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  • Journal article
    Lossi NS, Manoli E, Foerster A, Dajani R, Pape T, Freemont P, Filloux Aet al., 2021,

    The HsiB1C1 (TssB-TssC) complex of the pseudomonas aeruginosa Type VI secretion system forms a bacteriophage tail sheathlike structure

    , Journal of Biological Chemistry, Vol: 288, Pages: 7536-7548, ISSN: 0021-9258

    Protein secretion systems in Gram-negative bacteria evolved into a variety of molecular nanomachines. They are related to cell envelope complexes, which are involved in assembly of surface appendages or transport of solutes. They are classified as types, the most recent addition being the type VI secretion system (T6SS). The T6SS displays similarities to bacteriophage tail, which drives DNA injection into bacteria. The Hcp protein is related to the T4 bacteriophage tail tube protein gp19, whereas VgrG proteins structurally resemble the gp27/gp5 puncturing device of the phage. The tube and spike of the phage are pushed through the bacterial envelope upon contraction of a tail sheath composed of gp18. In Vibrio cholerae it was proposed that VipA and VipB assemble into a tail sheathlike structure. Here we confirm these previous data by showing that HsiB1 and HsiC1 of the Pseudomonas aeruginosa H1-T6SS assemble into tubules resulting from stacking of cogwheel-like structures showing predominantly 12-fold symmetry. The internal diameter of the cogwheels is ∼100 Å, which is large enough to accommodate an Hcp tube whose external diameter has been reported to be 85 Å. The N-terminal 212 residues of HsiC1 are sufficient to form a stable complex with HsiB1, but the C terminus of HsiC1 is essential for the formation of the tubelike structure. Bioinformatics analysis suggests that HsiC1 displays similarities to gp18-like proteins in its C-terminal region. In conclusion, we provide further structural and mechanistic insights into the T6SS and show that a phage sheathlike structure is likely to be a conserved element across all T6SSs.

  • Journal article
    Kelwick RJR, Ricci L, Chee SM, Bell D, Webb A, Freemont Pet al., 2019,

    Cell-free prototyping strategies for enhancing the sustainable production of polyhydroxyalkanoates bioplastics

    , Synthetic Biology, Vol: 3, ISSN: 2397-7000

    The polyhydroxyalkanoates (PHAs) are microbially-produced biopolymers that could potentially be used as sustainable alternatives to oil-derived plastics. However, PHAs are currently more expensive to produce than oil-derived plastics. Therefore, more efficient production processes would be desirable. Cell-free metabolic engineering strategies have already been used to optimise several biosynthetic pathways and we envisioned that cell-free strategies could be used for optimising PHAs biosynthetic pathways. To this end, we developed several Escherichia coli cell-free systems for in vitro prototyping PHAs biosynthetic operons, and also for screening relevant metabolite recycling enzymes. Furthermore, we customised our cell-free reactions through the addition of whey permeate, an industrial waste that has been previously used to optimise in vivo PHAs production. We found that the inclusion of an optimal concentration of whey permeate enhanced relative cell-free GFPmut3b production by ∼50%. In cell-free transcription-translation prototyping reactions, GC-MS quantification of cell-free 3-hydroxybutyrate (3HB) production revealed differences between the activities of the Native ΔPhaC_C319A (1.18 ±0.39 µM), C104 ΔPhaC_C319A (4.62 ±1.31 µM) and C101 ΔPhaC_C319A (2.65 ±1.27 µM) phaCAB operons that were tested. Interestingly, the most active operon, C104 produced higher levels of PHAs (or PHAs monomers) than the Native phaCAB operon in both in vitro and in vivo assays. Coupled cell-free biotransformation/transcription-translation reactions produced greater yields of 3HB (32.87 ±6.58 µM) and these reactions were also used to characterise a Clostridium propionicum Acetyl-CoA recycling enzyme. Together, these data demonstrate that cell-free approaches complement in vivo workflows for identifying additional strategies for optimising PHAs production.

  • Conference paper
    Girvan P, Teng X, Brooks NJ, Baldwin GS, Ying Let al., 2019,

    Redox Kinetics of the Amyloid-Beta-Copper Complex and Its Biological Implications

    , 63rd Annual Meeting of the Biophysical-Society, Publisher: CELL PRESS, Pages: 28A-28A, ISSN: 0006-3495
  • Journal article
    Silhan J, Zhao Q, Boura E, Thomson H, Förster A, Tang CM, Freemont PS, Baldwin GSet al., 2018,

    Structural basis for recognition and repair of the 3'-phosphate by NExo, a base excision DNA repair nuclease from Neisseria meningitidis

