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  • 1001.
    Westermark, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Yersinia-phagocyte interactions during early infection2013Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 1002.
    Westermark, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTRtg model for studies of bacterial gastrointestinal infections2012Ingår i: Immunology, ISSN 0019-2805, E-ISSN 1365-2567, Vol. 137, nr S1, s. 271-271Artikel i tidskrift (Övrigt vetenskapligt)
  • 1003.
    Westermark, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Immune response to diphtheria toxin-mediated depletion complicates the use of the CD11c-DTR(tg) model for studies of bacterial gastrointestinal infections2012Ingår i: Microbial Pathogenesis, ISSN 0882-4010, E-ISSN 1096-1208, Vol. 53, nr 3-4, s. 154-161Artikel i tidskrift (Refereegranskat)
    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.

  • 1004.
    Westermark, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Yersinia pseudotuberculosis efficiently avoids polymorphonuclear neutrophils during early infectionManuskript (preprint) (Övrigt vetenskapligt)
  • 1005.
    Westermark, Linda
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Fahlgren, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection2014Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, nr 3, s. 1181-1191Artikel i tidskrift (Refereegranskat)
    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.

  • 1006. Widhe, M
    et al.
    Ekerfelt, C
    Jarefors, S
    Skogman, B H
    Peterson, E M
    Bergström, S
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Forsberg, P
    Ernerudh, J
    T-cell epitope mapping of the Borrelia garinii outer surface protein A in lyme neuroborreliosis.2009Ingår i: Scandinavian Journal of Immunology, ISSN 0300-9475, E-ISSN 1365-3083, Vol. 70, nr 2, s. 141-8Artikel i tidskrift (Refereegranskat)
    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.

  • 1007. Widhe, Mona
    et al.
    Jarefors, Sara
    Ekerfelt, Christina
    Vrethem, Magnus
    Bergström, Sven
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    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.2004Ingår i: J Infect Dis, ISSN 0022-1899, Vol. 189, nr 10, s. 1881-91Artikel i tidskrift (Refereegranskat)
    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.

  • 1008. Widhe, Mona
    et al.
    Skogman, Barbro Hedin
    Jarefors, Sara
    Eknefelt, Mattias
    Eneström, Gunilla
    Nordwall, Maria
    Ekerfelt, Christina
    Croner, Stefan
    Bergström, Sven
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Forsberg, Pia
    Ernerudh, Jan
    Up-regulation of Borrelia-specific IL-4- and IFN-gamma-secreting cells in cerebrospinal fluid from children with Lyme neuroborreliosis.2005Ingår i: Int Immunol, ISSN 0953-8178, Vol. 17, nr 10, s. 1283-91Artikel i tidskrift (Refereegranskat)
    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.

  • 1009. Wieser, Andreas
    et al.
    Storz, Enno
    Liegl, Gabriele
    Peter, Annabell
    Pritsch, Michael
    Shock, Jonathan
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Schubert, Soeren
    Efficient quantification and characterization of bacterial outer membrane derived nano-particles with flow cytometric analysis2014Ingår i: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 304, nr 8, s. 1032-1037Artikel i tidskrift (Refereegranskat)
    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.

  • 1010.
    Wikberg, Maria L.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    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 vivo2002Ingår i: European Journal of Biochemistry, Vol. 269, nr 20, s. 4921-4929Artikel i tidskrift (Refereegranskat)
  • 1011.
    Wiklund, Peder
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Geriatrik. Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Rehabiliteringsmedicin.
    Nordström, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Rehabiliteringsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin.
    Högström, Magnus
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin.
    Alfredson, Håkan
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin.
    Engström, Patrik
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Gustafsson, Thomas
    Karolinska universitetslaboratoriet, Klinisk kemi.
    Franks, Paul
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Medicin.
    Nordström, Peter
    Umeå universitet, Medicinska fakulteten, Institutionen för kirurgisk och perioperativ vetenskap, Idrottsmedicin. Umeå universitet, Medicinska fakulteten, Institutionen för samhällsmedicin och rehabilitering, Geriatrik.
    High impact loading on the skeleton is associated with a decrease in glucose levels in young men2012Ingår i: Clinical Endocrinology, ISSN 0300-0664, E-ISSN 1365-2265, Vol. 77, nr 6, s. 823-827Artikel i tidskrift (Refereegranskat)
    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.

