The structure and function of the beta-barrel assembly machinery: an Achilles heel of Gram-negative pathogens

Antibiotics have revolutionized healthcare since their discovery in the middle of the last century, and antibiotics and other antimicrobial drugs have become an integral part of modern medicine, which we rely on to tackle bacterial infections. The use of antibiotics, however, has increased enormously, both in healthcare and in agriculture. Unfortunately, this has led to huge rise in antimicrobial resistance (AMR), where the bacteria that cause infections and are targeted by antibiotics become resistant to the drugs that we rely on to kill them. The emergence of AMR poses an urgent threat to society and a massive and growing problem for human health. In addition to the vast array of diseases caused by bacterial infection, hospital-acquired infections are an ever-increasing threat to our health. Approximately 300,000 patients a year are affected by hospital-acquired infections in England, costing the NHS in excess of one billion pounds a year. As AMR spreads, the bacteria that cause these infections are now becoming resistant to almost all of the drugs in our antibiotic arsenal. There is thus an urgent need to discover new antibiotics, and it is on this topic that our grant application is focused.

A major class of bacteria, the so-called Gram-negative bacteria, contains pathogens causing diseases in humans that include cholera, plague and gonorrhoea. Gram-negative bacteria are also responsible for many hospital-acquired infections, especially pneumonia and urinary tract infections. These include bacteria in the genera Acinetobacter, Escherichia, Haemophilus, Legionella, and Pseudomonas. We urgently need to develop new antibiotics able to prevent bacterial infection by hitting new targets in the bacterial armory: identifying and targeting the bacterial 'Achilles heel' to either render them susceptible to existing anti-bacterial agents or to kill them directly.

One such potential target is a protein complex called the beta-barrel assembly machinery, or BAM, which sits in the outer of two protective membranes that helps to shield Gram-negative bacteria from their environment (and from many antibiotics). We know that BAM is essential for bacterial survival and virulence. Its role is to insert new proteins that are made in the bacterial cell into this outer membrane. How BAM achieves this, however, is not known despite several 3D structures of the complex being solved using crystallography or cryo-electron microscopy in the last year. In the proposed work, we aim to use the very latest techniques, including cryo-electron microscopy, and single molecule FRET experiments to determine how OMPs are folded and inserted into the crowded bacterial outer membrane by BAM. Our aim is to determine the 3D structure of BAM caught in the act of inserting a protein into a membrane. Together with functional studies in test tubes and in the organism itself, our programme of research will reveal the molecular details of how BAM functions; how it recognizes its OMP substrates; how it folds OMPs into the crowded OM in the absence of an external energy source; and how it is organized in the bacterial OM. As well as enabling us to develop new methods to interrogate the function of this fascinating molecular machine, in the long term we aim to use the information gained to pave the way towards developing new routes to combating infections caused by Gram-negative pathogens.

The goal of this 5-year progamme grant is to deliver a step change in our understanding of how the proteins that comprise the outer membrane of E. coli are folded and inserted into the crowded outer membrane (OM): a feat achieved the beta-barrel assembly machinery (BAM). This integral membrane protein complex is comprised of five polypeptide chains in E. coli and is essential in all Gram-negative bacteria, including major human pathogens. Despite an explosion of structural information about BAM in the last 6 months, including our own 4.9 Å resolution cryo-EM structure, how BAM recognizes its substrate OMPs and catalyzes their folding into the OM remain unknown.

Here we will combine state-of-the-art cryo-EM, with ensemble and single molecule FRET, and functional assays of BAM in liposomes, nanodiscs, SMALPs and in E.coli, to reveal how this fascinating molecular machine functions. Using cross-linking and freeze trapping we will attempt to trap BAM caught in the act of OMP folding. We will also use cryo-ET to visualize BAM in 'OMP islands' in vivo and in a proposed supercomplex that spans the OM. The experiments build on a plethora of preliminary data that demonstrate the feasibility of the challenging experiments proposed. Specifically, we will address the following questions:

- How does the conformation of BAM change during its functional cycle?
- How does the native lipid environment affect BAM structure and function?
- How does BAM fold and assemble OMPs into the OM?
- How does BAM interact with the OMP-delivering chaperone SurA?
- What is the structure of BAM in situ in the bacterial OM?

Answering these questions will reveal fascinating new insights about this important molecular machine that is conserved from bacteria to man. Crucially, it will also provide opportunities for structure-based design of reagents able to inhibit BAM function, and thus to kill pathogenic microbes or render them susceptible to existing antimicrobial agents.

The overarching aim of this five-year MRC programme grant is to underpin future efforts to design new antibacterial agents exploiting a new understanding of the molecular mechanism of BAM in Gram-negative bacteria. We will discover how BAM recognizes, receives, folds and inserts OMPs of different sizes and sequences into the OM of E. coli. In the short-term, the impact of our research will be for researchers interested in OM biogenesis, membrane proteins, macromolecular machines and protein structure/function, in fields spanning biochemistry, biophysics, microbiology and structural biology (see Academic Beneficiaries).

Crucially, in addition to a fundamental research benefit, the work described focuses on the urgent and growing need to develop new reagents able to combat antibiotic-resistant pathogenic Gram-negative bacteria. All such bacteria have an Achilles heel in that they require the BAM complex, which is conserved across Gram-negative bacteria and is essential for viability and virulence. Importantly, BAM subunits are displayed, in part, on the outer surface of the bacterial OM and hence are readily accessible should protein-based antimicrobials be a way forward for the development of a new class of anti-microbial agents. The periplasmic space is also open to molecules <600 kDa in size via OMP porins (which are inserted by BAM), raising the prospect of inhibition of BAM with small molecules at both its extracellular- and periplasmic-facing domains.

Given the major importance of anti-microbial resistance to humankind, a major beneficiary of the research will be industries focusing on the urgent and unmet need for new anti-microbial agents. Medics needing successful treatments for patients presenting infections at home, in the work place, and importantly, in hospitals will also be beneficiaries, as will the whole of society in the developed and developing world that face the threats of pathogenic, antibiotic-resistant bacteria. Gram-negative bacteria pose major threats to human health and the wealth of the UK. For example, the cost of infections involving E.coli, K. pneumoniae, P. aeruginosa, A. baumannii, and Enterobacter sp. 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). The research programme we propose thus fits squarely within the remit of the MRC.

Finally, the research programme will train two researchers in an important and topical field using state-of-the-art approaches, including complex instrumentation, and requiring high level skills in computing and data analysis to understand the results obtained using the techniques described. The researchers employed will thus be well equipped for an industrial career in biotechnology-related fields, in sectors including health and medicine, pharmaceuticals, analytic biophysics and personal care. In addition the PDRAs will be trained in teamwork, problem solving, and communication across disciplines, having been exposed to research in a diverse, large and active research team. These skills are useful for a host of professions outside of the technical disciplines, including management, politics and government, business, and entrepreneurship. Hence the PDRAs employed will be highly employable across a number of different disciplines and environments, ensuring their successful career development, whichever route they choose to take.