    , Nucleic Acids Research, Vol: 46, Pages: 11980-11989, ISSN: 0305-1048

    NExo is an enzyme from Neisseria meningitidis that is specialized in the removal of the 3'-phosphate and other 3'-lesions, which are potential blocks for DNA repair. NExo is a highly active DNA 3'-phosphatase, and although it is from the class II AP family it lacks AP endonuclease activity. In contrast, the NExo homologue NApe, lacks 3'-phosphatase activity but is an efficient AP endonuclease. These enzymes act together to protect the meningococcus from DNA damage arising mainly from oxidative stress and spontaneous base loss. In this work, we present crystal structures of the specialized 3'-phosphatase NExo bound to DNA in the presence and absence of a 3'-phosphate lesion. We have outlined the reaction mechanism of NExo, and using point mutations we bring mechanistic insights into the specificity of the 3'-phosphatase activity of NExo. Our data provide further insight into the molecular origins of plasticity in substrate recognition for this class of enzymes. From this we hypothesize that these specialized enzymes lead to enhanced efficiency and accuracy of DNA repair and that this is important for the biological niche occupied by this bacterium.

  • Journal article
    Yu J, Knoppova J, Michoux F, Bialek W, Cota Segura E, Shukla M, Straskova A, Aznar G, Sobotka R, Komenda J, Murray J, Nixon PJet al., 2018,

    Ycf48 involved in the biogenesis of the oxygen-evolving photosystem II complex is a seven-bladed beta-propeller protein

    , Proceedings of the National Academy of Sciences, Vol: 115, Pages: E7824-E7833, ISSN: 0027-8424

    Robust photosynthesis in chloroplasts and cyanobacteria requires the participation of accessory proteins to facilitate the assembly and maintenance of the photosynthetic apparatus located within the thylakoid membranes. The highly conserved Ycf48 protein acts early in the biogenesis of the oxygen-evolving photosystem II (PSII) complex by binding to newly synthesized precursor D1 subunit and by promoting efficient association with the D2 protein to form a PSII reaction center (PSII RC) assembly intermediate. Ycf48 is also required for efficient replacement of damaged D1 during the repair of PSII. However, the structural features underpinning Ycf48 function remain unclear. Here we show that Ycf48 proteins encoded by the thermophilic cyanobacterium Thermosynechococcus elongatus and the red alga Cyanidioschyzon merolae form seven-bladed beta-propellers with the 19-aa insertion characteristic of eukaryotic Ycf48 located at the junction of blades 3 and 4. Knowledge of these structures has allowed us to identify a conserved “Arg patch” on the surface of Ycf48 that is important for binding of Ycf48 to PSII RCs but also to larger complexes, including trimeric photosystem I (PSI). Reduced accumulation of chlorophyll in the absence of Ycf48 and the association of Ycf48 with PSI provide evidence of a more wide-ranging role for Ycf48 in the biogenesis of the photosynthetic apparatus than previously thought. Copurification of Ycf48 with the cyanobacterial YidC protein insertase supports the involvement of Ycf48 during the cotranslational insertion of chlorophyll-binding apopolypeptides into the membrane.

  • Journal article
    Freemont PS, Salih O, He S, Planamente S, Stach L, MacDonald J, Manoli E, Scheres S, Filloux Aet al., 2018,

    Atomic Structure of Type VI Contractile Sheath from Pseudomonas aeruginosa

    , Structure, Vol: 26, Pages: 329-336.e3, ISSN: 0969-2126

    Pseudomonas aeruginosa has three type VI secretion systems (T6SSs), H1-, H2-, and H3-T6SS, each belonging to a distinct group. The two T6SS components, TssB/VipA and TssC/VipB, assemble to form tubules that conserve structural/functional homology with tail sheaths of contractile bacteriophages and pyocins. Here, we used cryoelectron microscopy to solve the structure of the H1-T6SS P. aeruginosa TssB1C1 sheath at 3.3 Å resolution. Our structure allowed us to resolve some features of the T6SS sheath that were not resolved in the Vibrio cholerae VipAB and Francisella tularensis IglAB structures. Comparison with sheath structures from other contractile machines, including T4 phage and R-type pyocins, provides a better understanding of how these systems have conserved similar functions/mechanisms despite evolution. We used the P. aeruginosa R2 pyocin as a structural template to build an atomic model of the TssB1C1 sheath in its extended conformation, allowing us to propose a coiled-spring-like mechanism for T6SS sheath contraction.

  • Journal article
    Mullineaux-Sanders C, Colins JW, Ruano-Gallego D, Levy M, Pevsner-Fischer M, Glegola-Madejska IT, Sagfors AM, Wong JLC, Elinav E, Crepin VF, Frankel GMet al., 2017,

    Citrobacter rodentium relies on commensals for colonization of the colonic mucosa

    , Cell Reports, Vol: 21, Pages: 3381-3389, ISSN: 2211-1247

    We investigated the role of commensals at the peak of infection with the colonic mouse pathogen Citrobacter rodentium. Bioluminescent and kanamycin (Kan)-resistant C. rodentium persisted avirulently in the cecal lumen of mice continuously treated with Kan. A single Kan treatment was sufficient to displace C. rodentium from the colonic mucosa, a phenomenon not observed following treatment with vancomycin (Van) or metronidazole (Met). Kan, Van, and Met induce distinct dysbiosis, suggesting C. rodentium relies on specific commensals for colonic colonization. Expression of the master virulence regulator ler is induced in germ-free mice, yet C. rodentium is only seen in the cecal lumen. Moreover, in conventional mice, a single Kan treatment was sufficient to displace C. rodentium constitutively expressing Ler from the colonic mucosa. These results show that expression of virulence genes is not sufficient for colonization of the colonic mucosa and that commensals are essential for a physiological infection course.