  • 1012.
    Wikström Hultdin, Ulrika
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Stina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Allgardsson, Anders
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Stier, Günter
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Öhman, Anders
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010Ingår i: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 66, nr Pt 3, s. 337-341Artikel i tidskrift (Refereegranskat)
    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.

  • 1013.
    Wikström Hultdin, Ulrika
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Stina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Grundström, Christin
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Huang, Shenghua
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sauer-Eriksson, Elisabeth
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Structure of FocB: a member of a family of transcription factors regulating fimbrial adhesin expression in uropathogenic Escherichia coli2010Ingår i: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, nr 16, s. 3368-3381Artikel i tidskrift (Refereegranskat)
    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.

  • 1014. Wilhelmsson, Peter
    et al.
    Fryland, Linda
    Börjesson, Stefan
    Nordgren, Johan
    Bergström, Sven
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Ernerudh, Jan
    Forsberg, Pia
    Lindgren, Per-Eric
    Prevalence and diversity of Borrelia species in ticks that have bitten humans in Sweden2010Ingår i: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 48, nr 11, s. 4169-4176Artikel i tidskrift (Refereegranskat)
    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.

  • 1015. Wilson, Sara I
    et al.
    Edlund, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Neural induction: toward a unifying mechanism2001Ingår i: Nature Neuroscience, ISSN 1097-6256, E-ISSN 1546-1726, s. 1161-1168Artikel, forskningsöversikt (Refereegranskat)
    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.

  • 1016.
    Wirebrand, Lisa
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Madhushani, Anjana W. K.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Irie, Yasuhiko
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF6002018Ingår i: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 20, nr 1, s. 186-199Artikel i tidskrift (Refereegranskat)
    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.

  • 1017.
    Witek, Barbara
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    El Wakil, Abeer
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Nord, Christoffer
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Ahlgren, Ulf
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Eriksson, Maria
    Umeå universitet, Medicinska fakulteten, Umeå centrum för molekylär medicin (UCMM).
    Vernersson-Lindahl, Emma
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Helland, Aslaug
    Alexeyev, Oleg A.
    Umeå universitet, Medicinska fakulteten, Institutionen för medicinsk biovetenskap, Patologi.
    Hallberg, Bengt
    Palmer, Ruth H.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 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 Hypogonadism2015Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 10, nr 5, artikel-id e0123542Artikel i tidskrift (Refereegranskat)
    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.

  • 1018. Wolfstetter, Georg
    et al.
    Shirinian, Margret
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Stute, Christiana
    Grabbe, Caroline
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hummel, Thomas
    Baumgartner, Stefan
    Palmer, Ruth H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    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.2009Ingår i: Mechanisms of Development, ISSN 0925-4773, E-ISSN 1872-6356, Vol. 126, nr 8-9, s. 721-736Artikel i tidskrift (Refereegranskat)
    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.

  • 1019. Wolf-Watz, M
    et al.
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, T
    Structure and backbone dynamics of Apo-CBFbeta in solution.2001Ingår i: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, nr 38, s. 11423-11432Artikel i tidskrift (Refereegranskat)
    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.

  • 1020. Wolf-Watz, M
    et al.
    Xie, X Q
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Holm, M
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, T
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Härd, T
    Solution properties of the free and DNA-bound Runt domain of AML1.1999Ingår i: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 261, nr 1, s. 251-60Artikel i tidskrift (Refereegranskat)
    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.

  • 1021.
    Wolf-Watz, Magnus
    et al.
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Kemi.
    Bäckström, Stefan
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Grundström, Thomas
    Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sauer, Uwe
    Umeå centrum för molekylär patogenes (UCMP) (Teknisk-naturvetenskaplig fakultet).
    Härd, Torleif
    Chloride binding by the AML1/Runx1 transcription factor studied by NMR2001Ingår i: FEBS Letters, ISSN 0014-5793, Vol. 488, nr 1-2, s. 81-4Artikel i tidskrift (Refereegranskat)
    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.

