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Journal articleJennings LK, Storek KM, Ledvina HE, et al., 2015,
Pel is a cationic exopolysaccharide that cross-links extracellular DNA in the Pseudomonas aeruginosa biofilm matrix
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 112, Pages: 11353-11358, ISSN: 1091-6490Biofilm formation is a complex, ordered process. In the opportunistic pathogen Pseudomonas aeruginosa, Psl and Pel exopolysaccharides and extracellular DNA (eDNA) serve as structural components of the biofilm matrix. Despite intensive study, Pel’s chemical structure and spatial localization within mature biofilms remain unknown. Using specialized carbohydrate chemical analyses, we unexpectedly found that Pel is a positively charged exopolysaccharide composed of partially acetylated 1→4 glycosidic linkages of N-acetylgalactosamine and N-acetylglucosamine. Guided by the knowledge of Pel’s sugar composition, we developed a tool for the direct visualization of Pel in biofilms by combining Pel-specific Wisteria floribunda lectin staining with confocal microscopy. The results indicate that Pel cross-links eDNA in the biofilm stalk via ionic interactions. Our data demonstrate that the cationic charge of Pel is distinct from that of other known P. aeruginosa exopolysaccharides and is instrumental in its ability to interact with other key biofilm matrix components.
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Journal articleLuther PK, Burgoyne T, Morris E, 2015,
Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle
, Journal of Molecular Biology, Vol: 427, Pages: 3527-3537, ISSN: 1089-8638The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoiningsarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of anumber of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies.Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contractionand the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-sectionelectron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinalsections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles havewider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously beenused for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, wehave used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extractedand compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links,presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres.The reconstruction shows that the terminal 5–7 nm of the actin filaments within the Z-band is devoid of anyα-actinin links and is likely to be the location of capping protein CapZ.
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Journal articleSo EC, Mattheis C, Tate EW, et al., 2015,
Creating a customized intracellular niche: subversion of host cell signaling by Legionella type IV secretion system effectors
, Canadian Journal of Microbiology, Vol: 61, Pages: 617-635, ISSN: 1480-3275The Gram-negative facultative intracellular pathogen Legionella pneumophila infects a wide range of different protozoa in the environment and also human alveolar macrophages upon inhalation of contaminated aerosols. Inside its hosts, it creates a defined and unique compartment, termed the Legionella-containing vacuole (LCV), for survival and replication. To establish the LCV, L. pneumophila uses its Dot/Icm type IV secretion system (T4SS) to translocate more than 300 effector proteins into the host cell. Although it has become apparent in the past years that these effectors subvert a multitude of cellular processes and allow Legionella to take control of host cell vesicle trafficking, transcription, and translation, the exact function of the vast majority of effectors still remains unknown. This is partly due to high functional redundancy among the effectors, which renders conventional genetic approaches to elucidate their role ineffective. Here, we review the current knowledge about Legionella T4SS effectors, highlight open questions, and discuss new methods that promise to facilitate the characterization of T4SS effector functions in the future.
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Journal articleKopniczky M, moore S, freemont P, 2015,
Multilevel regulation and translational switches in synthetic biology
, IEEE Transactions on Biomedical Circuits and Systems, Vol: 9, Pages: 485-496, ISSN: 1940-9990In contrast to the versatility of regulatory mechanisms in natural systems, synthetic genetic circuits have been so far predominantly composed of transcriptionally regulated modules. This is about to change as the repertoire of foundational tools for post-transcriptional regulation is quickly expanding. We provide an overview of the different types of translational regulators: protein, small molecule and RNA responsive and we describe the new emerging circuit designs utilizing these tools. There are several advantages of achieving multilevel regulation via translational switches and it is likely that such designs will have the greatest and earliest impact in mammalian synthetic biology for regenerative medicine and gene therapy applications.
