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Dive into the research topics where Vassilis Koronakis is active.

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Featured researches published by Vassilis Koronakis.


Nature | 2000

Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export.

Vassilis Koronakis; Andrew Sharff; Eva Koronakis; Ben F. Luisi; Colin Hughes

Diverse molecules, from small antibacterial drugs to large protein toxins, are exported directly across both cell membranes of Gram-negative bacteria. This export is brought about by the reversible interaction of substrate-specific inner-membrane proteins with an outer-membrane protein of the TolC family, thus bypassing the intervening periplasm. Here we report the 2.1-Å crystal structure of TolC from Escherichia coli, revealing a distinctive and previously unknown fold. Three TolC protomers assemble to form a continuous, solvent-accessible conduit—a ‘channel-tunnel’ over 140 Å long that spans both the outer membrane and periplasmic space. The periplasmic or proximal end of the tunnel is sealed by sets of coiled helices. We suggest these could be untwisted by an allosteric mechanism, mediated by protein–protein interactions, to open the tunnel. The structure provides an explanation of how the cell cytosol is connected to the external environment during export, and suggests a general mechanism for the action of bacterial efflux pumps.


The EMBO Journal | 1998

Substrate-induced assembly of a contiguous channel for protein export from E.coli: reversible bridging of an inner-membrane translocase to an outer membrane exit pore.

Thirumaran Thanabalu; Eva Koronakis; Colin Hughes; Vassilis Koronakis

The toxin HlyA is exported from Escherichia coli, without a periplasmic intermediate, by a type I system comprising an energized inner‐membrane (IM) translocase of two proteins, HlyD and the traffic ATPase HlyB, and the outer‐membrane (OM) porin‐like TolC. These and the toxin substrate were expressed separately to reconstitute export and, via affinity tags on the IM proteins, cross‐linked in vivo complexes were isolated before and after substrate engagement. HlyD and HlyB assembled a stable IM complex in the absence of TolC and substrate. Both engaged HlyA, inducing the IM complex to contact TolC, concomitant with conformational change in all three exporter components. The IM–OM bridge was formed primarily by HlyD, which assembled to stable IM trimers, corresponding to the OM trimers of TolC. The bridge was transient, components reverting to IM and OM states after translocation. Mutant HlyB that bound, but did not hydrolyse ATP, supported IM complex assembly, substrate recruitment and bridging, but HlyA stalled in the channel. A similar picture was evident when the HlyD C‐terminus was masked. Export thus occurs via a contiguous channel which is formed, without traffic ATPase ATP hydrolysis, by substrate‐induced, reversible bridging of the IM translocase to the OM export pore.


The EMBO Journal | 1999

Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella.

Richard D. Hayward; Vassilis Koronakis

Salmonella causes severe gastroenteritis in humans, entering non‐phagocytic cells to initiate intracellular replication. Bacterial engulfment occurs by macropinocytosis, which is dependent upon nucleation of host cell actin polymerization and condensation (‘bundling’) of actin filaments into cables. This is stimulated by contact‐induced delivery of an array of bacterial effector proteins, including the four Sips (Salmonella invasion proteins). Here we show in vitro that SipC bundles actin filaments independently of host cell components, a previously unknown pathogen activity. Bundling is directed by the SipC N‐terminal domain, while additionally the C‐terminal domain nucleates actin polymerization, an activity so far known only in eukaryotic proteins. The ability of SipC to cause actin condensation and cytoskeletal rearrangements was confirmed in vivo by microinjection into cultured cells, although as SipC associates with lipid bilayers it is possible that these activities are normally directed from the host cell membrane. The data suggest a novel mechanism by which a pathogen directly modulates the cytoskeletal architecture of mammalian target cells.


Current Opinion in Microbiology | 2009

Salmonella takes control: effector-driven manipulation of the host.