  • 1022.
    Xie, Xiao-Qi
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Pardali, Evangelia
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Holm, Magnus
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Sideras, Paschalis
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Grundström, Thomas
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    AML and Ets proteins regulate the I alpha1 germ-line promoter.1999Ingår i: European Journal of Immunology, ISSN 0014-2980, E-ISSN 1521-4141, Vol. 29, nr 2, s. 488-498Artikel i tidskrift (Refereegranskat)
    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.

  • 1023.
    Yadav, Akhilesh K.
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Espaillat, Akbar
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Bacterial Strategies to Preserve Cell Wall Integrity Against Environmental Threats2018Ingår i: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, artikel-id 2064Artikel, forskningsöversikt (Refereegranskat)
    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.

  • 1024. Yamamoto, Shouji
    et al.
    Mitobe, Jiro
    Ishikawa, Takahiko
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (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 cholerae2014Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 91, nr 2, s. 326-347Artikel i tidskrift (Refereegranskat)
    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.

  • 1025. Yamaoka, Yoshio
    et al.
    Souchek, Julianne
    Odenbreit, Stefan
    Haas, Rainer
    Arnqvist, Anna
    Umeå universitet, Medicinska fakulteten, Institutionen för odontologi, Oral mikrobiologi. Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Borén, Thomas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    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.2002Ingår i: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, nr 6, s. 2244-2246Artikel i tidskrift (Refereegranskat)
    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.

  • 1026.
    Yamazaki, Yasuo
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Schönherr, Christina
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Varshney, Gaurav K.
    Dogru, Murat
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Palmer, Ruth H.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Goliath family E3 ligases regulate the recycling endosome pathway via VAMP3 ubiquitylation2013Ingår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 32, nr 4, s. 524-537Artikel i tidskrift (Refereegranskat)
    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.

  • 1027.
    Yang, Hairu
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Drosophila skeletal muscles regulate the cellular immune response against wasp infection2016Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 1028.
    Yang, Hairu
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hultmark, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Institute of Biomedical Technology, University of Tampere, Tampere, Finland.
    Drosophila muscles regulate the immune response against wasp infection via carbohydrate metabolism2017Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, artikel-id 15713Artikel i tidskrift (Refereegranskat)
    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.

  • 1029.
    Yang, Hairu
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hultmark, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    JAK/STAT and insulin signaling in Drosophila muscles regulate cellular immune responses against parasitoid wasp infectionManuskript (preprint) (Övrigt vetenskapligt)
  • 1030.
    Yang, Hairu
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hultmark, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). BioMediTech, University of Tampere, Tampere, Finland.
    Tissue communication in a systemic immune response of Drosophila.2016Ingår i: Fly, ISSN 1933-6942, Vol. 10, nr 3, s. 115-122Artikel i tidskrift (Refereegranskat)
    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.

  • 1031.
    Yang, Hairu
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Kronhamn, Jesper
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Ekstrom, Jens-Ola
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Korkut, Gul Gizem
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hultmark, Dan
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    JAK/STAT signaling in Drosophila muscles controls the cellular immune response against parasitoid infection2015Ingår i: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 16, nr 12, s. 1664-1672Artikel i tidskrift (Refereegranskat)
    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.

  • 1032.
    Yasmin, Lubna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Francis, Matthew S
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Ottmann, Christian
    Palmer, Ruth H
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Hauser, Alan R
    Hallberg, Bengt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins2010Ingår i: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, nr 2, s. 217-224Artikel i tidskrift (Refereegranskat)
    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.

  • 1033. Yeung, Kelvin
    et al.
    Boija, Ann
    Karlsson, Edvin
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 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 universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). 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-like2017Ingår i: eLIFE, E-ISSN 2050-084X, Vol. 6, artikel-id e23084Artikel i tidskrift (Refereegranskat)
    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.

