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  • 1.
    Aili, Margareta
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Isaksson, Elin L
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Carlsson, Sara E
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Regulation of Yersinia Yop-effector delivery by translocated YopE2008Inngår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 298, nr 3-4, 183-192 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The bacterial pathogen Yersinia pseudotuberculosis uses a type III secretion (T3S) system to translocate Yop effectors into eukaryotic cells. Effectors are thought to gain access to the cytosol via pores formed in the host cell plasma membrane. Translocated YopE can modulate this pore formation through its GTPase-activating protein (GAP) activity. In this study, we analysed the role of translocated YopE and all the other known Yop effectors in the regulation of effector translocation. Elevated levels of Yop effector translocation into HeLa cells occurred by YopE-defective strains, but not those defective for other Yop effectors. Only Yersinia devoid of YopK exhibits a similar hyper-translocation phenotype. Since both yopK and yopE mutants also failed to down-regulate Yop synthesis in the presence of eukaryotic cells, these data imply that translocated YopE specifically regulates subsequent effector translocation by Yersinia through at least one mechanism that involves YopK. We suggest that the GAP activity of YopE might be working as an intra-cellular probe measuring the amount of protein translocated by Yersinia during infection. This may be a general feature of T3S-associated GAP proteins, since two homologues from Pseudomonas aeruginosa, exoenzyme S (ExoS) and exoenzyme T (ExoT), can complement the hyper-translocation phenotypes of the yopE GAP mutant.

  • 2.
    Akopyan, Karen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Edgren, Tomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wang-Edgren, Helen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Rosqvist, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Translocation of surface-localized effectors in type III secretion2011Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, nr 4, 1639-1644 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Pathogenic Yersinia species suppress the host immune response by using a plasmid-encoded type III secretion system (T3SS) to translocate virulence proteins into the cytosol of the target cells. T3SS-dependent protein translocation is believed to occur in one step from the bacterial cytosol to the target-cell cytoplasm through a conduit created by the T3SS upon target cell contact. Here, we report that T3SS substrates on the surface of Yersinia pseudotuberculosis are translocated into target cells. Upon host cell contact, purified YopH coated on Y. pseudotuberculosis was specifically and rapidly translocated across the target-cell membrane, which led to a physiological response in the infected cell. In addition, translocation of externally added YopH required a functional T3SS and a specific translocation domain in the effector protein. Efficient, T3SS-dependent translocation of purified YopH added in vitro was also observed when using coated Salmonella typhimurium strains, which implies that T3SS-mediated translocation of extracellular effector proteins is conserved among T3SS-dependent pathogens. Our results demonstrate that polarized T3SS-dependent translocation of proteins can be achieved through an intermediate extracellular step that can be reconstituted in vitro. These results indicate that translocation can occur by a different mechanism from the assumed single-step conduit model.

  • 3. Aldick, Thomas
    et al.
    Bielaszewska, Martina
    Uhlin, Bernt Eric
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Humpf, Hans-Ulrich
    Wai, Sun Nyunt
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Teknat- och Medfak). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Karch, Helge
    Vesicular stabilization and activity augmentation of enterohaemorrhagic Escherichia coli haemolysin.2009Inngår i: Molecular microbiology, ISSN 1365-2958, Vol. 71, nr 6, 1496-508 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]
    Haemolysin from enterohaemorrhagic Escherichia coli (EHEC-Hly), a putative EHEC virulence factor, belongs to the RTX (repeat-in-toxin) family whose members rapidly inactivate themselves by self-aggregation. By investigating the status of EHEC-Hly secreted extracellularly, we found the toxin both in a free, soluble form and associated, with high tendency and independently of its acylation status, to outer membrane vesicles (OMVs) extruded by EHEC. We compared the interaction of both toxin forms with erythrocytes using scanning electron microscopy and binding assays. The OMV-associated toxin was substantially (80 times) more stable under physiological conditions than the free EHEC-Hly as demonstrated by prolonged haemolytic activity (half-life time 20 h versus 15 min). The haemolysis was preceded by calcium-dependent binding of OMVs carrying EHEC-Hly to erythrocytes; this binding was mediated by EHEC-Hly. We demonstrate that EHEC-Hly is a biologically active cargo in OMVs with dual roles: a cell-binding protein and a haemolysin. These paired functions produce a biologically potent form of the OMV-associated RTX toxin and augment its potential towards target cells. Our findings provide a general concept for stabilization of RTX toxins and open new insights into the biology of these important virulence factors.
  • 4.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Hermoso, Juan A.
    de Pedro, Miguel A.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Peptidoglycan Remodeling by the Coordinated Action of Multispecific Enzymes2014Inngår i: Microbial Drug Resistance, ISSN 1076-6294, E-ISSN 1931-8448, Vol. 20, nr 3, 190-198 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The peptidoglycan (PG) cell wall constitutes the main defense barrier of bacteria against environmental insults and acts as communication interface. The biochemistry of this macromolecule has been well characterized throughout the years but recent discoveries have unveiled its chemical plasticity under environmental stresses. Non-canonical D-amino acids (NCDAA) are produced and released to the extracellular media by diverse bacteria. Such molecules govern cell wall adaptation to challenging environments through their incorporation into the polymer, a widespread capability among bacteria that reveals the inherent catalytic plasticity of the enzymes involved in the cell wall metabolism. Here, we analyze the recent structural and biochemical characterization of Bsr, a new family of broad spectrum racemases able to generate a wide range of NCDAA. We also discuss the necessity of a coordinated action of PG multispecific enzymes to generate adequate levels of modification in the murein sacculus. Finally, we also highlight how this catalytic plasticity of NCDAA-incorporating enzymes has allowed the development of new revolutionary methodologies for the study of PG modes of growth and in vivo dynamics.

  • 5.
    Alvarez, Laura
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hernandez, Sara B
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ultra-Sensitive, High-Resolution Liquid Chromatography Methods for the High-Throughput Quantitative Analysis of Bacterial Cell Wall Chemistry and Structure2016Inngår i: Bacterial Cell Wall Homeostasis: Methods and Protocols / [ed] Hee-Jeon Hong, New York: Springer Science+Business Media B.V., 2016, Vol. 1440, 11-27 s.Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    High-performance liquid chromatography (HPLC) analysis has been critical for determining the structural and chemical complexity of the cell wall. However this method is very time consuming in terms of sample preparation and chromatographic separation. Here we describe (1) optimized methods for peptidoglycan isolation from both Gram-negative and Gram-positive bacteria that dramatically reduce the sample preparation time, and (2) the application of the fast and highly efficient ultra-performance liquid chromatography (UPLC) technology to muropeptide separation and quantification. The advances in both analytical instrumentation and stationary-phase chemistry have allowed for evolved protocols which cut run time from hours (2-3 h) to minutes (10-20 min), and sample demands by at least one order of magnitude. Furthermore, development of methods based on organic solvents permits in-line mass spectrometry (MS) of the UPLC-resolved muropeptides. Application of these technologies to high-throughput analysis will expedite the better understanding of the cell wall biology.

