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  • 301.
    Surowiec, Izabella
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gouveia-Figueira, Sandra
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Orikiiriza, Judy
    Lindquist, Elisabeth
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bonde, Mari
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Magambo, Jimmy
    Muhinda, Charles
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    The oxylipin and endocannabidome responses in acute phase Plasmodium falciparum malaria in children2017In: Malaria Journal, ISSN 1475-2875, E-ISSN 1475-2875, Vol. 16, article id 358Article in journal (Refereed)
    Abstract [en]

    Background: Oxylipins and endocannabinoids are low molecular weight bioactive lipids that are crucial for initiation and resolution of inflammation during microbial infections. Metabolic complications in malaria are recognized contributors to severe and fatal malaria, but the impact of malaria infection on the production of small lipid derived signalling molecules is unknown. Knowledge of immunoregulatory patterns of these molecules in malaria is of great value for better understanding of the disease and improvement of treatment regimes, since the action of these classes of molecules is directly connected to the inflammatory response of the organism.

    Methods: Detection of oxylipins and endocannabinoids from plasma samples from forty children with uncomplicated and severe malaria as well as twenty controls was done after solid phase extraction followed by chromatography mass spectrometry analysis. The stable isotope dilution method was used for compound quantification. Data analysis was done with multivariate (principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA (R)) and univariate approaches (receiver operating characteristic (ROC) curves, t tests, correlation analysis).

    Results: Forty different oxylipin and thirteen endocannabinoid metabolites were detected in the studied samples, with one oxylipin (thromboxane B2, TXB2) in significantly lower levels and four endocannabinoids (OEA, PEA, DEA and EPEA) at significantly higher levels in infected individuals as compared to controls according to t test analysis with Bonferroni correction. Three oxylipins (13-HODE, 9-HODE and 13-oxo-ODE) were higher in severe compared to uncomplicated malaria cases according to the results from multivariate analysis. Observed changes in oxylipin levels can be connected to activation of cytochrome P450 (CYP) and 5-lipoxygenase (5-LOX) metabolic pathways in malaria infected individuals compared to controls, and related to increased levels of all linoleic acid oxylipins in severe patients compared to uncomplicated ones. The endocannabinoids were extremely responsive to malaria infection with majority of this class of molecules found at higher levels in infected individuals compared to controls.

    Conclusions: It was possible to detect oxylipin and endocannabinoid molecules that can be potential biomarkers for differentiation between malaria infected individuals and controls and between different classes of malaria. Metabolic pathways that could be targeted towards an adjunctive therapy in the treatment of malaria were also pinpointed.

  • 302.
    Surowiec, Izabella
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Orikiiriza, Judy
    Karlsson, Elisabeth
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nelson, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bonde, Mari
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Kyamanwa, Patrick
    Karenzi, Ben
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Trygg, Johan
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Normark, Johan
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases. nfectious Diseases Institute, School of Medicine and Health Sciences, Makerere University, Uganda.
    Metabolic signature profiling as a diagnostic and prognostic tool in paediatric Plasmodium falciparum malaria2015In: Open Forum Infectious Diseases, ISSN 2328-8957, Vol. 2, no 2Article in journal (Refereed)
    Abstract [en]

    Background: Accuracy in malaria diagnosis and staging is vital in order to reduce mortality and post infectious sequelae. Herein we present a metabolomics approach to diagnostic staging of malaria infection, specifically Plasmodium falciparum infection in children. Methods: A group of 421 patients between six months and six years of age with mild and severe states of malaria with age-matched controls were included in the study, 107, 192 and 122 individuals respectively. A multivariate design was used as basis for representative selection of twenty patients in each category. Patient plasma was subjected to Gas Chromatography-Mass Spectrometry analysis and a full metabolite profile was produced from each patient. In addition, a proof-of-concept model was tested in a Plasmodium berghei in-vivo model where metabolic profiles were discernible over time of infection. Results: A two-component principal component analysis (PCA) revealed that the patients could be separated into disease categories according to metabolite profiles, independently of any clinical information. Furthermore, two sub-groups could be identified in the mild malaria cohort who we believe represent patients with divergent prognoses. Conclusion: Metabolite signature profiling could be used both for decision support in disease staging and prognostication.

  • 303.
    Svenmarker, Pontus
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Department of Physics, Lund University.
    Xu, Can T.
    Liu, Haichun
    Wu, Xia
    Andersson-Engels, Stefan
    Multispectral guided fluorescence diffuse optical tomography using upconverting nanoparticles2014In: Applied Physics Letters, ISSN 0003-6951, E-ISSN 1077-3118, Vol. 104, no 7, p. 073703-Article in journal (Refereed)
    Abstract [en]

    We report on improved image detectability for fluorescence diffuse optical tomography using upconverting nanoparticles doped with rare-earth elements. Core-shell NaYF4 : Yb3+/Er3+ @ NaYF4 upconverting nanoparticles were synthesized through a stoichiometric method. The Yb3+/Er3+ sensitizer-activator pair yielded two anti-Stokes shifted fluorescence emission bands at 540 nm and 660 nm, here used to a priori estimate the fluorescence source depth with sub-millimeter precision. A spatially varying regularization incorporated the a priori fluorescence source depth estimation into the tomography reconstruction scheme. Tissue phantom experiments showed both an improved resolution and contrast in the reconstructed images as compared to not using any a priori information.

  • 304. Syngkon, Aurelia
    et al.
    Elluri, Sridhar
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Koley, Hemanta
    Rompikuntal, Pramod K
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Saha, Dhira Rani
    Chakrabarti, Manoj K
    Bhadra, Rupak K
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Pal, Amit
    Studies on a novel serine protease of a ΔhapAΔprtV Vibrio cholerae O1 strain and its role in hemorrhagic response in the rabbit ileal loop model2010In: PloS one, ISSN 1932-6203, Vol. 5, no 9, p. e13122-Article in journal (Refereed)
    Abstract [en]

    Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.

  • 305.
    Taheri, Nayyer
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Host-pathogen interactions during Campylobacter and Yersinia infections2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The innate immune system is known for protecting the host against invading pathogens, for instance enteropathogens infecting the gastrointestinal tract. The production of e.g. antimicrobial peptides, cytokines, and chemokines by innate immune cells and intestinal epithelial cells contribute to bacterial clearance. Given the significance of this system in overall defense, pathogens affect and/or manipulate immune cells and responses in favor of their own survival. This thesis focuses on how the Gram-negative enteropathogenic bacteria Yersinia pseudotuberculosis and Campylobacter jejuni affect the host, either directly via type 3 secretion system (T3SS) effector proteins or via outer membrane vesicles (OMVs), and how host factors potentially affect their virulence.

    Yersinia pseudotuberculosis uses its T3SS to translocate virulence factors that disable various immune responses and subvert phagocytosis. Neutrophils are main target cells during Yersinia infection. They release granules that contain proteins with antimicrobial properties to the cell's exterior upon activation through a process called degranulation. We found that extracellular Y. pseudotuberculosis could prevent neutrophil degranulation upon cell contact. Prevention of degranulation was shown to be mediated via co-operative actions of the two anti-phagocytic Yersinia outer proteins YopH and YopE. Bacterial contact with neutrophils resulted in a transient inhibition of degranulation and further prevented degranulation upon subsequent contact with avirulent Y. pseudotuberculosis (lacking YopE and YopH) as well as Escherichia coli. Thus, Y. pseudotuberculosis impairs several neutrophil defense mechanisms to remain in the extracellular environment and to increase its survival during infection.

