• Haprolid Inhibits Tumor Growth of Hepatocellular Carcinoma through Rb/E2F and Akt/mTOR Inhibition.

      Xing, Jun; Bhuria, Vikas; Bui, Khac Cuong; Nguyen, Mai Ly Thi; Hu, Zexi; Hsieh, Chih-Jen; Wittstein, Kathrin; Stadler, Marc; Wilkens, Ludwig; Li, Jun; et al. (MDPI, 2020-03-06)
      The efficacy of haprolid was evaluated in human HCC cell lines (Huh-7, Hep3B and HepG2) and xenograft tumors (NMRI-Foxn1nu mice with injection of Hep3B cells). Cytotoxic activity of haprolid was determined by the WST-1 and crystal violet assay. Wound healing, transwell and tumorsphere assays were performed to investigate migration and invasion of HCC cells. Apoptosis and cell-cycle distribution were measured by flow cytometry. The effects of haprolid on the Rb/E2F and Akt/mTOR pathway were examined by immunoblotting and immunohistochemistry.
    • Hybridorubrins A-D, novel azaphilone heterodimers from stromata of Hypoxylon fragiforme and insights into the biosynthetic machinery for azaphilone diversification.

      Becker, Kevin; Pfütze, Sebastian; Kuhnert, Eric; Cox, Russell; Stadler, Marc; Surup, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Wiley-VCH, 2020-08-04)
      The diversity of azaphilones in stromatal extracts of the fungus Hypoxylon fragiforme was investigated and linked to their biosynthetic machineries using bioinformatics. Nineteen azaphilone-type compounds were isolated and characterized by NMR spectroscopy and mass spectrometry, with their absolute stereoconfigurations assigned using Mosher ester analysis and ECD spectroscopy. Four unprecedented bisazaphilones, named hybridorubrins A-D ( 1 - 4 ), were elucidated, in addition to new fragirubrins F-G ( 5 - 6 ) and various known mitorubrin derivatives. Only the hybridorubrins, which are composed of mitorubrin and fragirubrin moieties, exhibited strong inhibition of Staphylococcus aureus biofilm formation. Analysis of the genome of H. fragiforme revealed the presence of two separate biosynthetic gene clusters (BGC) hfaza1 and hfaza2 responsible for azaphilone formation. While the hfaza1 BGC likely encodes the assembly of the backbone and addition of fatty acid moieties to yield the ( R )-configured series of fragirubrins, the hfaza2 BGC contains the necessary genes to synthesise the widely distributed ( S )-mitorubrins. This study is the first example of two distant cross-acting fungal BGC collaborating to produce two families of azaphilones and bisazaphilones derived thereof.
    • Hymenosetin, a 3-decalinoyltetramic acid antibiotic from cultures of the ash dieback pathogen, Hymenoscyphus pseudoalbidus.

      Halecker, Sandra; Surup, Frank; Kuhnert, Eric; Mohr, Kathrin I; Brock, Nelson L; Dickschat, Jeroen S; Junker, Corina; Schulz, Barbara; Stadler, Marc; Helmholtz Centre for ifection research, Innhoffenstr. 7, D38124 Braunschweig, Germany. (2014-04)
      A 3-decalinoyltetramic acid, for which the trivial name hymenosetin is proposed, was isolated from crude extracts of a virulent strain of the ash dieback pathogen, Hymenoscyphus pseudoalbidus (="Chalara fraxinea"). This compound was produced only under certain culture conditions in submerged cultures of the fungus. Its planar structure was determined by NMR spectroscopy and by mass spectrometry. The absolute stereochemistry was assigned by CD spectroscopy and HETLOC data. Hymenosetin exhibited broad spectrum antibacterial and antifungal activities (including strong inhibition of MRSA), as well as moderate cytotoxic effects. So far, the metabolite proved inactive in assays for evaluation of phytotoxicity, whereas viridiol, another secondary metabolite known from H. pseudoalbidus, was regarded as phytotoxic principle of the pathogen against its host, Fraxinus excelsior. Further studies will show whether hymenosetin constitutes a defence metabolite that is produced by the pathogenic fungus to combat other microbes and fungi in the natural environment.
    • Hypoxylon pulicicidum sp. nov. (Ascomycota, Xylariales), a pantropical insecticide-producing endophyte.

