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  • 1051. Weineisen, Maria
    et al.
    Sjöbring, Ulf
    Fällman, Maria
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Andersson, Tommy
    Streptococcal M5 protein prevents neutrophil phagocytosis by interfering with CD11b/CD18 receptor-mediated association and signaling.2004In: J Immunol, ISSN 0022-1767, Vol. 172, no 6, p. 3798-807Article in journal (Refereed)
    Abstract [en]

    Group A streptococci (GAS) are common human pathogens that express major surface-associated virulence factors designated M proteins. In this study, we explored directly the cellular mechanisms behind their supposed ability to prevent phagocytosis. Isolated human neutrophils killed an M-negative GAS mutant (DeltaM5), but not the wild-type parent strain (M5). After 3 h, 3-4 times as many DeltaM5 as M5 bacteria were associated with the neutrophils, and more DeltaM5 than M5 bacteria were ingested. However, there was no statistically significant difference between DeltaM5 and M5 bacteria in regard to the percentage of the neutrophil-associated bacteria that were ingested, indicating that M5 protein prevents an adhesion receptor-dependent association with neutrophils and not the phagocytic machinery per se. Different Abs against CD11b/CD18 (CR3) blocked adhesion and killing of DeltaM5 bacteria, whereas the blocking of two other complement receptors, CD11c/CD18 (CR4) and CD35 (CR1), did not. The CD11b/CD18-mediated killing of DeltaM5 bacteria resulted in protein tyrosine phosphorylations and Cdc42 activation. Furthermore, inhibition of CD11b/CD18 receptor engagement or tyrosine kinase activity blocked the DeltaM5-induced activation of Cdc42 as well as the killing of these bacteria. We conclude that M5 protein interferes with the CD11b/CD18-dependent association between GAS and neutrophils, and thereby blocks subsequent ingestion of the bacteria.

  • 1052.
    Weise, Christoph F
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Login, Frédéric H
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ho, Oanh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 8, p. 1950-1961Article in journal (Refereed)
    Abstract [en]

    The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.

  • 1053. Wenig, Katja
    et al.
    Chatwell, Lorenz
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Björck, Lars
    Huber, Robert
    Sondermann, Peter
    Structure of the streptococcal endopeptidase IdeS, a cysteine proteinase with strict specificity for IgG.2004In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 50, p. 17371-17376Article in journal (Refereed)
    Abstract [en]

    Pathogenic bacteria have developed complex and diverse virulence mechanisms that weaken or disable the host immune defense system. IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a secreted cysteine endopeptidase from the human pathogen S. pyogenes with an extraordinarily high degree of substrate specificity, catalyzing a single proteolytic cleavage at the lower hinge of human IgG. This proteolytic degradation promotes inhibition of opsonophagocytosis and interferes with the killing of group A Streptococcus. We have determined the crystal structure of the catalytically inactive mutant IdeS-C94S by x-ray crystallography at 1.9-A resolution. Despite negligible sequence homology to known proteinases, the core of the structure resembles the canonical papain fold although with major insertions and a distinct substrate-binding site. Therefore IdeS belongs to a unique family within the CA clan of cysteine proteinases. Based on analogy with inhibitor complexes of papain-like proteinases, we propose a model for substrate binding by IdeS.

  • 1054. Werren, John H
    et al.
    Richards, Stephen
    Desjardins, Christopher A
    Niehuis, Oliver
    Gadau, Jürgen
    Colbourne, John K
    Beukeboom, Leo W
    Desplan, Claude
    Elsik, Christine G
    Grimmelikhuijzen, Cornelis J P
    Kitts, Paul
    Lynch, Jeremy A
    Murphy, Terence
    Oliveira, Deodoro C S G
    Smith, Christopher D
    van de Zande, Louis
    Worley, Kim C
    Zdobnov, Evgeny M
    Aerts, Maarten
    Albert, Stefan
    Anaya, Victor H
    Anzola, Juan M
    Barchuk, Angel R
    Behura, Susanta K
    Bera, Agata N
    Berenbaum, May R
    Bertossa, Rinaldo C
    Bitondi, Márcia M G
    Bordenstein, Seth R
    Bork, Peer
    Bornberg-Bauer, Erich
    Brunain, Marleen
    Cazzamali, Giuseppe
    Chaboub, Lesley
    Chacko, Joseph
    Chavez, Dean
    Childers, Christopher P
    Choi, Jeong-Hyeon
    Clark, Michael E
    Claudianos, Charles
    Clinton, Rochelle A
    Cree, Andrew G
    Cristino, Alexandre S
    Dang, Phat M
    Darby, Alistair C
    de Graaf, Dirk C
    Devreese, Bart
    Dinh, Huyen H
    Edwards, Rachel
    Elango, Navin
    Elhaik, Eran
    Ermolaeva, Olga
    Evans, Jay D
    Foret, Sylvain
    Fowler, Gerald R
    Gerlach, Daniel
    Gibson, Joshua D
    Gilbert, Donald G
    Graur, Dan
    Gründer, Stefan
    Hagen, Darren E
    Han, Yi
    Hauser, Frank
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hunter, Henry C
    Hurst, Gregory D D
    Jhangian, Shalini N
    Jiang, Huaiyang
    Johnson, Reed M
    Jones, Andrew K
    Junier, Thomas
    Kadowaki, Tatsuhiko
    Kamping, Albert
    Kapustin, Yuri
    Kechavarzi, Bobak
    Kim, Jaebum
    Kim, Jay
    Kiryutin, Boris
    Koevoets, Tosca
    Kovar, Christie L
    Kriventseva, Evgenia V
    Kucharski, Robert
    Lee, Heewook
    Lee, Sandra L
    Lees, Kristin
    Lewis, Lora R
    Loehlin, David W
    Logsdon, John M
    Lopez, Jacqueline A
    Lozado, Ryan J
    Maglott, Donna
    Maleszka, Ryszard
    Mayampurath, Anoop
    Mazur, Danielle J
    McClure, Marcella A
    Moore, Andrew D
    Morgan, Margaret B
    Muller, Jean
    Munoz-Torres, Monica C
    Muzny, Donna M
    Nazareth, Lynne V
    Neupert, Susanne
    Nguyen, Ngoc B
    Nunes, Francis M F
    Oakeshott, John G
    Okwuonu, Geoffrey O
    Pannebakker, Bart A
    Pejaver, Vikas R
    Peng, Zuogang
    Pratt, Stephen C
    Predel, Reinhard
    Pu, Ling-Ling
    Ranson, Hilary
    Raychoudhury, Rhitoban
    Rechtsteiner, Andreas
    Reese, Justin T
    Reid, Jeffrey G
    Riddle, Megan
    Robertson, Hugh M
    Romero-Severson, Jeanne
    Rosenberg, Miriam
    Sackton, Timothy B
    Sattelle, David B
    Schlüns, Helge
    Schmitt, Thomas
    Schneider, Martina
    Schüler, Andreas
    Schurko, Andrew M
    Shuker, David M
    Simões, Zilá L P
    Sinha, Saurabh
    Smith, Zachary
    Solovyev, Victor
    Souvorov, Alexandre
    Springauf, Andreas
    Stafflinger, Elisabeth
    Stage, Deborah E
    Stanke, Mario
    Tanaka, Yoshiaki
    Telschow, Arndt
    Trent, Carol
    Vattathil, Selina
    Verhulst, Eveline C
    Viljakainen, Lumi
    Wanner, Kevin W
    Waterhouse, Robert M
    Whitfield, James B
    Wilkes, Timothy E
    Williamson, Michael
    Willis, Judith H
    Wolschin, Florian
    Wyder, Stefan
    Yamada, Takuji
    Yi, Soojin V
    Zecher, Courtney N
    Zhang, Lan
    Gibbs, Richard A
    Functional and evolutionary insights from the genomes of three parasitoid Nasonia species.2010In: Science, ISSN 0036-8075, E-ISSN 1095-9203, ISSN 1095-9203 EISSN, Vol. 327, no 5963, p. 343-8Article in journal (Refereed)
    Abstract [en]

    We report here genome sequences and comparative analyses of three closely related parasitoid wasps: Nasonia vitripennis, N. giraulti, and N. longicornis. Parasitoids are important regulators of arthropod populations, including major agricultural pests and disease vectors, and Nasonia is an emerging genetic model, particularly for evolutionary and developmental genetics. Key findings include the identification of a functional DNA methylation tool kit; hymenopteran-specific genes including diverse venoms; lateral gene transfers among Pox viruses, Wolbachia, and Nasonia; and the rapid evolution of genes involved in nuclear-mitochondrial interactions that are implicated in speciation. Newly developed genome resources advance Nasonia for genetic research, accelerate mapping and cloning of quantitative trait loci, and will ultimately provide tools and knowledge for further increasing the utility of parasitoids as pest insect-control agents.

