Protein-protein interactions in the Gram-negative bacterial cell envelope
In my laboratory, we aim to understand how protein-protein interactions in bacteria help support the organisation, structure and stability of the outer membrane and how such interactions can also be exploited by protein antibiotics known as bacteriocins to kill bacterial cells. Our research typically incorporates a range of biochemical, biophysical, structural and cell-based techniques.
Bacteriocins are species-specific protein antibiotics that parasitize a variety of outer membrane and periplasmic proteins in Gram-negative bacteria. Our work centres on the entry mechanisms of colicins, which target Escherichia coli, pyocins, which target Pseudomonas aeruginosa and klebicins, which target Klebsiella pneumoniae. These toxins serve as important agents of competition within microbial communities. We study both nuclease (DNases, rRNases and tRNases) and pore-forming bacteriocins, which use their network of protein-protein interactions within the cell envelope to establish a translocon complex that delivers a toxic domain into the cell. Hence, bacteriocin translocation represents a highly simplified model for understanding energised cellular protein import. We recently demonstrated that Tol- and Ton- dependent bacteriocins translocate across the outer membrane by the same basic mechanism in which the unfolded toxin is pulled through a specific translocator protein and powered by the proton-motive force of the inner membrane.
Porin threading drives receptor disengagement and establishes active colicin E9 transport through Escherichia coli OmpF. a-e, sequence of events at the E. coli cell surface as BtuB-bound ColE9 threads its disorder domain through two of the pores of OmpF. Threading causes the toxin to disengage from its receptor BtuB and associate with the periplasmic protein TolB. ColE9-TolB is dragged unfolded through a single subunit of OmpF by the PMF-linked inner membrane complex of TolQ-TolR-TolA. From the periplasm, ColE9 translocates across the inner membrane to deliver its cytotoxic nuclease (not shown). See Francis et al (2021) EMBO Jand Housden et al (2013) Sciencefor further details.
Tol-Pal is a little understood complex that is required for the stable maintenance of the Gram-negative outer membrane and which is recruited to the cell division apparatus late during division. We discovered a unique ‘mobilisation-and-capture’ mechanism underpins Tol-Pal function whereby force transduction across the cell envelope enables the accumulation of the peptidoglycan-binding protein Pal at division septa, stabilising the invaginating outer membrane. More recently, we solved the cryo-electron microscopy structure for the TolQR stator complex and demonstrated that structural rigidity within the force transducer protein TolA maximises delivery of mechanical force to the outer membrane following association with this PMF-coupled motor.
Efficient mechanotransduction of force by TolA to the outer membrane requires secondary structure in its periplasm spanning domain. Tol-Pal is a ubiquitous five-protein assembly in Gram-negative bacteria; TolQ, TolR and TolA are inner membrane proteins, Pal is an outer membrane lipoprotein and TolB is a periplasmic protein that binds Pal. The inner membrane complex of TolQ-TolR-TolA exploits the proton motive force (PMF) to dissociate the TolB-Pal complex, releasing Pal to bind the cell wall. During cell division the inner membrane complex becomes localised to the divisome enabling Pal deposition to also be localised. Increased [Pal] at the septum stabilises the invaginating outer membrane of the dividing cell. SeeSzczepaniak et al (2020) Nat Communand Williams-Jones et al (2023) PNASfor further details.
The rise of multidrug resistant bacteria coupled with the lack of new classes of antibiotics in over 40 years means there is a pressing and urgent need for new antibiotics especially for molecules that target pathogenic Gram-negative bacteria. In collaboration with Prof Dan Walker at the University of Strathclyde, we have developed novel bacteriocin engineering pipelines that help avoid some of the traditional problems associated with using these potent toxins as antibiotics. These advances are the basis for a new spinout company, Glox Therapeutics Ltd, which is led by our CEO Dr James Clark.