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Journal articleYang Y, Darbari VC, Zhang N, et al., 2015,
Structures of the RNA polymerase-sigma(54) reveal new and conserved regulatory strategies
, Science, Vol: 349, Pages: 882-885, ISSN: 0036-8075Transcription by RNA polymerase (RNAP) in bacteria requires specific promoter recognition by σ factors. The major variant σ factor (σ54) initially forms a transcriptionally silent complex requiring specialized adenosine triphosphate–dependent activators for initiation. Our crystal structure of the 450-kilodalton RNAP-σ54 holoenzyme at 3.8 angstroms reveals molecular details of σ54 and its interactions with RNAP. The structure explains how σ54 targets different regions in RNAP to exert its inhibitory function. Although σ54 and the major σ factor, σ70, have similar functional domains and contact similar regions of RNAP, unanticipated differences are observed in their domain arrangement and interactions with RNAP, explaining their distinct properties. Furthermore, we observe evolutionarily conserved regulatory hotspots in RNAPs that can be targeted by a diverse range of mechanisms to fine tune transcription.
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Journal articleHedberg SHM, Heng JYY, Williams DR, et al., 2015,
Self-interaction chromatography of mAbs: accurate measurement of dead volumes
, Pharmaceutical Research, Vol: 32, Pages: 3975-3985, ISSN: 1573-904XPurpose: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination.Method: SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements. Results: It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns. Conclusion: It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume.
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ReportCurry S, Leen EN, Sorgeloos F, et al., 2015,
A conserved interaction between a C-terminal motif in Norovirus VPg and the HEAT-1 domain of eIF4G is essential for translation initiation
, Publisher: bioRxivTranslation initiation is a critical early step in the replication cycle of the positive-sense, single-stranded RNA genome of noroviruses, a major cause of gastroenteritis in humans. Norovirus RNA, which has neither a 5 ́ m7G cap nor an internal ribosome entry site (IRES), adopts an unusual mechanism to initiate protein synthesis that relies on interactions between the VPg protein covalently attached to the 5 ́-end of the viral RNA and eukaryotic initiation factors (eIFs) in the host cell. For murine norovirus (MNV) we previously showed that VPg binds to the middle fragment of eIF4G (4GM; residues 652-1132). Here we have used pull-down assays, fluorescence anisotropy, and isothermal titration calorimetry (ITC) to demonstrate that a stretch of ~20 amino acids at the C terminus of MNV VPg mediates direct and specific binding to the HEAT-1 domain within the 4GM fragment of eIF4G. Our analysis further reveals that the MNV C-terminus binds to eIF4G HEAT-1 via a motif that is conserved in all known noroviruses. Fine mutagenic mapping suggests that the MNV VPg C terminus may interact with eIF4G in a helical conformation. NMR spectroscopy was used to define the VPg binding site on eIF4G HEAT-1, which was confirmed by mutagenesis and binding assays. We have found that this site is non-overlapping with the binding site for eIF4A on eIF4G HEAT-1 by demonstrating that norovirus VPg can form ternary VPg-eIF4G-eIF4A complexes. The functional significance of the VPg-eIF4G interaction was shown by the ability of fusion proteins containing the C- terminal peptide of MNV VPg to inhibit translation of norovirus RNA but not cap- or IRES-dependent translation. These observations define important structural details of a functional interaction between norovirus VPg and eIF4G and reveal a binding interface that might be exploited as a target for antiviral therapy.
