precisely what these substrates are for different PBPs, and, astonishingly, how beta-lactam antibiotics interfere with this process. The SWaN Alliance brings together a multidisciplinary team of the UK's leading experts in PG biology, imaging and PG focussed chemistry to tackle these outstanding questions. Our aim is to build an integrated, multi-centre, multidisciplinary research programme as the UK hub of activity in cell wall biosynthesis. This hub will, in the first instance, develop new insight, and open up new ways to target PBPs, to sidestep the current mechanisms of resistance. We intend to decipher the fundamental mechanisms of PBPs at the structural, functional and cytoplasmic levels, including how PBPs interact with their substrates, and how the two PBP enzymatic (transpeptidase and transglycosylase) activities are co-ordinated. This will include, for the first time, determination of kinetic constants for the TP reaction of PBPs and the development of a dual quantitative continuous TP/TG assay. We will extend this new functional and structural insight, to explore interactions of PBPs with landmark beta-lactams, recently developed novel non-lactam PBP inhibitors, and control proteins. In doing so we will develop a transformative understanding of how PBPs interact with beta-lactams from which we will better understand the role of PBP alterations and substrate alterations in the emergence of target mediated resistance. All of this activity will reinvigorate PBPs as targets for drug discovery and development by industry and academia.

Planned Impact

Antibiotics have been a mainstay of human healthcare for over 70 years. However, the inexorable spread of antibiotic resistance limits their ability to prevent and to cure life-threatening diseases. One of the most important and enduring targets for antibiotics is the biosynthesis of the bacterial cell wall. The predominant structural component of the wall is peptidoglycan, the synthesis of which is the target of penicillin and other clinically-relevant antibiotics such as methicillin, cephalosporins and carbapenems. Peptidoglycan is polymerized and cross-linked by a family of enzymes called penicillin-binding-proteins (PBPs) that are the single most important family of antibiotic targets known. Given the pivotal importance of PBPs in human healthcare, it is astounding that we still understand little of how peptidoglycan is made, how this process is controlled and how antibiotics interfere with it at the biochemical, structural and cellular levels. This information is vitally important to (1) help inform global pharma with current lactam projects with new mechanistic and structural insight for further development and (2) underpin the search for non-lactam antibiotics that target PBPs, with the bold objective of side-stepping decades of beta-lactamase evolution in one leap.

This proposal brings together a globally unique group of recognised world leaders in complementary aspects of bacterial biochemistry, chemistry, genetics, physics and physiology in the area of peptidoglycan metabolism, structure and architecture. Our aim is to build from this proposal a nationally integrated, multi-centre, multidisciplinary programme of research to address the critical and unresolved understanding of PBPs in relevant Gram negative and Gram positive pathogens that is essential for future antibiotic discovery.

This will extend key competencies and capabilities of academia to support and engage effectively with the global biotechnology and pharmaceutical sector. As such the project will have diverse impacts within the UK and internationally. Some of the expected impacts will be relatively short-term (i.e. within the life-time of the grant itself) while we expect others to be medium- to long-term in nature. The PI and industry advisory panel, populated with world leading industry consultants, with recognised track records in antibiotic discovery, and senior representatives from current pharma partners, will ensure that impact activities are considered and acted upon throughout the project. This will provide exceptional opportunities to engage with industry and through workshops to expand this across the wider academic community.

Extension of a unique multidisciplinary multi-institutional training environment in cell wall biosynthesis to a wide cohort of students will be another important impact, including the development of training across the antibiotic discovery pipeline with a clear industry focus and specialist input from our industry consultants. The results will be of widespread academic and pharmaceutical interest, because of almost all efforts to date on PBP inhibition have focused on beta-lactams.

We have unprecedented support from global pharma, from PhD support to running screens and providing access to probe compounds, and engaging as members of our exceptional scientific advisory panel. All have a strong interest in using the pre-competitive information that will be generated. To help achieve this engagement we have well defined objectives and routes for further exploitation.

We have help from a political consultant to help engage and inform regional and national government alongside national activities with Antibiotic Research UK, Antibiotic Action and Antibiotic Discovery UK, and international activities via the Pew Trust and the Wellcome Trust.