  • 1055.
    Westermark, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Yersinia-phagocyte interactions during early infection2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Pathogenic Gram-negative Yersinia species preferentially target and inactivate phagocytic cells of the innate immune defense by translocation of effector Yersinia outer proteins (Yops) into the cells via a type III secretion system. This indicates that inactivation and avoidance of the early innate immune response is an efficient way for Yersinia species to avoid elimination and to cause diseases ranging from mild gastroenteritis (Y. pseudotuberculosis and Y. enterocolitica) to plague (Y. pestis). In this project, we aimed to study the interaction between enteropathogenic Y. pseudotuberculosis and phagocytic cells during early infection.

    In situ interaction studies on infected intestinal tissues showed that Y. pseudotuberculosis mainly interacts with dendritic cells (DCs) in lymphoid tissues of the intestine during initial infection. After massive recruitment of polymorphonuclear neutrophils (PMNs) to the infected tissues, wild-type (wt) bacteria also interacted with this phagocyte. In contrast to the wt, mutants lacking the anti-phagocytic effectors YopH and YopE are avirulent in mice and unable to spread systemically. Interestingly, our interaction assay showed that these mutants not only interacted with DCs, but also with PMNs during the initial stage of infection. Thus, indicating that Y. pseudotuberculosis can avoid interaction with PMNs during early infection and that this is Yop-dependent. In a phagocytosis assay Y. pseudotuberculosis was demonstrated to inhibit internalization by DCs in a YopE-dependent manner, while both YopH and YopE were shown to be involved in the blocking of phagocytosis by macrophages and PMNs. Thus, indicating that YopH has cell type-specific effects. To further investigate the role of DCs during initial stages of infection, a mouse DC depletion model (CD11c-DTRtg) was applied. However, the DTx-mediated depletion of DCs in CD11c-DTRtg mice induced neutrophilia and the model could not give a definite answer to whether DCs play an important role in either restricting or stimulating progression of Y. pseudotuberculosis infection. To investigate involvement of PMNs during early infection mice were injected with the depleting antibody α-Ly6G. In absence of PMNs wt, as well as yopH and yopE mutants became more virulent, which further supports the importance of these Yops for the ability of Y. pseudotuberculosis to disseminate from the initial infection sites in the intestine to cause systemic disease.

    In summary, our studies show that inhibiting internalization and maturation of DCs and avoiding phagocytosis by and interaction with macrophages and PMNs during the early stages of infection are important virulence strategies for Y. pseudotuberculosis to be able to colonize tissues, proliferate and disseminate systemically.

    Download full text (pdf)
    Linda Westermark - Yersinia-phagocyte interactions during early infection
  • 1056.
    Westermark, Linda
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTRtg model for studies of bacterial gastrointestinal infections2012In: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 137, no S1, p. 271-271Article in journal (Other academic)
  • 1057.
    Westermark, Linda
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTR(tg) model for studies of bacterial gastrointestinal infections2012In: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 53, no 3-4, p. 154-161Article in journal (Refereed)
    Abstract [en]

    Dendritic cells play an important role in the immune response against pathogens, as they are responsible for the activation and control of both innate and adaptive immune responses. The CD11c-DTR(tg) model, which allows transient elimination of dendritic cells by diphtheria toxin-treatment (DTx), has been extensively used to study the importance of this immune cell during steady-state and infection conditions in mice. Infecting dendritic cell-depleted mice orally with Yersinia pseudotuberculosis results in a markedly reduced level of infection compared with infection of non-depleted mice. We show here that it is not the lack of dendritic cells per se that is responsible for the reduced infection efficiency, instead it is an immune response induced by the DTx-treatment that prevents the bacteria from establishing colonization in Peyer's patches. The DTx-induced depletion initiates an immune response, with elevated serum levels of keratinocyte-derived cytokine (KC) and recruitment of polymorphonuclear neutrophils to dendritic cell-containing organs, such as Peyer's patches. Since the window for having an animal depleted of dendritic cells is limited in time for this model, the DTx-mediated effect on the immune system complicates the use of this model in studies of early events during bacterial infections.

  • 1058.
    Westermark, Linda
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia pseudotuberculosis efficiently avoids polymorphonuclear neutrophils during early infectionManuscript (preprint) (Other academic)
  • 1059.
    Westermark, Linda
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection2014In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 3, p. 1181-1191Article in journal (Refereed)
    Abstract [en]

    The human-pathogenic species of the Gram-negative genus Yersinia preferentially target and inactivate cells of the innate immune defense, suggesting that this is a critical step by which these bacteria avoid elimination and cause disease. In this study, bacterial interactions with dendritic cells, macrophages, and polymorphonuclear neutrophils (PMNs) in intestinal lymphoid tissues during early Yersinia pseudotuberculosis infection were analyzed. Wild-type bacteria were shown to interact mainly with dendritic cells, but not with PMNs, on day 1 postinfection, while avirulent yopH and yopE mutants interacted with PMNs as well as with dendritic cells. To unravel the role of PMNs during the early phase of infection, we depleted mice of PMNs by using an anti-Ly6G antibody, after which we could see more-efficient initial colonization by the wild-type strain as well as by yopH, yopE, and yopK mutants on day 1 postinfection. Dissemination of yopH, yopE, and yopK mutants from the intestinal compartments to mesenteric lymph nodes was faster in PMN-depleted mice than in undepleted mice, emphasizing the importance of effective targeting of PMNs by these Yersinia outer proteins (Yops). In conclusion, escape from interaction with PMNs due to the action of YopH, YopE, and YopK is a key feature of pathogenic Yersinia species that allows colonization and effective dissemination.

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    Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection
  • 1060. Widhe, M
    et al.
    Ekerfelt, C
    Jarefors, S
    Skogman, B H
    Peterson, E M
    Bergström, S
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Forsberg, P
    Ernerudh, J
    T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.2009In: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 70, no 2, p. 141-8Article in journal (Refereed)
    Abstract [en]

    We studied the T-cell reactivity to overlapping peptides of B. garinii OspA, in order to locate possible immunodominant T-cell epitopes in neuroborreliosis. Cells from cerebrospinal fluid (CSF) and blood from 39 patients with neuroborreliosis and 31 controls were stimulated with 31 overlapping peptides, and interferon-gamma secreting cells were detected by ELISPOT. The peptides OspA(17-36), OspA(49-68), OspA(105-124), OspA(137-156), OspA(193-212) and OspA(233-252) showed the highest frequency of positive responses, being positive in CSF from 38% to 50% of patients with neuroborreliosis. These peptides also elicited higher responses in CSF compared with controls (P = 0.004). CSF cells more often showed positive responses to these peptides than blood cells (P = 0.001), in line with a compartmentalization to the central nervous system. Thus, a set of potential T-cell epitopes were identified in CSF cells from patients with neuroborreliosis. Further studies may reveal whether these epitopes can be used diagnostically and studies involving HLA interactions may show their possible pathogenetic importance.

