• Interferon-beta expression and type I interferon receptor signaling of hepatocytes prevent hepatic necrosis and virus dissemination in Coxsackievirus B3-infected mice.

      Koestner, Wolfgang; Spanier, Julia; Klause, Tanja; Tegtmeyer, Pia-K; Becker, Jennifer; Herder, Vanessa; Borst, Katharina; Todt, Daniel; Lienenklaus, Stefan; Gerhauser, Ingo; Detje, Claudia N; Geffers, Robert; Langereis, Martijn A; Vondran, Florian W R; Yuan, Qinggong; van Kuppeveld, Frank J M; Ott, Michael; Staeheli, Peter; Steinmann, Eike; Baumgärtner, Wolfgang; Wacker, Frank; Kalinke, Ulrich; TWINCORE, Zentrum für experimentelle und klinischeInfektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany (2018-08-01)
      During Coxsackievirus B3 (CVB3) infection hepatitis is a potentially life threatening complication, particularly in newborns. Studies with type I interferon (IFN-I) receptor (IFNAR)-deficient mice revealed a key role of the IFN-I axis in the protection against CVB3 infection, whereas the source of IFN-I and cell types that have to be IFNAR triggered in order to promote survival are still unknown. We found that CVB3 infected IFN-β reporter mice showed effective reporter induction, especially in hepatocytes and only to a minor extent in liver-resident macrophages. Accordingly, upon in vitro CVB3 infection of primary hepatocytes from murine or human origin abundant IFN-β responses were induced. To identify sites of IFNAR-triggering we performed experiments with Mx reporter mice, which upon CVB3 infection showed massive luciferase induction in the liver. Immunohistological studies revealed that during CVB3 infection MX1 expression of hepatocytes was induced primarily by IFNAR-, and not by IFN-III receptor (IFNLR)-triggering. CVB3 infection studies with primary human hepatocytes, in which either the IFN-I or the IFN-III axis was inhibited, also indicated that primarily IFNAR-, and to a lesser extent IFNLR-triggering was needed for ISG induction. Interestingly, CVB3 infected mice with a hepatocyte-specific IFNAR ablation showed severe liver cell necrosis and ubiquitous viral dissemination that resulted in lethal disease, as similarly detected in classical IFNAR-/- mice. In conclusion, we found that during CVB3 infection hepatocytes are major IFN-I producers and that the liver is also the organ that shows strong IFNAR-triggering. Importantly, hepatocytes need to be IFNAR-triggered in order to prevent virus dissemination and to assure survival. These data are compatible with the hypothesis that during CVB3 infection hepatocytes serve as important IFN-I producers and sensors not only in the murine, but also in the human system.
    • Metatranscriptome Analysis of the Vaginal Microbiota Reveals Potential Mechanisms for Protection against Metronidazole in Bacterial Vaginosis.

      Deng, Zhi-Luo; Gottschick, Cornelia; Bhuju, Sabin; Masur, Clarissa; Abels, Christoph; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-27)
      Bacterial vaginosis (BV) is a prevalent multifactorial disease of women in their reproductive years characterized by a shift from the
    • Exome sequencing and case-control analyses identify RCC1 as a candidate breast cancer susceptibility gene.

      Riahi, Aouatef; Radmanesh, Hoda; Schürmann, Peter; Bogdanova, Natalia; Geffers, Robert; Meddeb, Rym; Kharrat, Maher; Dörk, Thilo; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-15)
      Breast cancer is a genetic disease but the known genes explain a minority of cases. To elucidate the molecular basis of breast cancer in the Tunisian population, we performed exome sequencing on six BRCA1/BRCA2 mutation-negative patients with familial breast cancer and identified a novel frameshift mutation in RCC1, encoding the Regulator of Chromosome Condensation 1. Subsequent genotyping detected the 19-bp deletion in additional 5 out of 153 (3%) breast cancer patients but in none of 400 female controls (p = 0.0015). The deletion was enriched in patients with a positive family history (5%, p = 0.0009) and co-segregated with breast cancer in the initial pedigree. The mutant allele was lost in 4/6 breast tumors from mutation carriers which may be consistent with the hypothesis that RCC1 dysfunction provides a selective disadvantage at the stage of tumor progression. In summary, we propose RCC1 as a likely breast cancer susceptibility gene in the Tunisian population.
    • Birth, evolution, and transmission of satellite-free mammalian centromeric domains.

