This is the institutional Repository of the Helmholtz Centre for Infection Research in Braunschweig/Germany (HZI), the Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken/Germany, the TWINCORE Zentrum für Exprerimentelle und Klinische Infektionsforschung, Hannover/Germany,Helmholtz-Institut für RNA-basierte Infektionsforschung (HIRI), Würzburg/Germany, Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) the Study Centre Hannover, Hannover/Germany and the Centre for Individualised Infection Medicine (CiiM).

 

  • C19orf66 is an interferon-induced inhibitor of HCV replication that restricts formation of the viral replication organelle

    Kinast, Volker; Plociennikowska, Agnieszka; Anggakusuma; Bracht, Thilo; Todt, Daniel; Brown, Richard J.P.; Boldanova, Tujana; Zhang, Yudi; Brüggemann, Yannick; Friesland, Martina; et al. (Elsevier, 2020-04-12)
    Background & Aims HCV is a positive-strand RNA virus that primarily infects human hepatocytes. Recent studies have reported that C19orf66 is expressed as an interferon (IFN)-stimulated gene; however, the intrinsic regulation of this gene within the liver as well as its antiviral effects against HCV remain elusive. Methods Expression of C19orf66 was quantified in both liver biopsies and primary human hepatocytes, with or without HCV infection. Mechanistic studies of the potent anti-HCV phenotype mediated by C19orf66 were conducted using state-of-the-art virological, biochemical and genetic approaches, as well as correlative light and electron microscopy and transcriptome and proteome analysis. Results Upregulation of C19orf66 mRNA was observed in both primary human hepatocytes upon HCV infection and in the livers of patients with chronic hepatitis C (CHC). In addition, pegIFNα/ribavirin therapy induced C19orf66 expression in patients with CHC. Transcriptomic profiling and whole cell proteomics of hepatoma cells ectopically expressing C19orf66 revealed no induction of other antiviral genes. Expression of C19orf66 restricted HCV infection, whereas CRIPSPR/Cas9 mediated knockout of C19orf66 attenuated IFN-mediated suppression of HCV replication. Co-immunoprecipitation followed by mass spectrometry identified a stress granule protein-dominated interactome of C19orf66. Studies with subgenomic HCV replicons and an expression system revealed that C19orf66 expression impairs HCV-induced elevation of phosphatidylinositol-4-phosphate, alters the morphology of the viral replication organelle (termed the membranous web) and thereby targets viral RNA replication. Conclusion C19orf66 is an IFN-stimulated gene, which is upregulated in hepatocytes within the first hours post IFN treatment or HCV infection in vivo. The encoded protein possesses specific antiviral activity against HCV and targets the formation of the membranous web. Our study identifies C19orf66 as an IFN-inducible restriction factor with a novel antiviral mechanism that specifically targets HCV replication.
  • Cows selected for divergent mastitis susceptibility display a differential liver transcriptome profile after experimental Staphylococcus aureus mammary gland inoculation

    Heimes, A.; Brodhagen, J.; Weikard, R.; Becker, D.; Meyerholz, M. M.; Petzl, W.; Zerbe, H.; Schuberth, H. J.; Hoedemaker, M.; Schmicke, M.; et al. (Elsevier, 2020-07-01)
    Infection and inflammation of the mammary gland, and especially prevention of mastitis, are still major challenges for the dairy industry. Different approaches have been tried to reduce the incidence of mastitis. Genetic selection of cows with lower susceptibility to mastitis promises sustainable success in this regard. Bos taurus autosome (BTA) 18, particularly the region between 43 and 59 Mb, harbors quantitative trait loci (QTL) for somatic cell score, a surrogate trait for mastitis susceptibility. Scrutinizing the molecular bases hereof, we challenged udders from half-sib heifers having inherited either favorable paternal haplotypes for somatic cell score (Q) or unfavorable haplotypes (q) with the Staphylococcus aureus pathogen. RNA sequencing was used for an in-depth analysis of challenge-related alterations in the hepatic transcriptome. Liver exerts highly relevant immune functions aside from being the key metabolic organ. Hence, a holistic approach focusing on the liver enabled us to identify challenge-related and genotype-dependent differentially expressed genes and underlying regulatory networks. In response to the S. aureus challenge, we found that heifers with Q haplotypes displayed more activated immune genes and pathways after S. aureus challenge compared with their q half-sibs. Furthermore, we found a significant enrichment of differentially expressed loci in the genomic target region on BTA18, suggesting the existence of a regionally acting regulatory element with effects on a variety of genes in this region. © 2020 American Dairy Science Association
  • An amphipathic peptide with antibiotic activity against multidrug-resistant Gram-negative bacteria.