  • 1061. Widhe, Mona
    et al.
    Jarefors, Sara
    Ekerfelt, Christina
    Vrethem, Magnus
    Bergström, Sven
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Forsberg, Pia
    Ernerudh, Jan
    Borrelia-specific interferon-gamma and interleukin-4 secretion in cerebrospinal fluid and blood during Lyme borreliosis in humans: association with clinical outcome.2004In: J Infect Dis, ISSN 0022-1899, Vol. 189, no 10, p. 1881-91Article in journal (Refereed)
    Abstract [en]

    The Borrelia-specific interferon (IFN)- gamma and interleukin (IL)-4 responses of 113 patients and control subjects were analyzed using the sensitive enzyme-linked immunospot method. Cerebrospinal fluid (CSF) and blood samples were obtained, during the course of disease, from patients with chronic or nonchronic neuroborreliosis (NB) and from control subjects without NB. Blood samples were obtained from patients with Lyme skin manifestations and from healthy blood donors. Early increased secretion of Borrelia-specific IFN- gamma (P<.05) and subsequent up-regulation of IL-4 (P<.05) were detected in the CSF cells of patients with nonchronic NB. In contrast, persistent Borrelia-specific IFN- gamma responses were observed in the CSF cells of patients with chronic NB (P<.05). In patients with erythema migrans, increased IFN- gamma (P<.001) was observed in blood samples obtained early during the course of disease, whereas increased IL-4 (P<.05) was observed after clearance. On the contrary, patients with acrodermatitis chronica atrophicans had Borrelia-specific IFN- gamma (P<.001), but not IL-4, detected in blood samples. The present data suggest that an initial IFN- gamma response, followed by up-regulation of IL-4, is associated with nonchronic manifestations, whereas a persistent IFN- gamma response may lead to chronic Lyme borreliosis.

  • 1062. Widhe, Mona
    et al.
    Skogman, Barbro Hedin
    Jarefors, Sara
    Eknefelt, Mattias
    Eneström, Gunilla
    Nordwall, Maria
    Ekerfelt, Christina
    Croner, Stefan
    Bergström, Sven
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Forsberg, Pia
    Ernerudh, Jan
    Up-regulation of Borrelia-specific IL-4- and IFN-gamma-secreting cells in cerebrospinal fluid from children with Lyme neuroborreliosis.2005In: Int Immunol, ISSN 0953-8178, Vol. 17, no 10, p. 1283-91Article in journal (Refereed)
    Abstract [en]

    The Lyme disease-pathogen Borrelia burgdorferi binds the complement inhibitor factor H (FH) to its outer surface protein E- (OspE) and BbA68-families of lipoproteins. In earlier studies, only serum-resistant strains of the genospecies B. burgdorferi sensu stricto or B. afzelii, but not serum-sensitive B. garinii strains, have been shown to bind FH. Since B. garinii often causes neuroborreliosis in man, we have readdressed the interactions of B. garinii with FH. B. garinii 50/97 strain did not express FH-binding proteins. By transforming the B. garinii 50/97 strain with an OspE-encoding gene from complement-resistant B. burgdorferi (ospE-297), its resistance to serum killing could be increased. OspE genes were detected and cloned from the B. garinii BITS, Pistoia and 40/97 strains by PCR and sequencing. The deduced amino acid sequences differed in an N-terminal lysine-rich FH-binding region from OspE sequences of resistant strains. Recombinant B. garinii BITS OspE protein was found to have a considerably lower FH-binding activity than the B. burgdorferi sensu stricto 297 OspE protein P21 (P21-297). Unlike bacteria that had been kept in culture for a long time, neurovirulent B. garinii strains from neuroborreliosis patients were found to express approximately 27-kDa FH-binding proteins. These were not recognized by polyclonal anti-OspE or anti-BbA68 antibodies. We conclude that B. garinii strains carry ospE genes but have a decreased expression of OspE proteins and a reduced ability to bind FH, especially when grown for prolonged periods in vitro. Recently isolated neuroinvasive B. garinii strains, however, can express FH-binding proteins, which may contribute to the virulence of neuroborreliosis-causing B. garinii strains.

  • 1063. Wieser, Andreas
    et al.
    Storz, Enno
    Liegl, Gabriele
    Peter, Annabell
    Pritsch, Michael
    Shock, Jonathan
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Schubert, Soeren
    Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis2014In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 304, no 8, p. 1032-1037Article in journal (Refereed)
    Abstract [en]

    There currently exists no efficient and easy method for size profiling and counting of membranous nano-scale particles, such as bacterial outer membrane vesicles (OMVs). We present here a cost-effective and fast method capable of profiling and counting small sample volumes of nano-scale membranous vesicles with standard laboratory equipment without the need for any washing steps. OMV populations of different bacterial species are compared and even subpopulations of OMVs can be identified after a simple labelling procedure. Counting is possible over three orders of magnitude without any changes to the protocol. Protein contaminations do not alter the described measurements.

  • 1064.
    Wikberg, Maria L.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew S.
    Peden, Alex
    Aili, Margareta
    Stefansson, Kristina
    Palmer, Ruth H.
    Aitken, Alastair
    Hallberg, Bengt
    A nonphosphorylated 14-3-3 binding motif on exoenzyme S that is functional in vivo2002In: European Journal of Biochemistry, Vol. 269, no 20, p. 4921-4929Article in journal (Refereed)
  • 1065.
    Wiklund, Peder
    et al.
    Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Geriatric Medicine. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Rehabilitation Medicine.
    Nordström, Anna
    Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Rehabilitation Medicine. Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Högström, Magnus
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Alfredson, Håkan
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine.
    Engström, Patrik
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gustafsson, Thomas
    Karolinska universitetslaboratoriet, Klinisk kemi.
    Franks, Paul
    Umeå University, Faculty of Medicine, Department of Public Health and Clinical Medicine, Medicine.
    Nordström, Peter
    Umeå University, Faculty of Medicine, Department of Surgical and Perioperative Sciences, Sports Medicine. Umeå University, Faculty of Medicine, Department of Community Medicine and Rehabilitation, Geriatric Medicine.
    High impact loading on the skeleton is associated with a decrease in glucose levels in young men2012In: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 77, no 6, p. 823-827Article in journal (Refereed)
    Abstract [en]

    Objective The skeleton has been suggested to be involved in energy metabolism through osteocalcin (OC), an osteoblast-specific molecule. The objective of this study was to investigate whether high impact exercise stimulating bone formation would lead to changes in glucose and lipid metabolism independent of cardiorespiratory effects, and if OC mediates this association.

    Design Prospective intervention study.

    Methods Fifty men aged 20-32 years were allocated to an intervention group or a control group. The intervention group completed six different types of jumps in sets of five, with the frequency of these exercises gradually increasing over 8 weeks. At baseline and after 8 weeks, glycerol concentrations were measured in fat tissue as a marker of lipolysis by using microdialysis. Blood samples were assayed for OC and markers of glucose and lipid metabolism. Physical activity was measured using an accelerometer.

    Results After adjustment for confounders at baseline and changes in physical activity during the intervention period, the intervention was associated with a decrease in levels of glucose (p = 0.04), adrenalin (p = 0.03) and OC (p=0.04) after adjusting for baseline levels and changes in physical activity. No other differences between the groups were significant, although the trends of the metabolic variables favored the intervention group.

    Conclusions The results of this study suggest that high impact loading on the skeleton may affect glucose metabolism independent of the level of aerobic exercise.

  • 1066.
    Wikström Hultdin, Ulrika
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Stina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Allgardsson, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Stier, Günter
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Öhman, Anders
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, no Pt 3, p. 337-341Article in journal (Refereed)
    Abstract [en]

    The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His6-tagged fusion protein was captured by Ni2+-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His6 tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 Å resolution was collected at 100 K using synchrotron radiation.

  • 1067.
    Wikström Hultdin, Ulrika
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Lindberg, Stina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Grundström, Christin
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Huang, Shenghua
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sauer-Eriksson, Elisabeth
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 16, p. 3368-3381Article in journal (Refereed)
    Abstract [en]

    In uropathogenic Escherichia coli, UPEC, different types of fimbriae are expressed in order to mediate interactions with host tissue. FocB belongs to the PapB family of transcription factors involved in the regulation of fimbriae gene clusters. Recent findings suggest that members from this family of proteins may form different protein-protein complexes and that they may exert both positive and negative effects on transcription of fimbriae genes. To elucidate the detailed function of FocB, we have determined its crystal structure at 1.4 Å resolution. FocB is an all alpha helical structure with a helix-turn-helix (HTH) motif. Interestingly, conserved residues important for DNA-binding are not located in the recognition helix of the HTH-motif, but in the preceding helix. Results from protein-DNA binding studies indicated that FocB interacts with minor groove of its cognate DNA, which also points to a DNA-interaction unusual for this motif. Packing interactions in the crystals gave two plausible dimerization interfaces. Conserved residues known to be important for protein oligomerization are present at both interfaces, suggesting that both sites play a role in a functional FocB protein.