      Nergadze, Solomon G; Piras, Francesca M; Gamba, Riccardo; Corbo, Marco; Cerutti, Federico; McCarter, Joseph G W; Cappelletti, Eleonora; Gozzo, Francesco; Harman, Rebecca M; Antczak, Douglas F; Miller, Donald; Scharfe, Maren; Pavesi, Giulio; Raimondi, Elena; Sullivan, Kevin F; Giulotto, Elena; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Mammalian centromeres are associated with highly repetitive DNA (satellite DNA), which has so far hindered molecular analysis of this chromatin domain. Centromeres are epigenetically specified, and binding of the CENPA protein is their main determinant. In previous work, we described the first example of a natural satellite-free centromere on
    • Distribution and Evolution of Peroxisomes in Alveolates (Apicomplexa, Dinoflagellates, Ciliates).

      Ludewig-Klingner, Ann-Kathrin; Michael, Victoria; Jarek, Michael; Brinkmann, Henner; Petersen, Jörn; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      The peroxisome was the last organelle to be discovered and five decades later it is still the Cinderella of eukaryotic compartments. Peroxisomes have a crucial role in the detoxification of reactive oxygen species, the beta-oxidation of fatty acids, and the biosynthesis of etherphospholipids, and they are assumed to be present in virtually all aerobic eukaryotes. Apicomplexan parasites including the malaria and toxoplasmosis agents were described as the first group of mitochondriate protists devoid of peroxisomes. This study was initiated to reassess the distribution and evolution of peroxisomes in the superensemble Alveolata (apicomplexans, dinoflagellates, ciliates). We established transcriptome data from two chromerid algae (Chromera velia, Vitrella brassicaformis), and two dinoflagellates (Prorocentrum minimum, Perkinsus olseni) and identified the complete set of essential peroxins in all four reference species. Our comparative genome analysis provides unequivocal evidence for the presence of peroxisomes in Toxoplasma gondii and related genera. Our working hypothesis of a common peroxisomal origin of all alveolates is supported by phylogenetic analyses of essential markers such as the import receptor Pex5. Vitrella harbors the most comprehensive set of peroxisomal proteins including the catalase and the glyoxylate cycle and it is thus a promising model organism to investigate the functional role of this organelle in Apicomplexa.
    • Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

      Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene; Helmholtz-Zentrum for Infektion Research GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-01-01)
      Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated.
    • Decreased production of class-switched antibodies in neonatal B cells is associated with increased expression of miR-181b.

      Glaesener, Stephanie; Jaenke, Christine; Habener, Anika; Geffers, Robert; Hagendorff, Petra; Witzlau, Katrin; Imelmann, Esther; Krueger, Andreas; Meyer-Bahlburg, Almut; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018)
      The increased susceptibility to infections of neonates is caused by an immaturity of the immune system as a result of both qualitative and quantitative differences between neonatal and adult immune cells. With respect to B cells, neonatal antibody responses are known to be decreased. Accountable for this is an altered composition of the neonatal B cell compartment towards more immature B cells. However, it remains unclear whether the functionality of individual neonatal B cell subsets is altered as well. In the current study we therefore compared phenotypical and functional characteristics of corresponding neonatal and adult B cell subpopulations. No phenotypic differences could be identified with the exception of higher IgM expression in neonatal B cells. Functional analysis revealed differences in proliferation, survival, and B cell receptor signaling. Most importantly, neonatal B cells showed severely impaired class-switch recombination (CSR) to IgG and IgA. This was associated with increased expression of miR-181b in neonatal B cells. Deficiency of miR-181b resulted in increased CSR. With this, our results highlight intrinsic differences that contribute to weaker B cell antibody responses in newborns.
    • Global micro RNA expression profiling in the liver biopsies of Hepatitis B Virus infected patients suggests specific miRNA signatures for viral persistence and hepatocellular injury.