    Elliott, Alysha G; Huang, Johnny X; Neve, Søren; Zuegg, Johannes; Edwards, Ingrid A; Cain, Amy K; Boinett, Christine J; Barquist, Lars; Lundberg, Carina Vingsbo; Steen, Jason; et al. (2020-06-23)
    Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3–4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the ‘no observable adverse effect level’ (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models
  • Targeting zonulin and intestinal epithelial barrier function to prevent onset of arthritis.

    Tajik, Narges; Frech, Michael; Schulz, Oscar; Schälter, Fabian; Lucas, Sébastien; Azizov, Vugar; Dürholz, Kerstin; Steffen, Franziska; Omata, Yasunori; Rings, Andreas; et al. (Springer Nature, 2020-04-24)
    Gut microbial dysbiosis is associated with the development of autoimmune disease, but the mechanisms by which microbial dysbiosis affects the transition from asymptomatic autoimmunity to inflammatory disease are incompletely characterized. Here, we identify intestinal barrier integrity as an important checkpoint in translating autoimmunity to inflammation. Zonulin family peptide (zonulin), a potent regulator for intestinal tight junctions, is highly expressed in autoimmune mice and humans and can be used to predict transition from autoimmunity to inflammatory arthritis. Increased serum zonulin levels are accompanied by a leaky intestinal barrier, dysbiosis and inflammation. Restoration of the intestinal barrier in the pre-phase of arthritis using butyrate or a cannabinoid type 1 receptor agonist inhibits the development of arthritis. Moreover, treatment with the zonulin antagonist larazotide acetate, which specifically increases intestinal barrier integrity, effectively reduces arthritis onset. These data identify a preventive approach for the onset of autoimmune disease by specifically targeting impaired intestinal barrier function.
  • Critical Role of Zur and SmtB in Zinc Homeostasis of Mycobacterium smegmatis.

    Goethe, Elke; Laarmann, Kristin; Lührs, Janita; Jarek, Michael; Meens, Jochen; Lewin, Astrid; Goethe, Ralph (2020-04-21)
    Zinc homeostasis is crucial for bacterial cells, since imbalances affect viability. However, in mycobacteria, knowledge of zinc metabolism is incomplete. Mycobacterium smegmatis (MSMEG) is an environmental, nonpathogenic Mycobacterium that is widely used as a model organism to study mycobacterial metabolism and pathogenicity. How MSMEG maintains zinc homeostasis is largely unknown. SmtB and Zur are important regulators of bacterial zinc metabolism. In mycobacteria, these regulators are encoded by an operon, whereas in other bacterial species, SmtB and Zur are encoded on separate loci. Here, we show that the smtB-zur operon is consistently present within the genus Mycobacterium but otherwise found only in Nocardia, Saccharothrix, and Corynebacterium diphtheriae By RNA deep sequencing, we determined the Zur and SmtB regulons of MSMEG and compared them with transcriptional responses after zinc starvation or excess. We found an exceptional genomic clustering of genes whose expression was strongly induced by zur deletion and zinc starvation. These genes encoded zinc importers such as ZnuABC and three additional putative zinc transporters, including the porin MspD, as well as alternative ribosomal proteins. In contrast, only a few genes were affected by deletion of smtB and zinc excess. The zinc exporter ZitA was most prominently regulated by SmtB. Moreover, transcriptional analyses in combination with promoter and chromatin immunoprecipitation assays revealed a special regulation of the smtB-zur operon itself: an apparently zinc-independent, constitutive expression of smtB-zur resulted from sensitive coregulation by both SmtB and Zur. Overall, our data revealed yet unknown peculiarities of mycobacterial zinc homeostasis.IMPORTANCE Zinc is crucial for many biological processes, as it is an essential cofactor of enzymes and a structural component of regulatory and DNA binding proteins. Hence, all living cells require zinc to maintain constant intracellular levels. However, in excess, zinc is toxic. Therefore, cellular zinc homeostasis needs to be tightly controlled. In bacteria, this is achieved by transcriptional regulators whose activity is mediated via zinc-dependent conformational changes promoting or preventing their binding to DNA. SmtB and Zur are important antagonistically acting bacterial regulators in mycobacteria. They sense changes in zinc concentrations in the femtomolar range and regulate transcription of genes for zinc acquisition, storage, and export. Here, we analyzed the role of SmtB and Zur in zinc homeostasis in Mycobacterium smegmatis Our results revealed novel insights into the transcriptional processes of zinc homeostasis in mycobacteria and their regulation.

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