  • Journal article
    Zhang X, Aramayo RJ, Willhoft O, Ayala R, Bythell-Douglas R, Wigley DBet al., 2017,

    CryoEM structures of the human INO80 chromatin remodelling complex

    , Nature Structural and Molecular Biology, Vol: 25, Pages: 37-44, ISSN: 1545-9985

    Access to chromatin for processes such as DNA repair and transcription requires the sliding of nucleosomes along DNA. The multi-subunit INO80 chromatin remodelling complex has a particular role in DNA repair. Here we present the cryo electron microscopy structures of the active core complex of human INO80 at 9.6 Å with portions at 4.1 Å resolution along with reconstructions of combinations of subunits. Together these structures reveal the architecture of the INO80 complex, including Ino80 and actin-related proteins, which is assembled around a single Tip49a (RUVBL1) and Tip49b (RUVBL2) AAA+ heterohexamer. An unusual spoked-wheel structural domain of the Ino80 subunit is engulfed by this heterohexamer and the intimate association of this Ino80 domain with the heterohexamer is at the core of the complex. We also identify a cleft in RUVBL1 and RUVBL2, which forms a major interaction site for partner proteins and likely communicates partner-interactions with its nucleotide binding sites.

  • Journal article
    Noguchi Y, yuan Z, Bai L, Schneider S, Zhao G, Stillman B, Speck C, Li Het al., 2017,

    Cryo-EM structure of Mcm2-7 double-hexamer on DNA suggests a lagging strand DNA extrusion model

    , Proceedings of the National Academy of Sciences, Vol: 114, Pages: E9529-E9538, ISSN: 0027-8424

    During replication initiation, the core component of the helicase—the Mcm2-7 hexamer—is loaded on origin DNA as a double hexamer (DH). The two ring-shaped hexamers are staggered, leading to a kinked axial channel. How the origin DNA interacts with the axial channel is not understood, but the interaction could provide key insights into Mcm2-7 function and regulation. Here, we report the cryo-EM structure of the Mcm2-7 DH on dsDNA and show that the DNA is zigzagged inside the central channel. Several of the Mcm subunit DNA-binding loops, such as the oligosaccharide–oligonucleotide loops, helix 2 insertion loops, and presensor 1 (PS1) loops, are well defined, and many of them interact extensively with the DNA. The PS1 loops of Mcm 3, 4, 6, and 7, but not 2 and 5, engage the lagging strand with an approximate step size of one base per subunit. Staggered coupling of the two opposing hexamers positions the DNA right in front of the two Mcm2–Mcm5 gates, with each strand being pressed against one gate. The architecture suggests that lagging-strand extrusion initiates in the middle of the DH that is composed of the zinc finger domains of both hexamers. To convert the Mcm2-7 DH structure into the Mcm2-7 hexamer structure found in the active helicase, the N-tier ring of the Mcm2-7 hexamer in the DH-dsDNA needs to tilt and shift laterally. We suggest that these N-tier ring movements cause the DNA strand separation and lagging-strand extrusion.

  • Journal article
    Wen KY, Cameron L, Chappell J, Jensen K, Bell DJ, Kelwick R, Kopniczky M, Davies JC, Filloux A, Freemont PSet al., 2017,

    A Cell-Free Biosensor for Detecting Quorum Sensing Molecules in P. aeruginosa-Infected Respiratory Samples.

    , ACS Synthetic Biology, Vol: 6, Pages: 2293-2301, ISSN: 2161-5063

    Synthetic biology designed cell-free biosensors are a promising new tool for the detection of clinically relevant biomarkers in infectious diseases. Here, we report that a modular DNA-encoded biosensor in cell-free protein expression systems can be used to measure a bacterial biomarker of Pseudomonas aeruginosa infection from human sputum samples. By optimizing the cell-free system and sample extraction, we demonstrate that the quorum sensing molecule 3-oxo-C12-HSL in sputum samples from cystic fibrosis lungs can be quantitatively measured at nanomolar levels using our cell-free biosensor system, and is comparable to LC-MS measurements of the same samples. This study further illustrates the potential of modular cell-free biosensors as rapid, low-cost detection assays that can inform clinical practice.