Publications

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Bern M (2017) Towards an automated analysis of bacterial peptidoglycan structure. in Analytical and bioanalytical chemistry

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Bottomley AL (2017) Coordination of Chromosome Segregation and Cell Division in . in Frontiers in microbiology

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Cahill ST (2019) Studies on the inhibition of AmpC and other ß-lactamases by cyclic boronates. in Biochimica et biophysica acta. General subjects

 
Description Attendance at All Parliamentary Group for AMR
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
Impact APPG on Antibiotics - 'How close are we to meeting the urgent need for new antibiotics?'
 
Description Accelerate CHNUK AMR discovery: Establishing joint China/UK training and research platforms enabling highthroughput fragment based inhibitor discovery
Amount £1,000,000 (GBP)
Funding ID MR/P007503/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 05/2019
 
Description Coventry General Charities
Amount £157,000 (GBP)
Organisation General Charity of the City of Coventry 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2018 
End 09/2022
 
Description Newton UK china ANMR hubs
Amount £3,000,000 (GBP)
Funding ID MR/S014934/1 
Organisation University of Warwick 
Sector Academic/University
Country United Kingdom
Start 01/2019 
End 12/2021
 
Description Wellcome Trust Innovation Award
Amount £100,000 (GBP)
Funding ID 109676/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2016 
End 06/2017
 
Description Wellcome Trust Pathfinder Award
Amount £100,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 03/2017
 
Title Automated analysis of LC-MS/MS PG fragments 
Description We have reported a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of peptidoglycan fragments using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact The strategy described allows a non-biased, ultra sensitive and rapid analysis of peptidoglycan LC-MS/MS data. We have published the proof of concept of this approach and demonstrated that it has the potential to detect an unexpected diversity of peptidoglycan structures. This will allow to elucidate the role of a large number of enzymes involved in peptidoglycan remodelling previously unknown. Thanks to the completeness of the method developed, we can now envisage to study in great details how peptidoglycan structure changes following treatment by cell wall targeting antibiotics or during pathogenesis. Thanks to this breakthrough, we can now envisage studying very dilute peptidoglycan samples (for example, from individual abscesses or in individual organs from infected animals such as mice). This is expected to reduce the number of animals required to carry out such studies. 
 
Title Flow cytometry assay to quantify single-cell binding of proteins to bacterial cell surfaces 
Description Using the high throughput flow cytometer purchased with the funding, we have developed an assay which allows quantification of protein binding to bacterial cells. We have now set up the proof of concept with using a single LysM peptidoglycan binding domain (LysMA1) as a model. This assay invloves: - LysM labelling; covalent FITC or a translational fusion with a fluorescent protein can be used - incubation in the presence of cells - measurement of fluorescence associated with individual cells by flow cytometry We have defined the conditions (protein concentrations, cell densities, buffer conditions, flow cytometry parameters) which give a binding dose-response to saturation. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact This assay is instrumental to quantify protein-peptidoglycan interactions. It will allow us to measure the impact of peptidoglycan structure and cell wall components on binding (using isogenic mutants as substrates). We will also be able to compare the binding affinity of distinct LysM domains to the same cells. Beyond this project, this represents a technique widely applicable to any surface protein. 
 
Description H3D drug discovery University of Cape Town 
Organisation University of Cape Town
Country South Africa 
Sector Academic/University 
PI Contribution Input of reagents assays and protein structures
Collaborator Contribution Input of medicinal chemistry know-how and chemical matter
Impact Interpersonal exchange of postdocs and to be PhD students
Start Year 2017
 
Description Oxfrod chemistry 
Organisation University of Oxford
Department Chemistry Research Laboratory
Country United Kingdom 
Sector Academic/University 
PI Contribution We have discussed this project and set up a collaboration with Professor Chris Schofield at Oxford to look at the affinity of beta-lactam dimers and polymers with Beta-lactamase enzymes.
Collaborator Contribution They will provide access to equipment and reagents to support this collaboration as well as host the student
Impact too early
Start Year 2017
 
Description 10 Downing Street 2018 Chinese New Year Celebrations 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Invitation from the PM to attend event to showcase UK China collaborative AMR research
Year(s) Of Engagement Activity 2018
URL https://www.gov.uk/government/news/pm-hosts-chinese-new-year-reception-at-downing-street
 
Description BBSRC Strategic Partnership Visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact A team from the BBSRC executive visited the University on 10 May 2018 as a part of our
strategic partnership. More than 70 Warwick staff from various departments joined in
the open sessions.