  • 6.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Farag, Salah
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Genetically engineered frameshifted YopN-TyeA chimeras influence type III secretion system function in Yersinia pseudotuberculosis2013Inngår i: PLoS ONE, ISSN 1932-6203, Vol. 8, nr 10, e77767- s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

  • 7.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gurung, Jyoti
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Costa, Tiago
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ruuth, Kristina
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Zavialov, Anton
    Joint Biotechnology Laboratory, Department of Chemistry, University of Turku, Turku, Finland.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis2016Inngår i: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, 66Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopNW279G mutant lost all ability to bind TyeA. The TyeA residue F8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

  • 8.
    Amer, Ayad
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Åhlund, Monika
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Bröms, Jeanette
    Department of Medical Countermeasures, Swedish Defense Research Agency, Division of NBC12 Defense, Umeå, Sweden.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Francis, Matthew
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Impact of the N-terminal secretor domain on YopD translocator function in Yersinia pseudotuberculosis type III secretion2011Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, nr 23, 6683-6700 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

  • 9.
    Andersson, Christopher
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Regulatory pathways and virulence inhibition in Listeria monocytogenes2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Listeria monocytogenes is a rod-shaped Gram positive bacterium. It generally exist ubiquitously in nature, where it lives as a saprophyte. Occasionally it however enters the food chain, from where it can be ingested by humans and cause gastro-intestinal distress. In immunocompetent individuals L. monocytogenes is generally cleared within a couple of weeks, but in immunocompromised patients it can progress to listeriosis, a potentially life-threatening infection in the central nervous system. If the infected individual is pregnant, the bacteria can cross the placental barrier and infect the fetus, possibly leading to spontaneous abortion.

    The infectivity of L. monocytogenes requires a certain set of genes, and the majority of them is dependent on the transcriptional regulator PrfA. The expression and activity of PrfA is controlled at several levels, and has traditionally been viewed to be active at 37 °C (virulence conditions) where it bind as a homodimer to a “PrfA-box” and induces the expression of the downstream gene.

    One of these genes is ActA, which enables intracellular movement by recruiting an actin polymerizing protein complex. When studying the effects of a blue light receptor we surprisingly found an effect of ActA at non-virulent conditions, where it is required for the bacteria to properly react to light exposure.

    To further study the PrfA regulon we tested deletion mutants of several PrfA-regulated virulence genes in chicken embryo infection studies. Based on these studies we could conclude that the chicken embryo model is a viable complement to traditional murine models, especially when investigating non-traditional internalin pathogenicity pathways. We have also studied the effects of small molecule virulence inhibitors that, by acting on PrfA, can inhibit L. monocytogenes infectivity in cell cultures with concentrations in the low micro-molar range.

  • 10.
    Andersson, Christopher
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Gripenland, Jonas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Using the chicken embryo to assess virulence of Listeria monocytogenes and to model other microbial infections2015Inngår i: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 10, nr 8, 1155-1164 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Microbial infections are a global health problem, particularly as microbes are continually developing resistance to antimicrobial treatments. An effective and reliable method for testing the virulence of different microbial pathogens is therefore a useful research tool. This protocol describes how the chicken embryo can be used as a trustworthy, inexpensive, ethically desirable and quickly accessible model to assess the virulence of the human bacterial pathogen Listeria monocytogenes, which can also be extended to other microbial pathogens. We provide a step-by-step protocol and figures and videos detailing the method, including egg handling, infection strategies, pathogenicity screening and isolation of infected organs. From the start of incubation of the fertilized eggs, the protocol takes <4 weeks to complete, with the infection part taking only 3 d. We discuss the appropriate controls to use and potential adjustments needed for adapting the protocol for other microbial pathogens.

  • 11.
    Andersson, Emma K.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bengtsson, Christoffer
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Evans, Margery L.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sellstedt, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Anders E.G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hufnagel, David A.
    Bhattacharya, Moumita
    Tessier, Peter M.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Chapman, Matthew R.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). University of Michigan, USA..
    Modulation of curli assembly and pellicle biofilm formation by chemical and protein chaperones.2013Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 20, nr 10, 1245-1254 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by “chemical chaperones.”

  • 12.
    Andersson, Emma K.
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bengtsson, Christoffer
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Evans, Margery L.
    Chorell, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sellstedt, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Lindgren, Anders E.G.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Hufnagel, David A.
    Bhattacharya, Moumita
    Tessier, PeterM.
    Wittung-Stafshede, Pernilla
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Chapman, Matthew R.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). University of Michigan, USA.
    Modulation of Curli Assembly and Pellicle Biofilm Formation by Chemical and Protein Chaperones2013Inngår i: Chemistry and Biology, ISSN 1074-5521, E-ISSN 1879-1301, Vol. 20, nr 10, 1245-1254 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Enteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by "chemical chaperones."

  • 13.
    Andersson, Emma K
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Mei, Ya-Fang
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Wadell, Göran
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Adenovirus interactions with CD46 on transgenic mouse erythrocytes2010Inngår i: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 402, nr 1, 20-25 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Hemagglutination is an established method but has not been used previously to determine the efficacy of virus binding to a specific cellular receptor. Here we have utilized CD46-expressing erythrocytes from a transgenic mouse to establish whether and to what extent the species B adenoviruses (Ads) as well as Ad37 and Ad49 of species D can interact with CD46. A number of different agglutination patterns, and hence CD46 interactions, could be observed for the different adenovirus types. In this system Ad7p, Ad11a, and Ad14 did not agglutinate mouse erythrocytes at all. Hemagglutination of CD46 expressing erythrocytes with high efficiency was observed for the previously established CD46 users Ad11p and Ad35 as well as for the less investigated Ad34. Ad50 agglutinated with moderate efficiency. Ad16, Ad21 and Ad49 gave incomplete agglutination. Ad16 was the only adenovirus that could be eluted. No specific CD46 interaction could be observed for Ad3p or for Ad37.

  • 14.
    Andersson, Hans
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Olsson, Roger
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Reactions between Grignard reagents and heterocyclic N-oxides: stereoselective synthesis of substituted pyridines, piperidines, and piperazines2011Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 9, 337-346 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    In this perspective we discuss the recent developments of stereoselective synthesis of substituted pyridines, piperidines, and piperazines from cheap and commercially readily available starting materials. Pyridine N-oxides and pyrazine N-oxides are reacted with alkyl, aryl, alkynyl and vinyl Grignard reagents to give a diverse set of heterocycles in high yields. Optically active substituted piperazines are obtained by an asymmetric reaction from pyrazine N-oxides using sparteine as chiral ligand. In addition, a stereoselective synthesis of dienal-oximes from the reaction between pyridine N-oxides and Grignard reagents is presented, which results in a useful intermediate for the synthesis of a diverse set of compounds.

