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  • 101.
    Chandra, Naresh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University.
    The glycobiology of human adenovirus infections: implications for tropism and treatment2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Human adenoviruses (HAdVs) are common human pathogens, causing gastrointestinal, ocular, and respiratory infections on a regular basis. Epidemic keratoconjunctivitis (EKC) is a severe ocular infection for which no approved antivirals are available. HAdV-D37 is one of the causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. HAdV-D37 interacts with SA via the knob domain of the trimeric virus fiber protein, containing three SA-binding sites. HAdV-D37 also bind to glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, using biochemical and cell-based assays, the impact of GAGs on HAdV-D37 infection (paper I) was investigated. We found that HAdV-D37 interacts with both soluble and cell-surface sulfated GAGs via the knob domain of the viral fiber protein. Remarkably, removal of heparan sulfate (HS; a type of GAG) from human corneal epithelial (HCE) cells by heparinase III enhanced HAdV-D37 infection. We propose that sulfated GAGs in bodily secretions and on plasma membranes function as decoy receptors that prevent the virus from binding to SA-containing receptors and inhibit subsequent virus infection. We also found abundant HS in the basement membrane of the human corneal epithelium. We suggest that this layer of HS functions as a barrier to sub-epithelial infection of HAdV-D37. Based on this finding, we hypothesized that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Here, the antiviral effect of suramin (a known GAG-mimetic) and its analogs against HAdV-D37 (paper II) was evaluated. Interestingly, all compounds displayed antiviral effects by inhibiting the binding of HAdV-D37 to HCE cells. The antiviral effect of suramin was HAdV species-specific. We report for the first time that virus binding to cell-surface decoy receptor constitutes a potential target for antiviral drug development.

    HAdVs are the major cause of infectious conjunctivitis, constituting up to 75% of all conjunctivitis cases worldwide. Species B HAdV type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause EKC. Recently, HAdV-D53, -D54, and -D56 have emerged as new EKC-causing agents. HAdV-E4 causes both PCF and EKC. SA-containing glycans have been established as cellular receptors for HAdV-D37. By means of cell-based assays, we investigated if ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells (paper III). It was found that SA-containing glycans function as cellular receptors for five (HAdV-D8, -D37, -D53, -D54, and -D64) out of six EKC-causing species D HAdVs. We showed that these viruses interact with SAs via the knob domain of the viral fiber protein. HAdV-E4 and -D56 infection of cells was independent of SAs. Surprisingly, HCE cells were completely refractory to HAdV-B3 infection. A trivalent sialic acid (TSA) derivative ME0462 (compound 17a in paper II), designed to bind to SA-binding sites on HAdV-D37 fiber knob, also showed potent antiviral activity against several EKC-causing HAdVs. This suggests that ME0462 can be used as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs. Surface plasmon resonance (SPR) analysis confirmed a direct interaction between ME0462 and fiber knobs of EKC-causing HAdVs.

    Recently, a TSA derivative (ME0322; designed to bind to SA-binding sites on HAdV-D37 fiber knob) was shown potent antiviral against HAdV-D37 in vitro. To improve the antiviral potency of this compound, six new TSA derivatives were synthesized and their inhibitory effects were evaluated against HAdV-D37 (paper IV). Interestingly, the best compound 17a was found approximately three orders of magnitude more potent (IC50 (binding) = 1.4 nM, IC50 (infection) = 2.9 nM) than ME0322 (IC50 in µM range). SPR data showed that HAdV-D37 fiber knob binds to TSA compounds with high affinities. Structural data revealed the trivalent binding mode of all newly synthesized TSA compounds to HAdV-D37 fiber knob. Ophthalmic toxicity of compound 17a (best compound) was also investigated in rabbits without any sign of toxicity.

    HAdV-D36 is a member of species D HAdV and has the ability to infect a broad range of animals, which is unusual for HAdVs. Another remarkable feature of HAdV-D36 is that this virus induces obesity in experimental animals. Several epidemiological studies highlighted a link between HAdV-D36 and human obesity. There is no information about the cellular receptor usage by HAdV-D36. Using structural biology and cell-based approaches, we investigated the cellular receptor(s) for HAdV-D36 (paper V).  We show that HAdV-D36 attaches to host cells (via the fiber knob) using the coxsackie and adenovirus receptor (CAR), SA-containing glycans, and one or more unknown proteins or glycoproteins. Using glycan microarray, we found that HAdV-D36 displays binding preference to a rare SA-variant: 4-O,5-N-diacetylneuraminic acid (Neu4,5Ac2), over the more common SA (in humans) i.e. 5-N-acetylneuraminic acid (Neu5Ac). Structural analysis of HAdV-D36 fiber knob:Neu4,5Ac2 complex explained this preference. To date, Neu4,5Achas not been detected in humans, although it is synthesized by many domestic and livestock animals. Our results indicate that HAdV-D36 has evolved to utilize a specialized set of cellular receptors that coincide with a unique host range and pathogenicity profile.

    These studies provide insights into multiple roles of glycans in HAdV infection cycle and highlight the therapeutic potential of glycans/glycan-mimetics in HAdV-D37 infection.

  • 102.
    Chandra, Naresh
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Imhof, Sophie
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Caraballo, Rémi
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Elofsson, Mikael
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Sialic Acid-Containing Glycans as Cellular Receptors for Ocular Human Adenoviruses: Implications for Tropism and Treatment2019In: Viruses, ISSN 1999-4915, E-ISSN 1999-4915, Vol. 11, no 5, article id 395Article in journal (Refereed)
    Abstract [en]

    Human adenoviruses (HAdV) are the most common cause of ocular infections. Species B human adenovirus type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause epidemic keratoconjunctivitis (EKC). Recently, HAdV-D53, -D54, and -D56 emerged as new EKC-causing agents. HAdV-E4 is associated with both PCF and EKC. We have previously demonstrated that HAdV-D37 uses sialic acid (SA)-containing glycans as cellular receptors on human corneal epithelial (HCE) cells, and the virus interaction with SA is mediated by the knob domain of the viral fiber protein. Here, by means of cell-based assays and using neuraminidase (a SA-cleaving enzyme), we investigated whether ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells. We found that HAdV-E4 and -D56 infect HCE cells independent of SAs, whereas HAdV-D53 and -D64 use SAs as cellular receptors. HAdV-D8 and -D54 fiber knobs also bound to cell-surface SAs. Surprisingly, HCE cells were found resistant to HAdV-B3 infection. We also demonstrated that the SA-based molecule i.e., ME0462, designed to bind to SA-binding sites on the HAdV-D37 fiber knob, efficiently prevents binding and infection of several EKC-causing HAdVs. Surface plasmon resonance analysis confirmed a direct interaction between ME0462 and fiber knobs. Altogether, we demonstrate that SA-containing glycans serve as receptors for multiple EKC-causing HAdVs, and, that SA-based compound function as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs.

  • 103.
    Chapman, K B
    et al.
    Umeå University, Faculty of Medicine, Microbiology. Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD.
    Byström, Anders S
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Boeke, J D
    Initiator methionine tRNA is essential for Ty1 transposition in yeast1992In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 89, no 8, p. 3236-3240Article in journal (Refereed)
    Abstract [en]

    The yeast retrotransposon Ty1 transposes through an RNA intermediate by a mechanism similar to that of retroviral reverse transcription and integration. Ty1 RNA contains a putative minus strand primer binding site (-PBS) that is complementary to the 3' acceptor stem of the initiator methionine tRNA (tRNA(iMet)). Here we demonstrate that the tRNA(iMet) is used as a primer for Ty1 reverse transcription. Mutations in the Ty1 element that alter 5 of 10 nucleotides that are complementary to the tRNA(iMet) abolish Ty1 transposition, even though they are silent with regard to Ty1 protein coding. We have constructed a yeast strain lacking wild-type tRNA(iMet) that is dependent on a mutant derivative of tRNA(iMet) that has an altered acceptor stem sequence, engineered to restore homology with the Ty1 -PBS mutant. The compensatory mutations made in the tRNA(iMet) alleviate the transposition defect of the Ty1 -PBS mutant. The mutant and wild-type tRNA(iMet) are enriched within Ty1 virus-like particles irrespective of complementarity to the Ty1 -PBS. Thus, complementarity between the Ty1 -PBS and tRNA(iMet) is essential for transposition but is not necessary for packaging of the tRNA inside virus-like particles.