  • 1068. Wilhelmsson, Peter
    et al.
    Fryland, Linda
    Börjesson, Stefan
    Nordgren, Johan
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ernerudh, Jan
    Forsberg, Pia
    Lindgren, Per-Eric
    Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden2010In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 48, no 11, p. 4169-4176Article in journal (Refereed)
    Abstract [en]

    Members of the genus Borrelia are among the most common infectious agents causing tick-borne disease in humans worldwide. Here, we developed a Light Upon eXtension (LUX) real-time PCR assay that can detect and quantify Borrelia species in ticks that have fed on humans, and we applied the assay to 399 such ticks. Borrelia PCR-positive ticks were identified to species level by sequencing the products of conventional PCR performed using Borrelia group-specific primers. There was a 19% prevalence of Borrelia spp. in the detached ticks, and the number of spirochetes per Borrelia PCR-positive tick ranged from 2.0 x 10(2) to 4.9 x 10(5), with a median of 7.8 x 10(3) spirochetes. Adult ticks had a significantly larger number of spirochetes, with a median of 8.4 x 10(3) compared to the median of nymphs of 4.4 x 10(3). [corrected] Adult ticks also exhibited a higher prevalence of Borrelia (33%) than nymphs (14%). Among the identified species, Borrelia afzelii was found to predominate (61%) and was followed by B. garinii (23%), B. valaisiana (13%), B. burgdorferi sensu stricto (1%), B. lusitaniae (1%), and B. miyamotoi-like (1%). Also, 3% of the ticks were coinfected with multiple strains of B. afzelii. Notably, this is the first report of B. lusitaniae being detected in ticks in Sweden. Our LUX real-time PCR assay proved to be more sensitive than a corresponding TaqMan assay. In conclusion, the novel LUX real-time PCR method is a rapid and sensitive tool for detection and quantification of Borrelia spp. in ticks.

  • 1069. Wilson, Sara I
    et al.
    Edlund, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Neural induction: toward a unifying mechanism2001In: Nature Neuroscience, ISSN 1097-6256, E-ISSN 1546-1726, p. 1161-1168Article, review/survey (Refereed)
    Abstract [en]

    Neural induction constitutes the initial step in the generation of the vertebrate nervous system. In attempting to understand the principles that underlie this process, two key issues need to be resolved. When is neural induction initiated, and what is the cellular source and molecular nature of the neural inducing signal(s)? Currently, these aspects of neural induction seem to be very different in amphibian and amniote embryos. Here we highlight the similarities and the differences, and we propose a possible unifying mechanism.

  • 1070.
    Wirebrand, Lisa
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Madhushani, Anjana W. K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Irie, Yasuhiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Shingler, Victoria
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF6002018In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, no 1, p. 186-199Article in journal (Refereed)
    Abstract [en]

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids.

  • 1071.
    Witek, Barbara
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    El Wakil, Abeer
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nord, Christoffer
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Ahlgren, Ulf
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Eriksson, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Vernersson-Lindahl, Emma
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Helland, Aslaug
    Alexeyev, Oleg A.
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Pathology.
    Hallberg, Bengt
    Palmer, Ruth H.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg.
    Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism2015In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, no 5, article id e0123542Article in journal (Refereed)
    Abstract [en]

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

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  • 1072. Woldemeskel, Selamawit Abi
    et al.
    Daitch, Allison K.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Panis, Gael
    Zeinert, Rilee
    Gonzalez, Diego
    Smith, Erika
    Collier, Justine
    Chien, Peter
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Viollier, Patrick H.
    Goley, Erin D.
    The conserved transcriptional regulator CdnL is required for metabolic homeostasis and morphogenesis in Caulobacter2020In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 16, no 1, article id e1008591Article in journal (Refereed)
    Abstract [en]

    Bacterial growth and division require regulated synthesis of the macromolecules used to expand and replicate components of the cell. Transcription of housekeeping genes required for metabolic homeostasis and cell proliferation is guided by the sigma factor sigma(70). The conserved CarD-like transcriptional regulator, CdnL, associates with promoter regions where sigma(70) localizes and stabilizes the open promoter complex. However, the contributions of CdnL to metabolic homeostasis and bacterial physiology are not well understood. Here, we show that Caulobacter crescentus cells lacking CdnL have severe morphological and growth defects. Specifically, Delta cdnL cells grow slowly in both rich and defined media, and are wider, more curved, and have shorter stalks than WT cells. These defects arise from transcriptional downregulation of most major classes of biosynthetic genes, leading to significant decreases in the levels of critical metabolites, including pyruvate, alpha-ketoglutarate, ATP, NAD(+), UDP-N-acetyl-glucosamine, lipid II, and purine and pyrimidine precursors. Notably, we find that Delta cdnL cells are glutamate auxotrophs, and Delta cdnL is synthetic lethal with other genetic perturbations that limit glutamate synthesis and lipid II production. Our findings implicate CdnL as a direct and indirect regulator of genes required for metabolic homeostasis that impacts morphogenesis through availability of lipid II and other metabolites. Author summary To grow and divide, bacteria must accumulate precursor molecules to support duplication and expansion of cellular materials. One mechanism by which bacteria do this is by regulating the expression of genes whose products are important for production of these molecules. How gene expression is maintained or altered to support synthesis of appropriate molecules to balance growth with nutrient availability is not fully understood. In this paper, we describe the role of a regulator of gene expression called CdnL in maintaining levels of molecules required for bacterial growth and reproduction. CdnL broadly impacts the levels of genes required for most biosynthetic processes. CdnL's broad impact on transcription has downstream consequences on growth rate, cell shape, and nutrient requirements for growth. We report that CdnL is particularly important for maintaining levels of the amino acid glutamate and the cell wall precursor lipid II, each of which is critical for supporting proper growth and cell morphology. Our results implicate CdnL as a broadly conserved regulator of metabolic homeostasis, growth, and cell shape in bacteria.

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  • 1073. Wolfstetter, Georg
    et al.
    Shirinian, Margret
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Stute, Christiana
    Grabbe, Caroline
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hummel, Thomas
    Baumgartner, Stefan
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Holz, Anne
    Fusion of circular and longitudinal muscles in Drosophila is independent of the endoderm but further visceral muscle differentiation requires a close contact between mesoderm and endoderm.2009In: Mechanisms of Development, ISSN 0925-4773, E-ISSN 1872-6356, Vol. 126, no 8-9, p. 721-736Article in journal (Refereed)
    Abstract [en]

    In this study we describe the morphological and genetic analysis of the Drosophila mutant gürtelchen (gurt). gurt was identified by screening an EMS collection for novel mutations affecting visceral mesoderm development and was named after the distinct belt shaped visceral phenotype. Interestingly, determination of visceral cell identities and subsequent visceral myoblast fusion is not affected in mutant embryos indicating a later defect in visceral development. gurt is in fact a new huckebein (hkb) allele and as such exhibits nearly complete loss of endodermal derived structures. Targeted ablation of the endodermal primordia produces a phenotype that resembles the visceral defects observed in huckebein(gürtelchen) (hkb(gurt)) mutant embryos. It was shown previously that visceral mesoderm development requires complex interactions between visceral myoblasts and adjacent tissues. Signals from the neighbouring somatic myoblasts play an important role in cell type determination and are a prerequisite for visceral muscle fusion. Furthermore, the visceral mesoderm is known to influence endodermal migration and midgut epithelium formation. Our analyses of the visceral phenotype of hkb(gurt) mutant embryos reveal that the adjacent endoderm plays a critical role in the later stages of visceral muscle development, and is required for visceral muscle elongation and outgrowth after proper myoblast fusion.