Emma J. McGhie; Lyndsey C. Brawn; Peter J. Hume; Daniel Humphreys; Vassilis Koronakis

Salmonella pathogenesis relies upon the delivery of over thirty specialised effector proteins into the host cell via two distinct type III secretion systems. These effectors act in concert to subvert the host cell cytoskeleton, signal transduction pathways, membrane trafficking and pro-inflammatory responses. This allows Salmonella to invade non-phagocytic epithelial cells, establish and maintain an intracellular replicative niche and, in some cases, disseminate to cause systemic disease. This review focuses on the actions of the effectors on their host cell targets during each stage of Salmonella infection.


Proceedings of the National Academy of Sciences of the United States of America | 2009

The assembled structure of a complete tripartite bacterial multidrug efflux pump

Martyn F. Symmons; Evert Bokma; Eva Koronakis; Colin E. Hughes; Vassilis Koronakis

Bacteria like Escherichia coli and Pseudomonas aeruginosa expel drugs via tripartite multidrug efflux pumps spanning both inner and outer membranes and the intervening periplasm. In these pumps a periplasmic adaptor protein connects a substrate-binding inner membrane transporter to an outer membrane-anchored TolC-type exit duct. High-resolution structures of all 3 components are available, but a pump model has been precluded by the incomplete adaptor structure, because of the apparent disorder of its N and C termini. We reveal that the adaptor termini assemble a β-roll structure forming the final domain adjacent to the inner membrane. The completed structure enabled in vivo cross-linking to map intermolecular contacts between the adaptor AcrA and the transporter AcrB, defining a periplasmic interface between several transporter subdomains and the contiguous β-roll, β-barrel, and lipoyl domains of the adaptor. With short and long cross-links expressed as distance restraints, the flexible linear topology of the adaptor allowed a multidomain docking approach to model the transporter–adaptor complex, revealing that the adaptor docks to a transporter region of comparative stability distinct from those key to the proposed rotatory pump mechanism, putative drug-binding pockets, and the binding site of inhibitory DARPins. Finally, we combined this docking with our previous resolution of the AcrA hairpin–TolC interaction to develop a model of the assembled tripartite complex, satisfying all of the experimentally-derived distance constraints. This AcrA3-AcrB3-TolC3 model presents a 610,000-Da, 270-Å-long efflux pump crossing the entire bacterial cell envelope.


Molecular Microbiology | 2004

Interactions underlying assembly of the Escherichia coli AcrAB–TolC multidrug efflux system

Thierry Touzé; Jeyanthy Eswaran; Evert Bokma; Eva Koronakis; Colin Hughes; Vassilis Koronakis

The major Escherichia coli multidrug efflux pump AcrAB–TolC expels a wide range of antibacterial agents. Using in vivo cross‐linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate‐induced assembly of the closely related TolC‐dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross‐linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo. Confirming this view, AcrA could be cross‐linked independently to AcrB and TolC in vivo, and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C‐terminal half of AcrA, but binding to TolC required interaction of N‐ and C‐terminal polypeptides spanning the lipoyl‐like domains predicted to present the intervening coiled‐coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug‐independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.


The EMBO Journal | 1989

Isolation and analysis of the C-terminal signal directing export of Escherichia coli hemolysin protein across both bacterial membranes.

Vassilis Koronakis; Eva Koronakis; Colin Hughes

We have studied the C‐terminal signal which directs the complete export of the 1024‐amino‐acid hemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Isolation and sequencing of homologous hlyA genes from the related bacteria Proteus vulgaris and Morganella morganii revealed high primary sequence divergence in the three HlyA C‐termini and highlighted within the extreme terminal 53 amino acids the conservation of three contiguous sequences, a potential 18‐amino‐acid amphiphilic alpha‐helix, a cluster of charged residues, and a weakly hydrophobic terminal sequence rich in hydroxylated residues. Fusion of the C‐terminal 53 amino acid sequence to non‐exported truncated Hly A directed wild‐type export but export was radically reduced following independent disruption or progressive truncation of the three C‐terminal features by in‐frame deletion and the introduction of translation stop codons within the 3′ hlyA sequence. The data indicate that the HlyA C‐terminal export signal comprises multiple components and suggest possible analogies with the mitochondrial import signal. Hemolysis assays and immunoblotting confirmed the intracellular accumulation of non‐exported HlyA proteins and supported the view that export proceeds without a periplasmic intermediate. Comparison of cytoplasmic and extracellular forms of an independently exported extreme C‐terminal 194 residue peptide showed that the signal was not removed during export.