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Journal articleRosenberg MF, Bikadi Z, Hazai E, et al., 2015,
Three-dimensional structure of the human breast cancer resistance protein (BCRP/ABCG2) in an inward-facing conformation
, Acta Crystallographica Section D - Biological Crystallography, Vol: 71, Pages: 1725-1735, ISSN: 0907-4449ABCG2 is an efflux drug transporter that plays an important role in drug resistance and drug disposition. In this study, the first three-dimensional structure of human full-length ABCG2 analysed by electron crystallography from two-dimensional crystals in the absence of nucleotides and transported substrates is reported at 2 nm resolution. In this state, ABCG2 forms a symmetric homodimer with a noncrystallographic twofold axis perpendicular to the two-dimensional crystal plane, as confirmed by subtomogram averaging. This configuration suggests an inward-facing configuration similar to murine ABCB1, with the nucleotide-binding domains (NBDs) widely separated from each other. In the three-dimensional map, densities representing the long cytoplasmic extensions from the transmembrane domains that connect the NBDs are clearly visible. The structural data have allowed the atomic model of ABCG2 to be refined, in which the two arms of the V-shaped ABCG2 homodimeric complex are in a more closed and narrower conformation. The structural data and the refined model of ABCG2 are compatible with the biochemical analysis of the previously published mutagenesis studies, providing novel insight into the structure and function of the transporter.Keywords: ABCG2; BCRP; ABC transporter; ATP-binding cassette transporter; cryo-electron microscopy; three-dimensional structure from two-dimensional crystals.
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Journal articleCampbell KLS, Lapidot T, Williams DR, 2015,
Foaming of CO2-loaded amine solvents degraded thermally under stripper conditions
, Industrial & Engineering Chemistry Research, Vol: 54, Pages: 7751-7755, ISSN: 1520-5045Foaming of amine solutions remains a problem for natural gas sweetening and post-combustion carbon capture. New amine-based solutions are being developed to replace monoethanolamine (MEA). This work tested the foaminess of MEA and three alternatives (methyldiethanolamine (MDEA), 1-(2-aminoethyl)piperazine (AEPZ), and 2-amino-2-methyl-1-propanol (AMP)) before and after thermal degradation; two methods were used to describe the foaminess. Foam was only formed after thermal degradation. The first method suggests foaminess, where AEPZ > MDEA > MEA; AMP, by contrast, did not conform to this model and formed a stable foam. The second method, using liquid physical properties, found that solutions that contained more degradation products (MEA, MDEA, AMP) showed different foaminess than those that did not (i.e., changing the chemistry during degradation strongly impacts the foaminess, which is observed). The foaming of these degraded samples demonstrates complexity that cannot be replicated by simple model solutions. Therefore, this study is more representative of the foaming behavior that is observed in industrial cases.
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Journal articleMoscoso JA, Schramke H, Zhang Y, et al., 2015,
Binding of cyclic Di-AMP to the staphylococcus aureus sensor kinase KdpD occurs via the universal stress protein domain and downregulates the expression of the Kdp potassium transporter
, Journal of Bacteriology, ISSN: 1098-5530Nucleotide signalling molecules are important intracellular messengers that regulate a wide range of biological functions. The human pathogen Staphylococcus aureus produces the signalling nucleotide cyclic di-adenosine monophosphate (c-di-AMP). This molecule is common among Gram-positive bacteria and in many organisms essential for survival under standard laboratory growth conditions. In this study, we investigated the interaction of c-di-AMP with the S. aureus KdpD protein. The sensor kinase KdpD forms a two-component signalling system with the response regulator KdpE and regulates the expression of the kdpDE genes and the kdpFABC operon coding for the Kdp potassium transporter components. Here, we show that the S. aureus KdpD protein binds c-di-AMP specifically and with an affinity in the micromolar range through its universal stress protein (USP) domain. This domain is located within the N-terminal cytoplasmic region of KdpD and amino acids of a conserved SxS-X20-FTAxY motif are important for this binding. We further show that KdpD2, a second KdpD protein found in some S. aureus strains, also binds c-di-AMP and our bioinformatics analysis indicates that a subclass of KdpD proteins in c-di-AMP-producing bacteria has evolved to bind this signalling nucleotide. Finally, we show that c-di-AMP binding to KdpD inhibits the up-regulation of the kdpFABC operon under salt stress, thus indicating that c-di-AMP is a negative regulator of potassium uptake in S. aureus. IMPORTANCE: Staphylococcus aureus is an important human pathogen and major cause of food poisoning in western countries. A common method for food preservation is the use of salt to drive dehydration. This study sheds light on the regulation of potassium uptake in Staphylococcus aureus, an important aspect of this bacterium's ability to tolerate high levels of salt. We show that the signalling nucleotide c-di-AMP binds to a regulatory component of the Kdp potassium uptake system and that this binding has an inh