Presentation of Research and Virtual Reality system
Year(s) Of Engagement Activity 2018
URL https://warwick.ac.uk/fac/sci/lifesci/intranet/staffpg/support/comms/slsupdate/sls_update_june_2018....
 
Description China UK People to People Exchange Royal Society December 6th 2017 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact As PI for the CHNUK research grant I represent the wider UK-China AMR programme at the UK China People to People Dialogue in London on 6 December at the Royal Society.

There was space for 15 of the highest profile collaborations and the FCO recommended the programme as a whole, but specifically that the CHNUK to be featured. As described by the FCO at the time "The programme is obviously a key part of the UK's current portfolio of research collaborations, and the exhibition stands need to be visual. A lot of the other stands will be full of robots and other toys so I need something that would stand out, having seen some of the X-ray crystallography images I thought this might be eye catching enough.

There will be a walk through of the exhibition by UK and Chinese Ministers, including Madame Liu Yandong and hopefully Greg Clark on their way in to the building and hopefully they will stop and talk to several of the exhbitors and do photos but we don't know what will catch their eye just yet. You would be free to join the rest of the event after the Ministers have left, perhaps a colleague could attend the stand during the breaks etc."
Year(s) Of Engagement Activity 2017
URL https://www.gov.uk/government/topical-events/uk-china-high-level-people-to-people-dialogue-2017
 
Description MRC Milennium Medal Showcase of AMR research Palace of Westminster 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Presentation of MRC and RCUK funded AMR research to Peers and Politicians at the Palace of Westminster as part of the 2018 MRC Millennium Medal Award event
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=n2Z6Xxs8aA0
 
Description Pint of Science public outreach 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Pint of Science is an annual science festival that takes place every May and brings researchers to your local pub to present their scientific discoveries.
Year(s) Of Engagement Activity 2018
URL https://pintofscience.co.uk/event/life-is-the-name-of-the-game
 
Description Press release + interview BBC Sheffield to comment on published article (Salamaga et al., PLoS Pathogens, 2017) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Following the publication of the research article (Salamaga et al.,2017), a press release has been published by the university of Sheffield media team. This led to an interview with BBC Sheffield (31/07/2017), an article in the national media support Metro ("killer bug too small for antibiotics"), an article in AOL online and international media (India and Taiwan).
Year(s) Of Engagement Activity 2016,2017
 
Description Public Science Event Warwick University 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact On Thursday 26 April 2018, the School of Life Sciences hosted its fourth public
science evening of the 2017/18 academic year (the ninth since the programme
began in 2016). This evening was a panel discussion focussing on three topics of
interest, highlighted by the public from previous public science evenings. These
were: Epigenetics, Environment & Sustainability and Medical Microbiology
Year(s) Of Engagement Activity 2018
URL https://warwick.ac.uk/fac/sci/lifesci/intranet/staffpg/support/comms/slsupdate/sls_update_june_2018....
 
Description Radio 4 3 part drama production 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Radio drama highlighting factors driving antibiotic resistance and global consequences - raising public awareness at the National level
Year(s) Of Engagement Activity 2017
URL http://www.bbc.co.uk/programmes/b08g7y1l
 
Description UKRI Superheroes vs Superbugs live at the Science Museum 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact On 25 April, leading researchers from the MRC and UK research councils will join the Science Museum's free, adults-only, late opening of the Superbugs: The Fight for Our Livesopens in new window exhibition. The exhibition, which is sponsored by UKRI, looks at the causes and challenges of antimicrobial resistance (AMR), and why previously effective drugs are no longer winning the battle against some infections.
Mega Magnification - Be transported into the hidden world of bacteria. Use VR to shrink yourself, meet mighty bacterial proteins and reveal their microscopic secrets.
Year(s) Of Engagement Activity 2018
URL https://mrc.ukri.org/news/browse/ukri-superheroes-vs-superbugs-live-at-the-science-museum/