  • 104.
    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). Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Institute for Biology, Humboldt University, Berlin, Germany.
    Spotlight on... Emmanuelle Charpentier2018In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 365, no 4, article id fnx271Article in journal (Other academic)
  • 105.
    Charpentier, Emmanuelle
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Marraffini, Luciano A.
    Editorial overview: Novel technologies in microbiology: Recent advances in techniques in microbiology2014In: Current Opinion in Microbiology, ISSN 1369-5274, E-ISSN 1879-0364, Vol. 19, p. VIII-XArticle in journal (Refereed)
  • 106. Chauhan, Deepika
    et al.
    Srivastava, Pulkit Anupam
    Ritzl, Barbara
    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).
    Yennamalli, Ragothaman M.
    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).
    Priyadarshini, Richa
    Amino Acid-Dependent Alterations in Cell Wall and Cell Morphology of Deinococcus indicus DR12019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1449Article in journal (Refereed)
    Abstract [en]

    Deinococcus radiodurans exhibits growth medium-dependent morphological variation in cell shape, but there is no evidence whether this phenomenon is observed in other members of the Deinococcaceae family. In this study, we isolated a red-pigmented, aerobic, Deinococcus indicus strain DR1 from Dadri wetland, India. This D. indicus strain exhibited cell-morphology transition from rod-shaped cells to multi-cell chains in a growth-medium-dependent fashion. In response to addition of 1% casamino acids in the minimal growth medium, rod-shaped cells formed multi-cell chains. Addition of all 20 amino acids to the minimal medium was able to recapitulate the phenotype. Specifically, a combination of L-methionine, L-lysine, L-aspartate, and L-threonine caused morphological alterations. The transition from rod shape to multi-cell chains is due to delay in daughter cell separation after cell division. Minimal medium supplemented with L-ornithine alone was able to cause cell morphology changes. Furthermore, a comparative UPLC analysis of PG fragments isolated from D. indicus cells propagated in different growth media revealed alterations in the PG composition. An increase in the overall cross-linkage of PG was observed in muropeptides from nutrient-rich TSB and NB media versus PYE medium. Overall our study highlights that environmental conditions influence PG composition and cell morphology in D. indicus.

  • 107.
    Claesson, Rolf
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Gudmundson, Jan
    Östersund, Sweden.
    Höglund-Åberg, Carola
    Umeå University, Faculty of Medicine, Department of Odontology.
    Haubek, Dorte
    Aarhus, Denmark.
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Detection of a 640-bp deletion in the Aggregatibacter actinomycetemcomitans leukotoxin promoter region in isolates from an adolescent of Ethiopian origin2015In: Journal of Oral Microbiology, ISSN 2000-2297, E-ISSN 2000-2297, Vol. 7, article id 26974Article in journal (Refereed)
    Abstract [en]

    The expression of the leukotoxin of Aggregatibacter actinomycetemcomitans is regulated by the leukotoxin promoter. A 530-bp deletion or an 886-bp insertion sequence (IS) element in this region has earlier been described in highly leukotoxic isolates. Here, we report on highly leukotoxic isolate with a 640-bp deletion, which was detected in an adolescent of Ethiopian origin.

  • 108.
    Claesson, Rolf
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Höglund-Åberg, Carola
    Umeå University, Faculty of Medicine, Department of Odontology.
    Haubek, Dorte
    Johansson, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Age-related prevalence and characteristics of Aggregatibacter actinomycetemcomitans in periodontitis patients living in Sweden2017In: Journal of Oral Microbiology, ISSN 2000-2297, E-ISSN 2000-2297, Vol. 9, article id 1334504Article in journal (Refereed)
    Abstract [en]

    Background: The presence of Aggregatibacter actinomycetemcomitans in patients with periodontitis has been extensively studied for decades. Objective: To study the prevalence of A. actinomycetemcomitans in younger and older periodontitis patients and to genetically characterize isolates of this bacterium. Design: Data from microbiological analyses of 3459 subgingival plaque samples collected from 1445 patients, 337 'younger' patients (<= 35 yrs) and 1108 'older' patients (>35 yrs) during 15 years (2000-2014), has been summerized. Isolates of A. actinomycetemcomitans were serotyped, leukotoxin promoter typed (JP2 and non JP2) and arbitrarily primed PCR (APPCR) genotyped. The origin of the JP2 genotype detected in the study population was determined. Results: The prevalence of A. actinomycetemcomitans was higher among younger than older patients and samples from the younger patients contained higher proportions of the bacterium. Serotype b was more prevalent among younger patients and the majorty of these isolates was from the same AP-PCR genotype. The JP2 genotype was detected in 1.2% of the patients, and the majority of these carriers were of non-African origin. Conslusions: For presence and charcteristics of A. actinomycetemcomitans in clinical samples the age of the carriers were a discriminating factor. Additional, apparently non- African carriers of the JP2 genotype of A. actinomycetemcomitans were identified.

  • 109.
    Claesson, Rolf
    et al.
    Umeå University, Faculty of Medicine, Department of Odontology.
    Sjögren, Ulf
    Umeå University, Faculty of Medicine, Department of Odontology.
    Esberg, Anders
    Umeå University, Faculty of Medicine, Department of Odontology.
    Brundin, Malin
    Umeå University, Faculty of Medicine, Department of Odontology.
    Granlund, Margareta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Actinomyces radicidentis and Actinomyces haliotis, coccoid Actinomyces species isolated from the human oral cavity2017In: Anaerobe, ISSN 1075-9964, E-ISSN 1095-8274, Vol. 48, p. 19-26Article in journal (Refereed)
    Abstract [en]

    There are few reports on the bacterial species Actinomyces radicidentis in the literature. In this study, putative A. radicidentis isolates were collected from 16 root canal samples from 601 examined patients. The isolates were examined by biochemical tests, 16S rRNA gene sequencing, Arbitrarily-primed (AP-) PCR, antibiotic susceptibility testing, and MALDI-TOF analyses. In parallel, two A. radicidentis reference strains and two putative A. radicidentis isolates from United Kingdom were tested. Sixteen of the 18 isolates were confirmed as A. radicidentis. The remaining two isolates, both of which were isolated from root canals (one from Sweden and the other from the UK), but were identified as Actinomyces haliotis by sequencing ∼ 1300 base pairs of the 16S rRNA-gene. This isolates had a divergent, but between them similar, AP-PCR pattern, and a common distribution of sequence signatures in the 16S rRNA gene, but were not identified by MALDI-TOF. A. haliotis is a close relative to A. radicidentis, hitherto only been described from a sea-snail. The identity of A. haliotis was confirmed by a phylogenetic tree based on 16S rRNA gene sequences with species specific sequences included, and by additional biochemical tests. The examined bacteria exhibited similar antibiotic susceptibility patterns when tested for 10 separate antibiotic classes with E-tests (bioMérieux). The MIC90 for β-lactams (benzylpenicillin and cefuroxime) and vancomycin was 0.5 mg/L, for colistin and ciprofloxacin 8 mg/mL and for the other antibiotic classes ≤ 25 mg/mL The isolation of A. haliotis from infected dental root canals cast doubt on the accepted opinion that all Actinomyces infections have an endogenous source.

  • 110. Clementi, Emily A.
    et al.
    Wilhelm, Kristina R.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Schleucher, Juergen
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Morozova-Roche, Ludmilla A.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Hakansson, Anders P.
    A Complex of Equine Lysozyme and Oleic Acid with Bactericidal Activity against Streptococcus pneumoniae2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 11, p. Article Number: UNSP e80649-Article in journal (Refereed)
    Abstract [en]

    HAMLET and ELOA are complexes consisting of oleic acid and two homologous, yet functionally different, proteins with cytotoxic activities against mammalian cells, with HAMLET showing higher tumor cells specificity, possibly due to the difference in propensity for oleic acid binding, as HAMLET binds 5-8 oleic acid molecules per protein molecule and ELOA binds 11-48 oleic acids. HAMLET has been shown to possess bactericidal activity against a number of bacterial species, particularly those with a respiratory tropism, with Streptococcus pneumoniae displaying the greatest degree of sensitivity. We show here that ELOA also displays bactericidal activity against pneumococci, which at lower concentrations shows mechanistic similarities to HAMLET's bactericidal activity. ELOA binds to S. pneumoniae and causes perturbations of the plasma membrane, including depolarization and subsequent rupture, and activates an influx of calcium into the cells. Selective inhibition of calcium channels and sodium/calcium exchange activity significantly diminished ELOA's bactericidal activity, similar to what we have observed with HAMLET. Finally, ELOA-induced death was also accompanied by DNA fragmentation into high molecular weight fragments - an apoptosis-like morphological phenotype that is seen during HAMLET-induced death. Thus, in contrast to different mechanisms of eukaryote cell death induced by ELOA and HAMLET, these complexes are characterized by rather similar activities towards bacteria. Although the majority of these events could be mimicked using oleic acid alone, the concentrations of oleic acid required were significantly higher than those present in the ELOA complex, and for some assays, the results were not identical between oleic acid alone and the ELOA complex. This indicates that the lipid, as a common denominator in both complexes, is an important component for the complexes' bactericidal activities, while the proteins are required both to solubilize and/or present the lipid at the bacterial membrane and likely to confer other and separate functions during the bacterial death.

  • 111.
    Connolly-Andersen, Anne-Marie
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Thunberg, Therese
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Endothelial activation and repair during hantavirus infection: association with disease outcome2014In: Open forum infectious diseases, ISSN 2328-8957, Vol. 1, no 1, article id ofu027Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Endothelial activation and dysfunction play a central role in the pathogenesis of sepsis and viral hemorrhagic fevers. Hantaviral disease is a viral hemorrhagic fever and is characterized by capillary dysfunction, although the underlying mechanisms for hantaviral disease are not fully elucidated.

