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).

 

  • Characterization of the Stereoselective P450 Enzyme BotCYP Enables the Biosynthesis of the Bottromycin Core Scaffold.

    Adam, Sebastian; Franz, Laura; Milhim, Mohammed; Bernhardt, Rita; Kalinina, Olga V; Koehnke, Jesko; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (American Chemical Society, 2020-11-28)
    Bottromycins are ribosomally synthesized and post-translationally modified peptide natural product antibiotics that are effective against high-priority human pathogens such as methicillin-resistant Staphylococcus aureus. The total synthesis of bottromycins involves at least 17 steps, with a poor overall yield. Here, we report the characterization of the cytochrome P450 enzyme BotCYP from a bottromycin biosynthetic gene cluster. We determined the structure of a close BotCYP homolog and used our data to conduct the first large-scale survey of P450 enzymes associated with RiPP biosynthetic gene clusters. We demonstrate that BotCYP converts a C-terminal thiazoline to a thiazole via an oxidative decarboxylation reaction and provides stereochemical resolution for the pathway. Our data enable the two-pot in vitro production of the bottromycin core scaffold and may allow the rapid generation of bottromycin analogues for compound development.
  • Protecting-Group-Mediated Diastereoselective Synthesis of C4'-Methylated Uridine Analogs and Their Activity against the Human Respiratory Syncytial Virus.

    Köllmann, Christoph; Sake, Svenja M; Jones, Peter G; Pietschmann, Thomas; Werz, Daniel B; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (American Chemical Society, 2020-02-26)
    Adjusting the protecting group strategy, from an alkyl ether to a bidentate ketal at the carbohydrate backbone of uridine, facilitates a switchable diastereoselective α- or β-C4'/C5'-spirocyclopropanation. Using these spirocyclopropanated nucleosides as key intermediates, we synthesized a variety of C4'-methylated d-ribose and l-lyxose-configured uridine derivatives by a base-mediated ring-opening of the spirocyclopropanol moiety. Investigations of antiviral activity against the human respiratory syncytial virus were carried out for selected derivatives, showing moderate activity.
  • Active equine parvovirus-hepatitis infection is most frequently detected in Austrian horses of advanced age.

    Badenhorst, Marcha; de Heus, Phebe; Auer, Angelika; Tegtmeyer, Birthe; Stang, Alexander; Dimmel, Katharina; Tichy, Alexander; Kubacki, Jakub; Bachofen, Claudia; Steinmann, Eike; et al. (Wiley, 2021-03-11)
    ckground: Equine parvovirus-hepatitis (EqPV-H) research is in its infancy. Information regarding prevalence, geographical distribution, genetic diversity, pathogenesis and risk factors enhances understanding of this potentially fatal infection. Objectives: Determining the prevalence of EqPV-H in Austrian equids. Investigating factors increasing probability of infection, liver-associated biochemistry parameters, concurrent equine hepacivirus (EqHV) infection and phylogenetic analysis of Austrian EqPV-H variants. Study design: Cross-sectional study. Methods: Sera from 259 horses and 13 donkeys in Austria were analysed for anti-EqPV-H VP1-specific antibodies by luciferase immunoprecipitation system (LIPS) and EqPV-H DNA by nested polymerase chain reaction (PCR). Associations between infection status, sex and age were described. Glutamate dehydrogenase (GLDH), gamma-glutamyl transferase (GGT), bile acids and albumin concentrations were compared between horses with active infection and PCR-negative horses. PCR targeting partial EqPV-H NS1 was performed and phylogenetic analysis of Austrian EqPV-H variants was conducted. Complete coding sequences (CDS) of four Austrian variants were determined by next-generation sequencing (NGS) and compared with published sequences. Results: Horses' EqPV-H seroprevalence was 30.1% and DNA prevalence was 8.9%. One horse was co-infected with EqHV. Significantly, higher probability of active EqPV-H infection was identified in 16- to 31-year-old horses, compared with 1- to 8-year-old horses (P = 0.002; OR = 8.19; 95% CI = 1.79 to 37.50) and 9- to 15-year-old horses (P = 0.03; OR = 2.96; 95% CI = 1.08 to 8.17). Liver-associated plasma parameters were not significantly different between horses with active infection and controls. Austrian EqPV-H variants revealed high similarity to sequences worldwide. No evidence of EqPV-H was detected in donkeys. Main limitations: Equids' inclusion depended upon owner consent. There was only one sampling point per animal and the sample of donkeys was small. Conclusions: EqPV-H antibodies and DNA are frequently detected in Austrian horses, without associated hepatitis in horses with active infection. The risk of active EqPV-H infection increases with increasing age. Phylogenetic evidence supports close relation of EqPV-H variants globally, including Austrian variants.
  • The exo-β-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases.

    Müller, Maraike; Calvert, Matthew; Hottmann, Isabel; Kluj, Robert Maria; Teufel, Tim; Balbuchta, Katja; Engelbrecht, Alicia; Selim, Khaled A; Xu, Qingping; Borisova, Marina; et al. (Elsevier, 2021-03-05)
    Endo-β-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the β-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-β-N-acetylmuramidases, which catalyze an exo-lytic cleavage of β-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-β-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl β-MurNAc and the peptidoglycan-derived disaccharide MurNAc-β-1,4-GlcNAc. Together with the exo-β-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.
  • Transcriptome analysis following neurotropic virus infection reveals faulty innate immunity and delayed antigen presentation in mice susceptible to virus-induced demyelination.

    Ciurkiewicz, Malgorzata; Floess, Stefan; Beckstette, Michael; Kummerfeld, Maren; Baumgärtner, Wolfgang; Huehn, Jochen; Beineke, Andreas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley & Sons Ltd., 2021-07-06)
    Viral infections of the central nervous system cause acute or delayed neuropathology and clinical consequences ranging from asymptomatic courses to chronic, debilitating diseases. The outcome of viral encephalitis is partially determined by genetically programed immune response patterns of the host. Experimental infection of mice with Theiler's murine encephalomyelitis virus (TMEV) causes diverse neurologic diseases, including TMEV-induced demyelinating disease (TMEV-IDD), depending on the used mouse strain. The aim of the present study was to compare initial transcriptomic changes occurring in the brain of TMEV-infected SJL (TMEV-IDD susceptible) and C57BL/6 (TMEV-IDD resistant) mice. Animals were infected with TMEV and sacrificed 4, 7, or 14 days post infection. RNA was isolated from brain tissue and analyzed by whole-transcriptome sequencing. Selected differences were confirmed on a protein level by immunohistochemistry. In mock-infected SJL and C57BL/6 mice, >200 differentially expressed genes (DEGs) were detected. Following TMEV-infection, the number of DEGs increased to >700. Infected C57BL/6 mice showed a higher expression of transcripts related to antigen presentation via major histocompatibility complex (MHC) I, innate antiviral immune responses and cytotoxicity, compared with infected SJL animals. Expression of many of those genes was weaker or delayed in SJL mice, associated with a failure of viral clearance in this mouse strain. SJL mice showed prolonged elevation of MHC II and chemotactic genes compared with C57BL/6 mice, which presumably facilitates the induction of chronic demyelinating disease. In addition, elevated expression of several genes associated with immunomodulatory or -suppressive functions was observed in SJL mice. The exploratory study confirms previous observations in the model and provides an extensive list of new immunologic parameters potentially contributing to different outcomes of viral encephalitis in two mouse strains.

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