  • 1074. Wolf-Watz, M
    et al.
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, T
    Structure and backbone dynamics of Apo-CBFbeta in solution.2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 38, p. 11423-11432Article in journal (Refereed)
    Abstract [en]

    Runx proteins constitute a family of mammalian transcription factors that interact with DNA through their evolutionarily conserved Runt domain. CBFbeta, alternatively denoted PEBP2beta, is the non-DNA-binding heterodimer partner and acts to enhance the DNA binding affinity of Runx proteins. Runx proteins and CBFbeta are associated with a variety of biological functions and human diseases; they are, for example, together the most frequent targets for chromosomal rearrangements in acute human leukemias. We have determined the solution structure and characterized the backbone dynamics of C-terminally truncated fragments containing residues 1-141 of CBFbeta. The present apo-CBFbeta structure is very similar to that seen in a Runt-CBFbeta complex. An evaluation of backbone (15)N NMR relaxation parameters shows that CBFbeta is a rigid molecule with high order parameters throughout the backbone; the only regions displaying significant dynamics are a long loop and the C-terminal alpha-helix. A few residues display relaxation behavior indicating conformational exchange on microsecond to millisecond time scales, but only one of these is located at the Runt binding surface. Our structure and dynamics analysis of CBFbeta therefore suggests that the protein binds to Runt without large conformational changes or induced folding ("lock-and-key" interaction). The apo-CBFbeta structure presented here exhibits several significant differences with two other published NMR ensembles of very similar protein fragments. The differences are located in four regions outside of the central beta-barrel, whereas the beta-barrel itself is almost identical in the three NMR structures. The comparison illustrates that independently determined NMR structures may display rather large differences in backbone conformation in regions that appear to be well-defined in each of the calculated NMR ensembles.

  • 1075. Wolf-Watz, M
    et al.
    Xie, X Q
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Holm, M
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, T
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Härd, T
    Solution properties of the free and DNA-bound Runt domain of AML1.1999In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 261, no 1, p. 251-60Article in journal (Refereed)
    Abstract [en]

    The Runt domain is responsible for specific DNA and protein-protein interactions in a family of transcription factors which includes human AML1. Structural data on the Runt domain has not yet become available, possibly due to solubility and stability problems with expressed protein fragments. Here we describe the optimization and characterization of a 140-residue fragment, containing the Runt domain of AML1, which is suitable for structural studies. The fragment of AML1 including amino acids 46-185 [AML1 Dm(46-185)] contains a double cysteine-->serine mutation which does not affect Runt domain structure or DNA-binding affinity. Purified AML1 Dm(46-185) is soluble and optimally stable in a buffer containing 200 mm MgSO4 and 20 mm sodium phosphate at pH 6.0. Nuclear magnetic resonance and circular dichroism spectroscopy indicate that the Runt domain contains beta-sheet, but little or no alpha-helical secondary structure elements. The 45 N-terminal residues of AML1 are unstructured and removal of the N-terminal enhances sequence-specific DNA binding. The NMR spectrum of AML1 Dm(46-185) displays a favorable chemical shift dispersion and resolved NOE connectivities are readily identified, suggesting that a structure determination of this Runt domain fragment is feasible. A titration of 15N-labelled AML1 Dm(46-185) with a 14-bp cognate DNA duplex results in changes in the 15N NMR heteronuclear single quantum coherence spectrum which indicate the formation of a specific complex and structural changes in the Runt domain upon DNA binding.

  • 1076.
    Wolf-Watz, Magnus
    et al.
    Umeå University, Faculty of Science and Technology, Chemistry.
    Bäckström, Stefan
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Grundström, Thomas
    Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sauer, Uwe
    Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001In: FEBS Letters, ISSN 0014-5793, Vol. 488, no 1-2, p. 81-4Article in journal (Refereed)
    Abstract [en]

    It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.

  • 1077.
    Xie, Xiao-Qi
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Pardali, Evangelia
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Holm, Magnus
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Sideras, Paschalis
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Grundström, Thomas
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    AML and Ets proteins regulate the I alpha1 germ-line promoter.1999In: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 29, no 2, p. 488-498Article in journal (Refereed)
    Abstract [en]

    The immunoglobulin heavy chain (IgH) class switch recombination of B lymphocytes preferentially targets unrearranged IgH genes that have already been rendered transcriptionally active. Transcription of the germ-line IgH genes is controlled by intervening (I) regions upstream of their switch regions. The I alpha1 promoter activates transcription of the human germ-line C alpha1 gene for IgA1 and mediates the transforming growth factor (TGF)-beta1 responsiveness of this locus. Here we show that the I alpha1 promoter contains several binding sites for the AML/PEBP2/CBF family of transcription factors and that AML and Ets proteins are major regulators of the basal and TGF-beta-inducible promoter activity. Our data constitute a starting point for studies to elucidate the molecular mechanism by which TGF-beta regulates IgA production.

  • 1078.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Medicine, Department of Odontology.
    SSD1 modifies phenotypes of Elongator mutants2019In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983Article in journal (Refereed)
    Abstract [en]

    The translational decoding properties of tRNAs are influenced by post-transcriptional modification of nucleosides in their anticodon region. The Elongator complex promotes the first step in the formation of 5-methoxycarbonylmethyl (mcm(5)), 5-methoxycarbonylhydroxymethyl (mchm(5)), and 5-carbamoylmethyl (ncm(5)) groups on wobble uridine residues in eukaryotic cytosolic tRNAs. Elongator mutants in yeast, worms, plants, mice, and humans not only show a tRNA modification defect, but also a diverse range of additional phenotypes. Even though the phenotypes are almost certainly caused by the reduced functionality of the hypomodified tRNAs in translation, the basis for specific phenotypes is not well understood. Here, we discuss the recent finding that the phenotypes of Saccharomyces cerevisiae Elongator mutants are modulated by the genetic background. This background-effect is largely due to the allelic variation at the SSD1 locus, which encodes an mRNA-binding protein involved in post-transcriptional regulation of gene expression. A nonsense ssd1 allele is found in several wild-type laboratory strains and the presence of this allele aggravates the stress-induced phenotypes of Elongator mutants. Moreover, other phenotypes, such as the histone acetylation and telomeric gene silencing defects, are dependent on the mutant ssd1 allele. Thus, SSD1 is a genetic modifier of the phenotypes of Elongator-deficient yeast cells.

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  • 1079.
    Xu, Fu
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Byström, Anders
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Johansson, Marcus J. O.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    SSD1 suppresses phenotypes induced by the lack of Elongator-dependent tRNA modifications2019In: PLoS Genetics, ISSN 1553-7390, E-ISSN 1553-7404, Vol. 15, no 8, article id e1008117Article in journal (Refereed)
    Abstract [en]

    The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5 ) and 5-carbamoylmethyl (ncm5 ) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5 /ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5 /ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2- thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.

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  • 1080.
    Yadav, Akhilesh K.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Espaillat, Akbar
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bacterial Strategies to Preserve Cell Wall Integrity Against Environmental Threats2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2064Article, review/survey (Refereed)
    Abstract [en]

    Bacterial cells are surrounded by an exoskeleton-like structure, the cell wall, composed primarily of the peptidoglycan (PG) sacculus. This structure is made up of glycan strands cross-linked by short peptides generating a covalent mesh that shapes bacteria and prevents their lysis due to their high internal osmotic pressure. Even though the PG is virtually universal in bacteria, there is a notable degree of diversity in its chemical structure. Modifications in both the sugars and peptides are known to be instrumental for bacteria to cope with diverse environmental challenges. In this review, we summarize and discuss the cell wall strategies to withstand biotic and abiotic environmental insults such as the effect of antibiotics targeting cell wall enzymes, predatory PG hydrolytic proteins, and PG signaling systems. Finally we will discuss the opportunities that species-specific PG variability might open to develop antimicrobial therapies.