    Campylobacter jejuni lacks a T3SS and appears to use OMVs and flagella as its main secretion apparatus. During passage through the intestine C. jejuni is exposed to bile, an important physiological component and part of the natural barrier of the intestine, and ability to resist bile is advantageous for C. jejuni survival. We investigated how C. jejuni OMV production and protein content is affected by bile. The main invasion and colonization of C. jejuni occurs in the lower part of the intestine where the concentration of bile is low compared with the proximal intestine. The OMV proteomic profiles were radically altered when bacteria were grown in low concentration of bile corresponding to cecal concentrations. Twenty-five present of the detected proteins of OMVs showed an altered abundance in the presence of low concentration of bile. In contrast, the overall proteome of the bacteria was unaffected. Moreover, OMVs frombile-exposed bacteria could enhance adhesion as well as invasion of bacteria into intestinal epithelial cells, suggesting a role of OMVs to the virulence of C. jejuni in the gut. The body temperature differs between the asymptomatic avian carriers of C. jejuni and humans, which develop symptomatic disease. We investigated whether the bacterial growth temperature affects the OMV proteome and found that 59 proteins were differentially expressed at 37°C. Among the higher abundant proteins, significantly more proteins were predicted to be related to virulence. Thus, temperature has an impact on the property of the OMVs, and this might affect the outcome of infection by C. jejuni in different hosts.

    C. jejuni OMV interactions with innate immune cells were studied by analyses of OMV-mediated inflammasome activation. OMVs were found to induce ASC- and caspase-1-dependent inflammasome activation in murine and human macrophages and dendritic cells as well as in human neutrophils. While C. jejuni infection induced a low level of inflammasome-dependent cell death, OMV-induced inflammasome activation did not result in cell death. Thus, OMVs disseminate into tissue without bacteria can be a vehicle for virulence factors without inducing inflammatory cell death.

  • 306.
    Taheri, Nayyer
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia pseudotuberculosis Blocks Neutrophil Degranulation2016In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 84, no 12, p. 3369-3378Article in journal (Refereed)
    Abstract [en]

    Neutrophils are essential components of immunity and are rapidly recruited to infected or injured tissue. Upon their activation, neutrophils release granules to the cell's exterior, through a process called degranulation. These granules contain proteins with antimicrobial properties that help combat infection. The enteropathogenic bacterium Yersinia pseudotuberculosis successfully persists as an extracellular bacterium during infection by virtue of its translocation of virulence effectors (Yersinia outer proteins [Yops]) that act in the cytosol of host immune cells to subvert phagocytosis and proinflammatory responses. Here, we investigated the effect of Y. pseudotuberculosis on neutrophil degranulation upon cell contact. We found that virulent Y. pseudotuberculosis was able to prevent secondary granule release. The blocking effect was general, as the release of primary and tertiary granules was also reduced. Degranulation of secondary granules was also blocked in primed neutrophils, suggesting that this mechanism could be an important element of immune evasion. Further, wild-type bacteria conferred a transient block on neutrophils that prevented their degranulation upon contact with plasmid-cured, avirulent Y. pseudotuberculosis and Escherichia coli Detailed analyses showed that the block was strictly dependent on the cooperative actions of the two antiphagocytic effectors, YopE and YopH, suggesting that the neutrophil target structures constituting signaling molecules needed to initiate both phagocytosis and general degranulation. Thus, via these virulence effectors, Yersinia can impair several mechanisms of the neutrophil's antimicrobial arsenal, which underscores the power of its virulence effector machinery.

  • 307.
    Taheri, Nayyer
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Accumulation of virulence-associated proteins in Campylobacter jejuni Outer Membrane Vesicles at human body temperature2019In: Journal of Proteomics, ISSN 1874-3919, E-ISSN 1876-7737, Vol. 195, p. 33-40Article in journal (Refereed)
    Abstract [en]

    Campylobacter jejuni is the major cause of bacterial gastroenteritis in humans. In contrast, colonization in avian hosts is asymptomatic. Body temperature differs between human (37 °C) and avian (42 °C) hosts, and bacterial growth in 37 °C is therefore a potential cue for higher virulence properties during human infection. The proteome of the bacteria was previously shown to be altered by temperature. Here we investigated whether temperature has an effect on the C. jejuni outer membrane vesicle (OMV) proteome, as OMVs are considered to be bacterial vehicles for protein delivery and might play a role during infection. OMVs isolated from C. jejuni strain 81-176 grown at 37 °C and 42 °C were analyzed by LC-ESI-MS/MS. 181 proteins were detected in both sample groups, one protein was exclusively present, and three were absent in OMVs from 37 °C. Of the 181 proteins, 59 were differentially expressed; 30 proteins were detected with higher abundance, and 29 proteins with lower abundance at 37 °C. Among the more highly abundant proteins, significantly more proteins were predicted to be associated with virulence. These data show that temperature has an impact on the property of the OMVs, and this might affect the outcome of colonization/infection by C. jejuni in different hosts.

  • 308.
    Taheri, Nayyer
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Mahmud, A K M Firoj
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Sandblad, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Campylobacter jejuni bile exposure influences outer membrane vesicles protein content and bacterial interaction with epithelial cells2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 16996Article in journal (Refereed)
    Abstract [en]

    Campylobacter jejuni is a prevalent human pathogen and a major cause of bacterial gastroenteritis in the world. In humans, C. jejuni colonizes the intestinal tract and its tolerance to bile is crucial for bacteria to survive and establish infection. C. jejuni produces outer membrane vesicles (OMVs) which have been suggested to be involved in virulence. In this study, the proteome composition of C. jejuni OMVs in response to low concentration of bile was investigated. We showed that exposure of C. jejuni to low concentrations of bile, similar to the concentration in cecum, induced significant changes in the protein profile of OMVs released during growth without affecting the protein profile of the bacteria. This suggests that bile influences a selective packing of the OMVs after bacterial exposure to low bile. A low concentration of bile was found to increase bacterial adhesion to intestinal epithelial cells, likely by an enhanced hydrophobicity of the cell membrane following exposure to bile. The increased bacterial adhesiveness was not associated with increased invasion, instead bile exposure decreased C. jejuni invasion. OMVs released from bacteria upon exposure to low bile showed to increase both adhesion and invasion of non-bile-exposed bacteria into intestinal epithelial cells. These findings suggest that C. jejuni in environments with low concentrations of bile produce OMVs that facilitates colonization of the bacteria, and this could potentially contribute to virulence of C. jejuni in the gut.

