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  • 1. 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, 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.

  • 2.
    Di Martino, Maria Letizia
    et al.
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    Romilly, Cedric
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Colonna, Bianca
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    Prosseda, Gianni
    Sapienza Univ Roma, Ist Pasteur Italia Fdn Cenci Bolognetti, Dept Biol & Biotechnol C Darwin, Rome, Italy..
    One Gene and Two Proteins: a Leaderless mRNA Supports the Translation of a Shorter Form of the Shigella VirF Regulator2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, e01860-16Article in journal (Refereed)
    Abstract [en]

    VirF, an AraC-like activator, is required to trigger a regulatory cascade that initiates the invasive program of Shigella spp., the etiological agents of bacillary dysentery in humans. VirF expression is activated upon entry into the host and depends on many environmental signals. Here, we show that the virF mRNA is translated into two proteins, the major form, VirF(30) (30 kDa), and the shorter VirF(21) (21 kDa), lacking the N-terminal segment. By site-specific mutagenesis and toeprint analysis, we identified the translation start sites of VirF(30) and VirF(21) and showed that the two different forms of VirF arise from differential translation. Interestingly, in vitro and in vivo translation experiments showed that VirF(21) is also translated from a leaderless mRNA (llmRNA) whose 5' end is at position +309/+310, only 1 or 2 nucleotides upstream of the ATG84 start codon of VirF(21). The llmRNA is transcribed from a gene-internal promoter, which we identified here. Functional analysis revealed that while VirF(30) is responsible for activation of the virulence system, VirF(21) negatively autoregulates virF expression itself. Since VirF(21) modulates the intracellular VirF levels, this suggests that transcription of the llmRNA might occur when the onset of the virulence program is not required. We speculate that environmental cues, like stress conditions, may promote changes in virF mRNA transcription and preferential translation of llmRNA. IMPORTANCE Shigella spp. are a major cause of dysentery in humans. In bacteria of this genus, the activation of the invasive program involves a multitude of signals that act on all layers of the gene regulatory hierarchy. By controlling the essential genes for host cell invasion, VirF is the key regulator of the switch from the noninvasive to the invasive phenotype. Here, we show that the Shigella virF gene encodes two proteins of different sizes, VirF(30) and VirF(21), that are functionally distinct. The major form, VirF(30), activates the genes necessary for virulence, whereas the minor VirF(21), which shares the C-terminal two-thirds of VirF(30), negatively autoregulates virF expression itself. VirF(21) is transcribed from a newly identified gene-internal promoter and, moreover, is translated from an unusual leaderless mRNA. The identification of a new player in regulation adds complexity to the regulation of the Shigella invasive process and may help development of new therapies for shigellosis.

  • 3.
    Dicksved, Johan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Ellström, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engstrand, Lars
    Rautelin, Hilpi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology and Infectious Medicine.
    Susceptibility to Campylobacter Infection Is Associated with the Species Composition of the Human Fecal Microbiota2014In: mBio, ISSN 2150-7511, Vol. 5, no 5, e01212-14- p.Article in journal (Refereed)
    Abstract [en]

    The gut microbiota is essential for human health, but very little is known about how the composition of this ecosystem can influence and respond to bacterial infections. Here we address this by prospectively studying the gut microbiota composition before, during, and after natural Campylobacter infection in exposed poultry abattoir workers. The gut microbiota composition was analyzed with 16S amplicon sequencing of fecal samples from poultry abattoir workers during the peak season of Campylobacter infection in Sweden. The gut microbiota compositions were compared between individuals who became culture positive for Campylobacter and those who remained negative. Individuals who became Campylobacter positive had a significantly higher abundance of Bacteroides (P = 0.007) and Escherichia (P = 0.002) species than those who remained culture negative. Furthermore, this group had a significantly higher abundance of Phascolarctobacterium (P = 0.017) and Streptococcus (P = 0.034) sequences than the Campylobacter-negative group, which had an overrepresentation of Clostridiales (P = 0.017), unclassified Lachnospiraceae (P = 0.008), and Anaerovorax (P = 0.015) sequences. Intraindividual comparisons of the fecal microbiota compositions yielded small differences over time in Campylobacter-negative participants, but significant long-term changes were found in the Campylobacter-positive group (P < 0.005). The results suggest that the abundance of specific genera in the microbiota reduces resistance to Campylobacter colonization in humans and that Campylobacter infection can have long-term effects on the composition of the human fecal microbiota. IMPORTANCE Studies using mouse models have made important contributions to our understanding of the role of the gut microbiota in resistance to bacterial enteropathogen colonization. The relative abundances of Escherichia coli and Bacteroides species have been pointed out as important determinants of susceptibility to Gram-negative pathogens in general and Campylobacter infection in particular. In this study, we assessed the role of the human gut microbiota in resistance to Campylobacter colonization by studying abattoir workers that are heavily exposed to these bacteria. Individuals with a certain composition of the gut microbiota became culture positive for Campylobacter. As their microbiotas were characterized by high abundances of Bacteroides spp. and E. coli, well in line with the findings with mouse models, these bacterial species likely play an important role in colonization resistance also in humans.

  • 4.
    Engel, Philipp
    et al.
    Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
    Kwong, Waldan K.
    Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA; Department of Integrative Biology, University of Texas at Austin, Austin, Texas, USA.
    McFrederick, Quinn
    Department of Entomology, University of California, Riverside, California, USA.
    Anderson, Kirk E.
    USDA, Carl Hayden Bee Research Center, Tucson, Arizona, USA.
    Barribeau, Seth Michael
    Department of Biology, East Carolina University, Greenville, North Carolina, USA.
    Angus Chandler, James
    Department of Microbiology, California Academy of Sciences, San Francisco, California, USA.
    Cornman, R. Scott
    U.S. Geological Survey, Fort Collins Science Center, Fort Collins, Colorado, USA.
    Dainat, Jacques
    Linköping University, National Supercomputer Centre (NSC). Department of Medical Biochemistry and Microbiology Uppsala University, Uppsala, Sweden.
    de Miranda, Joachim R.
    Department of Ecology, Swedish University of Agricultural Sciences, Uppsala, Sweden.
    Doublet, Vincent
    Institute for Biology, Martin Luther University Halle-Wittenberg, Halle, Germany; German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany.
    Emery, Olivier
    Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland.
    Evans, Jay D.
    USDA, ARS Bee Research Laboratory, Beltsville, Maryland, USA.
    Farinelli, Laurent
    Fasteris SA, Switzerland.
    Flenniken, Michelle L.
    Department of Plant Sciences and Plant Pathology, Montana State University, Bozeman, Montana, USA.
    Granberg, Fredrik
    Department of Biomedical Sciences and Veterinary Public Health, Virology, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Grasis, Juris A.
    Department of Biology, North Life Sciences, San Diego State University, San Diego, California, USA.
    Gauthier, Laurent
    Department of Fundamental Microbiology, University of Lausanne, Lausanne, Switzerland; Ecology and Evolutionary Biology, Yale University, New Haven, Connecticut, USA.
    Hayer, Juliette
    Department of Animal Breeding and Genetics, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden.
    Koch, Hauke
    Department of Integrative Biology, University of Texas at Austin, Austin, Texas, USA; Royal Botanic Gardens, Kew, Richmond, Surrey, United Kingdom.
    Kocher, Sarah
    Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, Harvard University, Cambridge , Massachusetts , USA.
    Martinson, Vincent G.
    Department of Biology, University of Rochester, Rochester, New York, USA.
    Moran, Nancy
    Department of Integrative Biology, University of Texas at Austin, Austin, Texas, USA.
    Munoz-Torres, Monica
    Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley , California , USA.
    Newton, Irene
    Department of Biology, Indiana University, Bloomington, Indiana, USA.
    Paxton, Robert J.
    Institute for Biology, Martin Luther University Halle-Wittenberg, Halle, Germany; German Centre for Integrative Biodiversity Research (iDiv) Halle-Jena-Leipzig, Leipzig, Germany.
    Powell, Eli
    Department of Integrative Biology, University of Texas at Austin, Austin, Texas, USA.
    Sadd, Ben M.
    School of Biological Sciences, Illinois State University, Normal, Illinois, USA.
    Schmid-Hempel, Paul
    ETHZ Institut für Integrative Biologie, Zurich, Switzerland.
    Schmid-Hempel, Regula
    ETHZ Institut für Integrative Biologie, Zurich, Switzerland.
    Jin Song, Se
    University of Colorado at Boulder, Boulder, Colorado, USA.
    Schwarz, Ryan S.
    USDA, ARS Bee Research Laboratory, Beltsville, Maryland, USA.
    vanEngelsdorp, Dennis
    Department of Entomology, University of Maryland, College Park, Maryland, USA.
    Dainat, Benjamin
    Agroscope, Swiss Bee Research Centre, Bern, Switzerland; Bee Health Extension Service, Apiservice, Bern , Switzerland.
    The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 2, e02164-15Article, review/survey (Refereed)
    Abstract [en]