    METHODS: The temporal course of endothelial activation and repair were analyzed during Puumala hantavirus infection and associated with disease outcome and a marker for hypoxia, insulin-like growth factor binding protein 1 (IGFBP-1). The following endothelial activation markers were studied: endothelial glycocalyx degradation (syndecan-1) and leukocyte adhesion molecules (soluble vascular cellular adhesion molecule 1, intercellular adhesion molecule 1, and endothelial selectin). Cytokines associated with vascular repair were also analyzed (vascular endothelial growth factor, erythropoietin, angiopoietin, and stromal cell-derived factor 1).

    RESULTS: Most of the markers we studied were highest during the earliest phase of hantaviral disease and associated with clinical and laboratory surrogate markers for disease outcome. In particular, the marker for glycocalyx degradation, syndecan-1, was significantly associated with levels of thrombocytes, albumin, IGFBP-1, decreased blood pressure, and disease severity.

    CONCLUSIONS: Hantaviral disease outcome was associated with endothelial dysfunction. Consequently, the endothelium warrants further investigation when designing future medical interventions.

  • 112.
    Costa, Tiago
    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).
    Amer, Ayad
    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).
    Farag, Salah
    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).
    Wolf-Watz, Hans
    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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    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).
    Edgren, Tomas
    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, 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).
    Type III secretion translocon assemblies that attenuate Yersinia virulence2013In: Cellular Microbiology, ISSN 1462-5814, E-ISSN 1462-5822, Vol. 15, no 7, p. 1088-1110Article in journal (Refereed)
    Abstract [en]

    Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD(I262P) and YopD(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD(A263P) and YopD(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

  • 113.
    Costa, Tiago
    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). Department of Life Sciences, MRC Centre for Molecular Bacteriology and Infection, Imperial College, London, UK.
    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). Region Västerbotten.
    Farag, Salah
    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).
    Edgren, Tomas
    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). Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Center, Uppsala University, Uppsala, Sweden.
    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).
    Measurement of Yersinia translocon pore formation in erythrocytes2019In: Pathogenic Yersinia: methods and protocols / [ed] Viveka Vadyvaloo and Matthew B. Lawrenz, New York, NY, U.S.A.: Humana Press, 2019, p. 211-229Chapter in book (Refereed)
    Abstract [en]

    Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

  • 114.
    Costa, Tiago R. D.
    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).
    Amer, Ayad A. A.
    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).
    Farag, Salah I.
    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).
    Wolf-Watz, Hans
    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). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Fällman, Maria
    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).
    Fahlgren, Anna
    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).
    Edgren, Tomas
    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, 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).
    Active type III translocon assemblies that attenuate Yersinia virulenceManuscript (preprint) (Other academic)
    Abstract [en]

    Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted a-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localised Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopDA263P and YopDA270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.

  • 115.
    Costa, Tiago R. D.
    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).
    Carlsson, Katrin E.
    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).
    Edqvist, Petra J.
    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, 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).
    Influence of the LcrH chaperone on type III secretion system regulation in Yersinia pseudotuberculosisManuscript (preprint) (Other academic)
    Abstract [en]

    Human pathogenic Yersiniae share a common virulence plasmid that encodes for the Ysc-Yop type III secretion system (T3SS). Control of yop expression involves several pathways in which their cross-talk is not completely understood. LcrF, an AraC-like transcriptional activator, is required for temperature-dependent yop-transcription. In contrast, a repressive effect of the T3S chaperone LcrH and the cognate translocator substrate YopD occurs through binding to yop mRNA and inhibiting translation; a process that is also thought to involve LcrQ. Several homologous members of the LcrH family of translocator-class of T3S chaperones can act as a cofactor to amplify the activity of transcriptional activators analogous to LcrF. However, we show here in Y. pseudotuberculosis that LcrH does not induce LcrF-dependent transcription of target genes. Moreover, a full length DlcrH null mutant in which YopB and YopD are rapidly degraded is totally de-repressed for Yop synthesis even though the anti-activator LcrQ is forced to accumulate in the cytoplasm through rendering the Ysc-Yop T3SS non-functional or ectopically producing LcrQ in trans. Typically, this mutant cannot grow at 37°C. Thus, in all respects, the DlcrH null mutant mirrors the regulatory defects established for Yersinia lacking the translocator and anti-activator YopD. On the other hand, Y. pseudotuberculosis producing the LcrHE30G point mutant that is defective for YscY chaperone binding exhibits a mild regulatory defect that permits some growth at 37°C, but is blind to the cytoplasmic accumulation of LcrQ. Critically however, this mutant still responds to repression caused by YopD accumulation, which is stably produced and efficiently secreted by this strain. Thus, our work with LcrHE30G indicates an additional regulatory function of this versatile T3S chaperone that is independent of the LcrF transcription factor and the YopD anti-activator.

     

  • 116.
    Costa, Tiago R D
    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).
    Edqvist, Petra J
    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).
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Åhlund, 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).
    Forsberg, Åke
    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 Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    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).
    YopD self-assembly and binding to LcrV facilitate type III secretion activity by Yersinia pseudotuberculosis2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 33, p. 25269-25284Article in journal (Refereed)
    Abstract [en]

    YopD-like translocator proteins encoded by several Gram-negative bacteria are important for type III secretion-dependent delivery of anti-host effectors into eukaryotic cells. This probably depends on their ability to form pores in the infected cell plasma membrane, through which effectors may gain access to the cell interior. In addition, Yersinia YopD is a negative regulator essential for the control of effector synthesis and secretion. As a prerequisite for this functional duality, YopD may need to establish molecular interactions with other key T3S components. A putative coiled-coil domain and an alpha-helical amphipathic domain, both situated in the YopD C terminus, may represent key protein-protein interaction domains. Therefore, residues within the YopD C terminus were systematically mutagenized. All 68 mutant bacteria were first screened in a variety of assays designed to identify individual residues essential for YopD function, possibly by providing the interaction interface for the docking of other T3S proteins. Mirroring the effect of a full-length yopD gene deletion, five mutant bacteria were defective for both yop regulatory control and effector delivery. Interestingly, all mutations clustered to hydrophobic amino acids of the amphipathic domain. Also situated within this domain, two additional mutants rendered YopD primarily defective in the control of Yop synthesis and secretion. Significantly, protein-protein interaction studies revealed that functionally compromised YopD variants were also defective in self-oligomerization and in the ability to engage another translocator protein, LcrV. Thus, the YopD amphipathic domain facilitates the formation of YopD/YopD and YopD/LcrV interactions, two critical events in the type III secretion process.

  • 117.
    Croxatto, Antony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Pride, John
    Hardman, Andrea
    Williams, Paul
    Cámara, Miguel
    Milton, Debra L.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum2004In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 52, no 6, p. 1677-1689Article in journal (Refereed)
    Abstract [en]

    Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.

  • 118.
    Croxatto, Antony
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Weber, Barbara
    Chen, Chang
    Milton, Debra L
    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 Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Post-transcriptional regulation of the Vibrio anguillarum quorum-sensing regulator vanT by RpoSManuscript (Other academic)
  • 119. Danielsson, Jens
    et al.
    Awad, Wael
    Saraboji, Kadhirvel
    Kurnik, Martin
    Lang, Lisa
    Leinartaite, Lina
    Marklund, Stefan L
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry.
    Logan, Derek T
    Oliveberg, Mikael
    Global structural motions from the strain of a single hydrogen bond2013In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 110, no 10, p. 3829-3834Article in journal (Refereed)
    Abstract [en]

    The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the beta-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

  • 120. De Pascalis, Roberto
    et al.
    Chou, Alicia Y.
    Ryden, Patrik
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Kennett, Nikki J.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Elkins, Karen L.
    Models Derived from In Vitro Analyses of Spleen, Liver, and Lung Leukocyte Functions Predict Vaccine Efficacy against the Francisella tularensis Live Vaccine Strain (LVS)2014In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 5, no 2, article id e00936Article in journal (Refereed)
    Abstract [en]

    Currently, there are no licensed vaccines and no correlates of protection against Francisella tularensis, which causes tularemia. We recently demonstrated that measuring in vitro control of intramacrophage bacterial growth by murine F. tularensis-immune splenocytes, as well as transcriptional analyses, discriminated Francisella vaccines of different efficacies. Further, we identified potential correlates of protection against systemic challenge. Here, we extended this approach by studying leukocytes derived from lungs and livers of mice immunized by parenteral and respiratory routes with F. tularensis vaccines. Liver and lung leukocytes derived from intradermally and intranasally vaccinated mice controlled in vitro Francisella Live Vaccine Strain (LVS) intramacrophage replication in patterns similar to those of splenocytes. Gene expression analyses of potential correlates also revealed similar patterns in liver cells and splenocytes. In some cases (e. g., tumor necrosis factor alpha [TNF-alpha], interleukin 22 [IL-22], and granulocyte-macrophage colony-stimulating factor [GM-CSF]), liver cells exhibited even higher relative gene expression, whereas fewer genes exhibited differential expression in lung cells. In contrast with their strong ability to control LVS replication, splenocytes from intranasally vaccinated mice expressed few genes with a hierarchy of expression similar to that of splenocytes from intradermally vaccinated mice. Thus, the relative levels of gene expression vary between cell types from different organs and by vaccination route. Most importantly, because studies comparing cell sources and routes of vaccination supported the predictive validity of this coculture and gene quantification approach, we combined in vitro LVS replication with gene expression data to develop analytical models that discriminated between vaccine groups and successfully predicted the degree of vaccine efficacy. Thus, this strategy remains a promising means of identifying and quantifying correlative T cell responses.