      Singh, Avishek Kumar; Rooge, Sheetalnath Babasaheb; Varshney, Aditi; Vasudevan, Madavan; Bhardwaj, Ankit; Venugopal, Senthil Kumar; Trehanpati, Nirupama; Kumar, Manoj; Geffers, Robert; Kumar, Vijay; Sarin, Shiv Kumar; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-11-30)
      Hepatitis B virus (HBV) can manipulate the miRNA regulatory networks in infected cells to create a permissive environment for viral replication, cellular injury, disease onset and its progression. The aim of the present study was to understand the miRNA networks and their target genes in the liver of hepatitis B patients involved in HBV replication, liver injury and liver fibrosis. We investigated differentially expressed miRNAs by microarray in the liver biopsy samples from different stages of HBV infection and liver disease [immune tolerant (IT; n= 8); acute viral hepatitis (AVH; n=8); no fibrosis (n=16); early (F1+F2) (n=19) or late fibrosis (F3+F4) (n=14) and healthy controls (n=7)]. The miRNA expression levels were analyzed by the unsupervised principal component analysis (PCA) and hierarchical clustering. Analysis of miRNA-mRNA regulatory networks identified 17 miRNAs and 18 target gene interactions with four distinct nodes each representing a stage-specific gene regulation during disease progression. The IT group showed elevated miR-199a-5p, miR-221-3p and Let-7a-3p levels which could target genes involved in innate immune response and viral replication. In AVH group, miR-125b-5p and miR-3613-3p were up whereas miR-940 was down which might affect cell proliferation via STAT3 pathway. In early fibrosis, miR-34b-3p, miR-1224-3p and miR-1227-3p were up while miR-499a-5p was down which together, possibly mediate chronic inflammation. In advanced fibrosis, miR-1, miR-10b-5p, miR-96-5p, miR-133b and miR-671-5p were up while miR-20b-5p and miR-455-3p were down, possibly allowing chronic disease progression. Interestingly, only 8 of 17 liver-specific miRNAs exhibited a similar expression pattern in patient sera.
    • The Biofilm Inhibitor Carolacton Enters Gram-Negative Cells: Studies Using a TolC-Deficient Strain of Escherichia coli.

      Donner, Jannik; Reck, Michael; Bunk, Boyke; Jarek, Michael; App, Constantin Benjamin; Meier-Kolthoff, Jan P; Overmann, Jörg; Müller, Rolf; Kirschning, Andreas; Wagner-Döbler, Irene; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr, 7,38124 Braunschweig, Germany. (2017-11-01)
      The myxobacterial secondary metabolite carolacton inhibits growth of Streptococcus pneumoniae and kills biofilm cells of the caries- and endocarditis-associated pathogen Streptococcus mutans at nanomolar concentrations. Here, we studied the response to carolacton of an Escherichia coli strain that lacked the outer membrane protein TolC. Whole-genome sequencing of the laboratory E. coli strain TolC revealed the integration of an insertion element, IS5, at the tolC locus and a close phylogenetic relationship to the ancient E. coli K-12. We demonstrated via transcriptome sequencing (RNA-seq) and determination of MIC values that carolacton penetrates the phospholipid bilayer of the Gram-negative cell envelope and inhibits growth of E. coli TolC at similar concentrations as for streptococci. This inhibition is completely lost for a C-9 (R) epimer of carolacton, a derivative with an inverted stereocenter at carbon atom 9 [(S) → (R)] as the sole difference from the native molecule, which is also inactive in S. pneumoniae and S. mutans, suggesting a specific interaction of native carolacton with a conserved cellular target present in bacterial phyla as distantly related as Firmicutes and Proteobacteria. The efflux pump inhibitor (EPI) phenylalanine arginine β-naphthylamide (PAβN), which specifically inhibits AcrAB-TolC, renders E. coli susceptible to carolacton. Our data indicate that carolacton has potential for use in antimicrobial chemotherapy against Gram-negative bacteria, as a single drug or in combination with EPIs. Strain E. coli TolC has been deposited at the DSMZ; together with the associated RNA-seq data and MIC values, it can be used as a reference during future screenings for novel bioactive compounds. IMPORTANCE The emergence of pathogens resistant against most or all of the antibiotics currently used in human therapy is a global threat, and therefore the search for antimicrobials with novel targets and modes of action is of utmost importance. The myxobacterial secondary metabolite carolacton had previously been shown to inhibit biofilm formation and growth of streptococci. Here, we investigated if carolacton could act against Gram-negative bacteria, which are difficult targets because of their double-layered cytoplasmic envelope. We found that the model organism Escherichia coli is susceptible to carolacton, similar to the Gram-positive Streptococcus pneumoniae, if its multidrug efflux system AcrAB-TolC is either inactivated genetically, by disruption of the tolC gene, or physiologically by coadministering an efflux pump inhibitor. A carolacton epimer that has a different steric configuration at carbon atom 9 is completely inactive, suggesting that carolacton may interact with the same molecular target in both Gram-positive and Gram-negative bacteria.
    • Draft Genome Sequence of Zoonotic Streptococcus canis Isolate G361.