    As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the micro biome. The bee micro biome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee micro biome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

  • 5.
    Engel, Philipp
    et al.
    Univ Lausanne, Dept Fundamental Microbiol, Lausanne, Switzerland..
    Kwong, Waldan K.
    Yale Univ, Ecol & Evolutionary Biol, New Haven, CT USA.;Univ Texas Austin, Dept Integrat Biol, Austin, TX 78712 USA..
    McFrederick, Quinn
    Univ Calif Riverside, Dept Entomol, Riverside, CA 92521 USA..
    Anderson, Kirk E.
    USDA, Carl Hayden Bee Res Ctr, Tucson, AZ USA..
    Barribeau, Seth Michael
    E Carolina Univ, Dept Biol, Greenville, NC USA..
    Chandler, James Angus
    Calif Acad Sci, Dept Microbiol, San Francisco, CA 94118 USA.;Univ Calif Berkeley, Dept Mol & Cell Biol, 229 Stanley Hall, Berkeley, CA 94720 USA..
    Cornman, R. Scott
    US Geol Survey, Ft Collins Sci Ctr, Ft Collins, CO USA..
    Dainat, Jacques
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Linkopings Univ Victoria Westling, BILS, Linkoping, Sweden..
    de Miranda, Joachim R.
    Swedish Univ Agr Sci, Dept Ecol, Uppsala, Sweden..
    Doublet, Vincent
    Univ Halle Wittenberg, Inst Biol, D-06108 Halle, Germany.;German Ctr Integrat Biodivers Res iDiv, Leipzig, Germany..
    Emery, Olivier
    Univ Lausanne, Dept Fundamental Microbiol, Lausanne, Switzerland..
    Evans, Jay D.
    ARS, USDA, Bee Res Lab, Beltsville, MD USA..
    Farinelli, Laurent
    Fasteris SA, Plan Les Ouates, Switzerland..
    Flenniken, Michelle L.
    Montana State Univ, Dept Plant Sci & Plant Pathol, Bozeman, MT 59717 USA..
    Granberg, Fredrik
    BVF, SLU, Uppsala, Sweden..
    Grasis, Juris A.
    San Diego State Univ, Dept Biol, North Life Sci, San Diego, CA 92182 USA..
    Gauthier, Laurent
    Univ Lausanne, Dept Fundamental Microbiol, Lausanne, Switzerland.;Yale Univ, Ecol & Evolutionary Biol, New Haven, CT USA..
    Hayer, Juliette
    SLU, Inst Husdjursgenet, Uppsala, Sweden..
    Koch, Hauke
    Univ Texas Austin, Dept Integrat Biol, Austin, TX 78712 USA.;Royal Bot Gardens, Richmond, Surrey, England..
    Kocher, Sarah
    Harvard Univ, Dept Organism & Evolutionary Biol, Museum Comparat Zool, Cambridge, MA 02138 USA..
    Martinson, Vincent G.
    Univ Rochester, Dept Biol, Rochester, NY 14627 USA..
    Moran, Nancy
    Univ Texas Austin, Dept Integrat Biol, Austin, TX 78712 USA..
    Munoz-Torres, Monica
    Univ Calif Berkeley, Lawrence Berkeley Natl Lab, Environm Genom & Syst Biol Div, Berkeley, CA 94720 USA..
    Newton, Irene
    Indiana Univ, Dept Biol, Bloomington, IN USA..
    Paxton, Robert J.
    Univ Halle Wittenberg, Inst Biol, D-06108 Halle, Germany.;German Ctr Integrat Biodivers Res iDiv, Leipzig, Germany..
    Powell, Eli
    Univ Texas Austin, Dept Integrat Biol, Austin, TX 78712 USA..
    Sadd, Ben M.
    Illinois State Univ, Sch Biol Sci, Normal, IL 61761 USA..
    Schmid-Hempel, Paul
    ETHZ Inst Integrat Biol, Zurich, Switzerland..
    Schmid-Hempel, Regula
    ETHZ Inst Integrat Biol, Zurich, Switzerland..
    Song, Se Jin
    Univ Colorado, Boulder, CO 80309 USA..
    Schwarz, Ryan S.
    ARS, USDA, Bee Res Lab, Beltsville, MD USA..
    vanengelsdorp, Dennis
    Univ Maryland, Dept Entomol, College Pk, MD 20742 USA..
    Dainat, Benjamin
    Univ Lausanne, Dept Fundamental Microbiol, Lausanne, Switzerland.;Swiss Bee Resegman Ctr, Bern, Switzerland.;Apiservice, Bee Hlth Extens Serv, Bern, Switzerland.;Univ Calif Berkeley, Dept Mol & Cell Biol, 229 Stanley Hall, Berkeley, CA 94720 USA..
    The Bee Microbiome: Impact on Bee Health and Model for Evolution and Ecology of Host-Microbe Interactions2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 2, e02164Article, review/survey (Refereed)
    Abstract [en]

    As pollinators, bees are cornerstones for terrestrial ecosystem stability and key components in agricultural productivity. All animals, including bees, are associated with a diverse community of microbes, commonly referred to as the micro biome. The bee micro biome is likely to be a crucial factor affecting host health. However, with the exception of a few pathogens, the impacts of most members of the bee microbiome on host health are poorly understood. Further, the evolutionary and ecological forces that shape and change the microbiome are unclear. Here, we discuss recent progress in our understanding of the bee microbiome, and we present challenges associated with its investigation. We conclude that global coordination of research efforts is needed to fully understand the complex and highly dynamic nature of the interplay between the bee micro biome, its host, and the environment. High-throughput sequencing technologies are ideal for exploring complex biological systems, including host-microbe interactions. To maximize their value and to improve assessment of the factors affecting bee health, sequence data should be archived, curated, and analyzed in ways that promote the synthesis of different studies. To this end, the BeeBiome consortium aims to develop an online database which would provide reference sequences, archive metadata, and host analytical resources. The goal would be to support applied and fundamental research on bees and their associated microbes and to provide a collaborative framework for sharing primary data from different research programs, thus furthering our understanding of the bee microbiome and its impact on pollinator health.

  • 6.
    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 2150-7511, Vol. 6, no 1, 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.

