Our Research

Antimicrobial resistance is one of the most serious challenges to global health, yet the development of new molecules with novel mechanisms of action to combat resistance is lacking. Here, we report the discovery of molecular glue-like compounds that recruit TEM-family β-lactamases to the bacterial protease DegP for degradation. β-lactamase inhibitor tazobactam was found to accelerate degradation of TEM β-lactamases by DegP, which was further enhanced by linkerless incorporation of dipeptide motifs enriched among DegP substrates. The resulting molecular glue-like degraders showed improved synergy with β-lactam piperacillin against resistant E. coli compared to tazobactam, as well as good pharmacokinetic properties for oral dosing. Collectively, this work establishes periplasmic targeted protein degradation as a promising new mechanism for combating β-lactamase resistance.

Although the mechanism of phospholipid transport to build the OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant and collectively essential proteins mediating this process in Escherichia coli. YdbH is anchored to the inner membrane (IM) and its periplasmic β-sheet groove interacts directly with OM lipoprotein YnbE to form an intermembrane bridge. Our previous data supports a model where YnbE multimerizes to stack on the C-terminus of YdbH, and this multimerization is modulated by the periplasmic protein YdbL. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex. We resolve high-resolution structural data for YdbL and ascertain highly conserved interacting residues with the C-terminus of YnbE, which are critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL directly interacts with YnbE to ensure proper YdbH-YnbE intermembrane bridge formation for lipid transport.

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.

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

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