  • Journal article
    Berger C, Crepin V, Roumeliotis TI, Wright JC, Carson D, Pevsner-Fischer M, Furniss RCD, Dougan G, Bachash M, Yu L, Clements A, Collins JW, Elinav E, larrouy-maumus G, Choudhary JS, Frankel GMet al., 2017,

    Citrobacter rodentium subverts ATP flux 1 and cholesterol homeostasis in 2 intestinal epithelial cell in vivo

    , Cell Metabolism, Vol: 26, Pages: 738-752.e6, ISSN: 1550-4131

    The intestinal epithelial cells (IECs) that line the gut form a robust line of defense against ingested pathogens. We investigated the impact of infection with the enteric pathogen Citrobacter rodentium on mouse IEC metabolism using global proteomic and targeted metabolomics and lipidomics. The major signatures of the infection were upregulation of the sugar transporter Sglt4, aerobic glycolysis, and production of phosphocreatine, which mobilizes cytosolic energy. In contrast, biogenesis of mitochondrial cardiolipins, essential for ATP production, was inhibited, which coincided with increased levels of mucosal O2 and a reduction in colon-associated anaerobic commensals. In addition, IECs responded to infection by activating Srebp2 and the cholesterol biosynthetic pathway. Unexpectedly, infected IECs also upregulated the cholesterol efflux proteins AbcA1, AbcG8, and ApoA1, resulting in higher levels of fecal cholesterol and a bloom of Proteobacteria. These results suggest that C. rodentium manipulates host metabolism to evade innate immune responses and establish a favorable gut ecosystem.

  • Journal article
    Karampatzakis A, Song CZ, Allsopp LP, Filloux A, Rice SA, Cohen Y, Wohland T, Török Pet al., 2017,

    Probing the internal micromechanical properties of Pseudomonas aeruginosa biofilms by Brillouin imaging.

    , NPJ Biofilms Microbiomes, Vol: 3, ISSN: 2055-5008

    Biofilms are organised aggregates of bacteria that adhere to each other or surfaces. The matrix of extracellular polymeric substances that holds the cells together provides the mechanical stability of the biofilm. In this study, we have applied Brillouin microscopy, a technique that is capable of measuring mechanical properties of specimens on a micrometre scale based on the shift in frequency of light incident upon a sample due to thermal fluctuations, to investigate the micromechanical properties of an active, live Pseudomonas aeruginosa biofilm. Using this non-contact and label-free technique, we have extracted information about the internal stiffness of biofilms under continuous flow. No correlation with colony size was found when comparing the averages of Brillouin shifts of two-dimensional cross-sections of randomly selected colonies. However, when focusing on single colonies, we observed two distinct spatial patterns: in smaller colonies, stiffness increased towards their interior, indicating a more compact structure of the centre of the colony, whereas, larger (over 45 μm) colonies were found to have less stiff interiors.

  • Journal article
    freemont P, Stach L, 2017,

    The AAA+ ATPase p97, a cellular multi-tool

    , Biochemical Journal, Vol: 474, Pages: 2953-2976, ISSN: 1470-8728

    The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy.

  • Journal article
    Beckova M, Yu J, Krynicka V, Kozlo A, Shao S, Konik P, Komenda J, Murray JW, Nixon PJet al., 2017,

    Structure of Psb29/Thf1 and its association with the FtsH protease complex involved in photosystem II repair in cyanobacteria

    , Philosophical Transactions of the Royal Society B: Biological Sciences, Vol: 372, ISSN: 1471-2970

    One strategy for enhancing photosynthesis in crop plants is to improve the ability to repair photosystem II (PSII) in response to irreversible damage by light. Despite the pivotal role of thylakoid embedded FtsH protease complexes in the selective degradation of PSII subunits during repair, little is known about the factors involved in regulating FtsH expression. Here we show using the cyanobacterium Synechocystis sp. PCC 6803 that the Psb29 subunit, originally identified as a minor component of His tagged PSII preparations, physically interacts with FtsH complexes in vivo and is required for normal accumulation of the FtsH2/FtsH3 hetero oligomeric complex involved in PSII repair. We show using X ray crystallography that Psb29 from Thermosynechococcus elongatushas a unique fold consisting of a helical bundle and an extended C terminal helix and contains a highly conserved region that might be involved in binding to FtsH. A similar interaction is likely to occur in Arabidopsis chloroplasts between the Psb29 homologue, termed THF1, and the FTSH2/FTSH5 complex. The direct involvement of Psb29/THF1 in FtsH accumulation helps explain why THF1 is a target during the hypersensitive response in plants induced by pathogen infection. Downregulating FtsH function and the PSII repair cycle via THF1 would contribute to the production

  • Journal article
    Davies SK, Fearn S, Allsopp LP, Harrison F, Ware E, Diggle SP, Filloux A, McPhail DS, Bundy Jet al., 2017,

    Visualizing Antimicrobials in BacterialBiofilms: Three-Dimensional BiochemicalImaging Using TOF-SIMS