  • 15.
    Andersson, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Björnham, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik.
    Bullitt, Esther
    Department of Physiology and Biophysics, Boston University School of Medicine, 700 Albany St., Boston MA, USA.
    Svantesson, Mats
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Differentiating pili expressed by enterotoxigenic and uropathogenic escherichia coli with optical tweezersManuskript (preprint) (Annet vitenskapelig)
    Abstract [en]

    Enterotoxigenic Escherichia coli (ETEC) attach to the host epithelium in the intestinal tract via specific adhesion organelles expressed on the cell membrane. We investigate, by force measuring optical tweezers, the intrinsic biomechanical properties and kinetics of the colonization factor I (CFA/I) at a single pilus level. The measurements indicate that CFA/I pili are helix-like structures that can both be unraveled to a linearized polymer by applying a small external force, 7.5 ± 1.5 pN but also regain its helix-like structure when the applied force is reduced. The data confirm that layer-to-layer interactions, that stabilize the helix-like structure, are much weaker than the interactions found in pili expressed by Uropathogenic Escherichia coli (UPEC). It is also found, contrary to previous results assessed from UPEC pili, that the CFA/I undergo in some cases a sudden structural change, a force drop of ~2 pN, when unraveled from the helix-like configuration to an open helical linearized fiber. These data suggest a rotation of the filament about its helical axis, followed by a region in which the force required to extend the pili further increases rapidly. During this final elongation to a super-extended fiber, CFA/I pili do not show any structural transition as seen for UPEC pili. In addition, the CFA/I pili show faster kinetics than UPEC pili that allows for a larger dynamic regime of in vivo shear forces. The unfolding and refolding possibility points toward an organelle that has evolved to allow for dynamic damping of external forces and handling of harsh motion without breaking.

  • 16.
    Antti, Henrik
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Näsström, Elin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kouremenos, Konstantinos
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sundén-Cullberg, Jonas
    Guo, Yongzhi
    Moritz, Thomas
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Metabolic profiling for detection of staphylococcus aureus infection and antibiotic resistance2013Inngår i: PLoS ONE, ISSN 1932-6203, Vol. 8, nr 2, e56971- s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Due to slow diagnostics, physicians must optimize antibiotic therapies based on clinical evaluation of patients without specific information on causative bacteria. We have investigated metabolomic analysis of blood for the detection of acute bacterial infection and early differentiation between ineffective and effective antibiotic treatment. A vital and timely therapeutic difficulty was thereby addressed: the ability to rapidly detect treatment failures because of antibiotic-resistant bacteria. Methicillin-resistant (MRSA) and methicillin-sensitive (MSSA) were used and for infecting mice, while natural MSSA infection was studied in humans. Samples of bacterial growth media, the blood of infected mice and of humans were analyzed with combined Gas Chromatography/Mass Spectrometry. Multivariate data analysis was used to reveal the metabolic profiles of infection and the responses to different antibiotic treatments. experiments resulted in the detection of 256 putative metabolites and mice infection experiments resulted in the detection of 474 putative metabolites. Importantly, ineffective and effective antibiotic treatments were differentiated already two hours after treatment start in both experimental systems. That is, the ineffective treatment of MRSA using cloxacillin and untreated controls produced one metabolic profile while all effective treatment combinations using cloxacillin or vancomycin for MSSA or MRSA produced another profile. For further evaluation of the concept, blood samples of humans admitted to intensive care with severe sepsis were analyzed. One hundred thirty-three putative metabolites differentiated severe MSSA sepsis (n = 6) from severe sepsis (n = 10) and identified treatment responses over time. Combined analysis of human, , and mice samples identified 25 metabolites indicative of effective treatment of sepsis. Taken together, this study provides a proof of concept of the utility of analyzing metabolite patterns in blood for early differentiation between ineffective and effective antibiotic treatment in acute infections.

  • 17.
    Avican, Kemal
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Persistent infection by Yersinia pseudotuberculosis2015Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Enteropathogenic Yersinia species can infect many mammalian organs such as the small intestine, cecum, Peyer’s patches, liver, spleen, and lung and cause diseases that resemble a typhoid-like syndrome, as seen for other enteropathogens. We found that sublethal infection doses of Y. pseudotuberculosis gave rise to asymptomatic persistent infection in mice and identified the cecal lymphoid follicles as the primary site for colonization during persistence. Persistent Y. pseudotuberculosis is localized in the dome area, often in inflammatory lesions, as foci or as single cells, and also in neutrophil exudates in the cecal lumen. This new mouse model for bacterial persistence in cecum has potential as an investigative tool for deeper understanding of bacterial adaptation and host immune defense mechanisms during persistent infection. Here, we investigated the nature of the persistent infection established by Y. pseudotuberculosis in mouse cecal tissue using in vivo RNA-seq of bacteria during early and persistent stages of infection. Comparative analysis of the bacterial transcriptomes revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence in the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26°C. Genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, we show that ArcA, Fnr, FrdA, WrbA, RovA, and RfaH play critical roles in persistence. An extended investigation of the transcriptional regulator rfaH employing mouse infection studies, phenotypic characterizations, and RNA-seq transcriptomics analyses indicated that this gene product contributes to establishment of infection and confirmed that it regulates O-antigen biosynthesis genes in Y. pseudotuberculosis. The RNA-seq results also suggest that rfaH has a relatively global effect. Furthermore, we also found that the dynamics of the cecal tissue organization and microbial composition shows changes during different stages of the infection. Taken together, based on our findings, we speculate that this enteropathogen initiates infection by using its virulence factors in meeting the innate immune response in the cecal tissue. Later on, these factors lead to dysbiosis in the local microbiota and altered tissue organization. At later stages of the infection, the pathogen adapts to the environment in the cecum by reprogramming its transcriptome from a highly virulent mode to a more environmentally adaptable mode for survival and shedding. The in vivo transcriptomic analyses for essential genes during infections present strong candidates for novel targets for antimicrobials.

  • 18.
    Avican, Kemal
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Huss, Mikael
    Heroven, Ann Kathrin
    Beckstette, Michael
    Dersch, Petra
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Reprogramming of Yersinia from Virulent to Persistent Mode Revealed by Complex In Vivo RNA-seq Analysis2015Inngår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 11, nr 1, e1004600Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.