  • 1034.
    Yuan, Ming
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Antiphagocytosis by Yersinia pseudotuberculosis: role of the YopH target proteins2006Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 1035.
    Yuan, Ming
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Carlsson, Sara E.
    Fahlgren, Anna
    Fällman, Maria
    Mammalian actin-binding protein 1 influences spreading of macrophagesManuskript (Övrigt vetenskapligt)
  • 1036.
    Yuan, Ming
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Deleuil, Fabienne
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Interaction between the Yersinia tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein, Fyb2005Ingår i: Journal of Molecular Biology and Biotechnology, ISSN 1464-1801, E-ISSN 1660-2412, Vol. 9, nr 3-4, s. 214-223Artikel i tidskrift (Refereegranskat)
    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.

  • 1037.
    Yuan, Ming
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Mogemark, Lena
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Fällman, Maria
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Fyn binding protein, Fyb, interacts with mammalian actin binding protein, mAbp1.2005Ingår i: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 579, nr 11, s. 2339-2347Artikel i tidskrift (Refereegranskat)
    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.

  • 1038.
    Zhang, Hanqing
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Söderholm, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Sandblad, Linda
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wiklund, Krister
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Andersson, Magnus
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    DSeg: a dynamic image segmentation program to extract backbone patterns for filamentous bacteria and hyphae structures2019Ingår i: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 25, nr 3, s. 711-719Artikel i tidskrift (Refereegranskat)
    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.

  • 1039.
    Zhang, Zhen
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Aung, Kyaw Min
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Wai, Sun Nyunt
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Reversible senescence of human colon cancer cells after blockage of mitosis/cytokinesis caused by the CNF1 cyclomodulin from Escherichia coli2018Ingår i: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, artikel-id 17780Artikel i tidskrift (Refereegranskat)
    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.

  • 1040.
    Zlatkov, Nikola
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Regulatory mechanisms involved in pathoadaptation of extraintestinal pathogenic Escherichia coli2019Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    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.

  • 1041.
    Zlatkov, Nikola
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Absence of Global Stress Regulation in Escherichia coli Promotes Pathoadaptation and Novel c-di-GMP-dependent Metabolic Capability2019Ingår i: Scientific Reports, ISSN 2045-2322, Vol. 9, artikel-id 2600Artikel i tidskrift (Refereegranskat)
    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).

  • 1042.
    Zlatkov, Nikola
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Umeå Centre for Microbial Research (UCMR). Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS). Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    C-di-GMP-mediated Morphotypic Pathoadaptability of Neonatal Meningitis Escherichia coliManuskript (preprint) (Övrigt vetenskapligt)
  • 1043. Zocher, Georg
    et al.
    Mistry, Nitesh
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi.
    Frank, Martin
    Hähnlein-Schick, Irmgard
    Ekström, Jens-Ola
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Arnberg, Niklas
    Umeå universitet, Medicinska fakulteten, Institutionen för klinisk mikrobiologi, Virologi. Umeå universitet, Medicinska fakulteten, Molekylär Infektionsmedicin, Sverige (MIMS).
    Stehle, Thilo
    A sialic acid binding site in a human picornavirus2014Ingår i: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, nr 10, s. e1004401-Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 angstrom resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for alpha 2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that alpha 2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC.

  • 1044.
    Åberg, Anna
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    New insights into the role of ppGpp and DksA through their effect on transcriptional regulation of housekeeping and colonization related genes of Escherichia coli2008Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
    Abstract [en]

    Bacteria have the ability to sense different environmental signals. When an environmental stress is detected, bacteria rapidly adjust their gene expression profile to be able to survive and thrive. The transduction of such environmental signals often requires the coordinated involvement of several factors that constitute complex regulatory networks. Hence, depending on the combination of signals, a unique gene expression profile required to adapt to the specific stress conditions is generated. Proteins are the best-known regulatory factors. However, non-proteinaceous molecules are also important in signal-responsive control of bacterial gene expression. Alarmones are low molecular weight non-proteinaceous regulatory factors which can characteristically be rapidly turned-over to mediate instant changes in gene expression. One such alarmone is the modified nucleotide ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of this alarmone are expected to rapidly increase in response to any environmental stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that likewise directly targets RNA polymerase, has been suggested to be required for both the positive and negative regulation mediated by ppGpp in Escherichia coli.