    , mSphere, Vol: 2, ISSN: 2379-5042

    Bacterial biofilms are groups of bacteria that exist within a self-produced extracellular matrix, adhering to each other and usually to a surface. They grow on medical equipment and inserts such as catheters and are responsible for many persistent infections throughout the body, as they can have high resistance to many antimicrobials. Pseudomonas aeruginosa is an opportunistic pathogen that can cause both acute and chronic infections and is used as a model for research into biofilms. Direct biochemical methods of imaging of molecules in bacterial biofilms are of high value in gaining a better understanding of the fundamental biology of biofilms and biochemical gradients within them. Time of flight–secondary-ion mass spectrometry (TOF-SIMS) is one approach, which combines relatively high spatial resolution and sensitivity and can perform depth profiling analysis. It has been used to analyze bacterial biofilms but has not yet been used to study the distribution of antimicrobials (including antibiotics and the antimicrobial metal gallium) within biofilms. Here we compared two methods of imaging of the interior structure of P. aeruginosa in biological samples using TOF-SIMS, looking at both antimicrobials and endogenous biochemicals: cryosectioning of tissue samples and depth profiling to give pseudo-three-dimensional (pseudo-3D) images. The sample types included both simple biofilms grown on glass slides and bacteria growing in tissues in an ex vivo pig lung model. The two techniques for the 3D imaging of biofilms are potentially valuable complementary tools for analyzing bacterial infection.

  • Journal article
    Allsopp LP, Wood TE, Howard SA, Maggiorelli F, Nolan LM, Wettstadt S, Filloux Aet al., 2017,

    RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 114, Pages: 7707-7712, ISSN: 1091-6490

    The type VI secretion system (T6SS) is a weapon of bacterial warfare and host cell subversion. The Gram-negative pathogen Pseudomonas aeruginosa has three T6SSs involved in colonization, competition, and full virulence. H1-T6SS is a molecular gun firing seven toxins, Tse1–Tse7, challenging survival of other bacteria and helping P. aeruginosa to prevail in specific niches. The H1-T6SS characterization was facilitated through studying a P. aeruginosa strain lacking the RetS sensor, which has a fully active H1-T6SS, in contrast to the parent. However, study of H2-T6SS and H3-T6SS has been neglected because of a poor understanding of the associated regulatory network. Here we performed a screen to identify H2-T6SS and H3-T6SS regulatory elements and found that the posttranscriptional regulator RsmA imposes a concerted repression on all three T6SS clusters. A higher level of complexity could be observed as we identified a transcriptional regulator, AmrZ, which acts as a negative regulator of H2-T6SS. Overall, although the level of T6SS transcripts is fine-tuned by AmrZ, all T6SS mRNAs are silenced by RsmA. We expanded this concept of global control by RsmA to VgrG spike and T6SS toxin transcripts whose genes are scattered on the chromosome. These observations triggered the characterization of a suite of H2-T6SS toxins and their implication in direct bacterial competition. Our study thus unveils a central mechanism that modulates the deployment of all T6SS weapons that may be simultaneously produced within a single cell.

  • Journal article
    Glyde R, Ye F, Darbari V, Zhang N, Buck M, Zhang Xet al., 2017,

    Structures of RNA polymerase closed and intermediate complexes revealmechanisms of DNA opening and transcription initiation

    , Molecular Cell, Vol: 67, Pages: 106-116, ISSN: 1097-2765

    Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo.

  • Journal article
    Smith WD, Bardin E, Cameron L, Edmondson CL, Farrant KV, Martin I, Murphy RA, Soren O, Turnbull AR, Wierre-Gore N, Alton EW, Bundy JG, Bush A, Connett GJ, Faust SN, Filloux A, Freemont PS, Jones AL, Takats Z, Webb JS, Williams HD, Davies JCet al., 2017,

    Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis

    , FEMS Microbiology Letters, Vol: 364, ISSN: 0378-1097

    Pseudomonas aeruginosa opportunistically infects the airways of patients with cystic fibrosis and causes significant morbidity and mortality. Initial infection can often be eradicated though requires prompt detection and adequate treatment. Intermittent and then chronic infection occurs in the majority of patients. Better detection of P. aeruginosa infection using biomarkers may enable more successful eradication before chronic infection is established. In chronic infection P. aeruginosa adapts to avoid immune clearance and resist antibiotics via efflux pumps, β-lactamase expression, reduced porins and switching to a biofilm lifestyle. The optimal treatment strategies for P. aeruginosa infection are still being established, and new antibiotic formulations such as liposomal amikacin, fosfomycin in combination with tobramycin and inhaled levofloxacin are being explored. Novel agents such as the alginate oligosaccharide OligoG, cysteamine, bacteriophage, nitric oxide, garlic oil and gallium may be useful as anti-pseudomonal strategies, and immunotherapy to prevent infection may have a role in the future. New treatments that target the primary defect in cystic fibrosis, recently licensed for use, have been associated with a fall in P. aeruginosa infection prevalence. Understanding the mechanisms for this could add further strategies for treating P. aeruginosa in future.