  • 7. Felgner, S
    et al.
    Frahm, M
    Kocijancic, D
    Rohde, M
    Eckweiler, D
    Bielecka, A
    Bueno, Emilio
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Cava, Felipe
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
    Abraham, WR
    Curtiss, R
    Häussler, S
    Erhardt, M
    Weiss, S
    aroA-Deficient Salmonella enterica Serovar Typhimurium Is More Than a Metabolically Attenuated Mutant2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 5, e01220-16Article in journal (Refereed)
    Abstract [en]

    Recombinant attenuated Salmonella enterica serovar Typhimurium strains are believed to act as powerful live vaccine carriers that are able to elicit protection against various pathogens. Auxotrophic mutations, such as a deletion of aroA, are commonly introduced into such bacteria for attenuation without incapacitating immunostimulation. In this study, we describe the surprising finding that deletion of aroA dramatically increased the virulence of attenuated Salmonella in mouse models. Mutant bacteria lacking aroA elicited increased levels of the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) after systemic application. A detailed genetic and phenotypic characterization in combination with transcriptomic and metabolic profiling demonstrated that Delta aroA mutants display pleiotropic alterations in cellular physiology and lipid and amino acid metabolism, as well as increased sensitivity to penicillin, complement, and phagocytic uptake. In concert with other immunomodulating mutations, deletion of aroA affected flagellin phase variation and gene expression of the virulence-associated genes arnT and ansB. Finally, Delta aroA strains displayed significantly improved tumor therapeutic activity. These results highlight the importance of a functional shikimate pathway to control homeostatic bacterial physiology. They further highlight the great potential of Delta aroA-attenuated Salmonella for the development of vaccines and cancer therapies with important implications for host-pathogen interactions and translational medicine. 

    IMPORTANCE Recombinant attenuated bacterial vector systems based on genetically engineered Salmonella have been developed as highly potent vaccines. Due to the pathogenic properties of Salmonella, efficient attenuation is required for clinical applications. Since the hallmark study by Hoiseth and Stocker in 1981 (S. K. Hoiseth and B. A. D. Stocker, Nature 291:238-239, 1981, http://dx.doi.org/10.1038/291238a0), the auxotrophic Delta aroA mutation has been generally considered safe and universally used to attenuate bacterial strains. Here, we are presenting the remarkable finding that a deletion of aroA leads to pronounced alterations of gene expression, metabolism, and cellular physiology, which resulted in increased immunogenicity, virulence, and adjuvant potential of Salmonella. These results suggest that the enhanced immunogenicity of aroA-deficient Salmonella strains might be advantageous for optimizing bacterial vaccine carriers and immunotherapy. Accordingly, we demonstrate a superior performance of Delta aroA Salmonella in bacterium-mediated tumor therapy. In addition, the present study highlights the importance of a functional shikimate pathway to sustain bacterial physiology and metabolism.

  • 8. Gioti, Anastasia
    et al.
    Nystedt, Björn
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).
    Li, Wenjun
    Xu, Jun
    Andersson, Anna
    Averette, Anna F.
    Muench, Karin
    Wang, Xuying
    Kappauf, Catharine
    Kingsbury, Joanne M.
    Kraak, Bart
    Walker, Louise A.
    Johansson, Henrik J.
    Holm, Tina
    Lehtio, Janne
    Stajich, Jason E.
    Mieczkowski, Piotr
    Kahmann, Regine
    Kennell, John C.
    Cardenas, Maria E.
    Lundeberg, Joakim
    Saunders, Charles W.
    Boekhout, Teun
    Dawson, Thomas L.
    Munro, Carol A.
    de Groot, Piet W. J.
    Butler, Geraldine
    Heitman, Joseph
    Scheynius, Annika
    Genomic Insights into the Atopic Eczema-Associated Skin Commensal Yeast Malassezia sympodialis2013In: mBio, ISSN 2150-7511, Vol. 4, no 1, e00572-12- p.Article in journal (Refereed)
    Abstract [en]

    Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e. g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. IMPORTANCE Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

  • 9. Gioti, Anastasia
    et al.
    Nystedt, Björn
    Li, Wenjun
    Xu, Jun
    Andersson, Anna
    Averette, Anna F.
    Muench, Karin
    Wang, Xuying
    Kappauf, Catharine
    Kingsbury, Joanne M.
    Kraak, Bart
    Walker, Louise A.
    Johansson, Henrik J.
    Holm, Tina
    Lehtiö, Janne
    Stajich, Jason E.
    Mieczkowski, Piotr
    Kahmann, Regine
    Kennell, John C.
    Cardenas, Maria E.
    Lundeberg, Joakim
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Saunders, Charles W.
    Boekhout, Teun
    Dawson, Thomas L.
    Munro, Carol A.
    de Groot, Piet W. J.
    Butler, Geraldine
    Heitman, Joseph
    Scheynius, Annika
    Genomic Insights into the Atopic Eczema-Associated Skin Commensal Yeast Malassezia sympodialis2013In: mBio, ISSN 2150-7511, Vol. 4, no 1, e00572-12- p.Article in journal (Refereed)
    Abstract [en]

    Malassezia commensal yeasts are associated with a number of skin disorders, such as atopic eczema/dermatitis and dandruff, and they also can cause systemic infections. Here we describe the 7.67-Mbp genome of Malassezia sympodialis, a species associated with atopic eczema, and contrast its genome repertoire with that of Malassezia globosa, associated with dandruff, as well as those of other closely related fungi. Ninety percent of the predicted M. sympodialis protein coding genes were experimentally verified by mass spectrometry at the protein level. We identified a relatively limited number of genes related to lipid biosynthesis, and both species lack the fatty acid synthase gene, in line with the known requirement of these yeasts to assimilate lipids from the host. Malassezia species do not appear to have many cell wall-localized glycosylphosphatidylinositol (GPI) proteins and lack other cell wall proteins previously identified in other fungi. This is surprising given that in other fungi these proteins have been shown to mediate interactions (e. g., adhesion and biofilm formation) with the host. The genome revealed a complex evolutionary history for an allergen of unknown function, Mala s 7, shown to be encoded by a member of an amplified gene family of secreted proteins. Based on genetic and biochemical studies with the basidiomycete human fungal pathogen Cryptococcus neoformans, we characterized the allergen Mala s 6 as the cytoplasmic cyclophilin A. We further present evidence that M. sympodialis may have the capacity to undergo sexual reproduction and present a model for a pseudobipolar mating system that allows limited recombination between two linked MAT loci. IMPORTANCE Malassezia commensal yeasts are associated with a number of skin disorders. The previously published genome of M. globosa provided some of the first insights into Malassezia biology and its involvement in dandruff. Here, we present the genome of M. sympodialis, frequently isolated from patients with atopic eczema and healthy individuals. We combined comparative genomics with sequencing and functional characterization of specific genes in a population of clinical isolates and in closely related model systems. Our analyses provide insights into the evolution of allergens related to atopic eczema and the evolutionary trajectory of the machinery for sexual reproduction and meiosis. We hypothesize that M. sympodialis may undergo sexual reproduction, which has important implications for the understanding of the life cycle and virulence potential of this medically important yeast. Our findings provide a foundation for the development of genetic and genomic tools to elucidate host-microbe interactions that occur on the skin and to identify potential therapeutic targets.

  • 10. Greene, Sarah E.
    et al.
    Pinkner, Jerome S.
    Chorell, Erik
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Dodson, Karen W.
    Shaffer, Carrie L.
    Conover, Matt S.
    Livny, Jonathan
    Hadjifrangiskou, Maria
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Hultgren, Scott J.
    Pilicide ec240 Disrupts Virulence Circuits in Uropathogenic Escherichia coli2014In: mBio, ISSN 2150-7511, Vol. 5, no 6, UNSP e02038- p.Article in journal (Refereed)
    Abstract [en]

    Chaperone-usher pathway (CUP) pili are extracellular organelles produced by Gram-negative bacteria that mediate bacterial pathogenesis. Small-molecule inhibitors of CUP pili, termed pilicides, were rationally designed and shown to inhibit type 1 or P piliation. Here, we show that pilicide ec240 decreased the levels of type 1, P, and S piliation. Transcriptomic and proteomic analyses using the cystitis isolate UTI89 revealed that ec240 dysregulated CUP pili and decreased motility. Paradoxically, the transcript levels of P and S pilus genes were increased during growth in ec240, even though the level of P and S piliation decreased. In contrast, the most downregulated transcripts after growth in ec240 were from the type 1 pilus genes. Type 1 pilus expression is controlled by inversion of the fimS promoter element, which can oscillate between phase on and phase off orientations. ec240 induced the fimS phase off orientation, and this effect was necessary for the majority of ec240's inhibition of type 1 piliation. ec240 increased levels of the transcriptional regulators SfaB and PapB, which were shown to induce the fimS promoter phase off orientation. Furthermore, the effect of ec240 on motility was abolished in the absence of the SfaB, PapB, SfaX, and PapX regulators. In contrast to the effects of ec240, deletion of the type 1 pilus operon led to increased S and P piliation and motility. Thus, ec240 dysregulated several uropathogenic Escherichia coli (UPEC) virulence factors through different mechanisms and independent of its effects on type 1 pilus biogenesis and may have potential as an antivirulence compound. IMPORTANCE CUP pili and flagella play active roles in the pathogenesis of a variety of Gram-negative bacterial infections, including urinary tract infections mediated by UPEC. These are extremely common infections that are often recurrent and increasingly caused by antibiotic-resistant organisms. Preventing piliation and motility through altered regulation and assembly of these important virulence factors could aid in the development of novel therapeutics. This study increases our understanding of the regulation of these virulence factors, providing new avenues by which to target their expression.