  • 19.
    Avican, Ummehan
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Twin-arginine translocation in Yersinia: the substrates and their role in virulence2016Doktoravhandling, med artikler (Annet vitenskapelig)
    Abstract [en]

    Pathogenic Yersinia cause a manifold of diseases in humans ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to pneumonic and bubonic plague (Y. pestis), while all three have a common virulence strategy that relies on a well-studied type III secretion system and its effector proteins to colonize the host and evade immune responses. However, the role of other protein secretion and/or translocation systems in virulence of Yersinia species is not well known. In this thesis, we sought to investigate the contribution of twin-arginine translocation (Tat) pathway and its secreted substrates to the physiology and virulence of Y. pseudotuberculosis. Tat pathway uniquely exports folded proteins including virulence factors across the cytoplasmic membranes of bacteria. The proteins exported by Tat pathway contain a highly conserved twin-arginine motif in the N-terminal signal peptide. We found that the loss of Tat pathway causes a drastic change of the transcriptome of Y. pseudotuberculosis in stationary phase at environmental temperature with differential regulation of genes involved in virulence, carbon metabolism and stress responses. Phenotypic analysis revealed novel phenotypes of the Tat-deficient strain with defects in iron acquisition, acid resistance, copper oxidation and envelope integrity, which we were partly able to associate with the related Tat substrates. Moreover, increased glucose consumption and accumulation of intracellular fumarate were observed in response to inactivation of Tat pathway implicating a generic effect in cellular physiology. We evaluated the direct role of 22 in silico predicted Tat substrate mutants in the mouse infection model and found only one strain, ΔsufI, exhibited a similar degree of attenuation as Tat-deficient strain. Comparative in vivo characterization studies demonstrated a minor defect for ΔsufI in colonization of intestinal tissues compared to the Tat-deficient strain during early infection, whereas both SufI and TatC were required for dissemination from mesenteric lymph nodes and further systemic spread during late infection. This verifies that SufI has a major role in attenuation seen for the Tat deficient strain both during late infection and initial colonization. It is possible that other Tat substrates such as those involved in iron acquisition and copper resistance also has a role in establishing infection. Further phenotypic analysis indicated that SufI function is required for cell division and stress-survival. Transcriptomic analysis revealed that the highest number of differentially regulated genes in response to loss of Tat and SufI were involved in metabolism and transport. Taken together, this thesis presents a thorough analysis of the involvement of Tat pathway in the overall physiology and virulence strategies of Y. pseudotuberculosis. Finally, we propose that strong effects in virulence render TatC and SufI as potential targets for development of novel antimicrobial compounds

  • 20.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Avican, Kemal
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis of sufI and tatC mutants of Yersinia pseudotuberculosisManuskript (preprint) (Annet vitenskapelig)
  • 21.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, 2876-2886 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and Delta tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26 degrees C and 37 degrees C. The most significant changes in the transcriptome of the Delta tatC mutant were seen at 26 degrees C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCE In addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo. This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness.

  • 22.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Beckstette, Michael
    Heroven, Ann Kathrin
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Dersch, Petra
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Transcriptomic and phenotypic analysis reveals new functions for the Tat pathway in Yersinia pseudotuberculosis2016Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 198, nr 20, 2876-2886 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The Twin-arginine translocation (Tat) system mediates secretion of folded proteins that in bacteria, plants and archaea are identified via an N-terminal signal peptide. Tat systems are associated with virulence in many bacterial pathogens and our previous studies revealed that Tat deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analysed the global transcriptome of parental and ∆tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26oC and 37oC. The most significant changes in the transcriptome of the ∆tatC mutant were seen at 26oC during stationary phase growth and these included the altered expression of genes related to virulence, stress responses and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes including decreased YadA expression, impaired growth under iron-limiting and high copper conditions as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection.

  • 23.
    Avican, Ummehan
    et al.
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Doruk, Tugrul
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Östberg, Yngve
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    The Tat substrate SufI is critical for the ability of Yersinia pseudotuberculosis to cause systemic infection2017Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 85, nr 4, e00867-16Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important humanpathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a.sufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the.sufI and Delta tatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the Delta tatC mutant nor the Delta sufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the Delta tatC and Delta sufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the Delta tatC mutant.

  • 24.
    Axner, Ove
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Björnham, Oscar
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Castelain, Mickaël
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Klinth, Jeanna
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Koutris, Efstratios
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Schedin, Staffan
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för tillämpad fysik och elektronik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Assessing bacterial adhesion on an individual adhesin and single pili level using optical tweezers 2011Inngår i: Bacterial adhesion: chemistry, biology and physics / [ed] D. Line and A. Goldman, Berlin: Springer Berlin/Heidelberg, 2011, 301-313 s.Kapittel i bok, del av antologi (Fagfellevurdert)
    Abstract [en]

    Optical tweezers (OT) are a technique that, by focused laser light, can both manipulate micrometer sized objects and measure minute forces (in the pN range) in biological systems. The technique is therefore suitable for assessment of bacterial adhesion on an individual adhesin-receptor and single attachment organelle (pili) level. This chapter summarizes the use of OT for assessment of adhesion mechanisms of both non-piliated and piliated bacteria. The latter include the important helix-like pili expressed by uropathogenic Escherichia coli (UPEC), which have shown to have unique and intricate biomechanical properties. It is conjectured that the large flexibility of this type of pili allows for a redistribution of an external shear force among several pili, thereby extending the adhesion lifetime of bacteria. Systems with helix-like adhesion organelles may therefore act as dynamic biomechanical machineries, enhancing the ability of bacteria to withstand high shear forces originating from rinsing flows such as in the urinary tract. This implies that pili constitute an important virulence factor and a possible target for future anti-microbial drugs.

  • 25. Balonova, Lucie
    et al.
    Mann, Benjamin F
    Cerveny, Lukas
    Alley, William R, Jr
    Chovancova, Eva
    Forslund, Anna-Lena
    Salomonsson, Emelie N
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Damborsky, Jiri
    Novotny, Milos V
    Hernychova, Lenka
    Stulik, Jiri
    Characterization of protein glycosylation in Francisella tularensis subsp holarctica2012Inngår i: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 11, nr 7Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015016, 1-12, 2012.

  • 26.
    Bamyaci, Sarp
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ekestubbe, Sofie
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Nordfelth, Roland
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ertmann, Saskia
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    YopN is required for efficient translocation and virulence in Yersinia pseudotuberculosisManuskript (preprint) (Annet vitenskapelig)
  • 27. Berggren, Kristina
    et al.
    Vindebro, Reine
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bergström, Claes
    Spoerry, Christian
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Persson, Helena
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Fex, Tomas
    Kihlberg, Jan
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Luthman, Kristina
    3-aminopiperidine-based peptide analogues as the first selective noncovalent inhibitors of the bacterial cysteine protease IdeS2012Inngår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 55, nr 6, 2549-2560 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A series of eight peptides corresponding to the amino acid sequence of the hinge region of IgG and 17 newly synthesized peptide analogues containing a piperidine moiety as a replacement of a glycine residue were tested as potential inhibitors of the bacterial IgG degrading enzyme of Streptococcus pyogenes, IdeS. None of the peptides showed any inhibitory activity of IdeS, but several piperidine-based analogues were identified as inhibitors. Two different analysis methods were used: an SDS-PAGE based assay to detect IgG cleavage products and a surface plasmon resonance spectroscopy based assay to quantify the degree of inhibition. To investigate the selectivity of the inhibitors for IdeS, all compounds were screened against two other related cysteine proteases (SpeB and papain). The selectivity results show that larger analogues that are active inhibitors of IdeS are even more potent as inhibitors of papain, whereas smaller analogues that are active inhibitors of IdeS inhibit neither SpeB nor papain. Two compounds were identified that exhibit high selectivity against IdeS and will be used for further studies.

