Unravelling BamA Function Using Fluorescence & Single Molecule Force Experiments

The outer membrane (OM) of bacteria is an important outer coat which protects the bacterium from its surrounding environment. At the same time, the bacterium has to take up specific nutrients in order to grow and divide. To achieve this, the OM of one class of bacteria- the so-called called Gram negative type - is uniquely built of special lipids in which proteins that stabilise the OM and enable transport of nutrients into the bacterium reside. These Outer Membrane Proteins (so-called OMPs) are thus vital for bacterial growth and survival. Rather than being a sea of lipids with rare floating protein islands, the OM is now known to be cram-packed with OMPs, forming a very crowded environment. Bacteria continually make new OMPs as they grow and divide, and a fascinating nano-machine has evolved which is essential for these proteins to be inserted successfully into the crowded OM and to fold to the correct structure so that they can carry out their vital functions. This machine - called BAM (beta-barrel assembly machinery) - is the focus of this proposal. We propose to use the very latest biochemical and biophysical techniques to discover, for the first time, how this machinery works.

Many Gram negative bacteria are pathogenic, causing diseases in humans, animals and plants. Many such organisms have become, or are becoming, resistant to antibiotics that have so successfully protected us from the invasion of Gram negative bacteria since the discovery of penicillin >80 years ago. We now urgently need to develop new antibiotics able to prevent bacterial infection. BAM is one such exciting new target. How BAM functions, however, is not known. What is known is the structure of all of the protein parts (five in the case of the bacterium E.coli) opening the door to new experiments to work out how OMPs fold and how BAM allows this to happen efficiently in the bacterial OM. In the proposed work, we aim to use the very latest techniques, including fluorescence, FRET and single molecule 'pulling' experiments to unravel how OMPs fold and how BAM functions. Specifically we will focus on one component of BAM, known as BamA, which is the powerhouse for BAM-assisted OMP folding and assembly. Our aim is to understand the way in which this membrane protein functions by developing analytical methods with which we can monitor structural changes taking place during a folding or functional event. As well as providing new and fundamental information about how biology has evolved this clever nano-machine, in the long term we aim to use the information gained to pave the way towards developing new routes to combatting diseases caused by Gram negative organisms.

The proteins of the outer membrane of Gram negative bacteria (OMPs) perform a wealth of functions that are vital for OM integrity and cell viability. These proteins require the beta-barrel assembly machinery (BAM) to fold in the crowded OM. However, how BAM (a five-subunit, 203 kDa membrane protein complex) functions remains unknown. Determining how BAM folds OMPs is important both fundamentally and strategically since (i) BAM-mediated OMP folding must differ fundamentally from the much better understood folding mechanisms of cytosolic proteins and how these are assisted by ATP-dependent chaperones; and (ii) BAM is an attractive target for new antibiotics against Gram negative pathogens. Recent elucidation of the structure of BamA (itself an OMP), the BAM subunit responsible for OMP folding, has fuelled excitement into how this molecular machine works.
This application exploits recent developments in the applicants' laboratories in which we have (i) devised fluorescence methods able to measure the folding kinetics of OMPs ranging from 8-22 beta-strands into liposomes with excellent reproducibility and accuracy; (ii) expressed and purified BamA and the intact BAM complex and shown them to be functional in our in vitro folding assays; and (iii) developed methods able to measure protein:protein and protein:membrane interactions by dynamic force spectroscopy, enabling binding and folding/unfolding of single proteins to be measured with nm resolution. Using these methods and BamA, BamA mutants, and the BAM complex we propose here to:
(1) determine how BamA/BAM folds OMPs and inserts them into membranes and how the properties of the membrane affect BamA/BAM function; and
(2) determine how BamA/BAM alter the folding mechanisms of OMPs of different sequence and size.
Success will not only provide a step change in our understanding of a fascinating molecular machine, but will provide the platform for developing new routes to inhibit this essential folding machinery.

Economic and Societal Impact - The overarching aim of our programme of study is to understand the mechanism by which BamA, the vital central component of the BAM complex, inserts proteins into the outer membrane of E.coli. The short-term, immediate impact of this research will be for researchers in the fields of protein folding, membrane proteins and the molecular mechanism of protein complexes (see Academic Beneficiaries). Understanding the mechanism of the BAM complex will also enable routes to its inhibition and, therefore, to the development in the longer term of a novel class of antibiotics. BAM is an attractive target for the development of much needed new agents against Gram negative pathogens: it is found on the extracellular surface of all Gram negative bacteria; BamA (and BamD) are essential for cell viability, and both proteins are highly conserved across Gram negative organisms. Gram negative bacteria pose major threats to the health and wealth of the UK as they are major pathogens in humans (E.coli, K. pneumoniae, P. aeruginosa, A. baumannii, and Enterobacter sp), livestock (E.coli, D. nodosus, C. abortus and L. intracellularis) and plants (P. syringae, Xanthomonas sp. and E. amylovora). The cost of these infections and nosocomial infections with antibiotic resistant Gram negative bacteria is estimated to exceed 1.5 billion Euros in healthcare expenses and lost productivity each year in the EU (UK Five Year Antimicrobial Resistance Strategy 2013 to 2018, Depts of Health and Environment, Food and Rural Affairs, Sept 2013).

Given the urgent unmet need for new antibacterial therapeutics, we plan to exploit every opportunity for interacting with relevant industries and other interested parties as soon as possible during the proposed programme of study so that we can share results and exploit their potentials (see Pathway to Impact). This will be achieved through the traditional routes of publication, press releases and via the University and Astbury Centre web sites. In addition, we will organise a 1-day symposium held in Leeds for industry (approx. 30 delegates from large Pharma and SMEs) to increase visibility of our research to the pharmaceutical sector and ensure that we exploit our fundamental research to the full as soon as possible by developing potential translational opportunities. Success in understanding how BAM functions and how/whether the mechanism of action depends on the particular OMP substrate on the 3 year timescale of this grant would open the door to novel therapeutics in the longer term, of major cost and societal benefit to the UK via improved human and animal health and improved resistance to bacterial infections in plants. In turn this will enhance the competitiveness of the UK via improvements to the work-force, farming and opportunities in the pharmaceutical sector.

Training for non-academic careers - The trained fellow will be equipped for an industrial career in biotechnology-related fields, in sectors such as health and medicine, pharmaceuticals, analytic biophysics, chemical, food, and personal care. In addition the PDRA will be trained in teamwork, problem solving, and communication across disciplines having been exposed to research in a diverse, large active research laboratory. These skills are useful for a host of professions outside of the technical disciplines, including management, politics and government, business, and entrepreneurship. Hence the PDRA employed will be highly employable across a wide number of difference disciplines and environments, ensuring their successful career development in whichever route they choose to take.