    IMPORTANCE

    Identifying and quantifying correlates of protection is especially challenging for intracellular bacteria, including Francisella tularensis. F. tularensis is classified as a category A bioterrorism agent, and no vaccines have been licensed in the United States, but tularemia is a rare disease. Therefore, clinical trials to test promising vaccines are impractical. In this report, we further evaluated a novel approach to developing correlates by assessing T cell immune responses in lungs and livers of differentially vaccinated mice; these nonprofessional immune tissues are colonized by Francisella. The relative degree of vaccine efficacy against systemic challenge was reflected by the ability of immune T cells, particularly liver T cells, to control the intramacrophage replication of bacteria in vitro and by relative gene expression of several immunological mediators. We therefore developed analytical models that combined bacterial replication data and gene expression data. Several resulting models provided excellent discrimination between vaccines of different efficacies.

  • 121. De Pascalis, Roberto
    et al.
    Hahn, Andrew
    Brook, Helen M.
    Ryden, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Donart, Nathaniel
    Mittereder, Lara
    Frey, Blake
    Wu, Terry H.
    Elkins, Karen L.
    A panel of correlates predicts vaccine-induced protection of rats against respiratory challenge with virulent Francisella tularensis2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 5, article id e0198140Article in journal (Refereed)
    Abstract [en]

    There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat killed LVS, which were poorly protective. These genes included IFN-gamma, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5-7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.

  • 122.
    de Pedro, Miguel A.
    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).
    Cava, Felipe
    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).
    Structural constraints and dynamics of bacterial cell wall architecture2015In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, article id 449Article, review/survey (Refereed)
    Abstract [en]

    The peptidoglycan wall (PG) is a unique structure which confers physical strength and defined shape to bacteria. It consists of a net-like macromolecule of peptide interlinked glycan chains overlying the cell membrane. The structure and layout of the PG dictates that the wall has to be continuously modified as bacteria go through division, morphological differentiation, and adaptive responses. The PG is poorly known in structural terms. However, to understand morphogenesis a precise knowledge of glycan strand arrangement and of local effects of the different kinds of subunits is essential. The scarcity of data led to a conception of the PG as a regular, highly ordered structure which strongly influenced growth models. Here, we review the structure of the PG to define a more realistic conceptual framework. We discuss the consequences of the plasticity of murein architecture in morphogenesis and try to define a set of minimal structural constraints that must be fulfilled by any model to be compatible with present day information.

  • 123. Debruyne, L
    et al.
    Broman, T
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olsen, B
    Univ Uppsala Hosp, Dept Med Sci, Infect Dis Sect.
    On, SLW
    Vandamme, P
    Campylobacter volucris sp nov., isolated from black-headed gulls (Larus ridibundus)2010In: International Journal of Systematic and Evolutionary Microbiology, ISSN 1466-5026, E-ISSN 1466-5034, Vol. 60, no 8, p. 1870-1875Article in journal (Refereed)
    Abstract [en]

    During a study of the prevalence of Campylobacter jejuni in black-headed gulls (Larus ridibundus) in Sweden, three isolates, strains LMG 24379, LMG 24380(T) and LMG 24381, were initially identified as Campylobacter lari. Further characterization by both AFLP and whole-cell protein SDS-PAGE analyses revealed that they formed a distinct group in the genus Cam pylobacter. This unique position was confirmed by phenotypic characterization, 16S rRNA and hsp60 gene sequence analysis and DNA-DNA hybridizations. The combined data confirm that these isolates represent a novel species within the genus Campylobacter, for which the name Campylobacter volucris sp. nov. is proposed. The type strain is LMG 24380(T) (=CCUG 57498(T)).

  • 124. Desmarais, Samantha M.
    et al.
    Cava, Felipe
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    de Pedro, Miguel A.
    Huang, Kerwyn Casey
    Isolation and preparation of bacterial cell walls for compositional analysis by Ultra Performance Liquid Chromatography2014In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 83, p. UNSP e51183-Article in journal (Refereed)
    Abstract [en]

    The bacterial cell wall is critical for the determination of cell shape during growth and division, and maintains the mechanical integrity of cells in the face of turgor pressures several atmospheres in magnitude. Across the diverse shapes and sizes of the bacterial kingdom, the cell wall is composed of peptidoglycan, a macromolecular network of sugar strands crosslinked by short peptides. Peptidoglycan's central importance to bacterial physiology underlies its use as an antibiotic target and has motivated genetic, structural, and cell biological studies of how it is robustly assembled during growth and division. Nonetheless, extensive investigations are still required to fully characterize the key enzymatic activities in peptidoglycan synthesis and the chemical composition of bacterial cell walls. High Performance Liquid Chromatography (HPLC) is a powerful analytical method for quantifying differences in the chemical composition of the walls of bacteria grown under a variety of environmental and genetic conditions, but its throughput is often limited. Here, we present a straightforward procedure for the isolation and preparation of bacterial cell walls for biological analyses of peptidoglycan via HPLC and Ultra Performance Liquid Chromatography (UPLC), an extension of HPLC that utilizes pumps to deliver ultra-high pressures of up to 15,000 psi, compared with 6,000 psi for HPLC. In combination with the preparation of bacterial cell walls presented here, the low-volume sample injectors, detectors with high sampling rates, smaller sample volumes, and shorter run times of UPLC will enable high resolution and throughput for novel discoveries of peptidoglycan composition and fundamental bacterial cell biology in most biological laboratories with access to an ultracentrifuge and UPLC.

  • 125. Dever, Thomas E
    et al.
    Yang, Weimin
    Åström, Stefan
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Byström, Anders S
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Hinnebusch, A G
    Modulation of tRNA(iMet), eIF-2, and eIF-2B expression shows that GCN4 translation is inversely coupled to the level of eIF-2.GTP.Met-tRNA(iMet) ternary complexes1995In: Molecular and Cellular Biology, ISSN 0270-7306, E-ISSN 1098-5549, Vol. 15, no 11, p. 6351-6363Article in journal (Refereed)
    Abstract [en]

    To understand how phosphorylation of eukaryotic translation initiation factor (eIF)-2 alpha in Saccharomyces cerevisiae stimulates GCN4 mRNA translation while at the same time inhibiting general translation initiation, we examined the effects of altering the gene dosage of initiator tRNA(Met), eIF-2, and the guanine nucleotide exchange factor for eIF-2, eIF-2B. Overexpression of all three subunits of eIF-2 or all five subunits of eIF-2B suppressed the effects of eIF-2 alpha hyperphosphorylation on both GCN4-specific and general translation initiation. Consistent with eIF-2 functioning in translation as part of a ternary complex composed of eIF-2, GTP, and Met-tRNA(iMet), reduced gene dosage of initiator tRNA(Met) mimicked phosphorylation of eIF-2 alpha and stimulated GCN4 translation. In addition, overexpression of a combination of eIF-2 and tRNA(iMet) suppressed the growth-inhibitory effects of eIF-2 hyperphosphorylation more effectively than an increase in the level of either component of the ternary complex alone. These results provide in vivo evidence that phosphorylation of eIF-2 alpha reduces the activities of both eIF-2 and eIF-2B and that the eIF-2.GTP. Met-tRNA(iMet) ternary complex is the principal component limiting translation in cells when eIF-2 alpha is phosphorylated on serine 51. Analysis of eIF-2 alpha phosphorylation in the eIF-2-overexpressing strain also provides in vivo evidence that phosphorylated eIF-2 acts as a competitive inhibitor of eIF-2B rather than forming an excessively stable inactive complex. Finally, our results demonstrate that the concentration of eIF-2-GTP. Met-tRNA(iMet) ternary complexes is the cardinal parameter determining the site of reinitiation on GCN4 mRNA and support the idea that reinitiation at GCN4 is inversely related to the concentration of ternary complexes in the cell.

