Mechanisms of phospholipid decoy production

The declining efficacy of antibiotics is a major global healthcare concern that threatens advances in surgery, cancer chemotherapy and organ transplantation. In addition to dedicated mechanisms of antibiotic resistance, there is a growing appreciation of additional routes by which bacteria can survive exposure to antimicrobials. For example, our laboratory has recently discovered that Staphylococcus aureus releases phospholipid decoys during exposure to daptomycin, an antibiotic of last resort used to treat multi-drug-resistant Gram-positive bacteria. The release of decoys results in the inactivation of the antibiotic, enabling S. aureus to survive. We have since shown that this decoy mechanism exists in several other Gram-positive pathogens, and occurs via an active process that requires protein and lipid biosynthesis. Further work from our group has shown that a similar decoy mechanism exists in the Gram-negative bacterium Pseudomonas aeruginosa, which results in the inactivation of colistin, another antibiotic of last resort. However, in contrast to Gram-negative bacteria, the mechanism by which phospholipids are released from the membrane of Gram-positive bacteria is completely unknown and will form the focus of this work.

To address this aim, we will undertake a range of biochemical, genetic and imaging approaches. We will determine the membrane protein before and during daptomycin exposure to identify key proteins involved in phospholipid release. The roles of these proteins will be confirmed by generating deletion mutants, and/or by over-expressing relevant genes. We will track phospholipid biosynthesis and release using C14-labelled phospholipid precursors and image phospholipid release in bacterial cells to understand why individual bacteria live or die during daptomycin exposure. We will also capitalise on information from studies with Gram-negative bacteria, in which phospholipid release is well established and for which mechanistic information exists, to understand whether parallel processes exist in Gram-positive and negative bacteria to form a general defence against antibiotics. Ultimately, these studies will attempt to identify targets for small molecules that inhibit decoy production and thereby enhance the efficacy of daptomycin and colistin therapy.