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Professor Colin Hughes

Professor Colin Hughes

Professor of Microbiology

Deputy Head of Department

Director of Teaching

Fellow of Trinity College

Fellow of the Learned Society of Wales

Tripartite efflux pumps

Flagella

Toxin secretion

Department of Pathology
University of Cambridge
Tennis Court Road
Cambridge
CB2 1QP

Trinity College
Cambridge
CB2 1TQ

Office Phone: +44 (0)1223 333732 (Pathology) +44 (0)1223 338538 (Trinity)

Research Interests

Membrane machineries for toxin export and multidrug efflux

Hughes Research 1TolC-dependent membrane machineries export large virulence proteins like toxins, or eject antibiotics and other inhibitory agents, so are important to the survival of pathogens like E.coli, Pseudomonas, Neisseria, Serratia and Bordatella. These toxin export systems and multidrug resistance ‘pumps’ all have a tripartite structure, comprising an outer membrane-anchored TolC protein that projects across the inter-membrane periplasmic space to present an exit duct or 'trash chute', to molecules bound by inner membrane transporters. These are typically a traffic ATPase for protein export, and either an ATPase or a proton antiporter for efflux. The two apposed inner and outer membrane components are structurally and functionally linked by a periplasmic adaptor protein that is key to the recruitment and opening of the periplasmic entrance to the TolC exit channel. The assembled pumps span the entire bacterial cell envelope of both membranes and periplasmic space.

We have defined the toxin export signal and identified translocation stages and intermediates of the toxin export mechanism, and shown that when the transporter-adaptor complex is bound by export substrate it recruits TolC to effect reversible assembly. To establish the key features of the export and efflux mechanisms, we elucidated by crystallography high resolution structures of the common proteins, the TolC exit duct and the efflux pump adaptor. By extensive in vivo site-specific cross-linking and multidomain docking we established the interaction interfaces between the efflux pump exit duct, adaptor and antiporter. This has allowed us to use complex modelling to present the first view of a c.600,000 Da assembled multidrug efflux machinery. By combining electrophysiology of purified TolC open state variants inserted in lipid bilayers with the crystallography of closed and sequential open states we are elucidating the coiled coil movements underlying the ‘iris-like’ mechanism TolC entrance opening mechanism, and how this could be triggered and stabilized by the periplasmic hairpin of the efflux pump adaptor. Similar approaches have shown the TolC entrance can be liganded and blocked, indicating a potential way for pumps to be inhibited by novel drugs. This work is codirected by Prof. Koronakis.

The flagella subunit export pathway underlying assembly

Hughes Research 2Motile pathogenic bacteria build multi-component rotating flagella 'nanomachines' that act like helical 'propellers' on their surface to effect cell movement and population migration. In bacteria such as Salmonella, the flagellar filament comprises about 20,000 subunits, which pass through the central channel within the growing structure and polymerize under the flagella cap, following assembly of the flexible hook structure. Our research addresses the ordered process by which the flagellar structural subunits, those for the filament, cap, and filament-hook junction, are exported from the cytosol and delivered to the growing flagellar on the cell surface. This is achieved by a complex membrane export apparatus comprising over a dozen different proteins, including those of the integral inner membrane structure, the ATPase complex, and three chaperones that bind the major filament subunits, or minor cap or filament junction subunits. By assembling in vitro complexes and using genetics to create stalled intermediate export complexes we revealed how chaperones protect and pilot flagellar structural subunits to dock at an oligomeric membrane export ATPase. We have revealed an unusual mechanism in which the chaperones of the minor subunits are then selectively cycled at the ATPase complex by an escort protein, a process we suggest enhances subunit export and also facilitates the ordered stoichiometric assembly of the flagellum. This work is codirected by Dr Fraser.

Work is funded by parallel Programme Grants from the Wellcome Trust.

Key Publications

Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C (2000). Crystal structure of TolC central to multidrug efflux and protein export. 
Nature 405: 914

Andersen C, Koronakis E, Bokma E, Eswaran J, Humphreys D, Hughes C, Koronakis V (2002). Transition to the open state of the TolC periplasmic tunnel entrance. 
Proceedings of the National Academy of Sciences (USA) 99:11103-11108.

Higgins M, Boekma E, Koronakis E, Hughes C, Koronakis V (2004). Structure of the periplasmic component of a bacterial antibiotic efflux pump.
Proceedings of the National Academy of Sciences (USA) 101:9994-9999.

Thomas J, Stafford GP, Hughes C (2004). Docking of export chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. 
Proceedings of the National Academy of Sciences (USA) 101:3945-3950.

Higgins M, Eswaran J, Edwards P, Schertler G F X, Hughes C and Koronakis V (2004). Structure of the ligand-blocked periplasmic entrance of the bacterial multidrug efflux protein TolC 
Journal of Molecular Biology 342: 697

Evans L, Stafford G P, Ahmed S, Fraser G M, and Hughes C (2006). An escort mechanism for cycling export chaperones. 
Proceedings of the National Academy of Sciences (USA) 103: 17474

Stafford G P, Evans L, Krumscheid R, Dhillon P, Fraser G and Hughes C (2007). Sorting of early and late flagellar subunits after docking at the membrane ATPase of the type III export pathway. 
Journal of Molecular Biology 374: 877

Lobedanz S, Bokma E, Symmons M, Koronakis E, Hughes C, and Koronakis V (2007). A coiled-coil interface underlying TolC recruitment and pump assembly. 
Proceedings of the National Academy of Sciences (USA) 104: 4612

Symmons MF, Bokma E, Koronakis E, Hughes C, Koronakis V (2009). The assembled structure of a tripartite multidrug efflux pump. 
Proceedings of the National Academy of Sciences 1106: 7173

Pei XY, Hinchliffe P, Symmons MF, Koronakis E, Benz R, Hughes C, Koronakis V. (2011) Structures of sequential open states in a symmetrical opening transition of the TolC exit duct.
Proceedings of the National Academy of Sciences (USA) 108:2112-2117.

Evans LD, Poulter S, Terentjev EM, Hughes C, Fraser GM. (2013) A chain mechanism for flagellum growth. 
Nature 12;504(7479):287-90