This project focuses on characterising the fundamental process of outer membrane lipid transport in Gram-negative bacteria, a largely unknown process which is only now being revealed.
The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat requiring new targets and strategies to combat infection. Multidrug resistance is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The bacterial cell envelope has been and still remains one of the best targets for antimicrobials development. For a successful antibacterial drug development programme a detailed understanding of envelope structure and biogenesis is required. In Gram-negative bacteria the envelope is a complex, essential tripartite structure formed by two membranes sandwiching a peptidoglycan layer that together maintain the integrity and the shape of the cell. While the structure and composition of the bacterial cell envelope has been known for several years, details on its biogenesis have only recently emerged. These processes can be exploited for antimicrobial discovery as they are not present in humans. In particular the outer membrane has been focused on as it makes direct contact with the host. It is built by sophisticated and complex multi-protein machineries: proteins via the Bam machinery, lipoproteins via the Lol pathway and LPS via the Lpt pathway. Phospholipid (PL) transport however has remained elusive until now.
The MLA pathway – Retrograde lipid transport?
Gram-negative bacteria are generally more resistant than Gram-positive bacteria to antibiotics, detergents, and other toxic chemicals because of the sophisticated asymmetry of lipids in their outer membranes, lipopolysaccharides (LPS) in the outer leaflet, and phospholipids in the inner leaflet. Under stress conditions this asymmetry can be disrupted leading to shedding of LPS and phospholipid migration to the outer leaflet. These phospholipid bilayer patches lead to reduced barrier function and are detrimental to the cell. Recently a 6 protein pathway, the Mla pathway, has been identified that is suggested to actively prevent phospholipid accumulation at the cell surface. Knocking out any component of this pathway leads to OM barrier defects and increased susceptibility to antibiotic agents in addition to also abolishing virulence. Thus all components of the Mla pathway are important virulence factors. Furthermore recent evidence from the Knowles lab suggests that this pathway may also be able to transport lipid towards the outer membrane, and therefore provides the first evidence of a mechanism for how lipids get to the outer membrane.
At present little is known about how this pathway functions, but by using a multidisciplinary approach, working in the fields of both biophysics and molecular biology, including techniques such as NMR, X-ray crystallography, QCMD, FTIR and neutron reflectometry, this project will provide valuable insight in to the fundamental process of lipid transport and potentially identify druggable pockets and novel compounds that will not only abolish virulence but also impede restoration of a damaged OM and therefore increase the effectiveness of already available antibiotics.
Hughes, G.W. Hall, S.C.L, Laxton, C.S., Sridhar, P., Mahadi, A.H., Hatton, C. , Piggot, T.J., Jamshad, M., Spana, V., Cadby, I.T., Harding, C., Isom, G.L., Bryant, J.A., Parr, R.J., Yakub, Y., Jeeves, M., Huber, D., Henderson, I.R., Clifton, L.A., Lovering, A.L., Knowles, T.J. (2018) Evidence for phospholipid export from the gram-negative inner membrane: time to rethink the Mla pathway? Biorxiv doi: https://doi.org/10.1101/388546
Ekiert, D.C., Bhabha, G., Isom, G.L., Greenan, G., Ovchinnikov, S., Henderson, I.R., Cox, J.S., Vale, R.D. (2017) Architectures of Lipid Transport Systems for the Bacterial Outer Membrane. Cell 169(2):273-285
Ercan, B., Low, W.Y., Liu, X., Chng, SS. (2018) Characterization of Interactions and Phospholipid Transfer between Substrate Binding Proteins of the OmpC-Mla System. Biochemistry. doi: 10.1021/acs.biochem.8b00897 (Epub ahead of print)
Malinverni JC, Silhavy TJ. (2009) An ABC transport system that maintains lipid asymmetry in the gram-negative outer membrane. PNAS 106(19):8009-14