  • 11.
    Gullberg, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Albrecht, Lisa M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, Christoffer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selection of a multidrug resistance plasmid by sublethal levels of antibiotics and heavy metals2014In: mBio, ISSN 2150-7511, Vol. 5, no 5, e01918-14- p.Article in journal (Refereed)
    Abstract [en]

    How sublethal levels of antibiotics and heavy metals select for clinically important multidrug resistance plasmids is largely unknown. Carriage of plasmids generally confers substantial fitness costs, implying that for the plasmid-carrying bacteria to be maintained in the population, the plasmid cost needs to be balanced by a selective pressure conferred by, for example, antibiotics or heavy metals. We studied the effects of low levels of antibiotics and heavy metals on the selective maintenance of a 220-kbp extended-spectrum β-lactamase (ESBL) plasmid identified in a hospital outbreak of Klebsiella pneumoniae and Escherichia coli. The concentrations of antibiotics and heavy metals required to maintain plasmid-carrying bacteria, the minimal selective concentrations (MSCs), were in all cases below (almost up to 140-fold) the MIC of the plasmid-free susceptible bacteria. This finding indicates that the very low antibiotic and heavy metal levels found in polluted environments and in treated humans and animals might be sufficiently high to maintain multiresistance plasmids. When resistance genes were moved from the plasmid to the chromosome, the MSC decreased, showing that MSC for a specific resistance conditionally depends on genetic context. This finding suggests that a cost-free resistance could be maintained in a population by an infinitesimally low concentration of antibiotic. By studying the effect of combinations of several compounds, it was observed that for certain combinations of drugs each new compound added lowered the minimal selective concentration of the others. This combination effect could be a significant factor in the selection of multidrug resistance plasmids/bacterial clones in complex multidrug environments.

    IMPORTANCE: Antibiotic resistance is in many pathogenic bacteria caused by genes that are carried on large conjugative plasmids. These plasmids typically contain multiple antibiotic resistance genes as well as genes that confer resistance to biocides and heavy metals. In this report, we show that very low concentrations of single antibiotics and heavy metals or combinations of compounds can select for a large plasmid that carries resistance to aminoglycosides, β-lactams, tetracycline, macrolides, trimethoprim, sulfonamide, silver, copper, and arsenic. Our findings suggest that the low levels of antibiotics and heavy metals present in polluted external environments and in treated animals and humans could allow for selection and enrichment of bacteria with multiresistance plasmids and thereby contribute to the emergence, maintenance, and transmission of antibiotic-resistant disease-causing bacteria.

  • 12.
    Herlemann, Daniel P. R.
    et al.
    Leibniz Institute for Baltic Sea Research Warnemünde (IOW), Germany.
    Lundin, Daniel
    KTH Royal Institute of Technology.
    Labrenz, Matthias
    Leibniz Institute for Baltic Sea Research Warnemünde (IOW), Germany.
    Jürgens, Klaus
    Leibniz Institute for Baltic Sea Research Warnemünde (IOW), Germany.
    Zheng, Zongli
    Karolinska Institutet.
    Aspeborg, Henrik
    KTH Royal Institute of Technology.
    Andersson, Anders F.
    KTH Royal Institute of Technology.
    Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria2013In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 4, no 3, 1-9 p., e00569-12Article in journal (Refereed)
    Abstract [en]

    The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype’s distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates.

  • 13. Herlemann, Daniel P. R.
    et al.
    Lundin, Daniel
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Labrenz, Matthias
    Jürgens, Klaus
    Zheng, Zongli
    Aspeborg, Henrik
    KTH, School of Biotechnology (BIO), Glycoscience.
    Andersson, Anders F.
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Metagenomic De Novo Assembly of an Aquatic Representative of the Verrucomicrobial Class Spartobacteria2013In: mBio, ISSN 2150-7511, Vol. 4, no 3, e00569-12- p.Article in journal (Refereed)
    Abstract [en]

    The verrucomicrobial subdivision 2 class Spartobacteria is one of the most abundant bacterial lineages in soil and has recently also been found to be ubiquitous in aquatic environments. A 16S rRNA gene study from samples spanning the entire salinity range of the Baltic Sea indicated that, in the pelagic brackish water, a phylotype of the Spartobacteria is one of the dominating bacteria during summer. Phylogenetic analyses of related 16S rRNA genes indicate that a purely aquatic lineage within the Spartobacteria exists. Since no aquatic representative from the Spartobacteria has been cultured or sequenced, the metabolic capacity and ecological role of this lineage are yet unknown. In this study, we reconstructed the genome and metabolic potential of the abundant Baltic Sea Spartobacteria phylotype by metagenomics. Binning of genome fragments by nucleotide composition and a self-organizing map recovered the near-complete genome of the organism, the gene content of which suggests an aerobic heterotrophic metabolism. Notably, we found 23 glycoside hydrolases that likely allow the use of a variety of carbohydrates, like cellulose, mannan, xylan, chitin, and starch, as carbon sources. In addition, a complete pathway for sulfate utilization was found, indicating catabolic processing of sulfated polysaccharides, commonly found in aquatic phytoplankton. The high frequency of glycoside hydrolase genes implies an important role of this organism in the aquatic carbon cycle. Spatiotemporal data of the phylotype's distribution within the Baltic Sea indicate a connection to Cyanobacteria that may be the main source of the polysaccharide substrates. IMPORTANCE The ecosystem roles of many phylogenetic lineages are not yet well understood. One such lineage is the class Spartobacteria within the Verrucomicrobia that, despite being abundant in soil and aquatic systems, is relatively poorly studied. Here we circumvented the difficulties of growing aquatic Verrucomicrobia by applying shotgun metagenomic sequencing on a water sample from the Baltic Sea. By using a method based on sequence signatures, we were able to in silico isolate genome fragments belonging to a phylotype of the Spartobacteria. The genome, which represents the first aquatic representative of this clade, encodes a diversity of glycoside hydrolases that likely allow degradation of various complex carbohydrates. Since the phylotype cooccurs with Cyanobacteria, these may be the primary producers of the carbohydrate substrates. The phylotype, which is highly abundant in the Baltic Sea during summer, may thus play an important role in the carbon cycle of this ecosystem.