  • 126.
    di Summa, Pietro G
    et al.
    University of Manchester, University Hospital of Lausanne.
    Kalbermatten, Daniel F
    University Hospital of Basel.
    Raffoul, Wassim
    University Hospital of Lausanne.
    Terenghi, Giorgio
    University of Manchester.
    Kingham, Paul J
    Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB), Anatomy. University of Manchester.
    Extracellular matrix molecules enhance the neurotrophic effect of Schwann cell-like differentiated adipose-derived stem cells and increase cell survival under stress conditions2013In: Tissue Engineering. Part A, ISSN 1937-3341, E-ISSN 1937-335X, Vol. 19, no 3-4, p. 368-379Article in journal (Refereed)
    Abstract [en]

    Since the first reports of induction of adipose-derived stem cells (ASC) into neuronal and glial cell phenotypes, expectations have increased regarding their use in tissue engineering applications for nerve repair. Cell adhesion to extracellular matrix (ECM) is a basic feature of survival, differentiation, and migration of Schwann cells (SC) during nerve regeneration, and fibronectin and laminin are two key molecules of this process. Interaction between ECM and SC-like differentiated ASC (dASC) could potentially improve the neurotrophic potential of the stem cells. We have investigated the effect of ECM molecules on SC-like dASC in terms of proliferation, adhesion, and cell viability. Fibronectin and laminin did not affect the proliferation of dASC when compared with cell adherent tissue culture plastic, but significantly improved viability and cell attachment when dASC were exposed to apoptotic conditions. To assess the influence of the ECM molecules on dASC neurotrophic activity, dASC were seeded onto ECM-coated culture inserts suspended above dorsal root ganglia (DRG) sensory neurons. Neurite outgrowth of DRG neurons was enhanced when dASC were seeded on fibronectin and laminin when compared with controls. When DRG neurons and dASC were in direct contact on the various surfaces there was significantly enhanced neurite outgrowth and coculture with laminin-conditioned dASC produced the longest neurites. Compared with primary SCs, dASC grown on laminin produced similar levels of neurite outgrowth in the culture insert experiments but neurite length was shorter in the direct contact groups. Anti beta 1 integrin blocking antibody could inhibit baseline and dASC evoked neurite elongation but had no effect on outgrowth mediated by laminin-conditioned dASC. ECM molecules had no effect on the levels of nerve growth factor and brain-derived neurotrophic factor secretion from dASC. The results of the study suggest that ECM molecules can significantly improve the potential of dASC for nerve regeneration.

  • 127. Dicks, Matthew DJ
    et al.
    Spencer, Alexandra J
    Edwards, Nick J
    Wadell, Göran
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Bojang, Kalifa
    Gilbert, Sarah C
    Hill, Adrian VS
    Cottingham, Matthew G
    A novel chimpanzee adenovirus vector with low human seroprevalence: improved systems for vector derivation and comparative immunogenicity2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 7, p. e40385-Article in journal (Refereed)
    Abstract [en]

    Recombinant adenoviruses are among the most promising tools for vaccine antigen delivery. Recently, the development of new vectors has focused on serotypes to which the human population is less exposed in order to circumvent preexisting anti vector immunity. This study describes the derivation of a new vaccine vector based on a chimpanzee adenovirus, Y25, together with a comparative assessment of its potential to elicit transgene product specific immune responses in mice. The vector was constructed in a bacterial artificial chromosome to facilitate genetic manipulation of genomic clones. In order to conduct a fair head-to-head immunological comparison of multiple adenoviral vectors, we optimised a method for accurate determination of infectious titre, since this parameter exhibits profound natural variability and can confound immunogenicity studies when doses are based on viral particle estimation. Cellular immunogenicity of recombinant E1 E3-deleted vector ChAdY25 was comparable to that of other species E derived chimpanzee adenovirus vectors including ChAd63, the first simian adenovirus vector to enter clinical trials in humans. Furthermore, the prevalence of virus neutralizing antibodies (titre >1:200) against ChAdY25 in serum samples collected from two human populations in the UK and Gambia was particularly low compared to published data for other chimpanzee adenoviruses. These findings support the continued development of new chimpanzee adenovirus vectors, including ChAdY25, for clinical use.

  • 128. Dijk, Wieneke
    et al.
    Heine, Markus
    Vergnes, Laurent
    Boon, Mariëtte R.
    Schaart, Gert
    Hesselink, Matthijs K. C.
    Reue, Karen
    Lichtenbelt, Wouter D. van Marken
    Olivecrona, Gunilla
    Umeå University, Faculty of Medicine, Department of Medical Biosciences, Physiological chemistry.
    Rensen, Patrick C. N.
    Heeren, Joerg
    Kersten, Sander
    ANGPTL4 mediates shuttling of lipid fuel to brown adipose tissue during sustained cold exposure2015In: eLIFE, E-ISSN 2050-084X, Vol. 4, article id e08428Article in journal (Refereed)
    Abstract [en]

    Brown adipose tissue (BAT) activation via cold exposure is increasingly scrutinized as a potential approach to ameliorate cardio-metabolic risk. Transition to cold temperatures requires changes in the partitioning of energy substrates, re-routing fatty acids to BAT to fuel non-shivering thermogenesis. However, the mechanisms behind the redistribution of energy substrates to BAT remain largely unknown. Angiopoietin-like 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL) activity, is highly expressed in BAT. Here, we demonstrate that ANGPTL4 is part of a shuttling mechanism that directs fatty acids derived from circulating triglyceride-rich lipoproteins to BAT during cold. Specifically, we show that cold markedly down-regulates ANGPTL4 in BAT, likely via activation of AMPK, enhancing LPL activity and uptake of plasma triglyceride-derived fatty acids. In contrast, cold up-regulates ANGPTL4 in WAT, abolishing a cold-induced increase in LPL activity. Together, our data indicate that ANGPTL4 is an important regulator of plasma lipid partitioning during sustained cold.

  • 129.
    Dimova, Tanya
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Nagaeva, Olga
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Stenqvist, Ann-Christin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Hedlund, Malin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Kjellberg, Lennart
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Strand, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Sciences, Obstetrics and Gynaecology.
    Dehlin, Eva
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Mincheva-Nilsson, Lucia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Immunology.
    Maternal Foxp3 expressing CD4+ CD25+ and CD4+ CD25- regulatory T-cell populations are enriched in human early normal pregnancy decidua: a phenotypic study of paired decidual and peripheral blood samples.2011In: American Journal of Reproductive Immunology and Microbiology, ISSN 8755-8920, Vol. 66, no Suppl 1, p. 44-56Article in journal (Refereed)
    Abstract [en]

    PROBLEM: Regulatory T cells (Treg cells), a small subset of CD4(+) T cells maintaining tolerance by immunosuppression, are proposed contributors to the survival of the fetal semiallograft. We investigated Treg cells in paired decidual and peripheral blood (PB) samples from healthy women in early pregnancy and PB samples from non-pregnant women.

    METHOD OF STUDY: Distribution, location, cytokine mRNA, and phenotype were assessed in CD4(+) CD25(+) Treg cells from paired samples using immunohistochemistry, immunofluorescence, flow cytometry, and real-time quantitative RT-PCR.

    RESULTS: The presence and in situ distribution of CD4(+) Foxp3(+) Treg cells in decidua are hereby demonstrated for the first time. Three Foxp3(+) cell populations, CD4(+) CD25(++) Foxp3(+), CD4(+) CD25(+) Foxp3(+), and CD4(+) CD25(-) Foxp3(+), were enriched locally in decidua. In contrast, no statistically significant difference in numbers of circulating Treg cells between pregnant and non-pregnant women was found. The Foxp3(+) cells expressed the surface molecules CD45RO, CTLA-4, CD103, Neuropilin-1, LAG-3, CD62L, and TGFβ1 mRNA consistent with Treg phenotype. The population of CD4(+) CD25(-) Foxp3(+) cells, not described in human decidua before, was enriched 10-fold compared with PB in paired samples. Their cytokine expression was often similar to Th3 profile, and the Foxp3 mRNA expression level in CD4(+) CD25(-) cells was stable and comparable to that of CD4(+) CD25(+) Treg cells implying that the majority of CD4(+) CD25(-) Foxp3(+) cells might be naïve Treg cells.

    CONCLUSION: (i) There is a local enrichment of Treg cells in decidua (ii) The exclusive accumulation of decidual CD4(+) CD25(-) Foxp3(+) cells suggests an additional reservoir of Foxp3(+) naïve Treg cells that can be converted to 'classical' Treg cells in uterus.

  • 130. Dobler, G.
    et al.
    Bestehorn, M.
    Antwerpen, M.
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Complete Genome Sequence of a Low-Virulence Tick-Borne Encephalitis Virus Strain2016In: Microbiology Resource Announcements, ISSN 2576-098X, Vol. 4, no 5, article id e01145-16Article in journal (Refereed)
    Abstract [en]

    We report here the complete genome sequence (GenBank accession no. KX268728) of tick-borne encephalitis strain HB171/11, isolated from an Ixodes ricinus tick from a natural focus where human neurological disease is rare. The strain shows unique characteristics in neuroinvasiveness and neurovirulence.

