Unravelling the Bam complex

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 bottleneck 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.

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.

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.