  • 309.
    Taheri, Nayyer
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Campylobacter jejuni outer membrane vesicles activate inflammasome without inducing cell death in immune cellsManuscript (preprint) (Other academic)
  • 310.
    Tengel, Tobias
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Sethson, Ingmar
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Conformational analysis by CD and NMR spectroscopy of a peptide encompassing the amphipathic domain of YopD from Yersinia2002In: European Journal of Biochemistry, ISSN 0014-2956, E-ISSN 1432-1033, Vol. 269, no 15, p. 3659-3668Article in journal (Refereed)
    Abstract [en]

    To establish an infection, Yersinia pseudotuberculosis utilizes a plasmid-encoded type III secretion machine that permits the translocation of several anti-host factors into the cytosol of target eukaryotic cells. Secreted YopD is essential for this process. Pre-secretory stabilization of YopD is mediated by an interaction with its cognate chaperone, LcrH. YopD possesses LcrH binding domains located in the N-terminus and in a predicted amphipathic domain located near the C-terminus. This latter domain is also critical for Yersinia virulence. In this study, we designed synthetic peptides encompassing the C-terminal amphipathic domain of YopD. A solution structure of YopD278−300, a peptide that strongly interacted with LcrH, was obtained by NMR methods. The structure is composed of a well-defined amphipathic α helix ranging from Phe280 to Tyr291, followed by a type I β turn between residues Val292 and His295. The C-terminal truncated peptides, YopD278−292 and YopD271−292, lacked helical structure, implicating the β turn in helix stability. An interaction between YopD278−300 and its cognate chaperone, LcrH, was observed by NMR through line-broadening effects and chemical shift differences between the free peptide and the peptide–LcrH complex. These effects were not observed for the unstructured peptide, YopD278−292, which confirms that the α helical structure of the YopD amphipathic domain is a critical binding region of LcrH.

  • 311. Terceti, Mateus S.
    et al.
    Rivas, Amable J.
    Alvarez, Laura
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Noia, Manuel
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Osorio, Carlos R.
    rstB Regulates Expression of the Photobacterium damselae subsp damselae Major Virulence Factors Damselysin, Phobalysin P and Phobalysin C2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 582Article in journal (Refereed)
    Abstract [en]

    The marine pathogenic bacterium Photobacterium damselae subsp. damselae causes septicemia in marine animals and in humans. The pPHDD1 plasmid-encoded hemolysins damselysin (Dly) and phobalysin P (PhlyP), and the chromosome-encoded hemolysin phobalysin C (PhlyC) constitute its main virulence factors. However, the mechanisms by which expression of these three hemolysins is regulated remain unknown. Here we report the isolation of a mini-Tn10 transposon mutant which showed a strong impairment in its hemolytic activity. The transposon disrupted a putative sensor histidine kinase gene vda_000600 (rstB), which together with vda_000601 (rstA) is predicted to encode a putative two-component regulatory system. This system showed to be homologous to the Vibrio cholerae CarSR/VprAB and Escherichia coli RstAB systems. Reconstruction of the mutant by allelic exchange of rstB showed equal impairment in hemolysis, and complementation with a plasmid expressing rstAB restored hemolysis to wild-type levels. Remarkably, we demonstrated by promoter expression analyses that the reduced hemolysis in the rstB mutant was accompanied by a strong decrease in transcription activities of the three hemolysin genes dly (damselysin), hlyA(pl) (phobalysin P) and hlyA(ch) (phobalysin C). Thus, RstB, encoded in the small chromosome, regulates plasmid and chromosomal virulence genes. We also found that reduced expression of the three virulence genes correlated with a strong decrease in virulence in a sea bass model, demonstrating that RstB constitutes a master regulator of the three P. damselae subsp. damselae hemolysins and plays critical roles in the pathogenicity of this bacterium. This study represents the first evidence of a direct role of a RstAB-like system in the regulation of bacterial toxins.

  • 312.
    Thanikkal, Edvin J.
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kumar Gahlot, Dharmender
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Liu, Junfa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fredriksson Sundbom, Marcus
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Gurung, Jyoti M.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ruuth, Kristina
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Francis, Monika K.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Obi, Ikenna R.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Thompson, Karl M.
    Chen, Shiyun
    Dersch, Petra
    Francis, Matthew S.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The Yersinia pseudotuberculosis Cpx envelope stress system contributes to transcriptional activation of rovM2019In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, no 1, p. 37-57Article in journal (Refereed)
    Abstract [en]

    The Gram-negative enteropathogen Yersinia pseudotuberculosis possesses a number of regulatory systems that detect cell envelope damage caused by noxious extracytoplasmic stresses. The CpxA sensor kinase and CpxR response regulator two-component regulatory system is one such pathway. Active Cpx signalling upregulates various factors designed to repair and restore cell envelope integrity. Concomitantly, this pathway also down-regulates key determinants of virulence. In Yersinia, cpxA deletion accumulates high levels of phosphorylated CpxR (CpxR~P). Accumulated CpxR~P directly repressed rovA expression and this limited expression of virulence-associated processes. A second transcriptional regulator, RovM, also negatively regulates rovA expression in response to nutrient stress. Hence, this study aimed to determine if CpxR~P can influence rovA expression through control of RovM levels. We determined that the active CpxR~P isoform bound to the promoter of rovM and directly induced its expression, which naturally associated with a concurrent reduction in rovA expression. Site-directed mutagenesis of the CpxR~P binding sequence in the rovM promoter region desensitised rovM expression to CpxR~P. These data suggest that accumulated CpxR~P inversely manipulates the levels of two global transcriptional regulators, RovA and RovM, and this would be expected to have considerable influence on Yersinia pathophysiology and metabolism.

  • 313.
    Thanikkal, Edvin
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Mangu, Jagadish
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Francis, Matthew
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Interactions of the CpxA sensor kinase and cognate CpxR response regulator from Yersinia pseudotuberculosis2012In: BMC Research Notes, ISSN 1756-0500, E-ISSN 1756-0500, Vol. 5, no 1, p. 536-Article in journal (Refereed)
    Abstract [en]

    Background

    The CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.

    Results

    CpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the lamda cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.

    Conclusions

    This interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

  • 314.
    Thorslund, Sara E
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Edgren, Tomas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Pettersson, Jonas
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nordfelth, Roland
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sellin, Mikael E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Ivanova, Ekaterina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Isaksson, Elin L
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The RACK1 signaling scaffold protein selectively interacts with Yersinia pseudotuberculosis virulence function2011In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 6, no 2, p. e16784-Article in journal (Refereed)
    Abstract [en]

    Many Gram-negative bacteria use type III secretion systems to translocate effector proteins into host cells. These effectors interfere with cellular functions in a highly regulated manner resulting in effects that are beneficial for the bacteria. The pathogen Yersinia can resist phagocytosis by eukaryotic cells by translocating Yop effectors into the target cell cytoplasm. This is called antiphagocytosis, and constitutes an important virulence feature of this pathogen since it allows survival in immune cell rich lymphoid organs. We show here that the virulence protein YopK has a role in orchestrating effector translocation necessary for productive antiphagocytosis. We present data showing that YopK influences Yop effector translocation by modulating the ratio of the pore-forming proteins YopB and YopD in the target cell membrane. Further, we show that YopK that can interact with the translocators, is exposed inside target cells and binds to the eukaryotic signaling protein RACK1. This protein is engaged upon Y. pseudotuberculosis-mediated beta1-integrin activation and localizes to phagocytic cups. Cells with downregulated RACK1 levels are protected from antiphagocytosis. This resistance is not due to altered levels of translocated antiphagocytic effectors, and cells with reduced levels of RACK1 are still sensitive to the later occurring cytotoxic effect caused by the Yop effectors. Further, a yopK mutant unable to bind RACK1 shows an avirulent phenotype during mouse infection, suggesting that RACK1 targeting by YopK is a requirement for virulence. Together, our data imply that the local event of Yersinia-mediated antiphagocytosis involves a step where YopK, by binding RACK1, ensures an immediate specific spatial delivery of antiphagocytic effectors leading to productive inhibition of phagocytosis.

