Exploiting protein import to interrogate energy transduction through the bacterial cell envelope

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Bacteria are both friend and foe. It is estimated that our bodies contain more bacterial cells (the microbiome) than human cells. Bacteria are therefore vital to healthy living; for example, gut bacteria are essential to the digestion of food. However, many bacteria are causative agents of disease. Even those we host in our bodies can become opportunistic pathogens when exposed to different niches, as happens during surgery. The rise of antibiotic resistance amongst bacterial species has made this occurrence all the more frequent, raising the prospect that in the next 20-30 years routine hospital procedures and even giving birth will become hazardous because of the lack of effective antibiotics. The present application focuses on a group of protein molecules known as bacteriocins. These are naturally-occurring antimicrobials that bacteria produce during the warfare they wage with their neighbours to gain greater access to resources. Bacteriocins have the potential to be reconfigured to kill pathogenic bacteria but much still needs to be understood as to their mode of action. The bacteriocins investigated in this proposal target Gram-negative bacteria, in other words bacteria that have two membranes. The outer membrane is a unique asymmetric lipid bilayer that excludes many classes of antibiotics that are active against Gram-positive bacteria, which lack this additional membrane. Hence, the outer membrane is one of the reasons why Gram-negative bacteria are some of the most problematic in terms of antibiotic resistance. In recent years, we have learnt much about the underlying mechanisms of action of bacteriocins that kill Gram-negative bacteria, especially those that target E. coli, P. aeruginosa and K. pneumoniae. This recent work, much of it from Oxford and unpublished, has identified several critical pieces of information. First, bacteriocins often use a structural motif known as a beta-hairpin to dock onto the surface of the bacterium prior to transport. Second, bacteriocins have the potential to import significantly greater mass (1000x) than is normally permitted by the permeability filters of the outer membrane. These filters are proteins known as porins. Third, this property of bacteriocins is linked to their ability to tap into the energy of the cell, which is associated with the inner membrane of the bacterium. By tapping into this energy source, known as the proton motive force, bacteriocins catalyse their transport across the outer membrane, even carrying cargo molecules such as DNA and organic molecules.We will exploit these discoveries to understand the structural basis for bacteriocin beta-hairpin association with their porin receptors. We will also use the bacteriocins we've engineered to be much larger than normal to be able to attach them to polystyrene beads so that we can visualise the import of single bacteriocins in real time. By doing so, we can begin to interrogate the energetics of import, in other words the molecular mechanisms by which bacteriocins harness the proton motive force across the inner membrane to transport themselves across the outer membrane. Developing these new import assays will also tell us about the energy transduction systems themselves which are still shrouded in mystery. Finally, by achieving these goals we'll be laying the foundations for understanding the constraints the outer membrane places on bacteriocin entry so that we can engineer these future antimicrobials appropriately.