  • 131.
    Dongre, Mitesh
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Singh, Bhupender
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Aung, Kyaw Min
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Larsson, Per
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Miftakhova, Regina R.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Persson, Karina
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Askarian, Fatemeh
    Johannessen, Mona
    von Hofsten, Jonas
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM). Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
    Persson, Jenny L.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Erhardt, Marc
    Tuck, Simon
    Umeå University, Faculty of Medicine, Umeå Centre for Molecular Medicine (UCMM).
    Uhlin, Bernt Eric
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Wai, Sun Nyunt
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Flagella-mediated secretion of a novel Vibrio cholerae cytotoxin affecting both vertebrate and invertebrate hosts2018In: Communications Biology, ISSN 2399-3642, Vol. 1, article id 59Article in journal (Refereed)
    Abstract [en]

    Using Caenorhabditis elegans as an infection host model for Vibrio cholerae predator interactions, we discovered a bacterial cytotoxin, MakA, whose function as a virulence factor relies on secretion via the flagellum channel in a proton motive force-dependent manner. The MakA protein is expressed from the polycistronic makDCBA (motility-associated killing factor) operon. Bacteria expressing makDCBA induced dramatic changes in intestinal morphology leading to a defecation defect, starvation and death in C. elegans. The Mak proteins also promoted V. cholerae colonization of the zebrafish gut causing lethal infection. A structural model of purified MakA at 1.9 Å resolution indicated similarities to members of a superfamily of bacterial toxins with unknown biological roles. Our findings reveal an unrecognized role for V. cholerae flagella in cytotoxin export that may contribute both to environmental spread of the bacteria by promoting survival and proliferation in encounters with predators, and to pathophysiological effects during infections.

  • 132. Duffy, Margaret R.
    et al.
    Alonso-Padilla, Julio
    John, Lijo
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Chandra, Naresh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Khan, Selina
    Ballmann, Monika Z.
    Lipiec, Agnieszka
    Heemskerk, Evert
    Custers, Jerome
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Havenga, Menzo
    Baker, Andrew H.
    Lemckert, Angelique
    Generation and characterization of a novel candidate gene therapy and vaccination vector based on human species D adenovirus type 562018In: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 99, p. 135-147Article in journal (Refereed)
    Abstract [en]

    The vectorization of rare human adenovirus (HAdV) types will widen our knowledge of this family and their interaction with cells, tissues and organs. In this study we focus on HAdV-56, a member of human Ad species D, and create ease-of-use cloning systems to generate recombinant HAdV-56 vectors carrying foreign genes. We present in vitro transduction profiles for HAdV-56 in direct comparison to the most commonly used HAdV-5-based vector. In vivo characterizations demonstrate that when it is delivered intravenously (i.v.) HAdV-56 mainly targets the spleen and, to a lesser extent, the lungs, whilst largely bypassing liver transduction in mice. HAdV-56 triggered robust inflammatory and cellular immune responses, with higher induction of IFNγ, TNFα, IL5, IL6, IP10, MCP1 and MIG1 compared to HAdV-5 following i.v. administration. We also investigated its potential as a vaccine vector candidate by performing prime immunizations in mice with HAdV-56 encoding luciferase (HAdV-56-Luc). Direct comparisons were made to HAdV-26, a highly potent human vaccine vector currently in phase II clinical trials. HAdV-56-Luc induced luciferase 'antigen'-specific IFNγ-producing cells and anti-HAdV-56 neutralizing antibodies in Balb/c mice, demonstrating a near identical profile to that of HAdV-26. Taken together, the data presented provides further insight into human Ad receptor/co-receptor usage, and the first report on HAdV-56 vectors and their potential for gene therapy and vaccine applications.

  • 133.
    Duperthuy, Marylise
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Uhlin, Bernt Eric
    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).
    Biofilm Recruitment of Vibrio cholera by Matrix Proteolysis2015In: Trends in Microbiology, ISSN 0966-842X, E-ISSN 1878-4380, Vol. 23, no 11, p. 667-668Article in journal (Other academic)
  • 134.
    Edgren, Tomas
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Rosqvist, Roland
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Wolf-Watz, Hans
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Type III Secretion in Yersinia: Injectisome or Not?2012In: PLoS pathogens, ISSN 1553-7374, Vol. 8, no 5, p. e1002669-Article in journal (Refereed)
  • 135. Edqvist, Petra
    et al.
    Bröms, Jeanette E
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Steggo, Peter
    Forsberg, Åke
    Francis, Matthew
    Characterization of the tetratricopeptide repeats in type III secretion chaperones- mediators of substrate binding and specificityManuscript (Other academic)
  • 136.
    Edqvist, Petra J
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bröms, Jeanette E
    Åhlund, Monika K
    Forsberg, Åke
    Francis, Matthew S
    Functional insights into the YopD C-terminus through comprehensive site-directed mutagenesisManuscript (Other academic)
  • 137.
    Edqvist, Petra
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Olsson, Jan
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lavander, Moa
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Sundberg, Lena
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Forsberg, Åke
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Department of Microbiology, National Defense Research Agency, S-90182 Umeå.
    Wolf-Watz, Hans
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology).
    Lloyd, Scott
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    YscP and YscU Regulate Substrate Specificity of the Yersinia Type III Secretion System2003In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 185, no 7, p. 2259-2266Article in journal (Refereed)
    Abstract [en]

    Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37 degrees C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface. Upon eukaryotic cell contact, six effector proteins, called Yops, are translocated into the eukaryotic cell cytosol. Here, we show that a yscP mutant exports an increased amount of the needle component YscF to the bacterial cell surface but is unable to efficiently secrete effector Yops. Mutations in the cytoplasmic domain of the inner membrane protein YscU suppress the yscP phenotype by reducing the level of YscF secretion and increasing the level of Yop secretion. These results suggest that YscP and YscU coordinately regulate the substrate specificity of the Yersinia type III secretion system. Furthermore, we show that YscP and YscU act upstream of the cell contact sensor YopN as well as the inner gatekeeper LcrG in the pathway of substrate export regulation. These results further strengthen the strong evolutionary link between flagellar biosynthesis and type III synthesis.

  • 138.
    Edwin, Aaron
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Persson, Cecilia
    Mayzel, Maxim
    Wai, Sun Nyunt
    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).
    Öhman, Anders
    Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience, Clinical Neuroscience.
    Karlsson, B. Göran
    Sauer-Eriksson, A. Elisabeth
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Structure of the N-terminal domain of the metalloprotease PrtV from Vibrio cholerae2015In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 24, no 12, p. 2076-2080Article in journal (Refereed)
    Abstract [en]

    The metalloprotease PrtV from Vibrio cholerae serves an important function for the ability of bacteria to invade the mammalian host cell. The protein belongs to the family of M6 proteases, with a characteristic zinc ion in the catalytic active site. PrtV constitutes a 918 amino acids (102 kDa) multidomain pre-pro-protein that undergoes several N- and C-terminal modifications to form a catalytically active protease. We report here the NMR structure of the PrtV N- terminal domain (residues 23-103) that contains two short alpha-helices in a coiled coil motif. The helices are held together by a cluster of hydrophobic residues. Approximately 30 residues at the C-terminal end, which were predicted to form a third helical structure, are disordered. These residues are highly conserved within the genus Vibrio, which suggests that they might be functionally important.

  • 139. Eggenschwiler, Reto
    et al.
    Moslem, Mohsen
    Fráguas, Mariane Serra
    Galla, Melanie
    Papp, Oliver
    Naujock, Maximilian
    Fonfara, Ines
    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). Max Planck Institute for Infection Biology, Department of Regulation in Infection Biology, Berlin, Germany.
    Gensch, Ingrid
    Wähner, Annabell
    Beh-Pajooh, Abbas
    Mussolino, Claudio
    Tauscher, Marcel
    Steinemann, Doris
    Wegner, Florian
    Petri, Susanne
    Schambach, Axel
    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).
    Cathomen, Toni
    Cantz, Tobias
    Improved bi-allelic modification of a transcriptionally silent locus in patient-derived iPSC by Cas9 nickase2016In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 6, article id 38198Article in journal (Refereed)
    Abstract [en]

    Homology directed repair (HDR)-based genome editing via selectable long flanking arm donors can be hampered by local transgene silencing at transcriptionally silent loci. Here, we report efficient bi-allelic modification of a silent locus in patient-derived hiPSC by using Cas9 nickase and a silencing-resistant donor construct that contains an excisable selection/counter-selection cassette. To identify the most active single guide RNA (sgRNA)/nickase combinations, we employed a lentiviral vector-based reporter assay to determine the HDR efficiencies in cella. Next, we used the most efficient pair of sgRNAs for targeted integration of an improved, silencing-resistant plasmid donor harboring a piggyBac-flanked puro Delta tk cassette. Moreover, we took advantage of a dual-fluorescence selection strategy for bi-allelic targeting and achieved 100% counter-selection efficiency after bi-allelic excision of the selection/counter-selection cassette. Together, we present an improved system for efficient bi-allelic modification of transcriptionally silent loci in human pluripotent stem cells.