    This thesis describes dissection of the role of ppGpp and DksA on transcriptional regulation, primarily using the fim genetic determinant that encodes for the type 1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial adhesion/invasion of bladder cells, as well as in biofilm formation. We found that ppGpp regulates phase variation by increasing the sub-population of cells that express the fimbriae. The effect of ppGpp was ultimately traced to its role in transcription of the fimB gene that encodes a recombinase involved in the phase variation process (paper 1). In contrast, we unexpectedly found that lack of DksA causes an increase, rather than a decrease, in transcription from the fimB P2 promoter in vivo. However, in vitro transcription studies demonstrated that ppGpp and DksA, both independently and co-dependently, stimulate transcription from the fimB P2 promoter. These seemingly contradictory results from the in vivo and in vitro transcriptional studies were shown to be, at least in part, a consequence of the increased association of Gre-factors with RNA polymerase that can occur in the absence of DksA in vivo (paper 2).

    The results outlined above have implications for the role of ppGpp and/or DksA in global gene expression. Using gene expression profile (microarray analysis) during the transition from logarithmic to stationary phase of E. coli, we found that while most of the genes regulated by ppGpp and DksA are regulated in the same direction by the two factors, many were not. In addition to the fim genes, genes involved in flagella functioning, taxis responses, and a few genes encoding different transport systems are also differentially regulated in ppGpp- and DksA-deficient strains in vivo. Our results clearly indicate that the effect of deficiencies in ppGpp and DksA is far more complex than phenotypic similarity of the corresponding mutants anticipated by the proposed concerted action of ppGpp and DksA on gene expression (paper 2 & 3).

  • 1045.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Fernández, Jorge
    Sánchez, Alex
    Balsalobre, Carlos
    Global gene regulation by ppGpp and DksA in Escherichia coli: a transcriptomic approachManuskript (Övrigt vetenskapligt)
  • 1046.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Fernández-Vázquez, Jorge
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Cabrer-Panes, Juan David
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Sánchez, Alex
    Departament d'Estadística, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Balsalobre, Carlos
    Departament de Microbiologia, Universitat de Barcelona, Avgda. Diagonal 645, 08028 Barcelona, Spain.
    Similar and divergent effects of ppGpp and DksA deficiencies on transcription in Escherichia coli2009Ingår i: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 191, nr 10, s. 3226-3236Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The concerted action of ppGpp and DksA in transcription has been widely documented. In disparity with this model, phenotypic studies showed that ppGpp and DksA might also have independent and opposing roles in gene expression in Escherichia coli. In this study we used a transcriptomic approach to compare the global transcriptional patterns of gene expression in strains deficient in ppGpp (ppGpp(0)) and/or DksA (DeltadksA). Approximately 6 and 7% of all genes were significantly affected by more than twofold in ppGpp- and DksA-deficient strains, respectively, increasing to 13% of all genes in the ppGpp(0) DeltadksA strain. Although the data indicate that most of the affected genes were copositively or conegatively regulated by ppGpp and DksA, some genes that were independently and/or differentially regulated by the two factors were found. The large functional group of chemotaxis and flagellum synthesis genes were notably differentially affected, with all genes being upregulated in the DksA-deficient strain but 60% of them being downregulated in the ppGpp-deficient strain. Revealingly, mutations in the antipausing Gre factors suppress the upregulation observed in the DksA-deficient strain, emphasizing the importance of the secondary channel of the RNA polymerase for regulation and fine-tuning of gene expression in E. coli.