  • 14. Hurt, Aeron C.
    et al.
    Vijaykrishna, Dhanasekaran
    Butler, Jeffrey
    Baas, Chantal
    Maurer-Stroh, Sebastian
    Carolina Silva-de-la-Fuente, M.
    Medina-Vogel, Gonzalo
    Olsen, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infectious Diseases.
    Kelso, Anne
    Barr, Ian G.
    Gonzalez-Acuna, Daniel
    Detection of Evolutionarily Distinct Avian Influenza A Viruses in Antarctica2014In: mBio, ISSN 2150-7511, Vol. 5, no 3, e01098-14- p.Article in journal (Refereed)
    Abstract [en]

    Distinct lineages of avian influenza viruses (AIVs) are harbored by spatially segregated birds, yet significant surveillance gaps exist around the globe. Virtually nothing is known from the Antarctic. Using virus culture, molecular analysis, full genome sequencing, and serology of samples from Adelie penguins in Antarctica, we confirmed infection by H11N2 subtype AIVs. Their genetic segments were distinct from all known contemporary influenza viruses, including South American AIVs, suggesting spatial separation from other lineages. Only in the matrix and polymerase acidic gene phylogenies did the Antarctic sequences form a sister relationship to South American AIVs, whereas distant phylogenetic relationships were evident in all other gene segments. Interestingly, their neuraminidase genes formed a distant relationship to all avian and human influenza lineages, and the polymerase basic 1 and polymerase acidic formed a sister relationship to the equine H3N8 influenza virus lineage that emerged during 1963 and whose avian origins were previously unknown. We also estimated that each gene segment had diverged for 49 to 80 years from its most closely related sequences, highlighting a significant gap in our AIV knowledge in the region. We also show that the receptor binding properties of the H11N2 viruses are predominantly avian and that they were unable to replicate efficiently in experimentally inoculated ferrets, suggesting their continuous evolution in avian hosts. These findings add substantially to our understanding of both the ecology and the intra-and intercontinental movement of Antarctic AIVs and highlight the potential risk of an incursion of highly pathogenic AIVs into this fragile environment. IMPORTANCE Avian influenza viruses (AIVs) are typically maintained and spread by migratory birds, resulting in the existence of distinctly different viruses around the world. However, AIVs have not previously been detected in Antarctica. In this study, we characterized H11N2 viruses sampled from Adelie penguins from two geographically different sites in Antarctica and show that the segmented AIV genome diverged between 49 and 80 years ago from other AIVs, with several genes showing similarity and shared ancestry with H3N8 equine influenza viruses. This study provides the first insight into the ecology of AIVs in Antarctica and highlights the potential risk of an introduction of highly pathogenic AIVs into the continent.

  • 15. Kacar, Betül
    et al.
    Garmendia, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tuncbag, Nurcan
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hughes, Diarmaid
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Functional Constraints on Replacing an Essential Gene with Its Ancient and Modern Homologs.2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 4, e01276-17Article in journal (Refereed)
    Abstract [en]

    Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.

  • 16.
    Langereis, Jeroen D
    et al.
    Radboud University of Nijmegen, Netherlands .
    Stol, Kim
    Radboud University of Nijmegen, Netherlands .
    Schweda, Elke
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Twelkmeyer, Brigitte
    Karolinska Institute, Sweden .
    Bootsma, Hester J
    Radboud University of Nijmegen, Netherlands .
    de Vries, Stefan P W
    Radboud University of Nijmegen, Netherlands .
    Burghout, Peter
    Radboud University of Nijmegen, Netherlands .
    Diavatopoulos, Dimitri A
    Radboud University of Nijmegen, Netherlands .
    Hermans, Peter W M
    Radboud University of Nijmegen, Netherlands .
    Modified Lipooligosaccharide Structure Protects Nontypeable Haemophilus influenzae from IgM-Mediated Complement Killing in Experimental Otitis Media2012In: mBio, ISSN 2150-7511, Vol. 3, no 4Article in journal (Refereed)
    Abstract [en]

    Nontypeable Haemophilus influenzae (NTHi) is a Gram-negative, human-restricted pathogen. Although this bacterium typically colonizes the nasopharynx in the absence of clinical symptoms, it is also one of the major pathogens causing otitis media (OM) in children. Complement represents an important aspect of the host defense against NTHi. In general, NTHi is efficiently killed by complement-mediated killing; however, various resistance mechanisms have also evolved. We measured the complement resistance of NTHi isolates isolated from the nasopharynx and the middle ear fluids of OM patients. Furthermore, we determined the molecular mechanism of NTHi complement resistance. Complement resistance was strongly increased in isolates from the middle ear, which correlated with decreased binding of IgM. We identified a crucial role for the R2866_0112 gene in complement resistance. Deletion of this gene altered the lipooligosaccharide (LOS) composition of the bacterium, which increased IgM binding and complement-mediated lysis. In a novel mouse model of coinfection with influenza virus, we demonstrate decreased virulence for the R2866_0112 deletion mutant. These findings identify a mechanism by which NTHi modifies its LOS structure to prevent recognition by IgM and activation of complement. Importantly, this mechanism plays a crucial role in the ability of NTHi to cause OM. less thanbrgreater than less thanbrgreater thanIMPORTANCE Nontypeable Haemophilus influenzae (NTHi) colonizes the nasopharynx of especially young children without any obvious symptoms. However, NTHi is also a major pathogen in otitis media (OM), one of the most common childhood infections. Although this pathogen is often associated with OM, the mechanism by which this bacterium is able to cause OM is largely unknown. Our study addresses a key biological question that is highly relevant for child health: what is the molecular mechanism that enables NTHi to cause OM? We show that isolates collected from the middle ear fluid exhibit increased complement resistance and that the lipooligosaccharide (LOS) structure determines IgM binding and complement activation. Modification of the LOS structure decreased NTHi virulence in a novel NTHi-influenza A virus coinfection OM mouse model. Our findings may also have important implications for other Gram-negative pathogens harboring LOS, such as Neisseria meningitidis, Moraxella catarrhalis, and Bordetella pertussis.

  • 17. Lehours, P.
    et al.
    Vale, F. F.
    Bjursell, M. K.
    Melefors, O.
    Advani, R.
    Glavas, S.
    Guegueniat, J.
    Gontier, E.
    Lacomme, S.
    Alves Matos, A.
    Menard, A.
    Megraud, F.
    Engstrand, L.
    Andersson, Anders
    KTH, School of Biotechnology (BIO), Gene Technology. KTH, Centres, Science for Life Laboratory, SciLifeLab.
    Genome sequencing reveals a phage in Helicobacter pylori2011In: mBio, ISSN 2150-7511, Vol. 2, no 6, e00239- p.Article in journal (Refereed)
    Abstract [en]

     Helicobacter pylori chronically infects the gastric mucosa in more than half of the human population; in a subset of this population, its presence is associated with development of severe disease, such as gastric cancer. Genomic analysis of several strains has revealed an extensive H pylori pan-genome, likely to grow as more genomes are sampled. Here we describe the draft genome sequence (63 contigs; 26× mean coverage) of H pylori strain B45, isolated from a patient with gastric mucosa-associated lymphoid tissue (MALT) lymphoma. The major finding was a 24.6-kb prophage integrated in the bacterial genome. The prophage shares most of its genes (22/27) with prophage region II of Helicobacter acinonychis strain Sheeba. After UV treatment of liquid cultures, circular DNA carrying the prophage integrase gene could be detected, and intracellular tailed phage-like particles were observed in H pylori cells by transmission electron microscopy, indicating that phage production can be induced from the prophage. PCR amplification and sequencing of the integrase gene from 341 H pylori strains from different geographic regions revealed a high prevalence of the prophage (21.4%). Phylogenetic reconstruction showed four distinct clusters in the integrase gene, three of which tended to be specific for geographic regions. Our study implies that phages may play important roles in the ecology and evolution of H pylori.

