Our Research

To understand how Gram-negative bacteria build their outer membranes and protect themselves against antibiotics

The Gram-negative bacterial cell envelope and antimicrobial resistance

Antimicrobial resistant (AMR) micro-organisms cause a major global threat to modern medicine, resulting in over 700,000 deaths annually. Gram-negative, double-membraned bacteria are particularly difficult to treat, in part because their outer membrane (OM) acts as a barrier against antibiotics. Therefore, it is critical to understand how bacteria build and maintain their OM, potentially allowing for novel therapeutic approaches that disrupt this barrier.

            The Gram-negative bacterial cell envelope consists of an inner membrane (IM) and an OM separated by an aqueous periplasmic space. To build and maintain this OM, bacteria must transport lipids and hydrophobic proteins across the aqueous periplasm. Increasingly, evidence suggests that membrane components are transported via protein pathways that form shuttles or direct bridges between the IM and OM. We use a combination of structural biology, biochemistry and bacterial genetics to gain important insights into how bacteria build the OM.

The bacterial cell envelope consists of an outer membrane (OM) and an inner membrane (IM), separated by the periplasmic space, which houses the peptidoglycan cell wall. The OM itself is an asymmetric lipid bilayer with an outer leaflet of lipopolysaccharide (LPS) and an inner leaflet of phospholipid (PL). The cell envelope is scattered with proteins that are required for a range of important functions for the bacterial cell, including pathways involved transport of lipids and proteins to build and maintain the OM. Examples depicted are LetB and YhdP, which form tunnels and bridges across the periplasm, respectively.

Recent projects

  • LetB - Lipophilic Envelope Spanning Tunnel

    This ~3.5 Å cryo-EM structure of the E. coli MCE protein LetB reveals an ~0.6 megadalton complex that consists of seven stacked rings, with a central hydrophobic tunnel sufficiently long to span the periplasm. Lipids bind inside the tunnel, suggesting that it functions as a pathway for lipid transport. Cryo-EM structures in the open and closed states reveal a dynamic tunnel lining, with implications for gating or substrate translocation.

    Read the full story here: GL Isom*, N Coudray*, MR MacRae, CT McManus, DC Ekiert, G Bhabha (2020). LetB structure reveals a tunnel for lipid transport across the bacterial envelope. Cell

  • MlaFEDB phospholipid transporter

    We reveal the cryo-EM structure of lipid transporter MlaFEDB at 3.05 Å resolution. A continuous transport pathway extends from within MlaE, through the channel of MlaD, and into the periplasm. Two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. We show that phospholipids can indeed be detected at these locations within the bacterial cell, using an in vivo crosslinking method that we developed.

    Read the full story here: N Coudray*, GL Isom*, MR MacRae*, MN Saiduddin, G Bhabha and DC Ekiert (2020). Structure of bacterial phospholipid transporter MlaFEDB with substrate bound. eLife

  • YhdP phospholipid transporter

    YhdP has been implicated as a major protagonist in the transport of phospholipids to the outer membrane. Here, utilising AlphaFold, we observe YhdP to form an elongated assembly of 60 β strands that curve to form a continuous hydrophobic groove. This architecture is consistent with our negative stain electron microscopy data which reveals YhdP to be approximately 250 Å in length and thus sufficient to span the bacterial cell envelope. Furthermore, molecular dynamics simulations and in vivo bacterial growth assays indicate essential helical regions at the N- and C-termini of YhdP, that may embed into the inner and outer membranes respectively, reinforcing its envelope spanning nature. Our in vivo crosslinking data reveal phosphate-containing substrates captured along the length of the YhdP groove, providing direct evidence that YhdP transports phospholipids. This finding is congruent with our molecular dynamics simulations which demonstrate the propensity for inner membrane lipids to spontaneously enter the groove of YhdP. Collectively, our results support a model in which YhdP bridges the cell envelope, providing a hydrophobic environment for the transport of phospholipids to the outer membrane.

Latest publications

N Coudray*, GL Isom*, MR MacRae*, MN Saiduddin, G Bhabha and DC Ekiert (2020). Structure of bacterial phospholipid transporter MlaFEDB with substrate bound. eLife

LR Kolich*, Y Chang*, N Coudray*, SI Giacometti, MR MacRae, GL Isom, EM Teran, G Bhabha and DC Ekiert (2020). Structure of MlaFB uncovers novel mechanisms of ABC transporter regulation. eLife. PMID: 32602838

GL Isom*, N Coudray*, MR MacRae, CT McManus, DC Ekiert, G Bhabha (2020). LetB structure reveals a tunnel for lipid transport across the bacterial envelope. Cell. PMID: 32359438

GW Hughes, SC Hall, CS Laxton, P Sridhar, AH Mahadi, C Hatton, TJ Piggot, PJ Wotherspoon, AC Leney, DG Ward, M Jamshad, V Spana, IT Cadby, C Harding, GL Isom, JA Bryant, RJ Parr, Y Yakub, M Jeeves, D Huber, IR Henderson, LA Clifton, AL Lovering and TJ Knowles (2019). Evidence for phospholipid export from the bacterial inner membrane by the Mla ABC transport system. Nature Microbiology. PMID: 31235958

GL Isom, JL Rooke, CA Antunes, E Sheehan, TJ Wells, C Icke, AF Cunningham, JA Cole, IR Henderson and AE Rossiter (2018). Mammalian Cell Entry domains are required for bile resistance and virulence in Salmonella. Biorxiv

CA Antunes, EJ Richardson, J Quick, P Fuentes-utrilla, GL Isom, EC Goodall, J Möller, PA Hoskisson, AL Mattos-Guaraldi, AF Cunningham, NJ Loman, V Sangal, A Burkovski and IR Henderson (2018). Complete closed genome sequence of nontoxigenic invasive Corynebacterium diptheriae bv. mitis strain ISS 3319. Genome Announcements. PMID: 29437107

FC Morris, TJ Wells, JA Bryant, AE Shager, YR Sevastsyanovich, DJP Squire, J Marshall, GL Isom, J Rooke, R Maderbocus, TJ Knowles, M Overduin, AE Rossiter, AF Cunningham and IR Henderson (2018). YraP Contributes to Cell Envelope Integrity and Virulence of Salmonella enterica Serovar Typhimurium. Infection and Immunity. PMID: 30201701

GL Isom, NJ Davies, ZS Chong, JA Bryant, M Jamshad, M Sharif, AF Cunningham, TJ Knowles, SS Chng, JA Cole and IR Henderson (2017). MCE domain proteins: conserved inner membrane lipid-binding proteins required for outer membrane homeostasis. Scientific reports

DC Ekiert, G Bhabha, GL Isom, G Greenan, S Ovchinnikov, IR Henderson, JS Cox and RD Vale (2017). Architectures of lipid transport systems for the bacterial outer membrane. Cell. PMID: 28388411