      Bills, Gerald F; González-Menéndez, Victor; Martín, Jesús; Platas, Gonzalo; Fournier, Jacques; Peršoh, Derek; Stadler, Marc; Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Armilla, Granada, Spain. gerald.bills@medinaandalucia.es (2012)
      Nodulisporic acids (NAs) are indole diterpene fungal metabolites exhibiting potent systemic efficacy against blood-feeding arthropods, e.g., bedbugs, fleas and ticks, via binding to arthropod specific glutamate-gated chloride channels. Intensive medicinal chemistry efforts employing a nodulisporic acid A template have led to the development of N-tert-butyl nodulisporamide as a product candidate for a once monthly treatment of fleas and ticks on companion animals. The source of the NAs is a monophyletic lineage of asexual endophytic fungal strains that is widely distributed in the tropics, tentatively identified as a Nodulisporium species and hypothesized to be the asexual state of a Hypoxylon species.
    • Hypoxyvermelhotins A-C, new pigments from Hypoxylon lechatii sp. nov.

      Kuhnert, Eric; Heitkämper, Simone; Fournier, Jacques; Surup, Frank; Stadler, Marc; Dept of microbial drugs, Helmholtz-Centre for infection research, Inhoffenstr. 7, D-38124 Braunschweig, Germany. (2014-02)
      A new species of Hypoxylon was discovered, based on material collected in French Guiana and recognised on the basis of new combination of morpholological characters in comparison with type and authentic material of macroscopically similar taxa. These findings were corroborated by the rather isolated positions of its ITS-nrDNA and beta-tubulin DNA sequences in molecular phylogenies. However, the most salient feature of this fungus only became evident by a comparison of its stromatal HPLC profile, revealing several secondary metabolites that were hitherto not observed in stromata of any other member of the Xylariaceae. Part of the stromata were subsequently extracted to isolate these apparently specific components, using preparative chromatography. Five metabolites were obtained in pure state, and their chemical structures were elucidated by means of high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. They turned out to be tetramic acid derivatives of the so-called vermelhotin type. Aside from vermelhotin, previously isolated from cultures of endophytic fungi, we identified three novel congeners, for which the trivial names hypoxyvermelhotins A-C were proposed. Like vermelhotin, they constitute orange-red pigments and a preliminary biological characterisation revealed them to have rather strong cytotoxic and moderate to weak antimicrobial effects. These results further illustrate the high diversity of unique secondary metabolites in stromata of the hypoxyloid Xylariaceae, a family in which biological diversity seems to parallel the chemical diversity of their bioactive principles to a great extent.
    • Identification and genetics of 6-thioguanine secreted by Erwinia species and its interference with the growth of other bacteria.

      Wensing, A; Gernold, M; Jock, S; Jansen, R; Geider, K (2014-04)
      We identified a compound in culture supernatants of Erwinia species, such as Erwinia amylovora, E. pyrifoliae, E. billingiae, E. tasmaniensis, E. persicina and E. rhapontici absorbing at 340 nm, which was associated before with the yellow pigment produced by E. amylovora on media containing copper ions. The compound was purified from E. tasmaniensis strain Et1/99 supernatants by chromatography on Dowex-1 and Dowex-50 columns and identified by HPLC/MS and NMR analysis as 6-thioguanine (6TG). Its signal at 167 Da matched with the expected molecular mass. By random mutagenesis with miniTn5, we obtained mutants defective in the genes for pyrimidine and purine metabolism. A specific gene cluster with ycf genes described by us before, absent in the corresponding region of Escherichia coli, was identified in the genome sequence of three Erwinia species and named tgs region for thioguanine synthesis. Clones of the tgs gene cluster promoted 6TG synthesis and secretion in E. coli, when the bacteria were grown in minimal medium supplemented with amino acids. 6TG was bacteriostatic for E. coli and Salmonella typhimurium strains, with cell growth resumed after prolonged incubation. Similar results were obtained with P. agglomerans strains. Bacteria from the genus Pectobacterium were barely and Rahnella or Gibbsiella species were not inhibited by 6TG. Adenine and guanine relieved the toxic effect of 6TG on E. coli. Non-producing strains were fully virulent on host plants. 6TG synthesis may help erwinias to interfere with growth of some microorganisms in the environment.
    • Identification of species as producers of cyclodepsipeptide PF1022 A and resurrection of the genus as inferred from polythetic taxonomy.