  • 140.
    Ekestubbe, Sofie
    et al.
    Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Bröms, Jeanette E.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Edgren, Tomas
    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).
    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).
    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).
    Forsberg, Åke
    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).
    The amino-terminal part of the needle-tip translocator LcrV of Yersinia pseudotuberculosis is required for early targeting of YopH and in vivo virulence2016In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 6, article id 175Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) are dedicated to targeting anti-host effector proteins into the cytosol of the host cell to promote bacterial infection. Delivery of the effectors requires three specific translocator proteins, of which the hydrophilic translocator, LcrV, is located at the tip of the T3SS needle and is believed to facilitate insertion of the two hydrophobic translocators into the host cell membrane. Here we used Yersinia as a model to study the role of LcrV in T3SS mediated intracellular effector targeting. Intriguingly, we identified N-terminal IcrV mutants that, similar to the wild-type protein, efficiently promoted expression, secretion and intracellular levels of Yop effectors, yet they were impaired in their ability to inhibit phagocytosis by J774 cells. In line with this, the YopH mediated dephosphorylation of Focal Adhesion Kinase early after infection was compromised when compared to the wild type strain. This suggests that the mutants are unable to promote efficient delivery of effectors to their molecular targets inside the host cell upon host cell contact. The significance of this was borne out by the fact that the mutants were highly attenuated for virulence in the systemic mouse infection model. Our study provides both novel and significant findings that establish a role for LcrV in early targeting of effectors in the host cell.

  • 141.
    Ekström, Jens-Ola
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Habayeb, Mazen S
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Srivastava, Vaibhav
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Kieselbach, Thomas
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Wingsle, Gunnar
    Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC).
    Hultmark, Dan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Drosophila Nora virus capsid proteins differ from those of other picorna-like viruses2011In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 160, no 1-2, p. 51-58Article in journal (Refereed)
    Abstract [en]

    The recently discovered Nora virus from Drosophila melanogaster is a single-stranded RNA virus. Its published genomic sequence encodes a typical picorna-like cassette of replicative enzymes, but no capsid proteins similar to those in other picorna-like viruses. We have now done additional sequencing at the termini of the viral genome, extending it by 455 nucleotides at the 5' end, but no more coding sequence was found. The completeness of the final 12,333-nucleotide sequence was verified by the production of infectious virus from the cloned genome. To identify the capsid proteins, we purified Nora virus particles and analyzed their proteins by mass spectrometry. Our results show that the capsid is built from three major proteins, VP4A, B and C, encoded in the fourth open reading frame of the viral genome. The viral particles also contain traces of a protein from the third open reading frame, VP3. VP4A and B are not closely related to other picorna-like virus capsid proteins in sequence, but may form similar jelly roll folds. VP4C differs from the others and is predicted to have an essentially α-helical conformation. In a related virus, identified from EST database sequences from Nasonia parasitoid wasps, VP4C is encoded in a separate open reading frame, separated from VP4A and B by a frame-shift. This opens a possibility that VP4C is produced in non-equimolar quantities. Altogether, our results suggest that the Nora virus capsid has a different protein organization compared to the order Picornavirales.

  • 142. El Mouali, Youssef
    et al.
    Kim, Hyunhee
    Ahmad, Irfan
    Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.
    Brauner, Annelie
    Liu, Ying
    Skurnik, Mikael
    Galperin, Michael Y.
    Romling, Ute
    Stand-Alone EAL Domain Proteins Form a Distinct Subclass of EAL Proteins Involved in Regulation of Cell Motility and Biofilm Formation in Enterobacteria2017In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 199, no 18, article id e00179-17Article in journal (Refereed)
    Abstract [en]

    The second messenger cyclic dimeric GMP (c-di-GMP) is almost ubiquitous among bacteria as are the c-di-GMP turnover proteins, which mediate the transition between motility and sessility. EAL domain proteins have been characterized as c-di-GMP-specific phosphodiesterases. While most EAL domain proteins contain additional, usually N-terminal, domains, there is a distinct family of proteins with stand-alone EAL domains, exemplified by Salmonella enterica serovar Typhimurium proteins STM3611 (YhjH/PdeH), a c-di-GMP-specific phosphodiesterase, and the enzymatically inactive STM1344 (YdiV/CdgR) and STM1697, which regulate bacterial motility through interaction with the flagellar master regulator, FlhDC. We have analyzed the phylogenetic distribution of EAL-only proteins and their potential functions. Genes encoding EAL-only proteins were found in various bacterial phyla, although most of them were seen in proteobacteria, particularly enterobacteria. Based on the conservation of the active site residues, nearly all stand-alone EAL domains encoded by genomes from phyla other than proteobacteria appear to represent functional phosphodiesterases. Within enterobacteria, EAL-only proteins were found to cluster either with YhjH or with one of the subfamilies of YdiV-related proteins. EAL-only proteins from Shigella flexneri, Klebsiella pneumoniae, and Yersinia enterocolitica were tested for their ability to regulate swimming and swarming motility and formation of the red, dry, and rough (rdar) biofilm morphotype. In these tests, YhjH-related proteins S4210, KPN_01159, KPN_03274, and YE4063 displayed properties typical of enzymatically active phosphodiesterases, whereas S1641 and YE1324 behaved like members of the YdiV/STM1697 subfamily, with Yersinia enterocolitica protein YE1324 shown to downregulate motility in its native host. Of two closely related EAL-only proteins, YE2225 is an active phosphodiesterase, while YE1324 appears to interact with FlhD. These results suggest that in FlhDC-harboring beta-and gammaproteobacteria, some EAL-only proteins evolved to become catalytically inactive and regulate motility and biofilm formation by interacting with FlhDC. IMPORTANCE The EAL domain superfamily consists mainly of proteins with cyclic dimeric GMP-specific phosphodiesterase activity, but individual domains have been classified in three classes according to their functions and conserved amino acid signatures. Proteins that consist solely of stand-alone EAL domains cannot rely on other domains to form catalytically active dimers, and most of them fall into one of two distinct classes: catalytically active phosphodiesterases with well-conserved residues of the active site and the dimerization loop, and catalytically inactive YdiV/CdgR-like proteins that regulate bacterial motility by binding to the flagellar master regulator, FlhDC, and are found primarily in enterobacteria. The presence of apparently inactive EAL-only proteins in the bacteria that do not express FlhD suggests the existence of additional EAL interaction partners.

  • 143. Elbir, Haitham
    et al.
    Gimenez, Gregory
    Robert, Catherine
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cutler, Sally
    Raoult, Didier
    Drancourt, Michel
    Complete genome sequence of Borrelia crocidurae2012In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 194, no 14, p. 3723-3724Article in journal (Refereed)
    Abstract [en]

    We announce the draft genome sequence of Borrelia crocidurae (strain Achema). The 1,557,560-bp genome (27% GC content) comprises one 919,477-bp linear chromosome and 638,083-bp plasmids that together carry 1,472 open reading frames, 32 tRNAs, and three complete rRNAs, with almost complete colinearity between B. crocidurae and Borrelia duttonii chromosomes.

  • 144.
    Elbir, Haitham
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Larsson, Pär
    Division of CBRN Security and Defence, FOI Swedish Defence Research Agency, Umeå, Sweden.
    Normark, Johan
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Upreti, Mukunda
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Korenberg, Edward
    Gamaleya Research Institute for Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russian Federation.
    Larsson, Christer
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Genome Sequence of the Asiatic Species Borrelia persica2014In: Genome Announcements, ISSN 2169-8287, E-ISSN 2169-8287, Vol. 2, no 1, article id e01127-13Article in journal (Refereed)
    Abstract [en]

    We report the complete genome sequence of Borrelia persica, the causative agent of tick-borne relapsing fever borreliosis on the Asian continent. Its genome of 1,784,979 bp contains 1,850 open reading frames, three ribosomal RNAs, and 32 tRNAs. One clustered regularly interspaced short palindromic repeat (CRISPR) was detected.

  • 145.
    Eneslätt, Kjell
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Golovliov, Igor
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Rydén, Patrik
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
    Vaccine-mediated mechanisms controlling replication of Francisella tularensis in human peripheral blood mononuclear cells using a co-culture system2018In: Frontiers in Cellular and Infection Microbiology, E-ISSN 2235-2988, Vol. 8, article id 27Article in journal (Refereed)
    Abstract [en]

    Cell-mediated immunity (CMI) is normally required for efficient protection against intracellular infections, however, identification of correlates is challenging and they are generally lacking. Francisella tularensis is a highly virulent, facultative intracellular bacterium and CMI is critically required for protection against the pathogen, but how this is effectuated in humans is poorly understood. To understand the protective mechanisms, we established an in vitro co-culture assay to identify how control of infection of F. tularensis is accomplished by human cells and hypothesized that the model will mimic in vivo immune mechanisms. Non-adherent peripheral blood mononuclear cells (PBMCs) were expanded with antigen and added to cultures with adherent PBMC infected with the human vaccine strain, LVS, or the highly virulent SCHU S4 strain. Intracellular numbers of F. tularensis was followed for 72 h and secreted and intracellular cytokines were analyzed. Addition of PBMC expanded from naïve individuals, i.e., those with no record of immunization to F. tularensis, generally resulted in little or no control of intracellular bacterial growth, whereas addition of PBMC from a majority of F. tularensis-immune individuals executed static and sometimes cidal effects on intracellular bacteria. Regardless of infecting strain, statistical differences between the two groups were significant, P < 0.05. Secretion of 11 cytokines was analyzed after 72 h of infection and significant differences with regard to secretion of IFN-γ, TNF, and MIP-1β was observed between immune and naïve individuals for LVS-infected cultures. Also, in LVS-infected cultures, CD4 T cells from vaccinees, but not CD8 T cells, showed significantly higher expression of IFN-γ, MIP-1β, TNF, and CD107a than cells from naïve individuals. The co-culture system appears to identify correlates of immunity that are relevant for the understanding of mechanisms of the protective host immunity to F. tularensis.