  • 28.
    Bergström, Sven
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Zückert, Wolfram R
    Structure, function and biogenesis of the Borrelia cell envelope2010Inngår i: Borrelia, molecular biology, host interactions and pathogenesis / [ed] Eds DS Samuels and JD Radolf, Norfolk, UK: Caister Academic Press , 2010, 139-166 s.Kapittel i bok, del av antologi (Annet vitenskapelig)
  • 29.
    Bernardo-Garcia, Noelia
    et al.
    Department of Crystallography and Structural Biology, Instituto de Química Física "Rocasolano", CSIC, Madrid, Spain.
    Sanchez-Murcia, Pedro
    Univ Alcala De Henares, Area Farmacol, Dept Ciencias Biomed, Unidad Asociada I D I,CSIC, Madrid, Spain.
    Gago, Federico
    Univ Alcala De Henares, Area Farmacol, Dept Ciencias Biomed, Unidad Asociada I D I,CSIC, Madrid, Spain.
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Hermoso, Juan A.
    CSIC, Inst Quim Fis Rocasolano, Dept Crystallog & Struct Chem, Madrid, Spain.
    Structural Bioinformatics in Broad-Spectrum Racemases: a new path in anti-microbial research2016Inngår i: Current organic chemistry, ISSN 1385-2728, E-ISSN 1875-5348, Vol. 20, nr 11, 1222-1231 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    D-amino acids are essential components of the bacterial cell wall and play notable roles in microbiology as regulators, for example in sporulation, biofilm formation or interspecies communication. Racemases are the specific enzymes catalyzing the interconversion of L-amino acids to D-amino acids. While most of racemases are mono-specific, a family of broad-spectrum racemases that can racemize ten of the 19 natural chiral amino acids has been recently reported. These enzymes can interconvert radically different residues such as aliphatic and positively charged residues producing non-canonical D-amino acids. Crystal structures together with bioinformatics allowed identification of the residues defining the molecular footprint in broad-spectrum racemases, the specific features of their active sites and the structural basis of their promiscuity. Here we review the recent knowledge on this family compared with the well established of alanine racemases. This structural information is a prerequisite for the development of novel drugs against the important human pathogens for which broad-spectrum racemases play a key role.

  • 30. Bielig, H
    et al.
    Rompikuntal, Pramod Kumar
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Mitesh, Dongre
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Zurek, B
    Lindmark, B
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Ramstedt, Madeleine
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kufer, T A
    University of Cologne.
    NOD-like receptor activation by outer-membrane vesicles (OMVs) from non-O1 non-O139 Vibrio cholerae is modulated by the quorum sensing regulator HapR2011Inngår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 79, nr 4, 1418-1427 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. siRNA-mediated gene knock-down revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain, leucine rich repeat containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum sensing regulator HapR which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs in infective conditions. This work thus identified a new facet of how Vibrio affects host immune responses and defines a role for the quorum sensing machinery in this process.

  • 31.
    Björnfot, Ann-Catrin
    et al.
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Lavander, Moa
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Infektionssjukdomar.
    Forsberg, Åke
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wolf-Watz, Hans
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Autoproteolysis of YscU of Yersinia pseudotuberculosis is important for regulation of expression and secretion of Yop proteins2009Inngår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 13, 4259-4267 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    YscU of Yersinia can be autoproteolysed to generate a 10-kDa C-terminal polypeptide designated YscU(CC). Autoproteolysis occurs at the conserved N downward arrowPTH motif of YscU. The specific in-cis-generated point mutants N263A and P264A were found to be defective in proteolysis. Both mutants expressed and secreted Yop proteins (Yops) in calcium-containing medium (+Ca(2+) conditions) and calcium-depleted medium (-Ca(2+) conditions). The level of Yop and LcrV secretion by the N263A mutant was about 20% that of the wild-type strain, but there was no significant difference in the ratio of the different secreted Yops, including LcrV. The N263A mutant secreted LcrQ regardless of the calcium concentration in the medium, corroborating the observation that Yops were expressed and secreted in Ca(2+)-containing medium by the mutant. YscF, the type III secretion system (T3SS) needle protein, was secreted at elevated levels by the mutant compared to the wild type when bacteria were grown under +Ca(2+) conditions. YscF secretion was induced in the mutant, as well as in the wild type, when the bacteria were incubated under -Ca(2+) conditions, although the mutant secreted smaller amounts of YscF. The N263A mutant was cytotoxic for HeLa cells, demonstrating that the T3SS-mediated delivery of effectors was functional. We suggest that YscU blocks Yop release and that autoproteolysis is required to relieve this block.

  • 32.
    Björnham, Oscar
    et al.
    Swedish Defence Research Agency (FOI), Umeå, Sweden.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Theory for nonlinear dynamic force spectroscopy2017Inngår i: European Biophysics Journal, ISSN 0175-7571, E-ISSN 1432-1017, Vol. 46, nr 3, 225-233 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Dynamic force spectroscopy (DFS) is an experimental technique that is commonly used to assess information on the strength, energy landscape, and lifetime of noncovalent bio-molecular interactions. DFS traditionally requires an applied force that increases linearly with time so that the bio-complex under investigation is exposed to a constant loading rate. However, tethers or polymers can modulate the applied force in a nonlinear manner. For example, bacterial adhesion pili and polymers with worm-like chain properties are structures that show nonlinear force responses. In these situations, the theory for traditional DFS cannot be readily applied. In this work, we expand the theory for DFS to also include nonlinear external forces while still maintaining compatibility with the linear DFS theory. To validate the theory, we modeled a bio- complex expressed on a stiff, an elastic, and a worm-like chain polymer, using Monte Carlo methods, and assessed the corresponding rupture force spectra. It was found that the nonlinear DFS (NLDFS) theory correctly predicted the numerical results. We also present a protocol suggesting an experimental approach and analysis method of the data to estimate the bond length and the thermal off-rate.