  • Journal article
    Wigley DB, Willhoft O, McCormack EA, Aramayo R, Bythell-Douglas R, Ocloo L, Zhang Xet al., 2017,

    Cross-talk within a functional INO80 complex dimer regulates nucleosome sliding

    , eLife, Vol: 6, ISSN: 2050-084X

    Several chromatin remodellers have the ability to space nucleosomes on DNA. For ISWI remodellers, this involves an interplay between H4 histone tails, the AutoN and NegC motifs of the motor domains that together regulate ATPase activity and sense the length of DNA flanking the nucleosome. By contrast, the INO80 complex also spaces nucleosomes but is not regulated by H4 tails and lacks the AutoN and NegC motifs. Instead nucleosome sliding requires cooperativity between two INO80 complexes that monitor DNA length simultaneously on either side of the nucleosome during sliding. The C-terminal domain of the human Ino80 subunit (Ino80CTD) binds cooperatively to DNA and dimerisation of these domains provides crosstalk between complexes. ATPase activity, rather than being regulated, instead gradually becomes uncoupled as nucleosome sliding reaches an end point and this is controlled by the Ino80CTD. A single active ATPase motor within the dimer is sufficient for sliding.

  • Journal article
    Pallett MA, Crepin VF, Serafini N, Habibzay M, Kotik O, Sanchez-Garrido J, Di Santo J, Shenoy AR, Berger CN, Frankel GM, Pallett MA, Crepin VF, Serafini N, Habibzay M, Kotik O, Sanchez-Garrido J, Di Santo JP, Shenoy AR, Berger CN, Frankel Get al., 2017,

    Bacterial virulence factor inhibits caspase-4/11 activation in intestinal epithelial cells

    , Mucosal Immunology, Vol: 10, Pages: 602-612, ISSN: 1935-3456

    The human pathogen enteropathogenic Escherichia coli (EPEC), as well as the mouse pathogen Citrobacter rodentium, colonize the gut mucosa via attaching and effacing lesion formation and cause diarrheal diseases. EPEC and C. rodentium type III secretion system (T3SS) effectors repress innate immune responses and infiltration of immune cells. Inflammatory caspases such as caspase-1 and caspase-4/11 are crucial mediators of host defense and inflammation in the gut via their ability to process cytokines such as interleukin (IL)-1β and IL-18. Here we report that the effector NleF binds the catalytic domain of caspase-4 and inhibits its proteolytic activity. Following infection of intestinal epithelial cells (IECs) EPEC inhibited caspase-4 and IL-18 processing in an NleF-dependent manner. Depletion of caspase-4 in IECs prevented the secretion of mature IL-18 in response to infection with EPECΔnleF. NleF-dependent inhibition of caspase-11 in colons of mice prevented IL-18 secretion and neutrophil influx at early stages of C. rodentium infection. Neither wild-type C. rodentium nor C. rodentiumΔnleF triggered neutrophil infiltration or IL-18 secretion in Cas11 or Casp1/11-deficient mice. Thus, IECs have a key role in modulating early innate immune responses in the gut via a caspase-4/11—IL-18 axis, which is targeted by virulence factors encoded by enteric pathogens.

  • Journal article
    Yuan Z, Riera A, Bai L, Sun J, Nandi S, Spanos C, Chen ZA, Barbon M, Rappsilber J, Stillman B, Speck C, Li Het al., 2017,

    Structural basis of MCM2-7 replicative helicase loading by ORC-Cdc6 and Cdt1

    , Nature Structural & Molecular Biology, Vol: 24, Pages: 316-324, ISSN: 1545-9993

    To start DNA replication, the Origin Recognition Complex (ORC) and Cdc6 load a Mcm2-7 double hexamer onto DNA. Without ATP hydrolysis, ORC-Cdc6 recruits one Cdt1-bound Mcm2-7 hexamer, forming an ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) helicase loading intermediate. Here we report a 3.9Å structure of the OCCM on DNA. Flexible Mcm2-7 winged-helix domains (WHD) engage ORC-Cdc6. A three-domain Cdt1 configuration embraces Mcm2, Mcm4, and Mcm6, nearly half of the hexamer. The Cdt1 C-terminal domain extends to the Mcm6 WHD, which binds Orc4 WHD. DNA passes through the ORC-Cdc6 and Mcm2-7 rings. Origin DNA interaction is mediated by an a-helix in Orc4 and positively charged loops in Orc2 and Cdc6. The Mcm2-7 C-tier AAA+ ring is topologically closed by a Mcm5 loop that embraces Mcm2, but the N-tier ring Mcm2-Mcm5 interface remains open. This structure suggests loading mechanics of the first Cdt1-bound Mcm2-7 hexamer by ORC-Cdc6.