  • 1047.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Balsalobre, Carlos
    (p)ppGpp regulates type 1 fimbriation of Escherichia coli by modulating the expression of the site-specific recombinase FimB.2006Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 60, nr 6, s. 1520-1533Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    In this report we have examined the role of the regulatory alarmone (p)ppGpp on expression of virulence determinants of uropathogenic Escherichia coli strains. The ability to form biofilms is shown to be markedly diminished in (p)ppGpp-deficient strains. We present evidence (i) that (p)ppGpp tightly regulates expression of the type 1 fimbriae in both commensal and pathogenic E. coli isolates by increasing the subpopulation of cells that express the type 1 fimbriae; and (ii) that the effect of (p)ppGpp on the number of fimbrial expressing cells can ultimately be traced to its role in transcription of the fimB recombinase gene, whose product mediates inversion of the fim promoter to the productive (ON) orientation. Primer extension analysis suggests that the effect of (p)ppGpp on transcription of fimB occurs by altering the activity of only one of the two fimB promoters. Furthermore, spontaneous mutants with properties characteristic of ppGpp(0) suppressors restore fimB transcription and consequent downstream effects in the absence of (p)ppGpp. Consistently, the rpoB3770 allele also fully restores transcription of fimB in a ppGpp(0) strain and artificially elevated levels of FimB bypass the need for (p)ppGpp for type 1 fimbriation. Our findings suggest that the (p)ppGpp-stimulated expression of type 1 fimbriae may be relevant during the interaction of pathogenic E. coli with the host.

  • 1048.
    Åberg, Anna
    et al.
    Umeå universitet, Medicinsk fakultet, Molekylärbiologi (Medicinska fakulteten).
    Shingler, Victoria
    Umeå universitet, Teknisk-naturvetenskaplig fakultet, Molekylärbiologi (Teknisk-naturvetenskaplig fakultet).
    Balsalobre, Carlos
    Regulation of the fimB promoter: a case of differential regulation by ppGpp and DksA in vivo2008Ingår i: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 67, nr 6, s. 1223-1241Artikel i tidskrift (Refereegranskat)
  • 1049.
    Åberg, Veronica
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Fällman, Erik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Axner, Ove
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Institutionen för fysik.
    Uhlin, Bernt Eric
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Hultgren, Scott J.
    Department of Molecular Microbiology, Washington University in St. Louis School of Medicine, St. Louis,USA.
    Almqvist, Fredrik
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod2007Ingår i: Molecular BioSystems, ISSN 1742-206X, Vol. 3, s. 214-218Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The infectious ability of uropathogenic Escherichia coli relies on adhesive fibers, termed pili or fimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that are formed via a highly preserved assembly and secretion system called the chaperone-usher pathway. We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt both type 1 and P pilus biogenesis in E. coli. In this study, we show that the pilicides do not affect the structure, dynamics or function of the pilus rod. This was demonstrated by first suppressing the expression of P pili in E. coli by pilicide treatment and, next, measuring the biophysical properties of the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic force microscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers were determined by optical tweezers force measurements on individual pili and were found to be intact. The presented results establish a potential use of pilicides as chemical tools to study important biological processes e.g. adhesion, pilus biogenesis and the role of pili in infections and biofilm formation.

  • 1050. Åkesson, Per
    et al.
    Moritz, Linnea
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    Truedsson, Mikael
    Christensson, Bertil
    von Pawel-Rammingen, Ulrich
    Umeå universitet, Medicinska fakulteten, Institutionen för molekylärbiologi (Medicinska fakulteten).
    IdeS, a highly specific immunoglobulin G (IgG)-cleaving enzyme from Streptococcus pyogenes, is inhibited by specific IgG antibodies generated during infection2006Ingår i: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 74, nr 1, s. 497-503Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    IdeS, a recently discovered cysteine proteinase secreted by the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G. The fact that the enzyme targets one of the key molecules of the adapted immune response raised the question of whether an antibody response against IdeS could inhibit, i.e., neutralize, enzyme activity. Paired acute- and convalescent-phase serum samples from patients with pharyngotonsillitis (n = 10), bacteremia (n = 7), and erysipelas (n = 4) were analyzed. Antibodies with the ability to neutralize IdeS enzymatic activity were already found in two-thirds of acute-phase sera. However, patients who seroconverted to IdeS, in particular patients with pharyngotonsillitis and erysipelas, developed specific antibodies during convalescence with an increased capability to efficiently neutralize the enzymatic activity of IdeS. Also, the presence of neutralizing antibodies decreased the ability of IdeS to mediate bacterial survival in human immune blood. In patients with bacteremia, several acute-phase sera contained neutralizing antibodies, but no correlation was found to severity or outcome of invasive infections. Still, the fact that the human immune response targets the enzymatic activity of IdeS supports the view that the enzyme plays an important role during streptococcal infection.

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