  • 33.
    Boal, Frédéric
    et al.
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Puhar, Andrea
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Xuereb, Jean-Marie
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Kunduzova, Oksana
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    Sansonetti, Philippe J.
    INSERM U1202, Unite´ de Pathogénie Microbienne Moléculaire, Institut Pasteur, 75724 Paris Cedex 15, France.
    Payrastre, Bernard
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France; .
    Tronchére, Héléne
    INSERM U1048, I2MC and Universite´ Paul Sabatier, 31432 Toulouse, France.
    PI5P Triggers ICAM-1 Degradation in Shigella Infected Cells, Thus Dampening Immune Cell Recruitment2016Inngår i: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, nr 4, 750-759 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Shigella flexneri, the pathogen responsible for bacillary dysentery, has evolved multiple strategies to control the inflammatory response. Here, we show that Shigella subverts the subcellular trafficking of the intercellular adhesion molecule-1 (ICAM-1), a key molecule in immune cell recruitment, in a mechanism dependent on the injected bacterial enzyme IpgD and its product, the lipid mediator PI5P. Overexpression of IpgD, but not a phosphatase dead mutant, induced the internalization and the degradation of ICAM-1 in intestinal epithelial cells. Remarkably, addition of permeant PI5P reproduced IpgD effects and led to the inhibition of neutrophil recruitment. Finally, these results were confirmed in an in vivo model of Shigella infection where IpgD-dependent ICAM-1 internalization reduced neutrophil adhesion. In conclusion, we describe here an immune evasion mechanism used by the pathogen Shigella to divert the host cell trafficking machinery in order to reduce immune cell recruitment.

  • 34.
    Bonde, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Olofsson, Annelie
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Frost, Mikaela
    Jegerschöld, Caroline
    Karolinska Institutet, Sweden.
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structural analysis of the B. burgdorferi integral outer membrane protein, P13, in lipid bilayer NanodiscsManuskript (preprint) (Annet (populærvitenskap, debatt, mm))
  • 35.
    Bonde, Mari
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Östberg, Yngve
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Bunikis, Ignas
    Uppsala University.
    Nyunt Wai, Sun
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Effects of osmotic stress in P13 and P66 deficient Borrelia burgdorferi mutantsManuskript (preprint) (Annet (populærvitenskap, debatt, mm))
  • 36. Bosley, Katrine S
    et al.
    Botchan, Michael
    Bredenoord, Annelien L
    Carroll, Dana
    Charo, R Alta
    Charpentier, Emmanuelle
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Cohen, Ron
    Corn, Jacob
    Doudna, Jennifer
    Feng, Guoping
    Greely, Henry T
    Isasi, Rosario
    Ji, Weihzi
    Kim, Jin-Soo
    Knoppers, Bartha
    Lanphier, Edward
    Li, Jinsong
    Lovell-Badge, Robin
    Martin, G Steven
    Moreno, Jonathan
    Naldini, Luigi
    Pera, Martin
    Perry, Anthony C F
    Venter, J Craig
    Zhang, Feng
    Zhou, Qi
    CRISPR germline engineering--the community speaks.2015Inngår i: Nature Biotechnology, ISSN 1087-0156, E-ISSN 1546-1696, Vol. 33, nr 5, 478-486 s.Artikkel i tidsskrift (Fagfellevurdert)
  • 37.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, Ake
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Tetratricopeptide repeats are essential for PcrH chaperone function in Pseudomonas aeruginosa type III secretion.2006Inngår i: FEMS Microbiol Lett, ISSN 0378-1097, Vol. 256, nr 1, 57-66 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The type III secretion system (T3SS) is a specialized apparatus evolved by Gram-negative bacteria to deliver effector proteins into host cells, thus facilitating the establishment of an infection. Effector translocation across the target cell plasma membrane is believed to occur via pores formed by at least two secreted translocator proteins, the functions of which are dependent upon customized class II T3SS chaperones. Recently, three internal tetratricopeptide repeats (TPRs) were identified in this class of chaperones. Here, defined mutagenesis of the class II chaperone PcrH of Pseudomonas aeruginosa revealed these TPRs to be essential for chaperone activity towards the translocator proteins PopB and PopD and subsequently for the translocation of exoenzymes into host cells.

  • 38.
    Bröms, Jeanette E
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi.
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Forsberg, Ake
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Diminished LcrV secretion attenuates Yersinia pseudotuberculosis virulence.2007Inngår i: J Bacteriol, ISSN 0021-9193Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Many Gram negative bacterial pathogenicity factors that function beyond the outer membrane are secreted via a contact-dependent type III secretion system. Two types of substrates are predestined for this mode of secretion; anti-host effectors that are translocated directly into target cells and the translocators required for targeting of the effectors across the host cell membrane. N-terminal secretion signals are important for recognition of the protein cargo by the type III secretion machinery. Even though such signals are known for several effectors, a consensus signal sequence is not obvious. One of the tranclocators, LcrV, has been attributed other functions in addition to its role in translocation. These functions include regulation, presumably via interaction with LcrG inside the bacteria and immunomodulation via interaction with TLR2. Here we wanted to address the significance of the specific targeting of LcrV to the exterior for its function in regulation, effector targeting and virulence. The results, highlighting key N-terminal amino acids important for LcrV secretion, allowed us to dissect the role of LcrV in regulation from that in effector targeting/virulence. While only low levels of exported LcrV were required for in vitro effector translocation as deduced by a cell infection assay, fully functional export of LcrV was found to be a prerequisite for its role in virulence in the systemic murine infection model.

  • 39. Bui, Hue T B
    et al.
    Vo, Duy D
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Chau, Yen N T
    Tu, Cuc T K
    Mai, Hieu V
    Truong, Kiet V
    Facile Synthesis of 4-Oxo-4H-quinolizine-2-carboxamide Derivatives2015Inngår i: Synthetic Communications, ISSN 0039-7911, E-ISSN 1532-2432, Vol. 45, nr 24, 2861-2868 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A facile synthetic method for the construction of 2-substituted-4-oxo-4H-quinolizine-based core structure has been successfully developed. The synthesis made use of a one-pot Stobbe condensation followed by cyclization starting from the commercially available 2-pyridinecarbaldehyde. The structure of the formed 4-oxo-4H-quinolizine-2-carboxylate was fully confirmed by mass spectra, H-1 NMR and C-13 NMR, correlation spectrography, heteronuclear multiple bond correlation, and heteronuclear single quantum coherence (HSQC) spectra. The ethyl carboxylate moiety was then further functionalized via direct aminolysis by a range of amines to afford the corresponding 4-oxo-4H-quinolizine-2-carboxamides 4a-i in moderate to good yields.

