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  • 1.
    Alvarez, Francisco J.
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Ryman, Kicki
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Hooijmaijers, Cornelis
    Bulone, Vincent
    Ljungdahl, Per O.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Diverse Nitrogen Sources in Seminal Fluid Act in Synergy To Induce Filamentous Growth of Candida albicans2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 8, p. 2770-2780Article in journal (Refereed)
    Abstract [en]

    The pathogenic fungus Candida albicans is the leading cause of vulvovaginal candidiasis (VVC). VVC represents a major quality- of-life issue for women during their reproductive years, a stage of life where the vaginal epithelium is subject to periodic hormonally induced changes associated with menstruation and concomitant exposure to serum as well as potential intermittent contact with seminal fluid. Seminal fluid potently triggers Candida albicans to switch from yeastlike to filamentous modes of growth, a developmental response tightly linked to virulence. Conversely, vaginal fluid inhibits filamentation. Here, we used artificial formulations of seminal and vaginal fluids that faithfully mimic genuine fluids to assess the contribution of individual components within these fluids to filamentation. The high levels of albumin, amino acids, and N-acetylglucosamine in seminal fluid act synergistically as potent inducers of filamentous growth, even at atmospheric levels of CO2 and reduced temperatures (30 degrees C). Using a simplified in vitro model that mimics the natural introduction of seminal fluid into the vulvovaginal environment, a pulse of artificial seminal fluid (ASF) was found to exert an enduring potential to overcome the inhibitory efficacy of artificial vaginal fluid (AVF) on filamentation. These findings suggest that a transient but substantial change in the nutrient levels within the vulvovaginal environment during unprotected coitus can induce resident C. albicans cells to engage developmental programs associated with virulent growth.

  • 2.
    Baumgarten, Thomas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ytterberg, A. Jimmy
    Zubarev, Roman A.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress2018In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 12, article id e00270Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in E. coli.

    IMPORTANCE The bacterium Escherichia coli is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of E. coli, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.

  • 3. Chouaia, Bessem
    et al.
    Rossi, Paolo
    Montagna, Matteo
    Ricci, Irene
    Crotti, Elena
    Damiani, Claudia
    Epis, Sara
    Faye, Ingrid
    Stockholm University, Faculty of Science, Department of Genetics, Microbiology and Toxicology.
    Sagnon, N'Fale
    Alma, Alberto
    Favia, Guido
    Daffonchio, Daniele
    Bandi, Claudio
    Molecular Evidence for Multiple Infections as Revealed by Typing of Asaia Bacterial Symbionts of Four Mosquito Species2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 22, p. 7444-7450Article in journal (Refereed)
    Abstract [en]

    The recent increased detection of acetic acid bacteria (AAB) of the genus Asaia as symbionts of mosquitoes, such as Anopheles spp. and Aedes spp., prompted us to investigate the diversity of these symbionts and their relationships in different mosquito species and populations. Following cultivation-dependent and -independent techniques, we investigated the microbiota associated with four mosquito species, Anopheles stephensi, Anopheles gambiae, Aedes aegypti, and Aedes albopictus, which are important vectors of human and/or animal pathogens. Denaturing gradient gel electrophoresis (DGGE) analysis based on the 16S rRNA gene revealed the presence of several bacterial taxa, among which Asaia sequences were among the dominant in most of the samples. A collection of 281 Asaia isolates in cell-free media was established from individuals belonging to the four species. The isolates were typed by internal transcribed spacer (ITS)-PCR, tRNA-PCR, BOX-PCR, and randomly amplified polymorphic DNA (RAPD)-PCR, revealing that different Asaia strains are present in different mosquito populations, and even in single individuals.

  • 4. Daleke-Schermerhorn, Maria H.
    et al.
    Felix, Tristan
    Soprova, Zora
    ten Hagen-Jongman, Corinne M.
    Vikström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Xbrane Bioscience AB, Sweden.
    Majlessi, Laleh
    Beskers, Joep
    Follmann, Frank
    de Punder, Karin
    van der Wel, Nicole N.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pham, Thang V.
    Piersma, Sander R.
    Jimenez, Connie R.
    van Ulsen, Peter
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leclerc, Claude
    Jong, Wouter S. P.
    Luirink, Joen
    Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 18, p. 5854-5865Article in journal (Refereed)
    Abstract [en]

    Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating Delta tolR Delta tolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen-specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development.

  • 5. Fahlgren, Camilla
    et al.
    Hagström, Åke
    Nilsson, Douglas
    Stockholm University, Faculty of Science, Department of Applied Environmental Science (ITM).
    Zweifel, Ulla Li
    Annual Variations in the Diversity, Viability, and Origin of Airborne Bacteria2010In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 76, no 9, p. 3015-3025Article in journal (Refereed)
    Abstract [en]

    The presence of bacteria in aerosols has been known for centuries, but information on their identity and role in dispersing microbial traits is still limited. This study monitored the airborne bacterial community during an annual survey using samples collected from a 25-m tower near the Baltic Sea coast. The number of CFU was estimated using agar plates while the most probable number (MPN) of viable bacteria was estimated using dilution-to-extinction culturing assays (DCAs). The MPN and CFU values produced quantitatively similar results, displaying a pronounced seasonal pattern, with the highest numbers in winter. The most dominant bacteria growing in the DCAs all formed colonies on agar plates, were mostly pigmented (80%), and closely resembled (>97%) previously cultured bacteria based on their 16S rRNA gene sequences. 16S rRNA gene clone libraries were constructed on eight occasions during the survey; these revealed a highly diverse community with a few abundant operational taxonomic units (OTUs) and a long tail of rare OTUs. A majority of the cloned sequences (60%) were also most closely related to previously ""cultured"" bacteria. Thus, both culture-dependent and culture-independent techniques indicated that bacteria able to form colonies on agar plates predominate in the atmosphere. Both the DCAs and clone libraries indicated the dominance of bacteria belonging to the genera Sphingomonas sp. and Pseudomonas sp. on several sampling occasions. Potentially pathogenic strains as well as sequences closely resembling bacteria known to act as ice nuclei were found using both approaches. The origin of the sampled air mass was estimated using backward trajectories, indicating a predominant marine source.