      Eichhorn, Inga; van der Linden, Mark; Jarek, Michael; Fulde, Marcus; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-09-21)
      Here, we report the draft genome sequence of an SCM-positive Streptococcus canis strain, G361, isolated from a vaginal swab of a 40-year-old woman. The draft genome comprises 2,045,931 bp in 62 contigs.
    • Phylotranscriptomic consolidation of the jawed vertebrate timetree.

      Irisarri, Iker; Baurain, Denis; Brinkmann, Henner; Delsuc, Frédéric; Sire, Jean-Yves; Kupfer, Alexander; Petersen, Jörn; Jarek, Michael; Meyer, Axel; Vences, Miguel; Philippe, Hervé; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-09-01)
      Phylogenomics is extremely powerful but introduces new challenges as no agreement exists on "standards" for data selection, curation and tree inference. We use jawed vertebrates (Gnathostomata) as model to address these issues. Despite considerable efforts in resolving their evolutionary history and macroevolution, few studies have included a full phylogenetic diversity of gnathostomes and some relationships remain controversial. We tested a novel bioinformatic pipeline to assemble large and accurate phylogenomic datasets from RNA sequencing and find this phylotranscriptomic approach successful and highly cost-effective. Increased sequencing effort up to ca. 10Gbp allows recovering more genes, but shallower sequencing (1.5Gbp) is sufficient to obtain thousands of full-length orthologous transcripts. We reconstruct a robust and strongly supported timetree of jawed vertebrates using 7,189 nuclear genes from 100 taxa, including 23 new transcriptomes from previously unsampled key species. Gene jackknifing of genomic data corroborates the robustness of our tree and allows calculating genome-wide divergence times by overcoming gene sampling bias. Mitochondrial genomes prove insufficient to resolve the deepest relationships because of limited signal and among-lineage rate heterogeneity. Our analyses emphasize the importance of large curated nuclear datasets to increase the accuracy of phylogenomics and provide a reference framework for the evolutionary history of jawed vertebrates.
    • Complete Genome Sequence of JII-1961, a Bovine Mycobacterium avium subsp. paratuberculosis Field Isolate from Germany.

      Möbius, Petra; Nordsiek, Gabriele; Hölzer, Martin; Jarek, Michael; Marz, Manja; Köhler, Heike; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-24)
      Mycobacterium avium subsp. paratuberculosis causes Johne's disease in ruminants and was also detected in nonruminant species, including human beings, and in milk products. We announce here the 4.829-Mb complete genome sequence of the cattle-type strain JII-1961 from Germany, which is very similar to cattle-type strains recovered from different continents.
    • Divergent co-transcriptomes of different host cells infected with Toxoplasma gondii reveal cell type-specific host-parasite interactions.