  • 315.
    Thorslund, Sara E
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ermert, David
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Erttmann, Saskia F
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nilsson, Kristina
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hosseinzadeh, Ava
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Urban, Constantin F
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Role of YopK in Yersinia pseudotuberculosis Resistance Against Polymorphonuclear Leukocyte Defense2013In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 81, no 1, p. 11-22Article in journal (Refereed)
    Abstract [en]

    The enteropathogen Y. pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type three secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte (PMN, or neutrophil) defense. A yopK mutant, which is attenuated in the mouse infection model where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and ROS production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction PMN death, whereas the mutant caused a necrotic-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism where YopK functions to prevent unintended Yop delivery and thereby PMN disruption resulting in necrotic like cell death, which would enhance the inflammatory response favoring the host.

  • 316.
    Tiensuu, Teresa
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Guerreiro, Duarte N.
    de Oliveira, Ana Henriques
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    O'Byrne, Conor
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Flick of a switch: regulatory mechanisms allowing Listeria monocytogenes to transition from a saprophyte to a killer2019In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 165, no 8, p. 819-833Article, review/survey (Refereed)
    Abstract [en]

    In contrast to obligate intracellular pathogens that can remain in relatively stable host-associated environments, the soil-living bacterial pathogen Listeria monocytogenes has to sense and respond to physical and chemical cues in a variety of quite different niches. In particular, the bacterium has to survive the dramatic transition from its saprophytic existence to life within the host where nutritional stress, increased temperature, acidity, osmotic stress and the host defences present a new and challenging landscape. This review focuses on the sB and PrfA regulatory systems used by L. monocytogenes to sense the changing environment and implement survival mechanisms that help to overcome the disparate conditions within the host, but also to switch from a harmless saprophyte to an impressively effective pathogen.

  • 317. Toledo-Arana, Alejandro
    et al.
    Dussurget, Olivier
    Nikitas, Georgios
    Sesto, Nina
    Guet-Revillet, Hélène
    Balestrino, Damien
    Loh, Edmund
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Gripenland, Jonas
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Tiensuu, Teresa
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Vaitkevicius, Karolis
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Barthelemy, Mathieu
    Vergassola, Massimo
    Nahori, Marie-Anne
    Soubigou, Guillaume
    Régnault, Béatrice
    Coppée, Jean-Yves
    Lecuit, Marc
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cossart, Pascale
    The Listeria transcriptional landscape from saprophytism to virulence2009In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 459, no 7249, p. 950-956Article in journal (Refereed)
    Abstract [en]

    The bacterium Listeria monocytogenes is ubiquitous in the environment and can lead to severe food-borne infections. It has recently emerged as a multifaceted model in pathogenesis. However, how this bacterium switches from a saprophyte to a pathogen is largely unknown. Here, using tiling arrays and RNAs from wild-type and mutant bacteria grown in vitro, ex vivo and in vivo, we have analysed the transcription of its entire genome. We provide the complete Listeria operon map and have uncovered far more diverse types of RNAs than expected: in addition to 50 small RNAs (<500 nucleotides), at least two of which are involved in virulence in mice, we have identified antisense RNAs covering several open-reading frames and long overlapping 5' and 3' untranslated regions. We discovered that riboswitches can act as terminators for upstream genes. When Listeria reaches the host intestinal lumen, an extensive transcriptional reshaping occurs with a SigB-mediated activation of virulence genes. In contrast, in the blood, PrfA controls transcription of virulence genes. Remarkably, several non-coding RNAs absent in the non-pathogenic species Listeria innocua exhibit the same expression patterns as the virulence genes. Together, our data unravel successive and coordinated global transcriptional changes during infection and point to previously unknown regulatory mechanisms in bacteria.

  • 318. Tsatsaronis, James A.
    et al.
    Franch-Arroyo, Sandra
    Resch, Ulrike
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Extracellular Vesicle RNA: A Universal Mediator of Microbial Communication?2018In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 26, no 5, p. 401-410Article, review/survey (Refereed)
    Abstract [en]

    Both extracellular RNAs and extracellular vesicles (EVs) have recently garnered attention as novel mediators of intercellular communication in eukaryotes and prokaryotes alike. EVs not only permit export of RNA, but also facilitate delivery and trans-kingdom exchange of these and other biomolecules, for instance between microbes and their hosts. In this Opinion article, we propose that EV-mediated export of RNA represents a universal mechanism for interkingdom and intrakingdom communication that is conserved among bacterial, archaeal, and eukaryotic microbes. We speculate how microbes might use EV RNA to influence target cell gene expression or manipulate host immune responses.

  • 319.
    Urban, Constantin F.
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Nett, Jeniel E
    Neutrophil extracellular traps in fungal infection2019In: Seminars in Cell and Developmental Biology, ISSN 1084-9521, E-ISSN 1096-3634, Vol. 89, p. 47-57Article in journal (Refereed)
    Abstract [en]

    Fungal infections are a continuously increasing problem in modern health care. Understanding the complex biology of the emerging pathogens and unraveling the mechanisms of host defense may form the basis for the development of more efficient diagnostic and therapeutic tools. Neutrophils play a pivotal role in the defense against fungal pathogens. These phagocytic hunters migrate towards invading fungal microorganisms and eradicate them by phagocytosis, oxidative burst and release of neutrophil extracellular traps (NETs). In the last decade, the process of NET formation has received unparalleled attention, with numerous studies revealing the relevance of this neutrophil function for control of various mycoses. Here, we describe NET formation and summarize its role as part of the innate immune defense against fungal pathogens. We highlight factors influencing the formation of these structures and molecular mechanisms employed by fungi to impair the formation of NETs or subvert their antifungal effects.

  • 320. Urdaneta, Veronica
    et al.
    Hernandez, Sara B.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain.
    Casadesus, Josep
    Mutational and non mutational adaptation of Salmonella enterica to the gall bladder2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 5203Article in journal (Refereed)
    Abstract [en]

    During systemic infection of susceptible hosts, Salmonella enterica colonizes the gall bladder, which contains lethal concentrations of bile salts. Recovery of Salmonella cells from the gall bladder of infected mice yields two types of isolates: (i) bile-resistant mutants; (ii) isolates that survive lethal selection without mutation. Bile-resistant mutants are recovered at frequencies high enough to suggest that increased mutation rates may occur in the gall bladder, thus providing a tentative example of stress-induced mutation in a natural environment. However, most bile-resistant mutants characterized in this study show defects in traits that are relevant for Salmonella colonization of the animal host. Mutation may thus permit short-term adaptation to the gall bladder at the expense of losing fitness for transmission to new hosts. In contrast, non mutational adaptation may have evolved as a fitness-preserving strategy. Failure of RpoS(-) mutants to colonize the gall bladder supports the involvement of the general stress response in non mutational adaptation.