  • 18. Mellroth, Peter
    et al.
    Sandalova, Tatyana
    Kikhney, Alexey
    Vilaplana, Francisco
    KTH, School of Biotechnology (BIO), Glycoscience.
    Hesek, Dusan
    Lee, Mijoon
    Mobashery, Shahriar
    Normark, Staffan
    Svergun, Dmitri
    Henriques-Normark, Birgitta
    Achour, Adnane
    Structural and Functional Insights into Peptidoglycan Access for the Lytic Amidase LytA of Streptococcus pneumoniae2014In: mBio, ISSN 2150-7511, Vol. 5, no 1, e01120-13- p.Article in journal (Refereed)
    Abstract [en]

    The cytosolic N-acetylmuramoyl-L-alanine amidase LytA protein of Streptococcus pneumoniae, which is released by bacterial lysis, associates with the cell wall via its choline-binding motif. During exponential growth, LytA accesses its peptidoglycan substrate to cause lysis only when nascent peptidoglycan synthesis is stalled by nutrient starvation or beta-lactam antibiotics. Here we present three-dimensional structures of LytA and establish the requirements for substrate binding and catalytic activity. The solution structure of the full-length LytA dimer reveals a peculiar fold, with the choline-binding domains forming a rigid V-shaped scaffold and the relatively more flexible amidase domains attached in a trans position. The 1.05-angstrom crystal structure of the amidase domain reveals a prominent Y-shaped binding crevice composed of three contiguous subregions, with a zinc-containing active site localized at the bottom of the branch point. Site-directed mutagenesis was employed to identify catalytic residues and to investigate the relative impact of potential substrate-interacting residues lining the binding crevice for the lytic activity of LytA. In vitro activity assays using defined muropeptide substrates reveal that LytA utilizes a large substrate recognition interface and requires large muropeptide substrates with several connected saccharides that interact with all subregions of the binding crevice for catalysis. We hypothesize that the substrate requirements restrict LytA to the sites on the cell wall where nascent peptidoglycan synthesis occurs. IMPORTANCE Streptococcus pneumoniae is a human respiratory tract pathogen responsible for millions of deaths annually. Its major pneumococcal autolysin, LytA, is required for autolysis and fratricidal lysis and functions as a virulence factor that facilitates the spread of toxins and factors involved in immune evasion. LytA is also activated by penicillin and vancomycin and is responsible for the lysis induced by these antibiotics. The factors that regulate the lytic activity of LytA are unclear, but it was recently demonstrated that control is at the level of substrate recognition and that LytA required access to the nascent peptidoglycan. The present study was undertaken to structurally and functionally investigate LytA and its substrate-interacting interface and to determine the requirements for substrate recognition and catalysis. Our results reveal that the amidase domain comprises a complex substrate-binding crevice and needs to interact with a large-motif epitope of peptidoglycan for catalysis.

  • 19.
    Okumura, Cheryl
    et al.
    University of California.
    Anderson, Ericka L
    University of California.
    Döhrmann, Simon
    Tran, Dan N
    University of California.
    Olson, Joshua
    University of California.
    von Pawel-Rammingen, Ulrich
    Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Nizet, Victor
    University of California.
    IgG protease Mac/IdeS is not essential for phagocyte resistance or mouse virulence of M1T1 group A Streptococcus2013In: mBio, ISSN 2150-7511, Vol. 4, no 4, e00499-e00513 p.Article in journal (Refereed)
    Abstract [en]

    The Mac/IdeS protein of group A Streptococcus (GAS) is a secreted cysteine protease with cleavage specificity for IgG and is highly expressed in the GAS serotype M1T1 clone, which is the serotype most frequently isolated from patients with life-threatening invasive infections. While studies of Mac/IdeS with recombinant protein have shown that the protein can potentially prevent opsonophagocytosis of GAS by neutrophils, the role of the protein in immune evasion as physiologically produced by the living organism has not been studied. Here we examined the contribution of Mac/IdeS to invasive GAS disease by generating a mutant lacking Mac/IdeS in the hyperinvasive M1T1 background. While Mac/IdeS was highly expressed and proteolytically active in the hyperinvasive strain, elimination of the bacterial protease did not significantly influence GAS phagocytic uptake, oxidative-burst induction, cathelicidin sensitivity, resistance to neutrophil or macrophage killing, or pathogenicity in pre- or postimmune mouse infectious challenges. We conclude that in the highly virulent M1T1 background, Mac/IdeS is not essential for either phagocyte resistance or virulence. Given the conservation of Mac/IdeS and homologues across GAS strains, it is possible that Mac/IdeS serves another important function in GAS ecology or contributes to virulence in other strain backgrounds.

    IMPORTANCE Group A Streptococcus (GAS) causes human infections ranging from strep throat to life-threatening conditions such as flesh-eating disease and toxic shock syndrome. Common disease-associated clones of GAS can cause both mild and severe infections because of a characteristic mutation and subsequent change in the expression of several genes that develops under host immune selection. One of these genes encodes Mac/IdeS, a protease that has been shown to cleave antibodies important to the immune defense system. In this study, we found that while Mac/IdeS is highly expressed in hypervirulent GAS, it does not significantly contribute to the ability of the bacteria to survive white blood cell killing or produce invasive infection in the mouse. These data underscore the importance of correlating studies on virulence factor function with physiologic expression levels and the complexity of streptococcal pathogenesis and contribute to our overall understanding of how GAS causes disease.

  • 20.
    Olofsson, Annelie
    et al.
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Nygård Skalman, Lars
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Obi, Ikenna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Lundmark, Richard
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Arnqvist, Anna
    Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
    Uptake of Helicobacter pylori vesicles is facilitated by clathrin-dependent and clathrin-independent endocytic pathways2014In: mBio, ISSN 2150-7511, Vol. 5, no 3, e00979-14- p.Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: Bacteria shed a diverse set of outer membrane vesicles that function as transport vehicles to deliver effector molecules and virulence factors to host cells. Helicobacter pylori is a gastric pathogen that infects half of the world's population, and in some individuals the infection progresses into peptic ulcer disease or gastric cancer. Here we report that intact vesicles from H. pylori are internalized by clathrin-dependent endocytosis and further dynamin-dependent processes, as well as in a cholesterol-sensitive manner. We analyzed the uptake of H. pylori vesicles by gastric epithelial cells using a method that we refer to as quantification of internalized substances (qIS). The qIS assay is based on a near-infrared dye with a cleavable linker that enables the specific quantification of internalized substances after exposure to reducing conditions. Both chemical inhibition and RNA interference in combination with the qIS assay showed that H. pylori vesicles enter gastric epithelial cells via both clathrin-mediated endocytosis and additional endocytic processes that are dependent on dynamin. Confocal microscopy revealed that H. pylori vesicles colocalized with clathrin and dynamin II and with markers of subsequent endosomal and lysosomal trafficking. Interestingly, however, knockdown of components required for caveolae had no significant effect on internalization and knockdown of components required for clathrin-independent carrier (CLIC) endocytosis increased internalization of H. pylori vesicles. Furthermore, uptake of vesicles by both clathrin-dependent and -independent pathways was sensitive to depletion, but not sequestering, of cholesterol in the host cell membrane suggesting that membrane fluidity influences the efficiency of H. pylori vesicle uptake.

    IMPORTANCE: Bacterial vesicles act as long-distance tools to deliver toxins and effector molecules to host cells. Vesicles can cause a variety of host cell responses via cell surface-induced cell signaling or internalization. Vesicles of diverse bacterial species enter host cells via different endocytic pathways or via membrane fusion. With the combination of a fluorescence-based quantification assay that quantifies internalized vesicles in a large number of cells and either chemical inhibition or RNA interference, we show that clathrin-mediated endocytosis is the major pathway for uptake of Helicobacter pylori vesicles and that lipid microdomains of the host cell membrane affect uptake of vesicles via clathrin-independent pathways. Our results provide important insights about membrane fluidity and its important role in the complex process that directs the H. pylori vesicle to a specific endocytic pathway. Understanding the mechanisms that operate in vesicle-host interactions is important to fully recognize the impact of vesicles in pathogenesis.