      Swierzy, Izabela J; Händel, Ulrike; Kaever, Alexander; Jarek, Michael; Scharfe, Maren; Schlüter, Dirk; Lüder, Carsten G K; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-08-03)
      The apicomplexan parasite Toxoplasma gondii infects various cell types in avian and mammalian hosts including humans. Infection of immunocompetent hosts is mostly asymptomatic or benign, but leads to development of largely dormant bradyzoites that persist predominantly within neurons and muscle cells. Here we have analyzed the impact of the host cell type on the co-transcriptomes of host and parasite using high-throughput RNA sequencing. Murine cortical neurons and astrocytes, skeletal muscle cells (SkMCs) and fibroblasts differed by more than 16,200 differentially expressed genes (DEGs) before and after infection with T. gondii. However, only a few hundred of them were regulated by infection and these largely diverged in neurons, SkMCs, astrocytes and fibroblasts indicating host cell type-specific transcriptional responses after infection. The heterogeneous transcriptomes of host cells before and during infection coincided with ~5,400 DEGs in T. gondii residing in different cell types. Finally, we identified gene clusters in both T. gondii and its host, which correlated with the predominant parasite persistence in neurons or SkMCs as compared to astrocytes or fibroblasts. Thus, heterogeneous expression profiles of different host cell types and the parasites' ability to adapting to them may govern the parasite-host cell interaction during toxoplasmosis.
    • Dysbiosis in chronic periodontitis: Key microbial players and interactions with the human host.

      Deng, Zhi-Luo; Szafrański, Szymon P; Jarek, Michael; Bhuju, Sabin; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7., 38124 Braunschweig, Germany. (2017-06-16)
      Periodontitis is an extremely prevalent disease worldwide and is driven by complex dysbiotic microbiota. Here we analyzed the transcriptional activity of the periodontal pocket microbiota from all domains of life as well as the human host in health and chronic periodontitis. Bacteria showed strong enrichment of 18 KEGG functional modules in chronic periodontitis, including bacterial chemotaxis, flagellar assembly, type III secretion system, type III CRISPR-Cas system, and two component system proteins. Upregulation of these functions was driven by the red-complex pathogens and candidate pathogens, e.g. Filifactor alocis, Prevotella intermedia, Fretibacterium fastidiosum and Selenomonas sputigena. Nine virulence factors were strongly up-regulated, among them the arginine deiminase arcA from Porphyromonas gingivalis and Mycoplasma arginini. Viruses and archaea accounted for about 0.1% and 0.22% of total putative mRNA reads, respectively, and a protozoan, Entamoeba gingivalis, was highly enriched in periodontitis. Fourteen human transcripts were enriched in periodontitis, including a gene for a ferric iron binding protein, indicating competition with the microbiota for iron, and genes associated with cancer, namely nucleolar phosphoprotein B23, ankyrin-repeat domain 30B-like protein and beta-enolase. The data provide evidence on the level of gene expression in vivo for the potentially severe impact of the dysbiotic microbiota on human health.
    • Quorum sensing of Streptococcus mutans is activated by Aggregatibacter actinomycetemcomitans and by the periodontal microbiome.

      Szafrański, Szymon P; Deng, Zhi-Luo; Tomasch, Jürgen; Jarek, Michael; Bhuju, Sabin; Rohde, Manfred; Sztajer, Helena; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-20)
      The oral cavity is inhabited by complex microbial communities forming biofilms that can cause caries and periodontitis. Cell-cell communication might play an important role in modulating the physiologies of individual species, but evidence so far is limited.
    • Interleukin-2 improves amyloid pathology, synaptic failure and memory in Alzheimer's disease mice.

