Latest Publications
Complement-mediated killing of Escherichia coli by mechanical destabilization of the cell envelope
Georgina Benn, Christian Bortolini, David M Roberts, Alice L B Pyne, Seamus Holden, Bart W Hoogenboom
Complement proteins eliminate Gram-negative bacteria in the blood via the formation of membrane attack complex (MAC) pores in the outer membrane. However, it remains unclear how outer membrane poration leads to inner membrane permeation and cell lysis. Using atomic force microscopy (AFM) on living Escherichia coli (E. coli), we probed MAC-induced changes in the cell envelope and correlated these with subsequent cell death. We conclude that bacterial cell lysis is only an indirect effect of MAC formation; outer membrane poration leads to mechanical destabilization of the cell envelope, reducing its ability to contain the turgor pressure, leading to inner membrane permeation and cell death.
Structure of the MlaC-MlaD complex reveals molecular basis of periplasmic phospholipid transport
Peter Wotherspoon, Hannah Johnston, David J. Hardy, Rachel Holyfield, Soi Bui, Giedrė Ratkevičiūtė, Pooja Sridhar, Jonathan Colburn, Charlotte B. Wilson, Adam Colyer, Benjamin F. Cooper, Jack A. Bryant, Gareth W. Hughes, Phillip J. Stansfeld, Julien R. C. Bergeron & Timothy J. Knowles
The Maintenance of Lipid Asymmetry (Mla) pathway is a multicomponent system found in all gram-negative bacteria that contributes to virulence, vesicle blebbing and preservation of the outer membrane barrier function. Here, we report the structure of E. coli MlaC in complex with the MlaD hexamer in two distinct stoichiometries. Utilising in vivo complementation assays, an in vitro fluorescence-based transport assay, and molecular dynamics simulations, we confirm key residues, identifying the MlaD β6-β7 loop as essential for MlaCD function. We also provide evidence that phospholipids pass between the C-terminal helices of the MlaD hexamer to reach the central pore, providing insight into the trajectory of GPL transfer between MlaC and MlaD.
Membraneless channels sieve cations in ammonia-oxidizing marine archaea
Andriko von Kügelgen, C. Keith Cassidy, Sofie van Dorst, Lennart L. Pagani, Christopher Batters, Zephyr Ford, Jan Löwe, Vikram Alva, Phillip J. Stansfeld & Tanmay A. M. Bharat
Nitrosopumilus maritimus is an ammonia-oxidizing archaeon that is crucial to the global nitrogen cycle. A critical step for nitrogen oxidation is the entrapment of ammonium ions from a dilute marine environment at the cell surface and their subsequent channelling to the cell membrane of N. maritimus. Here we elucidate the structure of the molecular machinery responsible for this process, comprising the surface layer (S-layer), using electron cryotomography and subtomogram averaging from cells. We supplemented our in situ structure of the ammonium-binding S-layer array with a single-particle electron cryomicroscopy structure, revealing detailed features of this immunoglobulin-rich and glycan-decorated S-layer. This in situ structural study illuminates the biogeochemically essential process of ammonium binding and channelling, common to many marine microorganisms that are fundamental to the nitrogen cycle.
Biophysical basis of filamentous phage tactoid-mediated antibiotic tolerance in P. aeruginosa
Jan Böhning, Miles Graham, Suzanne C. Letham, Luke K. Davis, Ulrike Schulze, Phillip J. Stansfeld, Robin A. Corey, Philip Pearce, Abul K. Tarafder, Tanmay A. M. Bharat
Inoviruses are filamentous phages infecting numerous prokaryotic phyla. Inoviruses can self-assemble into mesoscale structures with liquid-crystalline order, termed tactoids, which protect bacterial cells in Pseudomonas aeruginosa biofilms from antibiotics. Here, we investigate the structural, biophysical, and protective properties of tactoids formed by the P. aeruginosa phage Pf4 and Escherichia coli phage fd. This study provides insights into how filamentous molecules protect bacteria from extraneous substances in biofilms and in host-associated infections.
A monomeric StayGold fluorescent protein
Esther Ivorra-Molla, Dipayan Akhuli, Martin B. L. McAndrew, William Scott, Lokesh Kumar, Saravanan Palani, Masanori Mishima, Allister Crow & Mohan K. Balasubramanian
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching. Despite favorable fluorescence properties, use of StayGold as a fluorescent tag is limited because it forms a natural dimer. Here we report the 1.6 Å structure of StayGold and generate a derivative, mStayGold, that retains the brightness and photostability of the original protein while being fully monomeric.
LipIDens: Simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.
T. Bertie Ansell, Wanling Song, Claire E. Coupland, Loic Carrique, Robin A. Corey, Anna L. Duncan, C. Keith Cassidy, Maxwell M. G. Geurts, Tim Rasmussen, Andrew B. Ward, Christian Siebold, Phillip J. Stansfeld, Mark S. P. Sansom
Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures.