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Albrecht R.,Max Planck Institute For Entwicklungsbiologie | Schutz M.,Institute For Medizinische Mikrobiologie Und Hygiene | Oberhettinger P.,Institute For Medizinische Mikrobiologie Und Hygiene | Faulstich M.,University of Würzburg | And 4 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2014

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA β-barrel and P5 domain was determined at 3Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an 'open' conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous β-barrel with impaired β1-β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer. © 2014 International Union of Crystallography.


Littlewood K.J.,Mapi Consultancy | Higashi K.,Mapi Consultancy | Jansen J.P.,Mapi Consultancy | Capkun-Niggli G.,Novartis | And 4 more authors.
Journal of Cystic Fibrosis | Year: 2012

Background: Various inhaled antibiotics are currently used for treating chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients, however their relative efficacies are unclear. We compared the efficacy of the inhaled antibiotics tobramycin (TIP, TIS-T, TIS-B), colistimethate sodium (colistin) and aztreonam lysine for inhalation (AZLI) based on data from randomised controlled trials. Methods: In the base case, efficacies of antibiotics were compared using a network meta-analysis of seven trials including change from baseline in forced expiratory volume in 1second (FEV1) % predicted, P. aeruginosa sputum density and acute exacerbations. Results: The tobramycin preparations, AZLI and colistin, showed comparable improvements in efficacy in terms of FEV1% predicted at 4. weeks; the difference in % change from baseline (95%CrI) for TIP was compared to TIS-T (- 0.55, -3.5;2.4), TIS-B (-0.64, -7.1;5.7), AZLI (3.64, -1.0;8.3) and colistin (5.77, -1.2;12.8). Conclusion: We conclude that all studied antibiotics have comparable efficacies for the treatment of chronic P. aeruginosa lung infection in CF. © 2012 European Cystic Fibrosis Society..


PubMed | Max Planck Institute For Entwicklungsbiologie, University of Würzburg, Institute For Medizinische Mikrobiologie Und Hygiene and University of Konstanz
Type: Journal Article | Journal: Acta crystallographica. Section D, Biological crystallography | Year: 2014

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the -barrel assembly machinery (BAM) for OMP recognition, folding and assembly. In Escherichia coli this function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of the E. coli BamA -barrel and P5 domain was determined at 3 resolution. These data add information beyond that provided in the recently published crystal structures of BamA from Haemophilus ducreyi and Neisseria gonorrhoeae and are a valuable basis for the interpretation of pertinent functional studies. In an `open conformation, E. coli BamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer. E. coli BamA is characterized by a discontinuous -barrel with impaired 1-16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.


Oberhettinger P.,Institute For Medizinische Mikrobiologie Und Hygiene | Schutz M.,Institute For Medizinische Mikrobiologie Und Hygiene | Raddatz G.,Max Planck Institute For Biologische Kybernetik | Keller H.,Max Planck Institute For Entwicklungsbiologie | And 2 more authors.
Plasmid | Year: 2011

Our laboratory strain Yersinia enterocolitica strain WA-314 biogroup 1B serotype O:8 displayed a different adhesion behavior to host cells compared to other Yersinia strains. To investigate whether this is based on differences in the gene content of the large pYV virulence plasmid which contains the major Yersinia adhesin YadA, we set out to sequence pYV WA-314. pYV WA-314 is very similar to pYV127/90, with a notable difference in the length of the Type III secretion system component YscP, which determines the needle length of the system. We found that we could improve the annotation of proteins previously described as "hypothetical" in pYV127/90 and other pYV plasmids, and show that pYV plasmids contain several and seemingly redundant plasmid partitioning and stabilization systems, explaining why these plasmids are not easily lost in laboratory cultures of Yersinia strains. © 2010 Elsevier Inc.


Raifer H.,University of Marburg | Mahiny A.J.,University of Marburg | Bollig N.,University of Marburg | Petermann F.,TU Munich | And 13 more authors.
European Journal of Immunology | Year: 2012

Apart from conventional CD4+ Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-β, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4-/-) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4-/- γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Pawlik G.,Albert Ludwigs University of Freiburg | Kulajta C.,Albert Ludwigs University of Freiburg | Sachelaru I.,Albert Ludwigs University of Freiburg | Schroder S.,Albert Ludwigs University of Freiburg | And 4 more authors.
Journal of Bacteriology | Year: 2010

Cytochrome oxidases are perfect model substrates for analyzing the assembly of multisubunit complexes because the need for cofactor incorporation adds an additional level of complexity to their assembly. cbb3-type cytochrome c oxidases (cbb3-Cox) consist of the catalytic subunit CcoN, the membrane-bound c-type cytochrome subunits CcoO and CcoP, and the CcoQ subunit, which is required for cbb3-Cox stability. Biogenesis of cbb3-Cox proceeds via CcoQP and CcoNO subcomplexes, which assemble into the active cbb3-Cox. Most bacteria expressing cbb3-Cox also contain the ccoGHIS genes, which encode putative cbb3-Cox assembly factors. Their exact function, however, has remained unknown. Here we analyzed the role of CcoH in cbb3-Cox assembly and showed that CcoH is a single spanning-membrane protein with an N-terminus-out-C-terminus-in (N out-Cin) topology. In its absence, neither the fully assembled cbb3-Cox nor the CcoQP or CcoNO subcomplex was detectable. By chemical cross-linking, we demonstrated that CcoH binds primarily via its transmembrane domain to the CcoP subunit of cbb3-Cox. A second hydrophobic stretch, which is located at the C terminus of CcoH, appears not to be required for contacting CcoP, but deleting it prevents the formation of the active cbb3-Cox. This suggests that the second hydrophobic domain is required for merging the CcoNO and CcoPQ subcomplexes into the active cbb 3-Cox. Surprisingly, CcoH does not seem to interact only transiently with the cbb3-Cox but appears to stay tightly associated with the active, fully assembled complex. Thus, CcoH behaves more like a bona fide subunit of the cbb3-Cox than an assembly factor per se. Copyright © 2010, American Society for Microbiology. All Rights Reserved.


Albesa-Jove D.,Imperial College London | Bertrand T.,Imperial College London | Carpenter E.P.,Imperial College London | Swain G.V.,Imperial College London | And 11 more authors.
Journal of Molecular Biology | Year: 2010

Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of ~275 Å. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes. © 2010 Elsevier Ltd.


Nordmann P.,French Institute of Health and Medical Research | Picazo J.J.,Hospital Clinico San Carlos | Mutters R.,Institute For Medizinische Mikrobiologie Und Hygiene | Korten V.,Marmara University | And 5 more authors.
Journal of Antimicrobial Chemotherapy | Year: 2011

Objectives: Doripenem is a new carbapenem recently introduced into Europe. The COMParative Activity of Carbapenem Testing (COMPACT) study compared the susceptibility of common Gram-negative bacilli causing serious infections in hospitalized patients with doripenem, imipenem and meropenem. Methods: Gram-negative isolates (4498 total: 2171 Pseudomonas species; 1910 Enterobacteriaceae; and 417 other Gram-negative bacilli) were collected from 80 centres in 16 countries in Europe, the Middle East and Africa during 2008-09. The MICs of doripenem, imipenem and meropenem were determined using Etest methodology and broth microdilution. Susceptibility was interpreted according to CLSI, EUCAST and FDA breakpoints. Results: The MIC 90s of doripenem, imipenem and meropenem for all isolates were 8, ≥64 and 32 mg/L, respectively. Doripenem had the lowest MIC 90 for Pseudomonas species at 16 mg/L, with imipenem and meropenem values of ≥64 mg/L. Enterobacteriaceae were highly susceptible to all three carbapenems, with MIC 90s of doripenem, imipenem and meropenem of 0.06, 0.5 and 0.12 mg/L, respectively. Other Gram-negative isolates, predominantly Acinetobacter baumannii, were resistant to all three carbapenems (MIC 90 ≥64 mg/L). Susceptibility to doripenem was observed in 14.9% of isolates resistant to imipenem and/or meropenem. Conclusions: Doripenem showed excellent activity against Gram-negative isolates; generally it was more active than imipenem and at least as good as meropenem. Against Pseudomonas species, doripenem was more active than both imipenem and meropenem, with doripenem susceptibility observed for some imipenemand/or meropenem-resistant isolates. © The Author 2011. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.


Luck C.,Institute For Medizinische Mikrobiologie Und Hygiene
Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz | Year: 2011

Legionellae are environmental bacteria that can be frequently isolated from technical water supply systems. The most prevalent species is Legionella pneumophila, especially serogroup 1. In the environment, legionellae multiply in amoebae. Since Legionella pneumonias cannot be distinguished from pneumonias caused by other microbial pathogens, special microbiological tests, e.g., urinary antigen assays, are essential to detect Legionella infections. All water supply systems to which the patient is exposed during the incubation time of 2 to 10 days might be the source of the infection. This can be confirmed or excluded by molecular typing of isolates from patients and the environment. The most commonly used techniques are monoclonal antibody typing and sequence-based typing (SBT). Some sequence types (ST) are frequently found among clinical strains but are seldom isolated from the environment, e.g., ST 23, 42, 47, 62, and 146. It is safe to assume that such strains are highly virulent. Conversely, it does not seem to be justified to dedicate the same awareness to all environmental Legionella strains. © 2011 Springer Medizin Verlag.


PubMed | Institute For Medizinische Mikrobiologie Und Hygiene
Type: Journal Article | Journal: Plasmid | Year: 2011

Our laboratory strain Yersinia enterocolitica strain WA-314 biogroup 1B serotype O:8 displayed a different adhesion behavior to host cells compared to other Yersinia strains. To investigate whether this is based on differences in the gene content of the large pYV virulence plasmid which contains the major Yersinia adhesin YadA, we set out to sequence pYV(WA-314). pYV(WA-314) is very similar to pYV127/90, with a notable difference in the length of the Type III secretion system component YscP, which determines the needle length of the system. We found that we could improve the annotation of proteins previously described as hypothetical in pYV127/90 and other pYV plasmids, and show that pYV plasmids contain several and seemingly redundant plasmid partitioning and stabilization systems, explaining why these plasmids are not easily lost in laboratory cultures of Yersinia strains.

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