  • 40. Bui, Hue Thi Buu
    et al.
    Ha, Quy Thi Kim
    Oh, Won Keun
    Vo, Duy Duc
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Chau, Yen Nguyen Tram
    Tu, Cuc Thi Kim
    Pham, Em Canh
    Tran, Phuong Thao
    Tran, Loan Thi
    Mai, Hieu Van
    Microwave assisted synthesis and cytotoxic activity evaluations of new benzimidazole derivatives2016Inngår i: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 57, nr 8, 887-891 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Twelve new 2-quinolizinylbenzimidazole and 2-naphthalylbenzimidazole derivatives with various 5- and 6-positioned substituents (aza, H, CH3, Cl, NO2, NH2, OCH3), have been synthesized in moderate to excellent yields via the condensation of 4-oxo-4H-quinolizinecarbaldehyde or naphthalenecarbaldehyde with substituted o-phenylenediamines, o-nitroaniline, and 2,3-pyridinediamine using sodium metabisulfite or sodium hydrosulfite under microwave irradiation. The new benzimidazole derivatives were screened for their cytotoxic activity against the human breast cancer cell line (MCF-7). The results showed on one hand that 2-(substituted quinolizinyl)-1H-benzimidazoles (12bf) were less active (3–6 fold) than the positive control Tamoxifen (CC50 = 6.52 μM), and on the other hand, among the 2-(substituted naphthalyl)-1H-benzimidazoles series (13af), compounds 6,7,8-trimethoxy-3-(5-chloro-1H-benzo[d]imidazol-2-yl)naphthalen-1-ol (13c) (CC50 = 7.48 μM) and 6,7,8-trimethoxy-3-(5-methoxy-1H-benzo[d]imidazol-2-yl)naphthalen-1-ol (13f) (CC50 = 6.43 μM) were found to be as active as Tamoxifen.

  • 41.
    Bäreclev, Caroline
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Vaitkevicius, Karolis
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Netterling, Sakura
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Johansson, Jörgen
    DExD-box RNA-helicases in Listeria monocytogenes are important for growth, ribosomal maturation, rRNA processing and virulence factor expression2014Inngår i: RNA Biology, ISSN 1547-6286, E-ISSN 1555-8584, Vol. 11, nr 11, 1458-1467 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    RNA-helicases are proteins required for the unwinding of occluding secondary RNA structures, especially at low temperatures. In this work, we have deleted all 4 DExD-box RNA helicases in various combinations in the Gram-positive pathogen Listeria monocytogenes. Our results show that 3 out of 4 RNA-helicases were important for growth at low temperatures, whereas the effect was less prominent at 37 degrees C. Over-expression of one RNA-helicase, Lmo1450, was able to overcome the reduced growth of the quadruple mutant strain at temperatures above 26 degrees C, but not at lower temperatures. The maturation of ribosomes was affected in different degrees in the various strains at 20 degrees C, whereas the effect was marginal at 37 degrees C. This was accompanied by an increased level of immature 23S rRNA precursors in some of the RNA-helicase mutants at low temperatures. Although the expression of the PrfA regulated virulence factors ActA and LLO decreased in the quadruple mutant strain, this strain showed a slightly increased infection ability. Interestingly, even though the level of the virulence factor LLO was decreased in the quadruple mutant strain as compared with the wild-type strain, the hly-transcript (encoding LLO) was increased. Hence, our results could suggest a role for the RNA-helicases during translation. In this work, we show that DExD-box RNA-helicases are involved in bacterial virulence gene-expression and infection of eukaryotic cells.

  • 42.
    Caraballo, Remi
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Larsson, Mikael
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Fysiologisk kemi.
    Nilsson, Stefan K.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Fysiologisk kemi.
    Ericsson, Madelene
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Fysiologisk kemi.
    Qian, Weixing
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tran, Nam Phuong Nguyen
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Kindahl, Tomas
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Svensson, Richard
    Uppsala, Sweden.
    Saar, Valeria
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Fysiologisk kemi.
    Artursson, Per
    Uppsala, Sweden.
    Olivecrona, Gunilla
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Fysiologisk kemi.
    Enquist, Per-Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Structure-activity relationships for lipoprotein lipase agonists that lower plasma triglycerides in vivo2015Inngår i: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 103, 191-209 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The risk of cardiovascular events increases in individuals with elevated plasma triglyceride (TG) levels, therefore advocating the need for efficient TG-lowering drugs. In the blood circulation, TG levels are regulated by lipoprotein lipase (LPL), an unstable enzyme that is only active as a non-covalently associated homodimer. We recently reported on a N-phenylphthalimide derivative (1) that stabilizes LPL in vitro, and moderately lowers triglycerides in vivo (Biochem. Biophys. Res. Common. 2014, 450, 1063). Herein, we establish structure activity relationships of 51 N-phenylphthalimide analogs of the screening hit 1. In vitro evaluation highlighted that modifications on the phthalimide moiety were not tolerated and that lipophilic substituents on the central phenyl ring were functionally essential. The substitution pattern on the central phenyl ring also proved important to stabilize LPL However, in vitro testing demonstrated rapid degradation of the phthalimide fragment in plasma which was addressed by replacing the phthalimide scaffold with other heterocyclic fragments. The in vitro potency was retained or improved and substance 80 proved stable in plasma and efficiently lowered plasma TGs in vivo. 2015 The Authors. Published by Elsevier Masson SAS.

  • 43.
    Caraballo, Rémi
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Saleeb, Michael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bauer, Johannes
    Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
    Liaci, Antonio-Manuel
    Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
    Chandra, Naresh
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Storm, Rickard J
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Frängsmyr, Lars
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Qian, Weixing
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Stehle, Thilo
    Interfaculty Institute of Biochemistry, University of Tübingen, Germany ; Department of Pediatrics, Vanderbilt University School of Medicine, USA.
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Elofsson, Mikael
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Triazole linker-based trivalent sialic acid inhibitors of adenovirus type 37 infection of human corneal epithelial cells2015Inngår i: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 13, nr 35, 9194-9205 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Adenovirus type 37 (Ad37) is one of the principal agents responsible for epidemic keratoconjunctivitis (EKC), a severe ocular infection that remains without any available treatment. Recently, a trivalent sialic acid derivative (ME0322, Angew. Chem. Int. Ed., 2011, 50, 6519) was shown to function as a highly potent inhibitor of Ad37, efficiently preventing the attachment of the virion to the host cells and subsequent infection. Here, new trivalent sialic acid derivatives were designed, synthesized and their inhibitory properties against Ad37 infection of the human corneal epithelial cells were investigated. In comparison to ME0322, the best compound (17a) was found to be over three orders of magnitude more potent in a cell-attachment assay (IC50 = 1.4 nM) and about 140 times more potent in a cell-infection assay (IC50 = 2.9nM). X-ray crystallographic analysis demonstrated a trivalent binding mode of all compounds to the Ad37 fiber knob. For the most potent compound ophthalmic toxicity in rabbits was investigated and it was concluded that repeated eye administration did not cause any adverse effects.

