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.