  • 321.
    Ur-Rehman, Tofeeq
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Department of Pharmacy, Quaid-i-Azam University, Islamabad, Pakistan .
    Nordfelth, Roland
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Blomgren, Anders
    Discovery Drug Metabolism, Pharmacokinetics, and Bioanalysis, AstraZeneca R&D Lund, Sweden.
    Zetterström, Caroline E
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Gylfe, Åsa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Preliminary pharmacokinetics of the bacterial virulence inhibitor N'-(3,5-dibromo-2-hydroxy-benzylidenene)-nicotinic acid hydrazide2012In: Advances in Yersinia Research, Springer, 2012, p. 349-356Chapter in book (Other academic)
    Abstract [en]

    Bacterial virulence inhibitors are potential novel drugs that may be used to treat infections. N′-(3,5-dibromo-2-hydroxy-benzylidene)-nicotinic acid hydrazide, ME0052, has been shown to inhibit type III secretion (T3S) and virulence in several Gram-negative enteric pathogens including Yersinia pseudotuberculosis. In vitro data suggest that ME0052 may be developed into drugs against bacterial gastroenteritis. Here we describe preliminary pharmacokinetics of ME0052 after intraperitoneal and subcutaneous administration in mice. The aim of this work was to identify suitable formulations and to determine pharmacokinetic parameters prior to testing in animal infection models. Peak plasma concentrations above the IC50 for virulence inhibition were achieved with high dose formulations and the elimination half-life was prolonged from 0.5 to 3.4 h using a poloxamer 407-based slow-release formulation.

  • 322.
    Vaitkevicius, Karolis
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Lindmark, Barbro
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rompikuntal, Pramod Kumar
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bhakdi, Sucharit
    Song, Tianyan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The PrtV protease in Vibrio cholerae influenses expression of the VCC/HlyA cytolytic activityManuscript (preprint) (Other (popular science, discussion, etc.))
  • 323.
    van Teeseling, Muriel C. F.
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    de Pedro, Miguel A.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Determinants of Bacterial Morphology: From Fundamentals to Possibilities for Antimicrobial Targeting2017In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 8, article id 1264Article in journal (Refereed)
    Abstract [en]

    Bacterial morphology is extremely diverse. Specific shapes are the consequence of adaptive pressures optimizing bacterial fitness. Shape affects critical biological functions, including nutrient acquisition, motility, dispersion, stress resistance and interactions with other organisms. Although the characteristic shape of a bacterial species remains unchanged for vast numbers of generations, periodical variations occur throughout the cell (division) and life cycles, and these variations can be influenced by environmental conditions. Bacterial morphology is ultimately dictated by the net-like peptidoglycan (PG) sacculus. The species-specific shape of the PG sacculus at any time in the cell cycle is the product of multiple determinants. Some morphological determinants act as a cytoskeleton to guide biosynthetic complexes spatiotemporally, whereas others modify the PG sacculus after biosynthesis. Accumulating evidence supports critical roles of morphogenetic processes in bacteria-host interactions, including pathogenesis. Here, we review the molecular determinants underlying morphology, discuss the evidence linking bacterial morphology to niche adaptation and pathogenesis, and examine the potential of morphological determinants as antimicrobial targets.

  • 324. Vanderlinde, Elizabeth M.
    et al.
    Strozen, Timothy G.
    Hernandez, Sara B.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Howard, S. Peter
    Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System2017In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, no 8, article id UNSP e00617-16Article in journal (Refereed)
    Abstract [en]

    In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the D, D-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG crosslinking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila. In a heterologous assembly model in E. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

  • 325. Varga, M G
    et al.
    Shaffer, C L
    Sierra, J C
    Suarez, G
    Piazuelo, M B
    Whitaker, M E
    Romero-Gallo, J
    Krishna, U S
    Delgado, A
    Gomez, M A
    Good, James A D
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Skaar, E P
    Correa, P
    Wilson, K T
    Hadjifrangiskou, M
    Peek, R M
    Pathogenic Helicobacter pylori strains translocate DNA and activate TLR9 via the cancer-associated cag type IV secretion system2016In: Oncogene, ISSN 0950-9232, E-ISSN 1476-5594, Vol. 35, no 48, p. 6262-6269Article in journal (Refereed)
    Abstract [en]

    Helicobacter pylori (H. pylori) is the strongest identified risk factor for gastric cancer, the third most common cause of cancer-related death worldwide. An H. pylori constituent that augments cancer risk is the strain-specific cag pathogenicity island, which encodes a type IV secretion system (T4SS) that translocates a pro-inflammatory and oncogenic protein, CagA, into epithelial cells. However, the majority of persons colonized with CagA+ H. pylori strains do not develop cancer, suggesting that other microbial effectors also have a role in carcinogenesis. Toll-like receptor 9 (TLR9) is an endosome bound, innate immune receptor that detects and responds to hypo-methylated CpG DNA motifs that are most commonly found in microbial genomes. High-expression tlr9 polymorphisms have been linked to the development of premalignant lesions in the stomach. We now demonstrate that levels of H. pylori-mediated TLR9 activation and expression are directly related to gastric cancer risk in human populations. Mechanistically, we show for the first time that the H. pylori cancer-associated cag T4SS is required for TLR9 activation and that H. pylori DNA is actively translocated by the cag T4SS to engage this host receptor. Activation of TLR9 occurs through a contact-dependent mechanism between pathogen and host, and involves transfer of microbial DNA that is both protected as well as exposed during transport. These results indicate that TLR9 activation via the cag island may modify the risk for malignancy within the context of H. pylori infection and provide an important framework for future studies investigating the microbial-epithelial interface in gastric carcinogenesis.

  • 326.
    Vdovikova, Svitlana
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Roles of membrane vesicles in bacterial pathogenesis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The production of membranous vesicles is observed to occur among organisms from all domains of the tree of life spanning prokaryotes (bacteria, archaea) and eukaryotes (plants, animals and fungi). Bacterial release of membrane-derived vesicles (MVs) has been studied most extensively in cases of Gram-negative species and implicating their outer membrane in formation of extracellular MVs. However, recent studies focusing on Gram-positive bacteria have established that they also undergo MV formation. Membrane vesicles are released during normal bacterial growth, they are derived from the bacterial membrane(s) and may function as transporters of different proteins, DNA and RNA to the neighbouring bacteria or to the cells of a mammalian host. The transport of virulence factors in a condensed manner via MVs to the host cells presumably protects these proteins from degradation and, thereby, targets the host cells in a specific manner.

    The aim of my thesis is to investigate secretion of MV-associated virulence factors and to study interactions of MVs produced by two selected Gram-negative and Gram-positive bacteria, i.e. Vibrio cholerae and Listeria monocytogenes, with eukaryotic host cells. Depending on whether the bacterium acts as an extracellular or intracellular pathogen, MVs may be considered to have specific functions, which may lead to the different outcomes of MV-host interactions.

