Unravelling the Bam complex

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

The growing resistance of micro-organisms to antimicrobial therapies, such as antibiotics, is a significant global public health issue. In Europe alone this is currently estimated to result in an additional 25,000 deaths each year, approximately 2.5 million avoidable days in hospital and an economic burden estimated to be at least £1.2 Billion per year. Resistance is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. A recent report by Professor Dame Sally Davies, the Government's Chief Medical Officer, likened it to a 'ticking time bomb' and warned that routine operations could become deadly in just 20 years if we lose the ability to fight infection.

The understanding of the Gram-negative bacterial cell envelope is critical to developing new antimicrobial agents. All Gram-negative bacteria possess two membranes that enclose the cell, separated by a space known as the periplasm. The outer of the two membranes is particularly resistant to the penetration of small molecules. The main function of this membrane is to form a semi-permeable layer that protects the bacterium from the environment but also to control the movement of molecules into and out of the cell and how the cell interacts with its environment. It is the proteins within this membrane that control these mechanisms by providing essential physiological, pathogenic and drug resistance functions and hence are the instruments of microbial warfare as they mediate many of the lethal processes responsible for infection and disease progression. Identifying compounds that can prevent the formation of this membrane could lead to the development of the next generation of antimicrobials.

Recently a single protein complex called the Bam complex was identified as being responsible for the folding and insertion of most if not all of the proteins into the outer membrane and hence has been identified as a key bottlenck in the formation of this membrane. Understanding how this complex works is therefore critical as the design of compounds that inhibit this process would inhibit outer membrane protein biogenesis and therefore essential physiological, pathogenic and drug resistance functions and could prove useful in combating diverse Gram-negative pathogens.

In this research project we plan to characterise the mechanistic processes the Bam complex undertakes in order to fold proteins into the outer membrane, we will identify ligand interaction sites and binding pockets that form during outer membrane protein insertion and thus provide valuable mechanistic insights that will aid in the discovery of molecular inhibitors and new classes of antimicrobial agents.

Technical Summary

To elucidate the mechanistic details of Bam complex mediated protein folding, we have developed two systems:
1. A surface tethered membrane bilayer system that allows real time monitoring of structural changes within the complex during Bam mediated protein folding.
2. A styrene maleic acid lipid particle (SMALP) system that encapsulates the complex and its lipid environment and provides a framework for probing lipid composition and also provides a novel way of structure determination by electron microscopy (EM).
Using these systems we have developed the following work packages:

A. Map the folding mechanism within the membrane. Using our surface tethered bilayer system in combination with selective deuteration and neutron reflectometry we will probe real time structural changes within the complex during chaperone binding and OMP folding, whilst stalled intermediates will allow us to characterise intermediates in the folding pathway. Quartz crystal microbalance will provide details on binding affinity, kinetics and loading. Analysing mutants and component removal will allow us to identify the role each plays in the folding process.

B. Elucidate atomic snapshots of the folding pathway. Using the latest generation cryo-EM in combination with SMALPs snapshots of the Bam folding pathway will be taken. SMALPs allow simple EM imaging of membrane proteins in all dimensions and will allow us to probe the folding event by A) using rapid freezing at set points during the folding cycle and B) utilising stalled intermediates. Structures will be resolved to identify the mode by which Bam mediated membrane insertion occurs.

C. Identify whether Bam modulates the local lipid environment. SMALPs will be used to extract the Bam complex and its associated lipid and LPS. This will be analysed by mass spectrometry and compared to the bulk outer membrane. The effect of component removal will be analysed in order to address the role each plays in lipid/LPS recuitment.

Planned Impact

Academic Impact:
The principal beneficiaries of this research are likely to be academic researchers in numerous fields. By identifying the mechanism by which the Bam complex functions, we will enhance the UK knowledge economy and contribute to the global understanding of outer membrane biogenesis and hence microbial pathogenesis, virulence and multidrug resistance and hence will be of interest to immunologists and bacteriologists. Furthermore the Bam complex is a new target for the development of novel antimicrobials, a goal desperately needed following the emergence of bacteria that are resistant to available antibiotics, especially with essentially few antibiotics under development or likely to be available for clinical use in the near future.
More generally, uncovering the key mechanism of outer membrane protein biogenesis will be of interest to anyone interested in biological membranes, protein folding, signalling, trafficking, secretion, drug discovery and bacterial physiology.