  • 21.
    Pettersson, John H. -O.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Norwegian Inst Publ Hlth, Domain Infect Control & Environm Hlth, Dept Infect Dis Epidemiol & Modelling Mol Biol, Oslo, Norway.
    Eldholm, Vegard
    Norwegian Inst Publ Hlth, Domain Infect Control & Environm Hlth, Dept Infect Dis Epidemiol & Modelling Mol Biol, Oslo, Norway..
    Seligman, Stephen J.
    Rockefeller Univ, St Giles Lab Human Genet Infect Dis, 1230 York Ave, New York, NY 10021 USA.;New York Med Coll, Dept Microbiol & Immunol, Valhalla, NY 10595 USA..
    Lundkvist, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Falconar, Andrew K.
    Univ Norte, Dept Med, Barranquilia, Colombia..
    Gaunt, Michael W.
    London Sch Hyg & Trop Med, London, England..
    Musso, Didier
    Inst Louis Malarde, Pole Rech Veilie Malad Infect Emergentes, Tahiti, Fr Polynesia..
    Nougairede, Antoine
    Aix Marseille Univ, UMR Emergence Pathol Virales EPV, INSERM 1207, IRA 190,EHESP, Marseille, France.;Inst Hosp Univ Mediterranee Infect, APHM Publ Hosp Marseille, Marseille, France..
    Charrel, Remi
    Aix Marseille Univ, UMR Emergence Pathol Virales EPV, INSERM 1207, IRA 190,EHESP, Marseille, France.;Inst Hosp Univ Mediterranee Infect, APHM Publ Hosp Marseille, Marseille, France..
    Gould, Ernest A.
    Aix Marseille Univ, UMR Emergence Pathol Virales EPV, INSERM 1207, IRA 190,EHESP, Marseille, France..
    de Lamballerie, Xavier
    Aix Marseille Univ, UMR Emergence Pathol Virales EPV, INSERM 1207, IRA 190,EHESP, Marseille, France.;Inst Hosp Univ Mediterranee Infect, APHM Publ Hosp Marseille, Marseille, France..
    How Did Zika Virus Emerge in the Pacific Islands and Latin America?2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 5, e01239-16Article in journal (Refereed)
    Abstract [en]

    The unexpected emergence of Zika virus (ZIKV) in the Pacific Islands and Latin America and its association with congenital Zika virus syndrome (CZVS) (which includes microcephaly) and Guillain-Barre syndrome (GBS) have stimulated wide-ranging research. High densities of susceptible Aedes spp., immunologically naive human populations, global population growth with increased urbanization, and escalation of global transportation of humans and commercial goods carrying vectors and ZIKV undoubtedly enhanced the emergence of ZIKV. However, flavivirus mutations accumulate with time, increasing the likelihood that genetic viral differences are determinants of change in viral phenotype. Based on comparative ZIKV complete genome phylogenetic analyses and temporal estimates, we identify amino acid substitutions that may be associated with increased viral epidemicity, CZVS, and GBS. Reverse genetics, vector competence, and seroepidemiological studies will test our hypothesis that these amino acid substitutions are determinants of epidemic and neurotropic ZIKV emergence.

  • 22.
    Resch, Ulrike
    et al.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Vascular Biology and Thrombosis Research, Medical University Vienna, Vienna, Austria.
    Tsatsaronis, James Anthony
    Le Rhun, Anais
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany.
    Stuebiger, Gerald
    Rohde, Manfred
    Kasvandik, Sergo
    Holzmeister, Susanne
    Tinnefeld, Philip
    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).
    Charpentier, Emmanuelle
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Department of Regulation in Infection Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany; Department of Regulation in Infection Biology, Max Planck Institute for Infection Biology, Berlin, Germany; Hannover Medical School, Hannover, Germany.
    A Two-Component Regulatory System Impacts Extracellular Membrane-Derived Vesicle Production in Group A Streptococcus2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, e00207-16Article in journal (Refereed)
    Abstract [en]

    Export of macromolecules via extracellular membrane-derived vesicles (MVs) plays an important role in the biology of Gram-negative bacteria. Gram-positive bacteria have also recently been reported to produce MVs; however, the composition and mechanisms governing vesiculogenesis in Gram-positive bacteria remain undefined. Here, we describe MV production in the Gram-positive human pathogen group A streptococcus (GAS), the etiological agent of necrotizing fasciitis and streptococcal toxic shock syndrome. M1 serotype GAS isolates in culture exhibit MV structures both on the cell wall surface and in the near vicinity of bacterial cells. A comprehensive analysis of MV proteins identified both virulence-associated protein substrates of the general secretory pathway in addition to "anchorless surface proteins." Characteristic differences in the contents, distributions, and fatty acid compositions of specific lipids between MVs and GAS cell membrane were also observed. Furthermore, deep RNA sequencing of vesicular RNAs revealed that GAS MVs contained differentially abundant RNA species relative to bacterial cellular RNA. MV production by GAS strains varied in a manner dependent on an intact two-component system, CovRS, with MV production negatively regulated by the system. Modulation of MV production through CovRS was found to be independent of both GAS cysteine protease SpeB and capsule biosynthesis. Our data provide an explanation for GAS secretion of macromolecules, including RNAs, lipids, and proteins, and illustrate a regulatory mechanism coordinating this secretory response. IMPORTANCE Group A streptococcus (GAS) is a Gram-positive bacterial pathogen responsible for more than 500,000 deaths annually. Establishment of GAS infection is dependent on a suite of proteins exported via the general secretory pathway. Here, we show that GAS naturally produces extracellular vesicles with a unique lipid composition that are laden with proteins and RNAs. Interestingly, both virulence-associated proteins and RNA species were found to be differentially abundant in vesicles relative to the bacteria. Furthermore, we show that genetic disruption of the virulence-associated two-component regulator CovRS leads to an increase in vesicle production. This study comprehensively describes the protein, RNA, and lipid composition of GAS-secreted MVs and alludes to a regulatory system impacting this process.

  • 23.
    Ruhe, Zachary C.
    et al.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Nguyen, Josephine Y.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Xiong, Jing
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA..
    Beck, Christina M.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Icahn Sch Med Mt Sinai, New York, NY 10029 USA..
    Perkins, Basil R.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Barnard Coll, Dept Biol, New York, NY USA..
    Low, David A.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA 93106 USA..
    Hayes, Christopher S.
    Univ Calif Santa Barbara, Dept Mol Cellular & Dev Biol, Santa Barbara, CA 93106 USA.;Univ Calif Santa Barbara, Biomol Sci & Engn Program, Santa Barbara, CA 93106 USA..
    CdiA Effectors Use Modular Receptor-Binding Domains To Recognize Target Bacteria2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 2, e00290-17Article in journal (Refereed)
    Abstract [en]

    Contact-dependent growth inhibition (CDI) systems encode CdiA effectors, which bind to specific receptors on neighboring bacteria and deliver C-terminal toxin domains to suppress target cell growth. Two classes of CdiA effectors that bind distinct cell surface receptors have been identified, but the molecular basis of receptor specificity is not understood. Alignment of BamA-specific CdiAEC93 from Escherichia coli EC93 and OmpC-specific CdiA(EC536) from E. coli 536 suggests that the receptor-binding domain resides within a central region that varies between the two effectors. In support of this hypothesis, we find that CdiA(EC93) fragments containing residues Arg1358 to Phe1646 bind specifically to purified BamA. Moreover, chimeric CdiA(EC93) that carries the corresponding sequence from CdiA(EC536) is endowed with OmpC-binding activity, demonstrating that this region dictates receptor specificity. A survey of E. coli CdiA proteins reveals two additional effector classes, which presumably recognize distinct receptors. Using a genetic approach, we identify the outer membrane nucleoside transporter Tsx as the receptor for a third class of CdiA effectors. Thus, CDI systems exploit multiple outer membrane proteins to identify and engage target cells. These results underscore the modularity of CdiA proteins and suggest that novel effectors can be constructed through genetic recombination to interchange different receptor-binding domains and toxic payloads. IMPORTANCE CdiB/CdiA two-partner secretion proteins mediate interbacterial competition through the delivery of polymorphic toxin domains. This process, known as contact-dependent growth inhibition (CDI), requires stable interactions between the CdiA effector protein and specific receptors on the surface of target bacteria. Here, we localize the receptor-binding domain to the central region of E. coli CdiA. Receptor-binding domains vary between CdiA proteins, and E. coli strains collectively encode at least four distinct effector classes. Further, we show that receptor specificity can be altered by exchanging receptor-binding regions, demonstrating the modularity of this domain. We propose that novel CdiA effectors are naturally generated through genetic recombination to interchange different receptor-binding domains and toxin payloads.