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  • 1081. Yamamoto, Shouji
    et al.
    Mitobe, Jiro
    Ishikawa, Takahiko
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Ohnishi, Makoto
    Watanabe, Haruo
    Izumiya, Hidemasa
    Regulation of natural competence by the orphan two-component system sensor kinase ChiS involves a non-canonical transmembrane regulator in Vibrio cholerae2014In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 91, no 2, p. 326-347Article in journal (Refereed)
    Abstract [en]

    In Vibrio cholerae, 41 chitin-inducible genes, including the genes involved in natural competence for DNA uptake, are governed by the orphan two-component system (TCS) sensor kinase ChiS. However, the mechanism by which ChiS controls the expression of these genes is currently unknown. Here, we report the involvement of a novel transcription factor termed TfoS' in this process. TfoS is a transmembrane protein that contains a large periplasmic domain and a cytoplasmic AraC-type DNA-binding domain, but lacks TCS signature domains. Inactivation of tfoS abolished natural competence as well as transcription of the tfoR gene encoding a chitin-induced small RNA essential for competence gene expression. A TfoS fragment containing the DNA-binding domain specifically bound to and activated transcription from the tfoR promoter. Intracellular TfoS levels were unaffected by disruption of chiS and coexpression of TfoS and ChiS in Escherichia coli recovered transcription of the chromosomally integrated tfoR::lacZ gene, suggesting that TfoS is post-translationally modulated by ChiS during transcriptional activation; however, this regulation persisted when the canonical phosphorelay residues of ChiS were mutated. The results presented here suggest that ChiS operates a chitin-induced non-canonical signal transduction cascade through TfoS, leading to transcriptional activation of tfoR.

  • 1082. Yamaoka, Yoshio
    et al.
    Souchek, Julianne
    Odenbreit, Stefan
    Haas, Rainer
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Odontology, Oral Microbiology. Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Borén, Thomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kodama, Tadashi
    Osato, Michael S
    Gutierrez, Oscar
    Kim, Jong G
    Graham, David Y
    Discrimination between cases of duodenal ulcer and gastritis on the basis of putative virulence factors of Helicobacter pylori.2002In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 6, p. 2244-2246Article in journal (Refereed)
    Abstract [en]

    The BabA, cagA, and vacA statuses of 827 Helicobacter pylori isolates were used in logistic regression models to discriminate duodenal ulcer from gastritis. Only BabA was a candidate for a universal virulence factor, but the low c statistic value (0.581) indicates that none of these factors were helpful in predicting the clinical presentation.

  • 1083.
    Yamazaki, Yasuo
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Schönherr, Christina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Varshney, Gaurav K.
    Dogru, Murat
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Palmer, Ruth H.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Goliath family E3 ligases regulate the recycling endosome pathway via VAMP3 ubiquitylation2013In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 32, no 4, p. 524-537Article in journal (Refereed)
    Abstract [en]

    Diverse cellular processes depend on endocytosis, intracellular vesicle trafficking, sorting and exocytosis, processes regulated post-transcriptionally by modifications such as phosphorylation and ubiquitylation. In addition to sorting to the lysosome, cargo is recycled to the plasma membrane via recycling endosomes. Here, we describe a role of the goliath gene family of protease-associated (PA) domain E3 ligases in regulating recycling endosome trafficking. The two Drosophila members of this family-Goliath and Godzilla(CG10277) - are located on endosomes, and both ectopic expression and loss-of-function lead to the accumulation of Rab5-positive giant endosomes. Furthermore, the human homologue RNF167 exhibits similar behaviour. We show that the soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein VAMP3 is a target of these ubiquitin ligases, and that recycling endosome trafficking is abrogated in response to their activity. Furthermore, mutation of the Godzilla ubiquitylation target lysines on VAMP3 abrogates the formation of enlarged endosomes induced by either Godzilla or RNF167. Thus, Goliath ubiquitin ligases play a novel role in regulating recycling endosome trafficking via ubiquitylation of the VAMP3 SNARE protein.

  • 1084.
    Yang, Hairu
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Drosophila skeletal muscles regulate the cellular immune response against wasp infection2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Drosophila melanogaster is widely used as a model organism to study the innate immune system because it lacks an adaptive immune response that could mask its innate immune response. The innate immune response of Drosophila primarily consists of humoral and cellular immune responses. The humoral immune response ismediated by antimicrobial peptides, and is induced by bacterial and fungal infections. The cellular immune response is mediated by blood cells (hemocytes), and is induced by bacterial and wasp infection. While the humoral immune response of Drosophila has been studied extensively, the cellular immune response is less well understood.

    In this work, I investigated the communication between different signaling pathways and tissues in Drosophila during infection by the parasitic wasp Leptopilina boulardi. I find that JAK/STAT signaling is strongly activated by wasp infection, in both hemocytes and (unexpectedly) larval skeletal muscles. This activation is mediated by the cytokines Upd2 and Upd3, which are secreted from circulating hemocytes. Deletion of upd2 or/and upd3 weakens the wasp-induced activation of JAK/STAT signaling in skeletal muscles and the cellular immune response to wasp infection, leading to reduced encapsulation of wasp eggs and a decrease in the number of circulating lamelloyctes. The suppression of JAK/STAT signaling also significantly weakens the cellular immune response in skeletal muscles, but not in fat bodies and hemocytes. However, the activation of this signaling in skeletal muscles has no obvious effect on the cellular immune response. Together, these results suggest that rather than being uninvolved bystanders, Drosophila skeletal musclesactively participate in cellular immune responses against wasp infection.

    To answer how Drosophila larval muscles participate cellular immune response, I min-screened the effects of several immune related signaling pathways in the muscles and the fat body on the cellular immune response. Interestingly, the cellular immune response was only significantly compromised by the suppression ofinsulin signaling in skeletal muscles, in a way that was veryreminiscent of the phenotypes induced by suppressing JAK/STAT signaling in muscles. While wasp infection activates JAK/STAT signaling in muscles, it has the opposite effect on insulin signaling. In addition, I find that insulin signaling in skeletal muscles can positively regulate JAK/STAT signaling. On the other hand, suppression of JAK/STAT signaling in muscles reduces insulin signaling locally in muscles and systemically in the fat body. Suppression of either insulin or JAK/STAT signaling in muscles leads to reductions in glycogen storage in muscles, the trehalose concentration in the hemolymph, and the frequency of feeding behavior. All these results indicate that JAK/STAT and insulin signaling in Drosophila skeletal muscles regulate cellular immune responses via their effects on carbohydrate metabolism. Our findings shed new light on the interactions between diabetes, metabolism, the immune system, and tissue communication.

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  • 1085.
    Yang, Hairu
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Institute of Biomedical Technology, University of Tampere, Tampere, Finland.
    Drosophila muscles regulate the immune response against wasp infection via carbohydrate metabolism2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 15713Article in journal (Refereed)
    Abstract [en]

    We recently found that JAK/STAT signaling in skeletal muscles is important for the immune response of Drosophila larvae against wasp infection, but it was not clear how muscles could affect the immune response. Here we show that insulin signaling is required in muscles, but not in fat body or hemocytes, during larval development for an efficient encapsulation response and for the formation of lamellocytes. This effect requires TOR signaling. We show that muscle tissue affects the immune response by acting as a master regulator of carbohydrate metabolism in the infected animal, via JAK/STAT and insulin signaling in the muscles, and that there is indirect positive feedback between JAK/STAT and insulin signaling in the muscles. Specifically, stimulation of JAK/STAT signaling in the muscles can rescue the deficient immune response when insulin signaling is suppressed. Our results shed new light on the interaction between metabolism, immunity, and tissue communication.

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  • 1086.
    Yang, Hairu
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    JAK/STAT and insulin signaling in Drosophila muscles regulate cellular immune responses against parasitoid wasp infectionManuscript (preprint) (Other academic)
  • 1087.
    Yang, Hairu
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). BioMediTech, University of Tampere, Tampere, Finland.
    Tissue communication in a systemic immune response of Drosophila.2016In: Fly, ISSN 1933-6942, Vol. 10, no 3, p. 115-122Article in journal (Refereed)
    Abstract [en]

    Several signaling pathways, including the JAK/STAT and Toll pathways, are known to activate blood cells (hemocytes) in Drosophila melanogaster larvae. They are believed to regulate the immune response against infections by parasitoid wasps, such as Leptopilina boulardi, but how these pathways control the hemocytes is not well understood. Here, we discuss the recent discovery that both muscles and fat body take an active part in this response. Parasitoid wasp infection induces Upd2 and Upd3 secretion from hemocytes, leading to JAK/STAT activation mainly in hemocytes and in skeletal muscles. JAK/STAT activation in muscles, but not in hemocytes, is required for an efficient encapsulation of wasp eggs. This suggests that Upd2 and Upd3 are important cytokines, coordinating different tissues for the cellular immune response in Drosophila. In the fat body, Toll signaling initiates a systemic response in which hemocytes are mobilized and activated hemocytes (lamellocytes) are generated. However, the contribution of Toll signaling to the defense against wasps is limited, probably because the wasps inject inhibitors that prevent the activation of the Toll pathway. In conclusion, parasite infection induces a systemic response in Drosophila larvae involving major organ systems and probably the physiology of the entire organism.

