Molecular grammar of SurA-client interactions in the periplasm of gram-negative bacteria

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Antibiotic resistance is projected to cause 10 million deaths per year by 2050, with gram-negative pathogens comprising 9 of the 12 bacteria that pose the greatest threat to human health, according to the World Health Organisation. These gram-negative pathogens have a unique outer membrane (OM) that acts as a first line of defence against an assault from potentially harmful molecules to the bacteria, such as antibiotics. As a result, the OM is essential for bacterial survival and is one reason why certain bacteria are resistant to different types of antibiotics. Finding ways to prevent correct assembly of the OM may therefore produce new routes to kill gram-negative bacteria, or make them more susceptible to existing antibiotics. However, the mechanism by with the OM is built remains mysterious, making its assembly difficult to target with therapeutics.Here we propose to determine how key proteins of the OM - so-called outer membrane proteins (or OMPs) - are folded into the OM to create the usually impenetrable cell wall. OMPs play essential roles in bacterial virulence and survival, so by understanding how OMPs are assembled into the OM it may be possible to develop new drugs that target this essential process. A key protein involved in ensuring OMPs reach the OM is a chaperone protein called SurA. SurA is an attractive target for the development of new drugs to control gram-negative pathogens because perturbing the chaperone function of SurA results in a loss of bacterial viability and virulence along with increased sensitivity to antibiotics. However, in order to target the chaperone function of SurA, further work is needed to understand its mechanism of action. We have recently discovered that two key sites on SurA are responsible for recognising OMPs. This is exciting, as it suggests that one or both of these sites could make good targets for new drug-like molecules. However, we still do not understand how each of these two binding sites contribute to OMP binding and chaperone function. Here we propose to use information from an array of complementary and cutting-edge experimental methodologies (including NMR spectroscopy, mass spectrometry, single molecular Forster resonance energy transfer, biochemistry/biophysics and bioinformatics) to understand how each of the newly discovered OMP binding sites on SurA recognises specific signals within its OMP clients. Further, we propose to determine how these two binding sites work together to bring about its chaperone function, in particular regarding SurA's role in protecting newly synthesised OMPs from aggregation and facilitating their delivery to the OM. This will uncover the molecular features of SurA that are essential for assisting in OMP biogenesis, which, in the future, could lead to new strategies to develop much-needed antibiotics that target gram-negative pathogens that threaten humans, plants and animals.