  • 44.
    Carlsson, Katrin E
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Liu, Junfa
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Extracytoplasmic-stress-responsive pathways modulate type III secretion in Yersinia pseudotuberculosis.2007Inngår i: Infect Immun, ISSN 0019-9567, Vol. 75, nr 8, 3913-24 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Three signal transduction pathways, the two-component systems CpxRA and BaeSR and the alternative sigma factor sigma(E), respond to extracytoplasmic stress that facilitates bacterial adaptation to changing environments. At least the CpxRA and sigma(E) pathways control the production of protein-folding and degradation factors that counter the effects of protein misfolding in the periplasm. This function also influences the biogenesis of multicomponent extracellular appendages that span the bacterial envelope, such as various forms of pili. Herein, we investigated whether any of these regulatory pathways in the enteropathogen Yersinia pseudotuberculosis affect the functionality of the Ysc-Yop type III secretion system. This is a multicomponent molecular syringe spanning the bacterial envelope used to inject effector proteins directly into eukaryotic cells. Disruption of individual components revealed that the Cpx and sigma(E) pathways are important for Y. pseudotuberculosis type III secretion of Yops (Yersinia outer proteins). In particular, a loss of CpxA, a sensor kinase, reduced levels of structural Ysc (Yersinia secretion) components in bacterial membranes, suggesting that these mutant bacteria are less able to assemble a functional secretion apparatus. Moreover, these bacteria were no longer capable of localizing Yops into the eukaryotic cell interior. In addition, a cpxA lcrQ double mutant engineered to overproduce and secrete Yops was still impaired in intoxicating cells. Thus, the Cpx pathway might mediate multiple influences on bacterium-target cell contact that modulate Yersinia type III secretion-dependent host cell cytotoxicity.

  • 45.
    Carlsson, Katrin E
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Liu, Junfa
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Edqvist, Petra J
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Francis, Matthew S
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet). Umeå universitet, Medicinsk fakultet, Umeå Centre for Microbial Research (UCMR).
    Influence of the Cpx extracytoplasmic-stress-responsive pathway on Yersinia sp.-eukaryotic cell contact.2007Inngår i: Infect Immun, ISSN 0019-9567, Vol. 75, nr 9, 4386-99 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The extracytoplasmic-stress-responsive CpxRA two-component signal transduction pathway allows bacteria to adapt to growth in extreme environments. It controls the production of periplasmic protein folding and degradation factors, which aids in the biogenesis of multicomponent virulence determinants that span the bacterial envelope. This is true of the Yersinia pseudotuberculosis Ysc-Yop type III secretion system. However, despite using a second-site suppressor mutation to restore Yop effector secretion by yersiniae defective in the CpxA sensor kinase, these bacteria poorly translocated Yops into target eukaryotic cells. Investigation of this phenotype herein revealed that the expression of genes which encode several surface-located adhesins is also influenced by the Cpx pathway. In particular, the expression and surface localization of invasin, an adhesin that engages beta1-integrins on the eukaryotic cell surface, are severely restricted by the removal of CpxA. This reduces bacterial association with eukaryotic cells, which could be suppressed by the ectopic production of CpxA, invasin, or RovA, a positive activator of inv expression. In turn, these infected eukaryotic cells then became susceptible to intoxication by translocated Yop effectors. In contrast, bacteria harboring an in-frame deletion of cpxR, which encodes the cognate response regulator, displayed an enhanced ability to interact with cell monolayers, as well as elevated inv and rovA transcription. This phenotype could be drastically suppressed by providing a wild-type copy of cpxR in trans. We propose a mechanism of inv regulation influenced by the direct negative effects of phosphorylated CpxR on inv and rovA transcription. In this fashion, sensing of extracytoplasmic stress by CpxAR contributes to productive Yersinia sp.-eukaryotic cell interactions.

  • 46.
    Cava, Felipe
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    de Pedro, Miguel A.
    Peptidoglycan plasticity in bacteria: emerging variability of the murein sacculus and their associated biological functions2014Inngår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 18, 46-53 s.Artikkel, forskningsoversikt (Fagfellevurdert)
    Abstract [en]

    The peptidoglycan (PG) sacculus once thought to be just a reinforcing, static and uniform structure, is fast becoming recognized as a dynamic cell constituent involved in every aspect of bacterial physiology. Recent advances showed that in addition to 'classical' tasks - as an essential element to define bacterial shape, size, division and resistance to osmotic stress the sacculus plays very important roles in many other fields. The very few chemical and structural changes that were once considered as bizarre, or maybe exotic exceptions, are now universally accepted as fundamental pieces in bacterial cell wall adaptation to different kinds of environmental stresses; immune response; intra-specific and inter-specific signalling and antibiotics, just to mention a few. Most, if not all, of these implications are a consequence of the enormous adaptability of PG metabolism to cope with changing conditions, a characteristic for which the term plasticity is proposed. Here we overview and comment on a number of recent contributions on the cell wall adaptive responses to environmental challenges that has greatly impacted the already high complexity of the PG biology field. These new evidences have revived the interest in PG plasticity as an exciting and trendy topic in current microbiology which considers this variability as the trustworthy picture of bacterial PG in nature.

  • 47.
    Cava, Felipe
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Kuru, Erkin
    Brun, Yves V
    de Pedro, Miguel A
    Modes of cell wall growth differentiation in rod-shaped bacteria2013Inngår i: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 16, nr 6, 731-737 s.Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    A bacterial cell takes on the challenge to preserve and reproduce its shape at every generation against a substantial internal pressure by surrounding itself with a mechanical support, a peptidoglycan cell wall. The enlargement of the cell wall via net incorporation of precursors into the pre-existing wall conditions bacterial growth and morphology. However, generation, reproduction and/or modification of a specific shape requires that the incorporation takes place at precise locations for a defined time period. Much has been learnt in the past few years about the biochemistry of the peptidoglycan synthesis process, but topological approaches to the understanding of shape generation have been hindered by a lack of appropriate techniques. Recent technological advances are paving the way for substantial progress in understanding the mechanisms of bacterial morphogenesis. Here we review the latest developments, focusing on the impact of new techniques on the precise mapping of cell wall growth sites.

  • 48.
    Chabes, Andrei
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk kemi och biofysik. Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Stillman, Bruce
    Constitutively high dNTP concentration inhibits cell cycle progression and the DNA damage checkpoint in yeast Saccharomyces cerevisiae.2007Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, Vol. 104, nr 4, 1183-8 s.Artikkel i tidsskrift (Fagfellevurdert)
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    CRISPR-Cas9: how research on a bacterial RNA-guided mechanism opened new perspectives in biotechnology and biomedicine2015Inngår i: EMBO Molecular Medicine, ISSN 1757-4676, E-ISSN 1757-4684, Vol. 7, nr 4, 363-365 s.Artikkel i tidsskrift (Fagfellevurdert)
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    Hess, Wolfgang R
    Editorial: RNA in bacteria2015Inngår i: FEMS Microbiology Reviews, ISSN 0168-6445, E-ISSN 1574-6976, Vol. 39, nr 3, 277-279 s.Artikkel i tidsskrift (Fagfellevurdert)
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