Molecular Microbiology | 1998

Salmonella InvG forms a ring‐like multimer that requires the InvH lipoprotein for outer membrane localization

Aimee M. Crago; Vassilis Koronakis

Salmonella species translocate virulence effector proteins from the bacterial cytoplasm into mammalian host cells by means of a type III secretion apparatus, encoded by the pathogenicity island‐1 (SPI‐1). Little is known about the assembly and structure of this secretion apparatus, but the InvG protein is essential and could be an outer membrane secretion channel for the effector proteins. We observed that in recombinant Escherichia coli, the yield of InvG was enhanced by co‐expression of InvH, and showed that mutation of invH decreased the level of InvG in wild‐type Salmonella typhimurium. In E. coli, InvG alone was able to form an SDS‐resistant multimer, but InvG localization to the outer membrane was dependent upon InvH, a lipoprotein itself located in the outer membrane, and no other SPI‐1 specific protein. InvG targeted to the outer membrane by InvH became accessible to extracellular protease. InvG and InvH did not, however, appear to form a stable complex. Electron microscopy of InvG membrane protein purified from E. coli revealed that it forms an oligomeric ring‐like structure with inner and outer diameters, 7 nm and 15 nm respectively.


Molecular Microbiology | 1997

RfaH and the ops element, components of a novel system controlling bacterial transcription elongation

Marc J. A. Bailey; Colin Hughes; Vassilis Koronakis

The RfaH protein controls the transcription of a specialized group of Escherichia coli and Salmonella operons that direct the synthesis, assembly and export of the lipopolysaccharide core, exopolysaccharide, F conjugation pilus and haemolysin toxin. RfaH is a specific regulator of transcript elongation; its loss increases transcription polarity in these operons without affecting initiation from the operon promoters. The operons of the RfaH‐dependent regulon contain a short conserved 5′ sequence, the ops element, deletion of which increases operon polarity to an extent similar to that caused by loss of RfaH. The ops element is also present upstream of polysaccharide gene clusters of Shigella flexneri, Yersinia enterocolitica, Vibrio cholerae and Klebsiella pneumoniae and the RP4 fertility operon of Pseudomonas aeruginosa, suggesting that this is a widely spread control system. The mechanistic coupling of RfaH and the ops element has been demonstrated in vitro and in vivo, and we suggest that the ops element recruits RfaH and potentially other factors to the RNA polymerase complex, modifying the complex to increase its processivity and allowing transcription to proceed over long distances.


The EMBO Journal | 2001

Cooperation between actin‐binding proteins of invasive Salmonella : SipA potentiates SipC nucleation and bundling of actin

Emma J. McGhie; Richard D. Hayward; Vassilis Koronakis

Pathogen‐induced remodelling of the host cell actin cytoskeleton drives internalization of invasive Salmonella by non‐phagocytic intestinal epithelial cells. Two Salmonella actin‐binding proteins are involved in internalization: SipC is essential for the process, while SipA enhances its efficiency. Using purified SipC and SipA proteins in in vitro assays of actin dynamics and F‐actin bundling, we demonstrate that SipA stimulates substantially SipC‐mediated nucleation of actin polymerization. SipA additionally enhances SipC‐ mediated F‐actin bundling, and SipC–SipA collaboration generates stable networks of F‐actin bundles. The data show that bacterial SipC and SipA cooperate to direct efficient modulation of actin dynamics, independently of host cell proteins. The ability of SipA to enhance SipC‐induced reorganization of the actin cytoskeleton in vivo was confirmed using semi‐ permeabilized cultured mammalian cells.

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Colin Hughes

University of Cambridge

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