  • 146.
    Eneslätt, Kjell
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Normark, Monica
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Björk, Rafael
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Rietz, Cecilia
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Zingmark, Carl
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Wolfraim, Lawrence A
    Stöven, Svenja
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Sjöstedt, Anders
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Signatures of T cells as correlates of immunity to Francisella tularensis2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 3, p. e32367-Article in journal (Refereed)
    Abstract [en]

    Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4(+) and/or CD8(+) T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naive). Significant differences were detected between either of the immune donor groups and naive individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-gamma, MCP-1, and MIP-1 beta. Expression of IFN-gamma, MIP-1 beta, and CD107a by CD4(+)CD45RO(+) or CD8(+) CD45RO(+) T cells correlated to antigen concentrations. In particular, IFN-gamma and MIP-1 beta strongly discriminated between immune and naive individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-alpha-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.

  • 147.
    Engdahl, Cecilia
    et al.
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Larsson, Pär
    Swedish Defense Research Agency, CBRN Defense and Security, Umeå, Sweden.
    Näslund, Jonas
    Swedish Defense Research Agency, CBRN Defense and Security, Umeå, Sweden.
    Bravo, Mayra
    Swedish Defense Research Agency, CBRN Defense and Security, Umeå, Sweden.
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Lundström, Jan O.
    Department of Ecology and Genetics, Evolutionary Biology Centre, Uppsala University, Uppsala, Sweden.
    Ahlm, Clas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Bucht, Göran
    Swedish Defense Research Agency, CBRN Defense and Security, Umeå, Sweden.
    Identification of Swedish mosquitoes based on molecular barcoding of the COI gene and SNP analysis2014In: Molecular Ecology Resources, ISSN 1755-098X, E-ISSN 1755-0998, Vol. 14, no 3, p. 478-488Article in journal (Refereed)
    Abstract [en]

    Mosquito-borne infectious diseases are emerging in many regions of the world. Consequently, surveillance of mosquitoes and concomitant infectious agents is of great importance for prediction and prevention of mosquito-borne infectious diseases. Currently, morphological identification of mosquitoes is the traditional procedure. However, sequencing of specified genes or standard genomic regions, DNA barcoding, has recently been suggested as a global standard for identification and classification of many different species. Our aim was to develop a genetic method to identify mosquitoes and to study their relationship. Mosquitoes were captured at collection sites in northern Sweden and identified morphologically before the cytochrome c oxidase subunit I (COI) gene sequences of 14 of the most common mosquito species were determined. The sequences obtained were then used for phylogenetic placement, for validation and benchmarking of phenetic classifications and finally to develop a hierarchical PCR-based typing scheme based on single nucleotide polymorphism sites (SNPs) to enable rapid genetic identification, circumventing the need for morphological characterization. The results showed that exact phylogenetic relationships between mosquito taxa were preserved at shorter evolutionary distances, but at deeper levels, they could not be inferred with confidence using COI gene sequence data alone. Fourteen of the most common mosquito species in Sweden were identified by the SNP/PCR-based typing scheme, demonstrating that genetic typing using SNPs of the COI gene is a useful method for identification of mosquitoes with potential for worldwide application.

  • 148.
    Engström, Patrik
    et al.
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Bailey, Leslie
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Önskog, Thomas
    Umeå University, Faculty of Science and Technology, Department of Mathematics and Mathematical Statistics.
    Bergström, Sven
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Johansson, Jörgen
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    A comparative study of RNA and DNA as internal gene expression controls early in the developmental cycle of Chlamydia pneumoniae2010In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 58, no 2, p. 244-253Article in journal (Refereed)
    Abstract [en]

    Many microbial pathogens invade and proliferate within host cells and the molecular mechanism underlying this behavior is currently being revealed for several bacterial species. Testing clinically relevant antibacterial compounds and elucidating their effects on gene expression requires adequate controls, especially when studying genetically intractable organisms such as Chlamydia spp., for which various gene fusions cannot be constructed. Until now, relative mRNA levels in Chlamydia have been measured using different internal gene expression controls, including 16S rRNA, mRNAs, and DNA. Here, we compared the advantages and disadvantages of various internal expression controls during the early phase of Chlamydia pneumoniae development. The relative abundance of target mRNAs varied using the different internal control RNAs. This was partly due to variation in the transcript stability of the RNA species. Also, seven out of nine of the analyzed RNAs increased fivefold or more between 2 and 14 h postinfection, while the amount of DNA and number of cells remained essentially unaltered. Our results suggest that RNA should not be used as a gene expression control during the early phase of Chlamydia development, and that intrinsic bacterial DNA is preferable for that purpose because it is stable, abundant, and its relative amount is generally correlated with bacterial numbers.

  • 149.
    Engström, Patrik
    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).
    Bergström, Malin
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Alfaro, Astrid C.
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Bahnan, Wael
    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).
    Almqvist, Fredrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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).
    Expansion of the Chlamydia trachomatis inclusion does not require bacterial replication2015In: International Journal of Medical Microbiology, ISSN 1438-4221, E-ISSN 1618-0607, Vol. 305, no 3, p. 378-382Article in journal (Refereed)
    Abstract [en]

    Chlamydia trachomatis replication takes place inside of a host cell, exclusively within a vacuole known as the inclusion. During an infection, the inclusion expands to accommodate the increasing numbers of C. trachomatis. However, whether inclusion expansion requires bacterial replication and/or de novo protein synthesis has not been previously investigated in detail. Therefore, using a chemical biology approach, we herein investigated C. trachomatis inclusion expansion under varying conditions in vitro. Under normal cell culture conditions, inclusion expansion correlated with C trachomatis replication. When bacterial replication was inhibited using KSK120: an inhibitor that targets C. trachomatis glucose metabolism, inclusions expanded even in the absence of bacterial replication. In contrast, when bacterial protein synthesis was inhibited using chloramphenicol, expansion of inclusions was blocked. Together, these data suggest that de novo protein synthesis is necessary, whereas bacterial replication is dispensable for C trachomatis inclusion expansion. (C) 2015 The Authors. Published by Elsevier GmbH.

  • 150.
    Engström, Patrik
    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).
    Krishnan, K. Syam
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Ngyuen, Bidong D.
    Chorell, Erik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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).
    Silver, Jim
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Bastidas, Robert J.
    Welch, Matthew D.
    Hultgren, Scott J.
    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).
    Valdivia, Raphael H.
    Almqvist, Fredrik
    Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Science and Technology, Department of Chemistry.
    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).
    A 2-Pyridone-Amide Inhibitor Targets the Glucose Metabolism Pathway of Chlamydia trachomatis2015In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 6, no 1, article id e02304-14Article in journal (Refereed)
    Abstract [en]

    In a screen for compounds that inhibit infectivity of the obligate intracellular pathogen Chlamydia trachomatis, we identified the 2-pyridone amide KSK120. A fluorescent KSK120 analogue was synthesized and observed to be associated with the C. trachomatis surface, suggesting that its target is bacterial. We isolated KSK120-resistant strains and determined that several resistance mutations are in genes that affect the uptake and use of glucose-6-phosphate (G-6P). Consistent with an effect on G-6P metabolism, treatment with KSK120 blocked glycogen accumulation. Interestingly, KSK120 did not affect Escherichia coli or the host cell. Thus, 2-pyridone amides may represent a class of drugs that can specifically inhibit C. trachomatis infection. IMPORTANCE Chlamydia trachomatis is a bacterial pathogen of humans that causes a common sexually transmitted disease as well as eye infections. It grows only inside cells of its host organism, within a parasitophorous vacuole termed the inclusion. Little is known, however, about what bacterial components and processes are important for C. trachomatis cellular infectivity. Here, by using a visual screen for compounds that affect bacterial distribution within the chlamydial inclusion, we identified the inhibitor KSK120. As hypothesized, the altered bacterial distribution induced by KSK120 correlated with a block in C. trachomatis infectivity. Our data suggest that the compound targets the glucose-6-phosphate (G-6P) metabolism pathway of C. trachomatis, supporting previous indications that G-6P metabolism is critical for C. trachomatis infectivity. Thus, KSK120 may be a useful tool to study chlamydial glucose metabolism and has the potential to be used in the treatment of C. trachomatis infections.

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