  • 24. Shaffer, Carrie L.
    et al.
    Good, James A. D.
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Kumar, Santosh
    Umeå University, Faculty of Science and Technology, Department of Chemistry.
    Krishnan, K. Syam
    Gaddy, Jennifer A.
    Loh, John T.
    Chappell, Joseph
    Almqvist, Fredrik
    Umeå University, Faculty of Science and Technology, Department of Chemistry. Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
    Cover, Timothy L.
    Hadjifrangiskou, Maria
    Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 2, e00221-16Article in journal (Refereed)
    Abstract [en]

    Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

  • 25. van Wijk, Xander M.
    et al.
    Dohrmann, Simon
    Hallström, Björn M.
    KTH, School of Biotechnology (BIO). Tech Univ Denmark, Denmark.
    Li, Shangzhong
    Voldborg, Bjorn G.
    Meng, Brandon X.
    McKee, Karen K.
    van Kuppevelt, Toin H.
    Yurchenco, Peter D.
    Palsson, Bernhard O.
    Lewis, Nathan E.
    Nizet, Victor
    Esko, Jeffrey D.
    Whole-Genome Sequencing of Invasion-Resistant Cells Identifies Laminin α2 as a Host Factor for Bacterial Invasion2017In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 8, no 1, e02128-16Article in journal (Refereed)
    Abstract [en]

    To understand the role of glycosaminoglycans in bacterial cellular invasion, xylosyltransferase-deficient mutants of Chinese hamster ovary (CHO) cells were created using clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-cas9) gene targeting. When these mutants were compared to the pgsA745 cell line, a CHO xylosyltransferase mutant generated previously using chemical mutagenesis, an unexpected result was obtained. Bacterial invasion of pgsA745 cells by group B Streptococcus (GBS), group A Streptococcus, and Staphylococcus aureus was markedly reduced compared to the invasion of wild-type cells, but newly generated CRISPR-cas9 mutants were only resistant to GBS. Invasion of pgsA745 cells was not restored by transfection with xylosyltransferase, suggesting that an additional mutation conferring panresistance to multiple bacteria was present in pgsA745 cells. Whole-genome sequencing and transcriptome sequencing (RNA-Seq) uncovered a deletion in the gene encoding the laminin subunit alpha 2 (Lama2) that eliminated much of domain L4a. Silencing of the long Lama2 isoform in wild-type cells strongly reduced bacterial invasion, whereas transfection with human LAMA2 cDNA significantly enhanced invasion in pgsA745 cells. The addition of exogenous laminin-alpha 2 beta 1 gamma 1/laminin-alpha 2 beta 2 gamma 1 strongly increased bacterial invasion in CHO cells, as well as in human alveolar basal epithelial and human brain microvascular endothelial cells. Thus, the L4a domain in laminin alpha 2 is important for cellular invasion by a number of bacterial pathogens. IMPORTANCE Pathogenic bacteria penetrate host cellular barriers by attachment to extracellular matrix molecules, such as proteoglycans, laminins, and collagens, leading to invasion of epithelial and endothelial cells. Here, we show that cellular invasion by the human pathogens group B Streptococcus, group A Streptococcus, and Staphylococcus aureus depends on a specific domain of the laminin alpha 2 subunit. This finding may provide new leads for the molecular pathogenesis of these bacteria and the development of novel antimicrobial drugs.

  • 26.
    Wang, Xiao
    et al.
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sjölinder, Mikael
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Gao, Yumin
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Wan, Yi
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Sjölinder, Hong
    Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute.
    Immune Homeostatic Macrophages Programmed by the Bacterial Surface Protein NhhA Potentiate Nasopharyngeal Carriage of Neisseria meningitidis2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 1, e01670-15Article in journal (Refereed)
    Abstract [en]

    Neisseria meningitidis colonizes the nasopharyngeal mucosa of healthy populations asymptomatically although the bacterial surface is rich in motifs that activate the host innate immunity. What determines the tolerant host response to this bacterium in asymptomatic carriers is poorly understood. We demonstrated that the conserved meningococcal surface protein, NhhA, orchestrates monocyte (Mo) differentiation specifically into macrophage-like cells with a CD200Rhi phenotype (NhhA-MΦ). In response to meningococcal stimulation, NhhA-MΦ failed to produce proinflammatory mediators. Instead, they upregulated IL-10 and Th2/Treg-attracting chemokines, such as CCL-17, CCL-18, and CCL-22. Moreover, NhhA-MΦ cells were highly efficient in eliminating bacteria. The in vivo validity of these findings was corroborated using a murine model challenged with N. meningitidis systematically or intranasally. NhhA-modulated immune response protected mice from septic shock; Mo/MΦ depletion abolished this protective effect. Intranasal administration of NhhA induced an anti-inflammatory response, which was associated with N. meningitidis persistence at the nasopharynx. In vitro studies demonstrated that NhhA-triggered Mo differentiation occurred upon engaged toll-like receptor (TLR)1/TLR2 signaling and ERK and JNK activation and required endogenously produced IL-10 and TNF-α. Our findings reveal a strategy that might be adopted by N. meningitidis to maintain asymptomatic nasopharyngeal colonization.

  • 27.
    Zwack, Erin
    et al.
    University of Pennsylvania, USA.
    Snyder, Annelise
    University of Pennsylvania, USA.
    Wynosky-Dolfi, Meghan
    University of Pennsylvania, USA.
    Ruthel, Gordon
    University of Pennsylvania, USA.
    Philip, Naomi
    University of Pennsylvania, USA.
    Marketon, Melanie
    Indiana University, USA.
    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).
    Bliska, James
    SUNY Stony Brook, USA.
    Brodsky, Igor
    University of Pennsylvania, USA.
    Inflammasome activation in response to the Yersinia type III secretion system requires hyperinjection of translocon proteins YopB and YopD2015In: mBio, ISSN 2150-7511, Vol. 6, no 1, e02095-14Article in journal (Refereed)
    Abstract [en]

    Type III secretion systems (T3SS) translocate effector proteins into target cells in order to disrupt or modulate host cell signaling pathways and establish replicative niches. However, recognition of T3SS activity by cytosolic pattern recognition receptors (PRRs) of the nucleotide-binding domain leucine rich repeat (NLR) family, either through detection of translocated products or membrane disruption, induces assembly of multiprotein complexes known as inflammasomes. Macrophages infected with Yersinia pseudotuberculosis strains lacking all known effectors or lacking the translocation regulator YopK induce rapid activation of both the canonical NLRP3 and noncanonical caspase-11 inflammasomes. While this inflammasome activation requires a functional T3SS, the precise signal that triggers inflammasome activation in response to Yersinia T3SS activity remains unclear. Effectorless strains of Yersinia as well as ΔyopK strains translocate elevated levels of T3SS substrates into infected cells. To dissect the contribution of pore formation and translocation to inflammasome activation, we took advantage of variants of YopD and LcrH that separate these functions of the T3SS. Notably, YopD variants that abrogated translocation but not pore-forming activity failed to induce inflammasome activation. Furthermore, analysis of individual infected cells revealed that inflammasome activation at the single-cell level correlated with translocated levels of YopB and YopD themselves. Intriguingly, LcrH mutants that are fully competent for effector translocation but produce and translocate lower levels of YopB and YopD also fail to trigger inflammasome activation. Our findings therefore suggest that hypertranslocation of YopD and YopB is linked to inflammasome activation in response to the Yersinia T3SS.

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