      Alves, Sandro; Churlaud, Guillaume; Audrain, Mickael; Michaelsen-Preusse, Kristin; Fol, Romain; Souchet, Benoit; Braudeau, Jérôme; Korte, Martin; Klatzmann, David; Cartier, Nathalie; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03-01)
      Interleukin-2 (IL-2)-deficient mice have cytoarchitectural hippocampal modifications and impaired learning and memory ability reminiscent of Alzheimer's disease. IL-2 stimulates regulatory T cells whose role is to control inflammation. As neuroinflammation contributes to neurodegeneration, we investigated IL-2 in Alzheimer's disease. Therefore, we investigated IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease relative to age-matched control individuals. We then treated APP/PS1ΔE9 mice having established Alzheimer's disease with IL-2 for 5 months using single administration of an AAV-IL-2 vector. We first found decreased IL-2 levels in hippocampal biopsies of patients with Alzheimer's disease. In mice, IL-2-induced systemic and brain regulatory T cells expansion and activation. In the hippocampus, IL-2 induced astrocytic activation and recruitment of astrocytes around amyloid plaques, decreased amyloid-β42/40 ratio and amyloid plaque load, improved synaptic plasticity and significantly rescued spine density. Of note, this tissue remodelling was associated with recovery of memory deficits, as assessed in the Morris water maze task. Altogether, our data strongly suggest that IL-2 can alleviate Alzheimer's disease hallmarks in APP/PS1ΔE9 mice with established pathology. Therefore, this should prompt the investigation of low-dose IL-2 in Alzheimer's disease and other neuroinflammatory/neurodegenerative disorders.
    • Dealing with salinity extremes and nitrogen limitation - an unexpected strategy of the marine bacterium Dinoroseobacter shibae.

      Kleist, Sarah; Ulbrich, Marcus; Bill, Nelli; Schmidt-Hohagen, Kerstin; Geffers, Robert; Schomburg, Dietmar; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-03)
      Having the right coping strategy for changes in osmolarity or desiccation is essential for the survival of every cell. So far, nothing is known about compatible solutes and the salt adaptation of the marine Rhodobacteraceae. The family member Dinoroseobacter shibae DFL12(T) is shown here to form the compatible solutes α-glucosylglycerol (GG) and α-glucosylglycerate (GGA). To our knowledge, this is the first experimental evidence for GGA formation within the α-proteobacteria. Together with glutamate and putrescine, these substances enable good growth in salinity ranging from 0.3% to 5%. A salinity of 5% leads to a biomass share of 7.6% of compatible solutes and the very low salt level of 0.3% results in an 18-fold increased putrescine concentration compared with environmental conditions. Additionally, the substitution of glutamate by GGA has been shown during exposure to nitrogen limitation and in the stationary growth phase of the organism. Salt shock transcriptome analysis of D. shibae has revealed the essential role of its 153 kb chromid, which carries the genes for GG biosynthesis and several transport and exchange systems. Within the family of Rhodobacteraceae, the genomic capability of forming GG and GGA is strictly restricted to marine family members.
    • The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.

      Poppe, Michael; Wittig, Sascha; Jurida, Liane; Bartkuhn, Marek; Wilhelm, Jochen; Müller, Helmut; Beuerlein, Knut; Karl, Nadja; Bhuju, Sabin; Ziebuhr, John; Schmitz, M Lienhard; Kracht, Michael; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr.7, 38124 Braunschweig, Germany. (2017-03)
      Coronavirus replication takes place in the host cell cytoplasm and triggers inflammatory gene expression by poorly characterized mechanisms. To obtain more insight into the signals and molecular events that coordinate global host responses in the nucleus of coronavirus-infected cells, first, transcriptome dynamics was studied in human coronavirus 229E (HCoV-229E)-infected A549 and HuH7 cells, respectively, revealing a core signature of upregulated genes in these cells. Compared to treatment with the prototypical inflammatory cytokine interleukin(IL)-1, HCoV-229E replication was found to attenuate the inducible activity of the transcription factor (TF) NF-κB and to restrict the nuclear concentration of NF-κB subunits by (i) an unusual mechanism involving partial degradation of IKKβ, NEMO and IκBα and (ii) upregulation of TNFAIP3 (A20), although constitutive IKK activity and basal TNFAIP3 expression levels were shown to be required for efficient virus replication. Second, we characterized actively transcribed genomic regions and enhancers in HCoV-229E-infected cells and systematically correlated the genome-wide gene expression changes with the recruitment of Ser5-phosphorylated RNA polymerase II and prototypical histone modifications (H3K9ac, H3K36ac, H4K5ac, H3K27ac, H3K4me1). The data revealed that, in HCoV-infected (but not IL-1-treated) cells, an extensive set of genes was activated without inducible p65 NF-κB being recruited. Furthermore, both HCoV-229E replication and IL-1 were shown to upregulate a small set of genes encoding immunomodulatory factors that bind p65 at promoters and require IKKβ activity and p65 for expression. Also, HCoV-229E and IL-1 activated a common set of 440 p65-bound enhancers that differed from another 992 HCoV-229E-specific enhancer regions by distinct TF-binding motif combinations. Taken together, the study shows that cytoplasmic RNA viruses fine-tune NF-κB signaling at multiple levels and profoundly reprogram the host cellular chromatin landscape, thereby orchestrating the timely coordinated expression of genes involved in multiple signaling, immunoregulatory and metabolic processes.
    • Tracking HCV protease population diversity during transmission and susceptibility of founder populations to antiviral therapy.