  • Journal article
    Nanev CN, Saridakis E, Chayen N, 2017,

    Protein crystal nucleation in pores

    , Scientific Reports, Vol: 7, ISSN: 2045-2322

    The most powerful method for protein structure determination is X-ray crystallography which relies on the availability of high quality crystals. Obtaining protein crystals is a major bottleneck, and inducing their nucleation is of crucial importance in this field. An effective method to form crystals is to introduce nucleation-inducing heterologous materials into the crystallization solution. Porous materials are exceptionally effective at inducing nucleation. It is shown here that a combined diffusion-adsorption effect can increase protein concentration inside pores, which enables crystal nucleation even under conditions where heterogeneous nucleation on flat surfaces is absent. Provided the pore is sufficiently narrow, protein molecules approach its walls and adsorb more frequently than they can escape. The decrease in the nucleation energy barrier is calculated, exhibiting its quantitative dependence on the confinement space and the energy of interaction with the pore walls. These results provide a detailed explanation of the effectiveness of porous materials for nucleation of protein crystals, and will be useful for optimal design of such materials.

  • Journal article
    Bernal P, Allsopp LP, Filloux AAM, Llamas MAet al., 2017,

    The Pseudomonas putida T6SS is a plant warden against phytopathogens

    , The ISME Journal, Vol: 11, Pages: 972-987, ISSN: 1751-7362

    Bacterial type VI secretion systems (T6SSs) are molecular weapons designed to deliver toxic effectors into prey cells. These nanomachines play an important role in inter-bacterial competition and provide advantages to T6SS active strains in polymicrobial environments. Here we analyse the genome of the biocontrol agent Pseudomonas putida KT2440 and identify three T6SS gene clusters (K1-, K2- and K3-T6SS). Besides, ten T6SS effector/immunity pairs were found, including putative nucleases and pore-forming colicins. We show that the K1-T6SS is a potent antibacterial device which secretes a toxic Rhs-type effector Tke2. Remarkably, P. putida eradicates a broad range of bacteria in a K1-T6SS-dependent manner, including resilient phytopathogens which demonstrates that the T6SS is instrumental to empower P. putida to fight against competitors. Furthermore, we observed a drastically reduced necrosis on the leaves of Nicotiana benthamiana during co-infection with P. putida and Xanthomonas campestris. Such protection is dependent on the activity of the P. putida T6SS. Many routes have been explored to develop biocontrol agents capable of manipulating the microbial composition of the rhizosphere and phyllosphere. Here we unveil a novel mechanism for plant biocontrol which needs to be considered for the selection of plant wardens whose mission is to prevent phytopathogen infections.

  • Journal article
    Ale A, Crepin VF, Collins, Constantinou N, Habibzay, Babtie AC, Frankel G, Stumpf MPet al., 2016,

    Model of host-pathogen Interaction dynamics links In vivo optical imaging and immune responses

    , Infection and Immunity, Vol: 85, ISSN: 1098-5522

    Tracking disease progression in vivo is essential for the development of treatments against bacterial infection. Optical imaging has become a central tool for in vivo tracking of bacterial population development and therapeutic response. For a precise understanding of in vivo imaging results in terms of disease mechanisms derived from detailed postmortem observations, however, a link between the two is needed. Here, we develop a model that provides that link for the investigation of Citrobacter rodentium infection, a mouse model for enteropathogenic Escherichia coli (EPEC). We connect in vivo disease progression of C57BL/6 mice infected with bioluminescent bacteria, imaged using optical tomography and X-ray computed tomography, to postmortem measurements of colonic immune cell infiltration. We use the model to explore changes to both the host immune response and the bacteria and to evaluate the response to antibiotic treatment. The developed model serves as a novel tool for the identification and development of new therapeutic interventions.

  • Journal article
    MacDonald J, Freemont PS, 2016,

    Computational protein design with backbone plasticity

    , Biochemical Society Transactions, Vol: 44, Pages: 1523-1529, ISSN: 1470-8752

    The computational algorithms used in the design of artificial proteins have become increasingly sophisticated in recent years, producing a series of remarkable successes. The most dramatic of these is the de novo design of artificial enzymes. The majority of these designs have reused naturally occurring protein structures as ‘scaffolds’ onto which novel functionality can be grafted without having to redesign the backbone structure. The incorporation of backbone flexibility into protein design is a much more computationally challenging problem due to the greatly increased search space, but promises to remove the limitations of reusing natural protein scaffolds. In this review, we outline the principles of computational protein design methods and discuss recent efforts to consider backbone plasticity in the design process.