Commercial Impact:
While we do not anticipate our research will produce commercially exploitable results immediately, in the medium term, the Bam complex represents a novel target for the development of antimicrobials. An important beneficiary would be the pharmaceutical industry, which would be given the ability to rationally design inhibitors of Bam complex function, thus providing new opportunities to attenuate bacteria in the pursuit of anti-infective agents. The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat, requiring new targets and strategies to combat infection. The Bam complex offers an accessible target for intervention against a variety of pathogenic Gram negative bacteria. For example, Gram-negative bacilli cause respiratory problems (Hemophilus influenzae, Pseudomonas aeruginosa), urinary problems (Escherichia coli, Proteus mirabilis), and gastrointestinal problems (Helicobacter pylori, Salmonella enteritidis) whilst Gram-negative cocci cause sexually transmitted disease such as Neisseria gonorrhoeae, and others meningitis, e.g. Neisseria meningitidis.

Societal Impact:
This proposal will impact upon society by improving our knowledge and understanding of the Gram-negative outer membrane, the essential organelle which protects all Gram-negative bacteria, with inserted proteins influencing pathogenicity, virulence and resistance. A recent report by Professor Dame Sally Davies, the Government's Chief Medical Examiner, likened antimicrobial resistance to a 'ticking time bomb' and warned that routine operations could become deadly in just 20 years if we lose the ability to fight infection. A new generation of antimicrobials against novel targets is therefore desperately required. Currently hospital acquired infections cost the NHS £1 billion a year and approximately 70% of all intensive care unit infections are the result of Gram-negative bacteria. The development of novel antibiotics is therefore likely to impact on every member of society, from those suffering from an infection, to the families of patients, carers and health professionals. We aim to uncover the key events in Bam complex function, the protein complex responsible for folding and inserting all outer membrane proteins. If these results can be exploited, the health benefits to society will be immense.
 
Description Gram-negative bacteria, for example E. coli (gut bacteria that in some cases can cause severe disease), Neisseria gonorrhoea (Causative agent of the sexually transmitted disease gonorrhoea) and Neisseria meningitidis (causative agent of bacterial meningitis) are surrounded by two lipid membranes that make them particularly resistant to antibiotic treatment. The outer membrane makes direct contact with the environment and hence us during infection. Understanding how this membrane forms is of critical importance as it could lead to the development of novel antibiotics that are desperately needed to treat the rising trend of antimicrobial resistance. Since 2000 many of the mechanisms have been identified but how lipids get to the outer membrane has remained an enigma. The research conducted during this grant has led to the discovery of the first mechanism of phospholipid transport towards the outer membrane in gram-negative bacteria. This pathway, known as the Mla pathway, was originally believed to transport lipids away from the outer membrane but through biochemical assays developed as part of this grant we have realised the pathway works in the opposite direction. This research is therefore one of the final keys in understanding the biogenesis of the outer membrane in gram-negative bacteria and could lead to the discovery of new antimicrobials against gram-negative bacteria that are desperately needed to combat the growing threat of antimicrobial resistance.
Exploitation Route Academic
Our research has opened up a completely new avenue in to research of the outer membrane of bacteria. The proteins involved have homologues in all gram-negative bacteria but also eukaryotes as well, thus the results of this research has far reaching relevance. The novel methods we have developed are applicable to almost all membrane protein systems. To highlight this research we plan to present this work at international conferences, seminars and within the press. Knowles also has numerous national and international collaborations, all will benefit from the use of this technology.
Pharma
Our research area focuses on novel mechanisms for the biogenesis of the outer membrane in gram-negative bacteria. Thus the proteins and pathways we study could be targeted for the development of novel antimicrobials. Knowles already has or has had numerous pharma collaborators. Knowles will engage with Pharma the results of this research to further develop interactions and collaborations.
Society
In the long term our research could lead to the development of novel antimicrobials that could be used to face the growing threat of antimicrobial resistance. Knowles will present the findings of this research at public engagement events, e.g. Think tank, cafe scientifique, open days etc.
Sectors Agriculture, Food and Drink,Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://www.biorxiv.org/content/10.1101/388546v1
 
Description Diamond User Committee
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
URL http://www.diamond.ac.uk/Users/DUC.html
 
Description ISIS neutron source user committee
Geographic Reach Multiple continents/international 
Policy Influence Type Participation in a advisory committee
URL https://www.isis.stfc.ac.uk/Pages/User-Committee.aspx
 
Description Crossing the periplasmic void, elucidating the mechanisms of phospholipid transport in Gram-negative bacteria
Amount £549,517 (GBP)
Funding ID BB/S017283/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2019 
End 08/2022
 