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Journal articleBosi E, Fondi M, Maida I, et al., 2015,
Genome-scale phylogenetic and DNA composition analyses of Antarctic Pseudoalteromonas bacteria reveal inconsistencies in current taxonomic affiliation
, Hydrobiologia, Vol: 761, Pages: 85-95, ISSN: 0018-8158Bacteria belonging to the Pseudoalteromonas genus have important ecological implications in marine environments, playing a role in the control of microbial community as producers of bioactive molecules endowed with antifouling activity and able to antagonize larvae, fungi and bacteria, including important human pathogens. For these reasons, representatives of this genus are very promising for biotechnological and biomedical applications. In this work, we used different genome-scale approaches to infer the taxonomy of 38 Pseudoalteromonas representatives (most of which isolated from Antarctica) and whose complete genome has been sequenced. We show that an accurate re-evaluation of the real taxonomic relationships of Pseudoalteromonas representatives is needed since many inconsistencies with the current taxonomic annotation were observed. Moreover, data obtained with different genome-scale methods are consistent, confirming the reliability of the genomic approaches. On the basis of these data, we propose a re-annotation for some Pseudoalteromonas species. This proposal should be validated in the future by comparing the phenotypes of these strains.
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Journal articleNguyen LT, Gumbart JC, Beeby M, et al., 2015,
Coarse-grained simulations of bacterial cell wall growth reveal that local coordination alone can be sufficient to maintain rod shape
, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 112, Pages: E3689-E3698, ISSN: 0027-8424- Author Web Link
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- Citations: 33
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Journal articleByrne B, Kazarian SG, Boulet-Audet M, 2015,
Cleaning-in-place for immunoaffinity resin monitored by in situ ATR-FTIR spectroscopy
, Analytical and Bioanalytical Chemistry, Vol: 407, Pages: 7111-7122, ISSN: 1618-2650In the next ten years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier Transform Infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs.
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Journal articleMersch D, Lee C-Y, Zhang JZ, et al., 2015,
Wiring of Photosystem II to Hydrogenase for Photoelectrochemical Water Splitting
, JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, Vol: 137, Pages: 8541-8549, ISSN: 0002-7863- Author Web Link
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- Citations: 186
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Journal articleBae HE, Gotfryd K, Thomas J, et al., 2015,
Deoxycholate-Based Glycosides (DCGs) for Membrane Protein Stabilisation
, CHEMBIOCHEM, Vol: 16, Pages: 1454-1459, ISSN: 1439-4227- Author Web Link
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- Citations: 5
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Conference paperPolo LM, Grundy G, Rulten S, et al., 2015,
New structural insights into PARP3 function
, 40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Publisher: WILEY-BLACKWELL, Pages: 397-397, ISSN: 1742-464X -
Journal articleBouffartigues E, Moscoso JA, Duchesne R, et al., 2015,
The absence of the Pseudomonas aeruginosa OprF protein leads to increased biofilm formation through variation in c-di-GMP level
, Frontiers in Microbiology, Vol: 6, ISSN: 1664-302XOprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.
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Journal articleFrankel GM, Habibzay M, Crepin-Sevenou V, et al., 2015,
Tir-induced actin remodeling triggers expression of CXCL1 in enterocytes and neutrophil recruitment during Citrobacter rodentium infection
, Infection and Immunity, Vol: 83, Pages: 3342-3354, ISSN: 1098-5522The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated leading to recruitment of Nck, activation of N-WASP and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382-462 or 478-547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478-547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium expressing Tir_Y451A/Y471A recruited significantly less neutrophils to the colon and triggered less colonic hyperplasia on day 14 post infection, compared to infection with the wild type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.