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  • 1088.
    Yang, Hairu
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kronhamn, Jesper
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ekstrom, Jens-Ola
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Korkut, Gul Gizem
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection2015In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 16, no 12, p. 1664-1672Article in journal (Refereed)
    Abstract [en]

    The role of JAK/STAT signaling in the cellular immune response of Drosophila is not well understood. Here, we show that parasitoid wasp infection activates JAK/STAT signaling in somatic muscles of the Drosophila larva, triggered by secretion of the cytokines Upd2 and Upd3 from circulating hemocytes. Deletion of upd2 or upd3, but not the related os (upd1) gene, reduced the cellular immune response, and suppression of the JAK/STAT pathway in muscle cells reduced the encapsulation of wasp eggs and the number of circulating lamellocyte effector cells. These results suggest that JAK/STAT signaling in muscles participates in a systemic immune defense against wasp infection.

  • 1089.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ottmann, Christian
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hauser, Alan R
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, no 2, p. 217-224Article in journal (Refereed)
    Abstract [en]

    14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.

  • 1090. Yeung, Kelvin
    et al.
    Boija, Ann
    Karlsson, Edvin
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of CBRN Security and Defence, FOI-Swedish Defence Research Agency, Umeå, Sweden.
    Holmqvist, Per-Henrik
    Tstskis, Yonit
    Nisoli, Ilaria
    Yap, Damian
    Lorzadeh, Alireza
    Moksa, Michelle
    Hirst, Martin
    Aparicio, Samuel
    Fanto, Manolis
    Stenberg, Per
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Division of CBRN Security and Defence, FOI-Swedish Defence Research Agency, Umeå, Sweden.
    Mannervik, Mattias
    McNeill, Helen
    Atrophin controls developmental signaling pathways via interactions with Trithorax-like2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e23084Article in journal (Refereed)
    Abstract [en]

    Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atros critical role in development and disease, relatively little is known about Atros binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.

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  • 1091.
    Yuan, Ming
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Antiphagocytosis by Yersinia pseudotuberculosis: role of the YopH target proteins2006Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1 integrins on a host cell via its surface protein invasin. This event stimulates signal transduction to the actin cytoskeleton of the eukaryotic cell, which allows the cell to engulf the bacterium that is attached to its surface. However, the pathogen Y. pseudotuberculosis can evade such phagocytosis by injecting virulence effectors that interfere with the antipathogenic machinery of the host cells. One of these virulence effectors is the tyrosine phosphatase YopH. Through its enzymatic activity, YopH blocks phagocytosis by affecting the signalling that is associated with cytoskeletal rearrangements.

    Cas is a substrate of YopH in both professional and non-professional phagocytes. We showed that YopH binds to the central substrate domain of Cas and that this interaction is required for YopH to target focal adhesion structures in host cells. We also demonstrated that YopH binds another substrate, FAK, through Cas. Moreover, we suggested that targeting of Cas is necessary for the cytotoxic effects mediated by YopH.

    The protein Fyb is specific to immune cells, and it has been identified as a substrate of YopH in macrophages. We discovered that both the N-terminal substrate-binding domain and the C-terminal catalytic region of YopH bind Fyb in a phosphotyrosine-dependent manner. Moreover, we observed that both the substrate-binding domain and the phosphatase activity of YopH are essential for the effects of this protein on macrophages, which include dephosphorylation of Fyb, blocking of phagocytosis, and cytotoxicity.

    The role of Fyb in macrophages is largely unknown, although there is evidence that this protein is involved in integrin-linked actin organization. We identified a novel interaction partner of Fyb, mAbp1, which is a protein that binds to F-actin. Studies in vitro indicated that mAbp1 binds to the N terminus of Fyb via a C-terminal SH3 domain. We also found that both Fyb and mAbp1 co-localize with F-actin at the leading edges of macrophages. Further studies suggested that mAbp1 influences the spreading of macrophages and the antiphagocytosis mediated by pathogenic Yersinia. These results support a role for Fyb in signalling that affects F-actin dynamics, and they also provide additional insight into the mechanisms involved. Fyb has been shown to form a complex with SKAP-HOM, another substrate of YopH in macrophages. Our data implied that the level of SKAP-HOM protein depends on the presence of Fyb, but the function of the Fyb/SKAP-HOM complex in macrophages has not been determined. However, since Fyb is the only known haematopoietic-specific substrate of YopH, it is possible that Fyb is involved in other antimicrobial functions.

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  • 1092.
    Yuan, Ming
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Carlsson, Sara E.
    Fahlgren, Anna
    Fällman, Maria
    Mammalian actin-binding protein 1 influences spreading of macrophagesManuscript (Other academic)
  • 1093.
    Yuan, Ming
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Deleuil, Fabienne
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb2005In: Journal of Molecular Biology and Biotechnology, ISSN 1464-1801, E-ISSN 1660-2412, Vol. 9, no 3-4, p. 214-223Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia species can evade phagocytosis by injecting virulence effectors that interfere with the phagocytic machinery of host cells. One of these virulence effectors is the protein tyrosine phosphatase YopH. Through its enzymatic activity, YopH interferes with the initial phagocytic process by affecting signalling for cytoskeletal rearrangements. Fyb (Fyn-binding protein), which is an immune cell-specific adaptor protein, has been identified as a substrate of YopH in macrophages. In this study, the interaction between YopH and Fyb is studied. We show that YopH binds to Fyb via different regions in both phosphotyrosine-dependent and phosphotyrosine-independent ways. The phosphotyrosine substrate binding N-terminal part (1-130) of YopH as well as the C-terminal catalytic region binds to Fyb in a phosphotyrosine-dependent manner. We also show that a central part of YopH (130-260) interacts with the Fyb C-terminus (548-783) in a phosphotyrosine-independent manner. Further, we demonstrate that the N-terminal binding region of YopH is important for YopH-mediated functions on macrophages such as dephosphorylation of Fyb, blockage of phagocytosis, and cytotoxic effects. Copyright (c) 2005 S. Karger AG, Basel.

  • 1094.
    Yuan, Ming
    et al.
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Mogemark, Lena
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine).
    Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1.2005In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, no 11, p. 2339-2347Article in journal (Refereed)
    Abstract [en]

    The immune cell specific protein Fyn-T binding protein (Fyb) has been identified as a target of the Yersinia antiphagocytic effector Yersinia outer protein H (YopH), but its role in macrophages is unknown. By using Fyb domains as bait to screen a mouse lymphoma cDNA library, we identified a novel interaction partner, mammalian actin binding protein 1 (mAbp1). We show that mAbp1 binds the Fyb N-terminal via its C-terminally located src homology 3 domain. The interaction between Fyb and mAbp1 is detected in macrophage lysates and the proteins co-localize with F-actin in the leading edge. Hence, mAbp1 is likely to constitute a downstream effector of Fyb involved in F-actin dynamics.

  • 1095. Zbornikova, Eva
    et al.
    Knejzlik, Zdenek
    Hauryliuk, Vasili
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). University of Tartu, Institute of Technology, Tartu,Estonia.
    Krasny, Libor
    Rejman, Dominik
    Analysis of nucleotide pools in bacteria using HPLC-MS in HILIC mode2019In: Talanta: The International Journal of Pure and Applied Analytical Chemistry, ISSN 0039-9140, E-ISSN 1873-3573, Vol. 205, article id 120161Article in journal (Refereed)
    Abstract [en]

    Nucleotides, nucleosides and their derivatives are present in all cells at varying concentrations that change with the nutritional, and energetic status of the cell. Precise measurement of the concentrations of these molecules is instrumental for understanding their regulatory effects. Such measurement is challenging due to the inherent instability of these molecules and, despite many decades of research, the reported values differ widely. Here, we present a comprehensive and easy-to-use approach for determination of the intracellular concentrations of > 25 target molecular species. The approach uses rapid filtration and cold acidic extraction followed by high performance liquid chromatography (HPLC) in the hydrophilic interaction liquid chromatography (HILIC) mode using zwitterionic columns coupled with UV and MS detectors. The method reliably detects and quantifies all the analytes expected to be observed in the bacterial cell and paves the way for future studies correlating their concentrations with biological effects.