  • Journal article
    MacDonald JT, Kabasakal BV, Godding D, Kraatz S, Henderson L, Barber J, Freemont PS, Murray JWet al., 2016,

    Synthetic beta-solenoid proteins with the fragment-free computational design of a beta-hairpin extension

    , Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: 10346-10351, ISSN: 1091-6490

    The ability to design and construct structures with atomic level precisionis one of the key goals of nanotechnology. Proteins offer anattractive target for atomic design, as they can be synthesized chemicallyor biologically, and can self-assemble. However the generalizedprotein folding and design problem is unsolved. One approach tosimplifying the problem is to use a repetitive protein as a scaffold.Repeat proteins are intrinsically modular, and their folding and structuresare better understood than large globular domains. Here, wehave developed a new class of synthetic repeat protein, based onthe pentapeptide repeat family of beta-solenoid proteins. We haveconstructed length variants of the basic scaffold, and computationallydesigned de novo loops projecting from the scaffold core. Theexperimentally solved 3.56 ˚A resolution crystal structure of one designedloop matches closely the designed hairpin structure, showingthe computational design of a backbone extension onto a syntheticprotein core without the use of backbone fragments from knownstructures. Two other loop designs were not clearly resolved in thecrystal structures and one loop appeared to be in an incorrect conformation.We have also shown that the repeat unit can accommodatewhole domain insertions by inserting a domain into one of the designedloops.

  • Journal article
    Furniss RCD, Slater S, Frankel G, Clements Aet al., 2016,

    Enterohaemorrhagic E. coli modulates an ARF6:Rab35 signalling axis to prevent recycling endosome maturation during infection

    , Journal of Molecular Biology, Vol: 428, Pages: 3399-3407, ISSN: 1089-8638

    Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC/EHEC) manipulate a plethora of host cell processes to establish infection of the gut mucosa. This manipulation is achieved via the injection of bacterial effector proteins into host cells using a Type III secretion system. We have previously reported that the conserved EHEC and EPEC effector EspG disrupts recycling endosome function, reducing cell surface levels of host receptors through accumulation of recycling cargo within the host cell. Here we report that EspG interacts specifically with the small GTPases ARF6 and Rab35 during infection. These interactions target EspG to endosomes and prevent Rab35-mediated recycling of cargo to the host cell surface. Furthermore, we show that EspG has no effect on Rab35-mediated uncoating of newly formed endosomes, and instead leads to the formation of enlarged EspG/TfR/Rab11 positive, EEA1/Clathrin negative stalled recycling structures. Thus, this paper provides a molecular framework to explain how EspG disrupts recycling whilst also reporting the first known simultaneous targeting of ARF6 and Rab35 by a bacterial pathogen.

  • Journal article
    Schuster C, Bellows L, Tosi T, Campeotto, Corrigan, Freemont P, Grundling Aet al., 2016,

    The second messenger c-di-AMP inhibits the osmolyte uptake system OpuC in Staphylococcus aureus

    , Science Signaling, Vol: 9, Pages: ra81-ra81, ISSN: 1945-0877

    Staphylococcus aureus is an important opportunistic human pathogen that is highly resistant to osmotic stresses. To survive an increase in osmolarity, bacteria immediately take up potassium ions and small organic compounds known as compatible solutes. The second messenger cyclic diadenosine monophosphate (c-di-AMP) reduces the ability of bacteria to withstand osmotic stress by binding to and inhibiting several proteins that promote potassium uptake. We identified OpuCA, the adenosine triphosphatase (ATPase) component of an uptake system for the compatible solute carnitine, as a c-di-AMP target protein in S. aureus and found that the LAC*ΔgdpP strain of S. aureus, which overproduces c-di-AMP, showed reduced carnitine uptake. The paired cystathionine-β-synthase (CBS) domains of OpuCA bound to c-di-AMP, and a crystal structure revealed a putative binding pocket for c-di-AMP in the cleft between the two CBS domains. Thus, c-di-AMP inhibits osmoprotection through multiple mechanisms.

  • Journal article
    Rasheed M, Garnett J, Perez-Dorado I, Muhl D, Filloux A, Matthews Set al., 2016,

    Crystal structure of the CupB6 adhesive tip from the chaperone-usher family of pili from Pseudomonas aeruginosa

    , Biochimica et Biophysica Acta - Protein Structure, Vol: 1864, Pages: 1500-1505, ISSN: 0005-2795

    Pseudomonas aeruginosa is a Gram-negative opportunistic bacterial pathogen that can cause chronicinfection of the lungs of cystic fibrosis patients. Chaperone-usher systems in P. aeruginosa are knownto translocate and assemble adhesive pili on the bacterial surface and contribute to biofilm formationwithin the host. Here, we report the crystal structure of the tip adhesion subunit CupB6 from thecupB1-6 gene cluster. The tip domain is connected to the pilus via the N-terminal donor strand fromthe main pilus subunit CupB1. Although the CupB6 adhesion domain bears structural features similarto other CU adhesins it displays an unusual polyproline helix adjacent to a prominent surface pocket,which are likely the site for receptor recognition.

  • Journal article
    Filloux A, Freemont P, 2016,

    Structural biology: baseplates in contractile machines

    , Nature Microbiology, Vol: 1, ISSN: 2058-5276

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