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Journal articleMatthews SJ, Miliara X, Garnett J, et al., 2015,
Structural insight into the TRIAP1/PRELI-like domain family of mitochondrial phospholipid transfer complexes
, EMBO Reports, Vol: 16, Pages: 824-835, ISSN: 1469-221XThe composition of the mitochondrial membrane is important for its architecture and proper function. Mitochondria depend on a tightly regulated supply of phospholipid via intra-mitochondrial synthesis and by direct import from the endoplasmic reticulum. The Ups1/PRELI-like family together with its mitochondrial chaperones (TRIAP1/Mdm35) represent a unique heterodimeric lipid transfer system that is evolutionary conserved from yeast to man. Work presented here provides new atomic resolution insight into the function of a human member of this system. Crystal structures of free TRIAP1 and the TRIAP1–SLMO1 complex reveal how the PRELI domain is chaperoned during import into the intermembrane mitochondrial space. The structural resemblance of PRELI-like domain of SLMO1 with that of mammalian phoshatidylinositol transfer proteins (PITPs) suggest that they share similar lipid transfer mechanisms, in which access to a buried phospholipid-binding cavity is regulated by conformationally adaptable loops.
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Journal articleMaskell DP, Renault L, Serrao E, et al., 2015,
Structural basis for retroviral integration into nucleosomes
, Nature, Vol: 523, Pages: 366-369, ISSN: 0028-0836Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome1, 2. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome–nucleosome interface involving both gyres of nucleosomal DNA and one H2A–H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A–H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration.
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Journal articleScanu T, Spaapen RM, Bakker JM, et al., 2015,
<i>Salmonella</i> Manipulation of Host Signaling Pathways Provokes Cellular Transformation Associated with Gallbladder Carcinoma
, CELL HOST & MICROBE, Vol: 17, Pages: 763-774, ISSN: 1931-3128- Author Web Link
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- Citations: 151
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Journal articleHengge R, Gruendling A, Jenal U, et al., 2015,
Bacterial signal transduction by cyclic Di-GMP and other nucleotide second messengers
, Journal of Bacteriology, Vol: 198, Pages: 15-26, ISSN: 1098-5530The first International Symposium on c-Di-GMP Signaling in Bacteria (22 to 25 March 2015, Harnack-Haus, Berlin, Germany) brought together 131 molecular microbiologists from 17 countries to discuss recent progress in our knowledge of bacterial nucleotide second messenger signaling. While the focus was on signal input, synthesis, degradation, and the striking diversity of the modes of action of the current second messenger paradigm, i.e., cyclic di-GMP (c-di-GMP), “classics” like cAMP and (p)ppGpp were also presented, in novel facets, and more recent “newcomers,” such as c-di-AMP and c-AMP-GMP, made an impressive appearance. A number of clear trends emerged during the 30 talks, on the 71 posters, and in the lively discussions, including (i) c-di-GMP control of the activities of various ATPases and phosphorylation cascades, (ii) extensive cross talk between c-di-GMP and other nucleotide second messenger signaling pathways, and (iii) a stunning number of novel effectors for nucleotide second messengers that surprisingly include some long-known master regulators of developmental pathways. Overall, the conference made it amply clear that second messenger signaling is currently one of the most dynamic fields within molecular microbiology, with major impacts in research fields ranging from human health to microbial ecology.