Description Bam complex 
Organisation University of Queensland
Country Australia 
Sector Academic/University 
PI Contribution Research - Protein structure analysis
Collaborator Contribution Research - TRADIS screening, mutagenesis, cell based assays.
Impact Numerous publications.
Start Year 2009
 
Description DsbD 
Organisation Imperial College London
Country United Kingdom 
Sector Academic/University 
PI Contribution Protein expression, purification and characterisation. lipidic cubic phase crystallisation and sample preparation for electron microscopy.
Collaborator Contribution MPL - Provision of LCP plates, trainining in LCP screening. Expertise. Use of Sec-Mals Leicester - expertise in electron microscopy. Structure determination by EM. Training and equipment usage.
Impact No outcomes yet.
Start Year 2018
 
Description DsbD 
Organisation Rutherford Appleton Laboratory
Department Membrane Protein Laboratory
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Protein expression, purification and characterisation. lipidic cubic phase crystallisation and sample preparation for electron microscopy.
Collaborator Contribution MPL - Provision of LCP plates, trainining in LCP screening. Expertise. Use of Sec-Mals Leicester - expertise in electron microscopy. Structure determination by EM. Training and equipment usage.
Impact No outcomes yet.
Start Year 2018
 
Description DsbD 
Organisation University of Leicester
Country United Kingdom 
Sector Academic/University 
PI Contribution Protein expression, purification and characterisation. lipidic cubic phase crystallisation and sample preparation for electron microscopy.
Collaborator Contribution MPL - Provision of LCP plates, trainining in LCP screening. Expertise. Use of Sec-Mals Leicester - expertise in electron microscopy. Structure determination by EM. Training and equipment usage.
Impact No outcomes yet.
Start Year 2018
 
Description EM Bam complex 
Organisation Sanofi
Department Genzyme Corporation
Country United States 
Sector Private 
PI Contribution Protein expression, purification, characterisation and activity assay. Provided Styrene maleic acid polymer free of charge for their research.
Collaborator Contribution Electron microscopy analysis of the protein complex produced.
Impact Dataset collected. Analysis not complete.
Start Year 2017
 
Description Mla pathway 
Organisation University of Birmingham
Department School of Biosciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Expertise in small angle scattering (SAXS), training in small angle scattering. Access to purification facilities, UV spectrophotometry. Data collection at SAXS facilities (ESRF, Grenoble, France). Data analysis.
Collaborator Contribution Expertise in protein crystallisation. Access to protein crystallisation facilities and equipment. Crystallisation consumables. Data collection and analysis.
Impact Paper and grant being submitted 2018
Start Year 2017
 
Description Muscle atrophy 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided expertise, equipment usage and training in protein expression and purification.
Collaborator Contribution Expertise in functional assays.
Impact Grant application - under review.
Start Year 2018
 
Description Rutherford appleton laboratory 
Organisation Science and Technologies Facilities Council (STFC)
Department ISIS Neutron and Muon Source
Country United Kingdom 
Sector Academic/University 
PI Contribution Research - producing samples for neutron study, developing new methods for surface deposition. Publication preparation
Collaborator Contribution Neutron science research Publication preparation
Impact Publication under review in Nature microbiology
Start Year 2013
 
Description Sec Pathway 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution NMR and SAXS spectroscopy studies. Proof reading
Collaborator Contribution Experimental support Proof reading Paper writing
Impact Two publications currently under review A third in preparation.
Start Year 2016
 
Description Willcox 
Organisation University of Birmingham
Department Institute of Cancer and Genomic Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Nuclear Magnetic resonance protein structure determination Publication writing.
Collaborator Contribution Surface plasmon resonance studies. Publication writing
Impact 3 Publications
Start Year 2010
 
Description Invited speaker - University of Kent 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact Invited speaker at Kent University to approximately 50-100 students/academics. This has led to a number of new collaborations using technology I have developed at University of Birmingham.
Year(s) Of Engagement Activity 2018
 
Description Invited speaker Gordon Research conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact An invited talk to a Gordon Research Conference to > 300 academics. This sparked questions and discussion afterwards and new collaborations have been initiated.
Year(s) Of Engagement Activity 2018
 
Description Media Video 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Participated in a media video advertising the University of Birmingham's Henry Wellcome Building for Biomolecular NMR.
Year(s) Of Engagement Activity 2018
 
Description NMSUM 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presented a seminar at the Neutron and Muon Users meeting 2017. Purpose was to highlight my research on the Bam complex and the novel methods we are using for studying it. The overall aim at this conference was to highlight how neutrons can be used to study membrane proteins. This will hopefully result in others adopting the methods I presented to study their own systems.
Year(s) Of Engagement Activity 2017