    V. cholerae transport systems for virulence factors include the Type VI secretion system and MVs (also referred to as the “Type 0” secretion system). We have identified that the biologically active form of PrtV protease in different V. cholerae serogroups is transported via MVs. PrtV protease is essential for V. cholerae environmental survival and protection from natural predator grazing. We demonstrated that PrtV is primarily translocated via the inner membrane to the periplasmic space, where it undergoes autoproteolysis, and the truncated version of PrtV protein is packaged inside the MVs and released from the surface of bacteria. MV-associated PrtV protease showed a contribution to bacterial resistance towards the antimicrobial peptide LL-37, thereby, enhancing bacterial survival by avoiding this innate immune defense of the host.

    We also studied another virulence factor of V. cholerae, the pore-forming toxin VCC, which was found to be transported by MVs. MV-associated VCC is biologically active and triggers an autophagic response in the target cells. We suggested that autophagy serves as a cellular defense mechanism against the MV-associated bacterial virulence factor of V. cholerae.

    Listeria monocytogenes is a Gram-positive intracellular and facultative anaerobic food-borne pathogen causing listeriosis. It causes only sporadic outbreaks in healthy individuals, however, it is dangerous for a fetus or newborn child, and for pregnant and immunocompromised people, leading to a deadly infection in one third of the cases. We have analyzed MVs produced by L. monocytogenes and their interaction with eukaryotic cells. Confocal microscopy analysis showed that MVs are internalized into HeLa and HEK293 cells and are accumulated in lysosomes. Moreover, L. monocytogenes produces MVs inside the host cells and even inside the phagosomes. We found that the major virulence factor of L. monocytogenes, the cholesterol-dependent pore-forming protein listeriolysin O (LLO), is entrapped inside the MVs and resides there in an oxidized inactive state. LLO is known to induce autophagy by making pores in the phagosomal membrane of targeted eukaryotic cells. In our studies, we have shown that MVs effectively abrogated autophagy induced by Torin1, by purified LLO or by another pore-forming toxin from V. cholerae. We also found that MVs promote bacterial intracellular survival inside mouse embryonic fibroblasts. In addition, MVs have been shown to have a strong protective activity against host cell necrosis initiated by pore-forming toxin. Taken together, these findings suggested that in vivo MVs production from L. monocytogenes might be a relevant strategy of bacteria to manipulate host responses and to promote bacterial survival inside the host cells. 

  • 327.
    Vdovikova, Svitlana
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Gilfillan, Siv
    Wang, Shixiong
    Dongre, Mitesh
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Hurtado, Antonio
    Modulation of gene transcription and epigenetics of colon carcinoma cells by bacterial membrane vesicles2018In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 7434Article in journal (Refereed)
    Abstract [en]

    Interactions between bacteria and colon cancer cells infuence the transcription of the host cell. Yet is it undetermined whether the bacteria itself or the communication between the host and bacteria is responsible for the genomic changes in the eukaryotic cell. Now, we have investigated the genomic and epigenetic consequences of co-culturing colorectal carcinoma cells with membrane vesicles from pathogenic bacteria Vibrio cholerae and non-pathogenic commensal bacteria Escherichia coli. Our study reveals that membrane vesicles from pathogenic and commensal bacteria have a global impact on the gene expression of colon-carcinoma cells. The changes in gene expression correlate positively with both epigenetic changes and chromatin accessibility of promoters at transcription start sites of genes induced by both types of membrane vesicles. Moreover, we have demonstrated that membrane vesicles obtained only from V. cholerae induced the expression of genes associated with epithelial cell diferentiation. Altogether, our study suggests that the observed genomic changes in host cells might be due to specifc components of membrane vesicles and do not require communication by direct contact with the bacteria.

  • 328.
    Vdovikova, Svitlana
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Luhr, Morten
    Szalai, Paula
    Skalman, Lars Nygård
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Francis, Monika K.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Engedal, Nikolai
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wai, Sun N.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death2017In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 7, article id 154Article in journal (Refereed)
    Abstract [en]

    Bacterial membrane vesicle (MV) production has been mainly studied in Gram-negative species. In this study, we show that Listeria monocytogenes, a Gram-positive pathogen that causes the food-borne illness listeriosis, produces MVs both in vitro and in vivo. We found that a major virulence factor, the pore-forming hemolysin listeriolysin O (LLO), is tightly associated with the MVs, where it resides in an oxidized, inactive state. Previous studies have shown that LLO may induce cell death and autophagy. To monitor possible effects of LLO and MVs on autophagy, we performed assays for LC3 lipidation and LDH sequestration as well as analysis by confocal microscopy of HEK293 cells expressing GFP-LC3. The results revealed that MVs alone did not affect autophagy whereas they effectively abrogated autophagy induced by pure LLO or by another pore-forming toxin from Vibrio cholerae, VCC. Moreover, Listeria monocytogenes MVs significantly decreased Torin1-stimulated macroautophagy. In addition, MVs protected against necrosis of HEK293 cells caused by the lytic action of LLO. We explored the mechanisms of LLO-induced autophagy and cell death and demonstrated that the protective effect of MVs involves an inhibition of LLO-induced pore formation resulting in inhibition of autophagy and the lytic action on eukaryotic cells. Further, we determined that these MVs help bacteria to survive inside eukaryotic cells (mouse embryonic fibroblasts). Taken together, these findings suggest that intracellular release of MVs from L. monocytogenes may represent a bacterial strategy to survive inside host cells, by its control of LLO activity and by avoidance of destruction from the autophagy system during infection.

  • 329. Vestö, Kim
    et al.
    Huseby, Douglas L.
    Snygg, Iina
    Wang, Helen
    Hughes, Diarmaid
    Rhen, Mikael
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Muramyl Endopeptidase Spr Contributes to Intrinsic Vancomycin Resistance in Salmonella enterica Serovar Typhimurium2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2941Article in journal (Refereed)
    Abstract [en]

    The impermeability barrier provided by the outer membrane of enteric bacteria, a feature lacking in Gram-positive bacteria, plays a major role in maintaining resistance to numerous antimicrobial compounds and antibiotics. Here we demonstrate that mutational inactivation of spr, coding for a muramyl endopeptidase, significantly sensitizes Salmonella enterica serovar Typhimurium to vancomycin without any accompanying apparent growth defect or outer membrane destabilization. A similar phenotype was not achieved by deleting the genes coding for muramyl endopeptidases MepA, PbpG, NlpC, YedA, or YhdO. The spr mutant showed increased autolytic behavior in response to not only vancomycin, but also to penicillin G, an antibiotic for which the mutant displayed a wild-type MIC. A screen for suppressor mutations of the spr mutant phenotype revealed that deletion of tsp (prc), encoding a periplasmic carboxypeptidase involved in processing Spr and PBP3, restored intrinsic resistance to vancomycin and reversed the autolytic phenotype of the spr mutant. Our data suggest that Spr contributes to intrinsic antibiotic resistance in S. Typhimurium without directly affecting the outer membrane permeability barrier. Furthermore, our data suggests that compounds targeting specific cell wall endopeptidases might have the potential to expand the activity spectrum of traditional Gram-positive antibiotics.