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Conference paperBernal P, Civantos C, Filloux A, et al., 2015,
Type VI secretion in the plant growth promoting rhizobacteria Pseudomonas putida
, FEMS Microbiology Congress 2015BackgroundBacterial type VI secretion systems (T6SSs) are recently discovered nanomachines used to inject effectors into prokaryotic or eukaryotic cells. Therefore, T6SSs are involved in both inter-bacterial competition and bacterial pathogenesis.ObjectivesThe aim is the study of the T6SS of Pseudomonas putida a soil bacterium with the capacity to colonise the root of crop plants. The colonisation by this bacterium provides growth advantages to the plant and, importantly, protection against plant pathogens. This makes P. putida a relevant biocontrol agent. Since T6SS is mainly used by environmental bacteria for interbacterial competition, one might speculate that T6SSs play a relevant role in the biocontrol properties of P. putida. Methods• in silico analysis of P. putida KT2440 genome • Competition assays to determine H1-T6SS activity and for the identification of H1-T6SS targets.• Regulatory studies: qRT-PCR, transcriptional fusionsConclusionsThe in silico analysis has revealed the existence of three putative T6SSs (H1, H2, and H3). The clusters contain the genes encoding the conserved core components and some accessories, including regulatory proteins and toxins-immunity pairs. Additional T6SS-related genes are found scattered on the chromosome.By competition assays we have determined that H1-T6SS is active and that mutants in H1-T6SS structural components lack the ability to kill model prey strains. Moreover, the system can be used to kill serious phytopathogens such as Pseudomonas syringae in in vitro assays. Interestingly, the H1-T6SS is induced in stationary phase and controlled by the global regulators RetS and GacS-GacA, and by two alternative sigma factors, RpoS and RpoN.
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Journal articleMartzoukou O, Karachaliou M, Yalelis V, et al., 2015,
Oligomerization of the UapA purine transporter Is critical for ER-exit, plasma membrane localization and turnover
, Journal of Molecular Biology, Vol: 427, Pages: 2679-2696, ISSN: 1089-8638Central to the process of transmembrane cargo trafficking is the successful folding and exit from the ER (endoplasmic reticulum) through packaging in COPII vesicles. Here, we use the UapA purine transporter of Aspergillus nidulans to investigate the role of cargo oligomerization in membrane trafficking. We show that UapA oligomerizes (at least dimerizes) and that oligomerization persists upon UapA endocytosis and vacuolar sorting. Using a validated bimolecular fluorescence complementation assay, we provide evidence that a UapA oligomerization is associated with ER-exit and turnover, as ER-retained mutants due to either modification of a Tyr-based N-terminal motif or partial misfolding physically associate but do not associate properly. Co-expression of ER-retained mutants with wild-type UapA leads to in trans plasma membrane localization of the former, confirming that oligomerization initiates in the ER. Genetic suppression of an N-terminal mutation in the Tyr motif and mutational analysis suggest that transmembrane α-helix 7 affects the oligomerization interface. Our results reveal that transporter oligomerization is essential for membrane trafficking and turnover and is a common theme in fungi and mammalian cells.
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Journal articleSana TG, Baumann C, Merdes A, et al., 2015,
Internalization of Pseudomonas aeruginosa Strain PAO1 into Epithelial Cells Is Promoted by Interaction of a T6SS Effector with the Microtubule Network.