      Khera, Tanvi; Todt, Daniel; Vercauteren, Koen; McClure, C Patrick; Verhoye, Lieven; Farhoudi, Ali; Bhuju, Sabin; Geffers, Robert; Baumert, Thomas F; Steinmann, Eike; Meuleman, Philip; Pietschmann, Thomas; Brown, Richard J P; Helmholtz Centre for infection research, Inhoffenstr. 7., 38124 Braunschweig, Germany. (2017-03)
      Due to the highly restricted species-tropism of Hepatitis C virus (HCV) a limited number of animal models exist for pre-clinical evaluation of vaccines and antiviral compounds. The human-liver chimeric mouse model allows heterologous challenge with clinically relevant strains derived from patients. However, to date, the transmission and longitudinal evolution of founder viral populations in this model have not been characterized in-depth using state-of-the-art sequencing technologies. Focusing on NS3 protease encoding region of the viral genome, mutant spectra in a donor inoculum and individual recipient mice were determined via Illumina sequencing and compared, to determine the effects of transmission on founder viral population complexity. In all transmissions, a genetic bottleneck was observed, although diverse viral populations were transmitted in each case. A low frequency cloud of mutations (<1%) was detectable in the donor inoculum and recipient mice, with single nucleotide variants (SNVs) > 1% restricted to a subset of nucleotides. The population of SNVs >1% was reduced upon transmission while the low frequency SNV cloud remained stable. Fixation of multiple identical synonymous substitutions was apparent in independent transmissions, and no evidence for reversion of T-cell epitopes was observed. In addition, susceptibility of founder populations to antiviral therapy was assessed. Animals were treated with protease inhibitor (PI) monotherapy to track resistance associated substitution (RAS) emergence. Longitudinal analyses revealed a decline in population diversity under therapy, with no detectable RAS >1% prior to therapy commencement. Despite inoculation from a common source and identical therapeutic regimens, unique RAS emergence profiles were identified in different hosts prior to and during therapeutic failure, with complex mutational signatures at protease residues 155, 156 and 168 detected. Together these analyses track viral population complexity at high-resolution in the human-liver chimeric mouse model post-transmission and under therapeutic intervention, revealing novel insights into the evolutionary processes which shape viral protease population composition at various critical stages of the viral life-cycle.
    • Complete Genome Sequences of Three Multidrug-Resistant Clinical Isolates of Streptococcus pneumoniae Serotype 19A with Different Susceptibilities to the Myxobacterial Metabolite Carolacton.

      Donner, Jannik; Bunk, Boyke; Schober, Isabel; Spröer, Cathrin; Bergmann, Simone; Jarek, Michael; Overmann, Jörg; Wagner-Döbler, Irene; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-02-16)
      The full-genome sequences of three drug- and multidrug-resistant Streptococcus pneumoniae clinical isolates of serotype 19A were determined by PacBio single-molecule real-time sequencing, in combination with Illumina MiSeq sequencing. A comparison to the genomes of other pneumococci indicates a high nucleotide sequence identity to strains Hungary19A-6 and TCH8431/19A.