  • 330. Vincents, Bjarne
    et al.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Björck, Lars
    Abrahamson, Magnus
    Enzymatic characterization of the streptococcal endopeptidase, IdeS, reveals that it is a cysteine protease with strict specificity for IgG cleavage due to exosite binding.2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 49, p. 15540-15549Article in journal (Refereed)
    Abstract [en]

    Streptococcus pyogenes, an important pathogen in humans, secretes an IgG specific endopeptidase named IdeS. To elucidate the mechanism that is responsible for this specificity, we have here characterized the activity of IdeS in detail. Both gamma chains of human IgG or its Fc fragment were cleaved in the hinge region after Gly236 by IdeS, but other proteins or synthetic peptides containing sequences such as the P(4)-P(1) segment in the IgG cleavage site, or long peptides resembling the IgG hinge, were not hydrolyzed at all. This is likely due to a second binding site interacting with the Fc part of IgG. The lack of IdeS activity on peptide substrates necessitated the development of an assay with IgG as the substrate for kinetic studies. IdeS showed a sigmoidal velocity curve at physiological IgG concentrations, and a declining enzyme rate at higher IgG concentrations. This atypical velocity curve suggests product inhibition and/or allosteric control, which again indicates the presence of an exosite involved in substrate binding. The pseudoequilibrium constant for IdeS hydrolysis of IgG was 90 microM. The enzyme exhibited activity in the pH range of 5.1-7.6, with an optimum at pH 6.6. IdeS was stable above pH 10 but not at acidic pH. It exhibited an activity maximum around 37 degrees C and a decreased thermal stability at 42 degrees C. Iodoacetate and iodoacetamide inhibited IdeS, as expected for a cysteine protease, and biochemical evidence verified this classification. E-64 and chicken cystatin, specific inhibitors of family C1 and C13 cysteine proteases, were without effect on enzyme activity, as were class specific serine, aspartic, and metallo protease inhibitors. No significant similarities were found in protein sequence comparisons with known enzyme families, suggesting that IdeS represents a novel family of cysteine proteases.

  • 331. Voss, Stephanie
    et al.
    Li, Fu
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
    Raetz, Andreas
    Roeger, Matthias
    Wu, Yao-Wen
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Chemical Genomics Centre of the Max Planck Society, Otto-Hahn-Strasse 15, 44227 Dortmund, Germany; Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
    Spatial Cycling of Rab GTPase, Driven by the GTPase Cycle, Controls Rab's Subcellular Distribution2019In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 4, p. 276-285Article in journal (Refereed)
    Abstract [en]

    Rab GTPases (>60 members in humans) function as master regulators of intracellular membrane trafficking. Correct and specific localization of Rab proteins is required for their function. How the distinct spatial distribution of Rab GTPases in the cell is regulated remains elusive. To globally assess the subcellular localization of Rab1, we determined kinetic parameters of two pathways that control the spatial cycles of Rabl, i.e., vesicular transport and GDP dissociation inhibitor (GDI)-mediated recycling. We demonstrate that the switching between GTP and GDP binding states, which is governed by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), GDI, and GDI displacement factor (GDF), is a major determinant of Rab1's ability to effectively cycle between cellular compartments and eventually its subcellular distribution. In silico perturbations of vesicular transport, GEFs, GAPs, GDI, and GDF using a mathematical model with simplified cellular geometries showed that these regulators play an important role in the subcellular distribution and activity of Rab1.

    The full text will be freely available from 2020-01-29 00:00
  • 332.
    Wang, He
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Avican, Kemal
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Erttmann, Saskia F.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Nuss, Aaron M.
    Dersch, Petra
    Fällman, Maria
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Edgren, Tomas
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Increased plasmid copy number is essential for Yersinia T3SS function and virulence2016In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 353, no 6298, p. 492-495Article in journal (Refereed)
    Abstract [en]

    Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmid-encoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria.

  • 333.
    Wang, Lixiao
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    Sauer, Uwe H
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Science and Technology).
    OnD-CRF: predicting order and disorder in proteins using [corrected] conditional random fields2008In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 24, no 11, p. 1401-1402Article in journal (Refereed)
    Abstract [en]

    MOTIVATION: Order and Disorder prediction using Conditional Random Fields (OnD-CRF) is a new method for accurately predicting the transition between structured and mobile or disordered regions in proteins. OnD-CRF applies CRFs relying on features which are generated from the amino acids sequence and from secondary structure prediction. Benchmarking results based on CASP7 targets, and evaluation with respect to several CASP criteria, rank the OnD-CRF model highest among the fully automatic server group. AVAILABILITY: http://babel.ucmp.umu.se/ond-crf/

  • 334.
    Weber, Barbara
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hasic, Medisa
    Chen, Chang
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Milton, Debra L
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum.2009In: Environmental Microbiology, ISSN 1462-2912, E-ISSN 1462-2920, Vol. 11, no 12, p. 3018-3028Article in journal (Refereed)
    Abstract [en]

    Type VI protein secretion systems (T6SS) are essential for virulence of several Gram-negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA-H (vibrio type six secretion) was sequenced and shown to encode eight proteins. VtsE-H are signature proteins found in other T6SSs, while VtsA-D are not associated with T6SS studied so far. In-frame deletions were made in each gene. Secretion of a haemolysin-co-regulated-like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE-H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE-H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress-response regulator RpoS and the quorum-sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA-H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.

  • 335.
    Weber, Barbara
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lindell, Kristoffer
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    El Qaidi, Samir
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hjerde, Erik
    Department of Chemistry, Faculty of Science and Technology, University of Tromsø, Tromsø 9037, Norway.
    Willassen, Nils-Peder
    Department of Chemistry, Faculty of Science and Technology, University of Tromsø, Tromsø 9037, Norway.
    Milton, Debra L
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum2011In: Microbiology, ISSN 1350-0872, E-ISSN 1465-2080, Vol. 157, no 12, p. 3324-3339Article in journal (Refereed)
    Abstract [en]

    Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qui, which encodes the ancestral quorum regulatory RNA Owl, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Orr sRNAs were identified. All four sRNAs were positively regulated by sigma(54) and the sigma(54)-dependent response regulator Van, and showed a redundant activity. The Orr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Orr sRNAs and stabilized van T mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Orr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.

  • 336. Webster, Steve J.
    et al.
    Brode, Sven
    Ellis, Lou
    Fitzmaurice, Timothy J.
    Elder, Matthew J.
    Gekara, Nelson O.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Tourlomousis, Panagiotis
    Bryant, Clare
    Clare, Simon
    Chee, Ronnie
    Gaston, Hill J. S.
    Goodall, Jane C.
    Detection of a microbial metabolite by STING regulates inflammasome activation in response to Chlamydia trachomatis infection2017In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 13, no 6, article id e1006383Article in journal (Refereed)
    Abstract [en]

    The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and noninfectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1 beta processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1 beta processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand D cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

  • 337.
    Weise, Christoph F
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Login, Frédéric H
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Ho, Oanh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Gröbner, Gerhard
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wolf-Watz, Magnus
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 8, p. 1950-1961Article in journal (Refereed)
    Abstract [en]

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

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

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

  • 339.
    Westermark, Linda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Yersinia-phagocyte interactions during early infection2013Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

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

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

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

  • 340.
    Westermark, Linda
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fahlgren, Anna
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Fällman, Maria
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Yersinia pseudotuberculosis Efficiently Escapes Polymorphonuclear Neutrophils during Early Infection2014In: Infection and Immunity, ISSN 0019-9567, E-ISSN 1098-5522, Vol. 82, no 3, p. 1181-1191Article in journal (Refereed)
    Abstract [en]

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

  • 341. Wiley, David J
    et al.
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Schesser, Kurt
    Induction of the Yersinia type 3 secretion system as an all-or-none phenomenon2007In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 373, no 1, p. 27-37Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner.