, mBio, Vol: 6, ISSN: 2150-7511Invasion of nonphagocytic cells through rearrangement of the actin cytoskeleton is a common immune evasion mechanism used by most intracellular bacteria. However, some pathogens modulate host microtubules as well by a still poorly understood mechanism. In this study, we aim at deciphering the mechanisms by which the opportunistic bacterial pathogen Pseudomonas aeruginosa invades nonphagocytic cells, although it is considered mainly an extracellular bacterium. Using confocal microscopy and immunofluorescence, we show that the evolved VgrG2b effector of P. aeruginosa strain PAO1 is delivered into epithelial cells by a type VI secretion system, called H2-T6SS, involving the VgrG2a component. An in vivo interactome of VgrG2b in host cells allows the identification of microtubule components, including the γ-tubulin ring complex (γTuRC), a multiprotein complex catalyzing microtubule nucleation, as the major host target of VgrG2b. This interaction promotes a microtubule-dependent internalization of the bacterium since colchicine and nocodazole, two microtubule-destabilizing drugs, prevent VgrG2b-mediated P. aeruginosa entry even if the invasion still requires actin. We further validate our findings by demonstrating that the type VI injection step can be bypassed by ectopic production of VgrG2b inside target cells prior to infection. Moreover, such uncoupling between VgrG2b injection and bacterial internalization also reveals that they constitute two independent steps. With VgrG2b, we provide the first example of a bacterial protein interacting with the γTuRC. Our study offers key insight into the mechanism of self-promoting invasion of P. aeruginosa into human cells via a directed and specific effector-host protein interaction. IMPORTANCE: Innate immunity and specifically professional phagocytic cells are key determinants in the ability of the host to control P. aeruginosa infection. However, among various virulence strategies, includi
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Journal articleGarnett JA, Diallo M, Matthews SJ, 2015,
Purification, crystallization and preliminary X-ray diffraction analysis of the Escherichia coli common pilus chaperone EcpB
, Acta Crystallographica Section F: Structural Biology Communications, Vol: 71, Pages: 676-679, ISSN: 2053-230XPili are key cell-surface components that allow the attachment of bacteria to both biological and abiotic solid surfaces, whilst also mediating interactions between themselves. In Escherichia coli, the common pilus (Ecp) belongs to an alternative chaperone-usher (CU) pathway that plays a major role in both early biofilm formation and host-cell adhesion. The chaperone EcpB is involved in the biogenesis of the filament, which is composed of EcpA and EcpD. Initial attempts at crystallizing EcpB using natively purified protein from the bacterial periplasm were not successful; however, after the isolation of EcpB under denaturing conditions and subsequent refolding, crystals were obtained at pH 8.0 using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.4 Å resolution. These crystals belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 62.65, c = 121.14 Å and one monomer in the asymmetric unit. Molecular replacement was unsuccessful, but selenomethionine-substituted protein and heavy-atom derivatives are being prepared for phasing. The three-dimensional structure of EcpB will provide invaluable information on the subtle mechanistic differences in biogenesis between the alternative and classical CU pathways. Furthermore, this is the first time that this refolding strategy has been used to purify CU chaperones, and it could be implemented in similar systems where it has not been possible to obtain highly ordered crystals.
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Journal articleChain B, Arnold J, Akthar S, et al., 2015,
A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody.
, PLOS One, Vol: 10, ISSN: 1932-6203The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.
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Journal articleMessens W, Bolton D, Frankel G, et al., 2015,
Defining pathogenic verocytotoxin-producing <i>Escherichia coli</i> (VTEC) from cases of human infection in the European Union, 2007-2010
, EPIDEMIOLOGY AND INFECTION, Vol: 143, Pages: 1652-1661, ISSN: 0950-2688- Author Web Link
- Cite
- Citations: 22
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Journal articleAxford D, Foadi J, Hu N-J, et al., 2015,
Structure determination of an integral membrane protein at room temperature from crystals <i>in situ</i>
, ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY, Vol: 71, Pages: 1228-1237, ISSN: 2059-7983- Author Web Link
- Cite
- Citations: 35
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Journal articleCheung PCW, Williams DR, 2015,
Separation of transition metals and chelated complexes in wastewaters
, Environmental Progress & Sustainable Energy, Vol: 34, Pages: 761-783, ISSN: 1944-7442This review responds to the ongoing needs of wastewater engineers tasked with treating aqueous solutions containing chelated complexes of metal ions, of which nickel citrate in electrodeless plating and copper–EDTA in electronic chip board manufacturing are two key examples. Because of the presence of these sequestering agents, metallic ions cannot be readily precipitated by alkalinity, making a compelling case for the discovery of alternative methods of treatment. This review is a critical appraisal of the varying degrees of success in separation process strategies deployed for the recovery of metallic ions under such challenging chemical conditions. Guidance is provided on making progress toward satisfactory industrial solutions to surmount these difficulties.
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