  • 6.
    Geueke, Birgit
    et al.
    Department of Environmental Microbiology, Swiss Federal Institute for Aquatic Science and Technology (Eawag), Dübendorf, Switzerland.
    Miska, Milena E.
    Department of Environmental Microbiology, Swiss Federal Institute for Aquatic Science and Technology (Eawag), Dübendorf, Switzerland.
    Poiger, Thomas
    Agroscope Changins-Wädenswil, Research Station ACW, Wädenswil, Switzerland.
    Rentsch, Daniel
    Laboratory for Functional Polymers, Swiss Federal Laboratories for Materials Science and Technology (EMPA), Dübendorf, Switzerland.
    Lal, Rup
    Department of Zoology, University of Delhi, Delhi, India.
    Holliger, Christof
    Laboratory for Environmental Biotechnology, School of Architecture, Civil and Environmental Engineering, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
    Kohler, Hans-Peter E.
    Department of Environmental Microbiology, Swiss Federal Institute for Aquatic Science and Technology (Eawag), Dübendorf, Switzerland.
    Enantioselective Dehydrochlorination of δ-Hexachlorocyclohexane and δ-Pentachlorocyclohexene by LinA1 and LinA2 from Sphingobium indicum B90A2013In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 79, no 19, p. 6180-6183Article in journal (Refereed)
    Abstract [en]

    ÎŽ-Hexachlorocyclohexane (ÎŽ-HCH), one of the prevalent isomers of technical HCH, was enantioselectively dehydrochlorinated by the dehydrochlorinases LinA1 and LinA2 from Sphingobium indicum B90A to the very same ÎŽ-pentachlorocyclohexene enantiomer. Racemic ÎŽ-pentachlorocyclohexene, however, was transformed with opposite enantioselectivities by the two enzymes. A transformation pathway based on an anti-1,2-elimination, followed by a syn-1,4-elimination and a subsequent syn-1,2-elimination is postulated.

  • 7.
    Hjelm, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Söderström, Bill
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vikström, David
    Jong, Wouter S. P.
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Autotransporter-Based Antigen Display in Bacterial Ghosts2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 2, p. 726-735Article in journal (Refereed)
    Abstract [en]

    Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage phi X174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-) electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

  • 8. Jacobson, Mark J.
    et al.
    Lin, Guangyun
    Tepp, William
    Dupuy, Jerome
    Stenmark, Pål
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Stevens, Raymond C.
    Johnson, Eric A.
    Purification, Modeling, and Analysis of Botulinum Neurotoxin Subtype A5 (BoNT/A5) from Clostridium botulinum Strain A6612222011In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 77, no 12, p. 4217-4222Article in journal (Refereed)
    Abstract [en]

    A Clostridium botulinum type A strain (A661222) in our culture collection was found to produce the botulinum neurotoxin subtype A5 (BoNT/A5). Its neurotoxin gene was sequenced to determine its degree of similarity to available sequences of BoNT/A5 and the well-studied BoNT/A1. Thirty-six amino acid differences were observed between BoNT/A5 and BoNT/A1, with the predominant number being located in the heavy chain. The amino acid chain of the BoNT/A from the A661222 strain was superimposed over the crystal structure of the known structure of BoNT/A1 to assess the potential significance of these differences-specifically how they would affect antibody neutralization. The BoNT/A5 neurotoxin was purified to homogeneity and evaluated for certain properties, including specific toxicity and antibody neutralization. This study reports the first purification of BoNTA5 and describes distinct differences in properties between BoNT/A5 and BoNT/A1.

  • 9.
    Neubeck, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Geological Sciences.
    Sun, Li
    Müller, Bettina
    Ivarsson, Magnus
    Hosgörmez, Hakan
    Özcan, Dogacan
    Broman, Curt
    Stockholm University, Faculty of Science, Department of Geological Sciences.
    Schnürer, Anna
    Microbial Community Structure in a Serpentine-Hosted Abiotic Gas Seepage at the Chimaera Ophiolite, Turkey2017In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 83, no 12, article id UNSP e03430Article in journal (Refereed)
    Abstract [en]

    The surface waters at the ultramafic ophiolitic outcrop in Chimaera, Turkey, are characterized by high pH values and high metal levels due to the percolation of fluids through areas of active serpentinization. We describe the influence of the liquid chemistry, mineralogy, and H-2 and CH4 levels on the bacterial community structure in a semidry, exposed, ultramafic environment. The bacterial and archaeal community structures were monitored using Illumina sequencing targeting the 16S rRNA gene. At all sampling points, four phyla, Proteobacteria, Actinobacteria, Chloroflexi, and Acidobacteria, accounted for the majority of taxa. Members of the Chloroflexi phylum dominated low-diversity sites, whereas Proteobacteria dominated high-diversity sites. Methane, nitrogen, iron, and hydrogen oxidizers were detected as well as archaea and metal-resistant bacteria. IMPORTANCE Our study is a comprehensive microbial investigation of the Chimaera ophiolite. DNA has been extracted from 16 sites in the area and has been studied from microbial and geochemical points of view. We describe a microbial community structure that is dependent on terrestrial, serpentinization-driven abiotic H-2, which is poorly studied due to the rarity of these environments on Earth.

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