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

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

  • 343.
    Wu, Yao-Wen
    et al.
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Li, Fu
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry. Chemical Genomics Centre of the Max Planck Society, Dortmund, Germany; Max Planck Institute of Molecular Physiology, Dortmund, Germany.
    Bacterial interaction with host autophagy2019In: Virulence, ISSN 2150-5594, E-ISSN 2150-5608, Vol. 10, no 1, p. 352-362Article, review/survey (Refereed)
    Abstract [en]

    Autophagy is a conserved and fundamental cellular process mainly to recycle or eliminate dysfunctional cellular organelles or proteins. As a response to cellular stress, autophagy is used as a defense mechanism to combat the infection with pathogenic bacteria. However, many intracellular bacteria have developed diverse mechanisms to evade recognition, to manipulate the autophagic pathway, and to hijack the autophagosomal compartment for replication. In this review, we discuss recent understandings on how bacteria interact with host autophagy.

  • 344.
    Wu, Yao-Wen
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Waldmann, Herbert
    Toward the role of cholesterol and cholesterol transfer protein in autophagosome biogenesis2019In: Autophagy, ISSN 1554-8627, E-ISSN 1554-8635, Vol. 15, no 12, p. 2167-2168Article in journal (Refereed)
    Abstract [en]

    A forward chemical genetic approach led to identification of autogramins as novel autophagy inhibitors. Autogramins selectively target the cholesterol transfer protein GRAMD1A (GRAM domain containing 1A). Autogramins compete with cholesterol binding to the StART domain of GRAMD1A, thereby inhibiting its cholesterol transfer activity. GRAMD1A associates with phosphatidylinositol monophosphate via its GRAM domain. GRAMD1A accumulates at autophagosome initiation sites upon starvation. This protein is involved in cholesterol distribution in response to starvation and is required for autophagosome biogenesis. Therefore, we identify a novel function of GRAMD1A and a new role of cholesterol in macroautophagy/autophagy.

  • 345.
    Yadav, Akhilesh K.
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Espaillat, Akbar
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bacterial Strategies to Preserve Cell Wall Integrity Against Environmental Threats2018In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 9, article id 2064Article, review/survey (Refereed)
    Abstract [en]

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

  • 346. Yang, Fan
    et al.
    Korban, Schuyler S.
    Pusey, P. Lawrence
    Elofsson, Michael
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Sundin, George W.
    Zhao, Youfu
    Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora2014In: Molecular plant pathology, ISSN 1464-6722, E-ISSN 1364-3703, Vol. 15, no 1, p. 44-57Article in journal (Refereed)
    Abstract [en]

    The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E.amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E.amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E.amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease.

  • 347.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Jansson, Anna L
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Panahandeh, Tooba
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Palmer, Ruth H
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine).
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology).
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Medical Biosciences, Pathology.
    Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity.2006In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 273, no 3, p. 638-646Article in journal (Refereed)
  • 348.
    Yasmin, Lubna
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Veesenmeyer, Jeffrey L
    Diaz, Maureen H
    Francis, Matthew S
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Ottmann, Christian
    Palmer, Ruth H
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Hauser, Alan R
    Hallberg, Bengt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins2010In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 427, no 2, p. 217-224Article in journal (Refereed)
    Abstract [en]

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

  • 349.
    Zakrisson, Johan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Tethered cells in fluid flows: beyond the Stokes’ drag force approach2015In: Physical Biology, ISSN 1478-3967, E-ISSN 1478-3975, Vol. 12, article id 056006Article in journal (Refereed)
    Abstract [en]

    Simulations of tethered cells in viscous sub-layers are frequently performed using the Stokes' drag force, but without taking into account contributions from surface corrections, lift forces, buoyancy, the Basset force, the cells' finite inertia, or added mass. In this work, we investigate to what extent such contributions, under a variety of hydrodynamic conditions, influence the force at the anchor point of a tethered cell and the survival probability of a bacterium that is attached to a host by either a slip or a catch bond via a tether with a few different biomechanical properties. We show that a consequence of not including some of these contributions is that the force to which a bond is exposed can be significantly underestimated; in general by similar to 32-46%, where the influence of the surface corrections dominate ( the parallel and normal correction coefficients contribute similar to 5-8 or similar to 23-26%, respectively). The Basset force is a major contributor, up to 20%, for larger cells and shear rates. The lift force and inertia contribute when cells with radii >3 mu m have shear rates>2000 s(-1). Buoyancy contributes significantly for cells with radii > 3 mu m for shear rates<10 s(-1). Since the lifetime of a bond depends strongly on the force, both the level of approximation and the biomechanical model of the tether significantly affect the survival probability of tethered bacteria. For a cell attached by a FimH-mannose bond and an extendable tether with a shear rate of 3000 s(-1), neglecting the surface correction coefficients or the Basset force can imply that the survival probability is overestimated by more than an order of magnitude. This work thus shows that in order to quantitatively assess bacterial attachment forces and survival probabilities, both the fluid forces and the tether properties need to be modeled accurately.

  • 350.
    Zakrisson, Johan
    et al.
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Wiklund, Krister
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Axner, Ove
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Andersson, Magnus
    Umeå University, Faculty of Science and Technology, Department of Physics. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    The shaft of the type 1 fimbriae regulates an externalforce to match the FimH catch bond2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 10, p. 2137-2148Article in journal (Refereed)
    Abstract [en]

    Type 1 fimbriae mediate adhesion of uropathogenic Escherichia coli to host cells. It has been hypothesized that due to their ability to uncoil under exposure to force, fimbriae can reduce fluid shear stress on the adhesin-receptor interaction by which the bacterium adheres to the surface. In this work, we develop a model that describes how the force on the adhesin-receptor interaction of a type 1 fimbria varies as a bacterium is affected by a time-dependent fluid flow mimicking in vivo conditions. The model combines in vivo hydrodynamic conditions with previously assessed biomechanical properties of the fimbriae. Numerical methods are used to solve for the motion and adhesion force under the presence of time-dependent fluid profiles. It is found that a bacterium tethered with a type 1 pilus will experience significantly reduced shear stress for moderate to high flow velocities and that the maximum stress the adhesin will experience is limited to ∼120 pN, which is sufficient to activate the conformational change of the FimH adhesin into its stronger state but also lower than the force required for breaking it under rapid loading. Our model thus supports the assumption that the type 1 fimbria shaft and the FimH adhesin-receptor interaction are optimized to each other, and that they give piliated bacteria significant advantages in rapidly changing fluidic environments.

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