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), Braunschweig Integrated Centre for Systems biology (BRICS), Centre for Structural Systems Biology (CSSB) and the Study Centre Hannover, Hannover/Germany.

 

  • Engineering of Streptomyces lividans for heterologous expression of secondary metabolite gene clusters.

    Ahmed, Yousra; Rebets, Yuriy; Estévez, Marta Rodríguez; Zapp, Josef; Myronovskyi, Maksym; Luzhetskyy, Andriy; HIPS, Helmholtz-Institut für Pharmazeutische Forschung Saarland, Universitätscampus E8.1 66123 Saarbrücken, Germany. (BioMed Central (BMC), 2020-01-09)
    BACKGROUND: Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS: In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION: The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.
  • Correlation between immunoglobulin dose administered and plasma neutralization of streptococcal superantigens in patients with necrotizing soft tissue infections.

    Bergsten, Helena; Madsen, Martin Bruun; Bergey, Francois; Hyldegaard, Ole; Skrede, Steinar; Arnell, Per; Oppegaard, Oddvar; Itzek, Andreas; Perner, Anders; Svensson, Mattias; et al. (Oxford University Press, 2020-01-09)
    Analyses of plasma collected pre- and post-administration of intravenous immunoglobulin G (IVIG) from patients with Group A Streptococcus necrotizing soft tissue infections demonstrated a negative correlation between IVIG dose and toxin-triggered T-cell proliferation (r=-0.67, p<0.0001). One 25g-dose IVIG was sufficient to yield patient plasma neutralizing activity against streptococcal superantigens.
  • Tracheal brush cells release acetylcholine in response to bitter tastants for paracrine and autocrine signaling.

    Hollenhorst, Monika I; Jurastow, Innokentij; Nandigama, Rajender; Appenzeller, Silke; Li, Lei; Vogel, Jörg; Wiederhold, Stephanie; Althaus, Mike; Empting, Martin; Altmüller, Janine; et al. (Wiley, 2020-01-01)
    For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter "taste" substances are most probably initiated by tracheal brush cells (BC). Our single-cell RNA-seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca2+]i in BC and subsequent ACh-release. ACh-release is regulated in an autocrine manner. While the muscarinic ACh-receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca2+]i in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5- and M3R-mediated. We show that ACh-release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
  • Phosphatidylcholine PC ae C44:6 in cerebrospinal fluid is a sensitive biomarker for bacterial meningitis.

    de Araujo, Leonardo Silva; Pessler, Kevin; Sühs, Kurt-Wolfram; Novoselova, Natalia; Klawonn, Frank; Kuhn, Maike; Kaever, Volkhard; Müller-Vahl, Kirsten; Trebst, Corinna; Skripuletz, Thomas; et al. (BioMed Central (BMC), 2020-01-07)
    BACKGROUND: The timely diagnosis of bacterial meningitis is of utmost importance due to the need to institute antibiotic treatment as early as possible. Moreover, the differentiation from other causes of meningitis/encephalitis is critical because of differences in management such as the need for antiviral or immunosuppressive treatments. Considering our previously reported association between free membrane phospholipids in cerebrospinal fluid (CSF) and CNS involvement in neuroinfections we evaluated phosphatidylcholine PC ae C44:6, an integral constituent of cell membranes, as diagnostic biomarker for bacterial meningitis. METHODS: We used tandem mass spectrometry to measure concentrations of PC ae C44:6 in cell-free CSF samples (n = 221) from patients with acute bacterial meningitis, neuroborreliosis, viral meningitis/encephalitis (herpes simplex virus, varicella zoster virus, enteroviruses), autoimmune neuroinflammation (anti-NMDA-receptor autoimmune encephalitis, multiple sclerosis), facial nerve and segmental herpes zoster (shingles), and noninflammatory CNS disorders (Bell's palsy, Tourette syndrome, normal pressure hydrocephalus). RESULTS: PC ae C44:6 concentrations were significantly higher in bacterial meningitis than in all other diagnostic groups, and were higher in patients with a classic bacterial meningitis pathogen (e.g. Streptococcus pneumoniae, Neisseria meningitidis, Staphylococcus aureus) than in those with less virulent or opportunistic pathogens as causative agents (P = 0.026). PC ae C44:6 concentrations were only moderately associated with CSF cell count (Spearman's ρ = 0.45; P = 0.009), indicating that they do not merely reflect neuroinflammation. In receiver operating characteristic curve analysis, PC ae C44:6 equaled CSF cell count in the ability to distinguish bacterial meningitis from viral meningitis/encephalitis and autoimmune CNS disorders (AUC 0.93 both), but had higher sensitivity (91% vs. 41%) and negative predictive value (98% vs. 89%). A diagnostic algorithm comprising cell count, lactate and PC ae C44:6 had a sensitivity of 97% (specificity 87%) and negative predictive value of 99% (positive predictive value 61%) and correctly diagnosed three of four bacterial meningitis samples that were misclassified by cell count and lactate due to low values not suggestive of bacterial meningitis. CONCLUSIONS: Increased CSF PC ae C44:6 concentrations in bacterial meningitis likely reflect ongoing CNS cell membrane stress or damage and have potential as additional, sensitive biomarker to diagnose bacterial meningitis in patients with less pronounced neuroinflammation.
  • Pigmentosins from Gibellula sp. As antibiofilm agents and a new glycosylated asperfuran from Cordyceps javanica

    Helaly, Soleiman E.; Kuephadungphan, Wilawan; Phainuphong, Patima; Ibrahim, Mahmoud A.A.; Tasanathai, Kanoksri; Mongkolsamrit, Suchada; Luangsa-Ard, Janet Jennifer; Phongpaichit, Souwalak; Rukachaisirikul, Vatcharin; Stadler, Marc; et al. (Beilstein Institut, 2019-12-16)
    n the course of our exploration of the Thai invertebrate-pathogenic fungi for biologically active metabolites, pigmentosin A (1) and a new bis(naphtho-α-pyrone) derivative, pigmentosin B (2), were isolated from the spider-associated fungus Gibellula sp. Furthermore, a new glycosylated asperfuran 3, together with one new (6) and two known (4 and 5) cyclodepsipeptides, was isolated from Cordyceps javanica. The pigmentosins 1 and 2 showed to be active against biofilm formation of Staphylococcus aureus DSM1104. The lack of toxicity toward the studied microorganism and cell lines of pigmentosin B (2), as well as the antimicrobial effect of pigmentosin A (1), made them good candidates for further development for use in combination therapy of infections involving biofilm-forming S. aureus. The structure elucidation and determination of the absolute configuration were accomplished using a combination of spectroscopy, including 1D and 2D NMR, HRMS, Mosher ester analysis, and comparison of calculated/experimental ECD spectra. A chemotaxonomic investigation of the secondary metabolite profiles using analytical HPLC coupled with diode array detection and mass spectrometry (HPLC–DAD–MS) revealed that the production of pigmentosin B (2) was apparently specific for Gibellula sp., while the glycoasperfuran 3 was specific for C. javanica.

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