  • 1096.
    Zhang, Hanqing
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Söderholm, Niklas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics.
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics.
    DSeg: a dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures2019In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 25, no 3, p. 711-719Article in journal (Refereed)
    Abstract [en]

    Analysis of numerous filamentous structures in an image is often limited by the ability of algorithms to accurately segment complex structures or structures within a dense population. It is even more problematic if these structures continuously grow when recording a time-series of images. To overcome these issues we present DSeg; an image analysis program designed to process time-series image data, as well as single images, to segment filamentous structures. The program includes a robust binary level-set algorithm modified to use size constraints, edge intensity, and past information. We verify our algorithms using synthetic data, differential interference contrast images of filamentous prokaryotes, and transmission electron microscopy images of bacterial adhesion fimbriae. DSeg includes automatic segmentation, tools for analysis, and drift correction, and outputs statistical data such as persistence length, growth rate, and growth direction. The program is available at Sourceforge.

  • 1097.
    Zhang, Zhen
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Aung, Kyaw Min
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Reversible senescence of human colon cancer cells after blockage of mitosis/cytokinesis caused by the CNF1 cyclomodulin from Escherichia coli2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 17780Article in journal (Refereed)
    Abstract [en]

    Cytotoxic necrotizing factor 1 (CNF1), a protein toxin produced by extraintestinal pathogenic Escherichia coli, activates the Rho-family small GTPases in eukaryotic cell, thereby perturbing multiple cellular functions. Increasing epidemiological evidence suggests a link between CNF1 and human inflammatory bowel disease and colorectal cancer. At the cellular level, CNF1 has been hypothesized to reprogram cell fate towards survival due to the role in perturbing cell cycle and apoptosis. However, it remains undetermined how cells survive from CNF1 intoxication. In this work, we show that CNF1 treatment blocks mitosis/cytokinesis, elicits endoreplication and polyploidisation in cultured human colon cancer cells, and drives them into reversible senescence, which provides a survival route for cells via depolyploidisation. Senescence in CNF1-treated cells is demonstrated with upregulation of several senescence markers including senescence-associated β-galactosidase activity, p53, p21 and p16, and concomitant inhibition of the retinoblastoma protein phosphorylation. Importantly, progeny derived from CNF1 treatment exhibit genomic instability exemplified by increased aneuploidy and become more resistant to CNF1, but not to 5-fluorouracil and oxaliplatin, the two agents commonly used in chemotherapeutic treatment for colorectal cancer. These observations display survival features of the cell after CNF1 treatment that may have implications for the potential role of CNF1 in carcinogenesis.

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  • 1098.
    Zlatkov, Nikola
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Regulatory mechanisms involved in pathoadaptation of extraintestinal pathogenic Escherichia coli2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Establishment of commensal bacteria within a new niche of their host usually promotes the transition from commensalism to pathogenicity. Extraintestinal Pathogenic Escherichia coli (ExPEC) represent different pathovars with biphasic lifestyle – they can reside in the gut as commensals or they can escape and cause diseases elsewhere in the human body. Depending on the disease they are linked to, ExPEC can be divided into Uropathogenic E. coli (UPEC), Newborn Meningitis-causing E. coli (NMEC) and Sepsis-associated E. coli (SEPEC).

    Pathoadaptive mutations linked to c-di-GMP signaling were investigated in the NMEC strain IHE3034 which lacks the main global stress regulator RpoS. We investigated the role of ycgG2 in the lifestyle of NMEC. Deletion of ycgG2, shown by us to encode an YcgG allozyme with c-di-GMP phosphodiesterase (PDE) activity, and the restored RpoS led to a decrease in the S-fimbriae, otherwise robustly produced in artificial urine, hinting that the urinary tract could serve as a habitat for NMEC. We showed that NMEC were capable of aerobic citrate utilization in the presence of a co-substrate - a property that normally does not exist in E. coli. Our data hint that this metabolic upgrade is enhanced by the lack, or reduced activity, of c-di-GMP PDEs. We also found that citrate utilization is a property of ExPEC, since we reconstituted it in E. coli UTI89 (a cystitis isolate) via inactivation of its RpoS, and since a set of pyelonephritis E. coli isolates use citrate aerobically in the presence of glucose. The main reason for this metabolic capability is the absence of RpoS which leads to the production of the citrate transporter CitT. Furthermore, we highlighted the deletion of the fec operon (required for the ferric citrate uptake) in a large group of different ExPEC strains and we showed that NMEC can use CitT for in vitro ferric citrate uptake dependent on YcgG2 as an alternative system.

    Another pathoadaptive mutation which influences the fitness of ExPEC is the clyA (cytolysin A) gene inactivation, resulting from different deletions in different ExPEC genomes. When we restored the clyA+ locus, the UPEC strain 536 displayed increased susceptibility to antimicrobial peptides and urea. We also showed that the ClyA expression in 536 was increased by the presence of the DNA-binding regulator SfaX and another stand-alone PDE similar to YcgG2, called SfaY. The results were further confirmed by ClyA downregulation in NMEC deficient in SfaY and SfaX.

    We also studied the role of sfaY - a gene coding for another stand-alone c-di-GMP PDE. The expression of sfaY is under the regulation of the main promoter of the horizontally acquired sfa gene cluster. The latter is responsible for the regulation and assembly of the virulence-associated S-fimbriae, via which ExPEC bacteria bind to sialylated receptors. We found that NMEC are competent for filamentation because of a c-di-GMP-dependent program under the control of a phase-variation event which selectively turns ‘ON’ the sfa promoter in a subpopulation of bacteria. When SfaY is present, c-di-GMP levels are reduced, thus inducing the SOS stress response via the canonical LexA-RecA pathway. The signaling resulted in an internal differentiation of the bacterial population into a subpopulation exhibiting a filamentous morphotype (bacteria with induced SOS stress response) and a subpopulation of small motile and non-motile bacteria. Hence, this molecular program could serve as a clue to explain the formation of the intracellular bacterial communities observed during urinary tract infection by UPEC.

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  • 1099.
    Zlatkov, Nikola
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Absence of Global Stress Regulation in Escherichia coli Promotes Pathoadaptation and Novel c-di-GMP-dependent Metabolic Capability2019In: Scientific Reports, ISSN 2045-2322, Vol. 9, article id 2600Article in journal (Refereed)
    Abstract [en]

    athoadaptive mutations linked to c-di-GMP signalling were investigated in neonatal meningitis-causing Escherichia coli (NMEC). The results indicated that NMEC strains deficient in RpoS (the global stress regulator) maintained remarkably low levels of c-di-GMP, a major bacterial sessility-motility switch. Deletion of ycgG2, shown here to encode a YcgG allozyme with c-di-GMP phosphodiesterase activity, and the restoration of RpoS led to a decrease in S-fimbriae, robustly produced in artificial urine, hinting that the urinary tract could serve as a habitat for NMEC. We showed that NMEC were skilled in aerobic citrate utilization in the presence of glucose, a property that normally does not exist in E. coli. Our data suggest that this metabolic novelty is a property of extraintestinal pathogenic E. coli since we reconstituted this ability in E. coli UTI89 (a cystitis isolate) via deactivation rpoS; additionally, a set of pyelonephritis E. coli isolates were shown here to aerobically use citrate in the presence of glucose. We found that the main reason for this metabolic capability is RpoS inactivation leading to the production of the citrate transporter CitT, exploited by NMEC for ferric citrate uptake dependent on YcgG2 (an allozyme with c-di-GMP phosphodiesterase activity).

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    fulltext
  • 1100.
    Zlatkov, Nikola
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    C-di-GMP-mediated Morphotypic Pathoadaptability of Neonatal Meningitis Escherichia coliManuscript (preprint) (Other academic)
1920212223 1051 - 1100 of 1119
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