      Wittstein, K; Cordsmeier, A; Lambert, C; Wendt, L; Sir, E B; Weber, J; Wurzler, N; Petrini, L E; Stadler, M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2020-01-26)
      Rosellinia (Xylariaceae) is a large, cosmopolitan genus comprising over 130 species that have been defined based mainly on the morphology of their sexual morphs. The genus comprises both lignicolous and saprotrophic species that are frequently isolated as endophytes from healthy host plants, and important plant pathogens. In order to evaluate the utility of molecular phylogeny and secondary metabolite profiling to achieve a better basis for their classification, a set of strains was selected for a multi-locus phylogeny inferred from a combination of the sequences of the internal transcribed spacer region (ITS), the large subunit (LSU) of the nuclear rDNA, beta-tubulin (TUB2) and the second largest subunit of the RNA polymerase II (RPB2). Concurrently, various strains were surveyed for production of secondary metabolites. Metabolite profiling relied on methods with high performance liquid chromatography with diode array and mass spectrometric detection (HPLC-DAD/MS) as well as preparative isolation of the major components after re-fermentation followed by structure elucidation using nuclear magnetic resonance (NMR) spectroscopy and high resolution mass spectrometry (HR-MS). Two new and nine known isopimarane diterpenoids were identified during our mycochemical studies of two selected Dematophora strains and the metabolites were tested for biological activity. In addition, the nematicidal cyclodepsipeptide PF1022 A was purified and identified from a culture of Rosellinia corticium, which is the first time that this endophyte-derived drug precursor has been identified unambiguously from an ascospore-derived isolate of a Rosellinia species. While the results of this first HPLC profiling were largely inconclusive regarding the utility of secondary metabolites as genus-specific chemotaxonomic markers, the phylogeny clearly showed that species featuring a dematophora-like asexual morph were included in a well-defined clade, for which the genus Dematophora is resurrected. Dematophora now comprises all previously known important plant pathogens in the genus such as D. arcuata, D. bunodes, D. necatrix and D. pepo, while Rosellinia s. str. comprises those species that are known to have a geniculosporium-like or nodulisporium-like asexual morph, or where the asexual morph remains unknown. The extensive morphological studies of L.E. Petrini served as a basis to transfer several further species from Rosellinia to Dematophora, based on the morphology of their asexual morphs. However, most species of Rosellinia and allies still need to be recollected in fresh state, cultured, and studied for their morphology and their phylogenetic affinities before the infrageneric relationships can be clarified.
    • Ijuhya vitellina sp. nov., a novel source for chaetoglobosin A, is a destructive parasite of the cereal cyst nematode Heterodera filipjevi.

      Ashrafi, Samad; Helaly, Soleiman; Schroers, Hans-Josef; Stadler, Marc; Richert-Poeggeler, Katja R; Dababat, Abdelfattah A; Maier, Wolfgang; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2017-01-01)
      Cyst nematodes are globally important pathogens in agriculture. Their sedentary lifestyle and long-term association with the roots of host plants render cyst nematodes especially good targets for attack by parasitic fungi. In this context fungi were specifically isolated from nematode eggs of the cereal cyst nematode Heterodera filipjevi. Here, Ijuhya vitellina (Ascomycota, Hypocreales, Bionectriaceae), encountered in wheat fields in Turkey, is newly described on the basis of phylogenetic analyses, morphological characters and life-style related inferences. The species destructively parasitises eggs inside cysts of H. filipjevi. The parasitism was reproduced in in vitro studies. Infected eggs were found to harbour microsclerotia produced by I. vitellina that resemble long-term survival structures also known from other ascomycetes. Microsclerotia were also formed by this species in pure cultures obtained from both, solitarily isolated infected eggs obtained from fields and artificially infected eggs. Hyphae penetrating the eggshell colonised the interior of eggs and became transformed into multicellular, chlamydospore-like structures that developed into microsclerotia. When isolated on artificial media, microsclerotia germinated to produce multiple emerging hyphae. The specific nature of morphological structures produced by I. vitellina inside nematode eggs is interpreted as a unique mode of interaction allowing long-term survival of the fungus inside nematode cysts that are known to survive periods of drought or other harsh environmental conditions. Generic classification of the new species is based on molecular phylogenetic inferences using five different gene regions. I. vitellina is the only species of the genus known to parasitise nematodes and produce microsclerotia. Metabolomic analyses revealed that within the Ijuhya species studied here, only I. vitellina produces chaetoglobosin A and its derivate 19-O-acetylchaetoglobosin A. Nematicidal and nematode-inhibiting activities of these compounds have been demonstrated suggesting that the production of these compounds may represent an adaptation to nematode parasitism.
    • Improved production of medium-chain-length Polyhydroxyalkanotes in glucose-based fed-batch cultivations of metabolically engineered Pseudomonas putida strains.

      Poblete-Castro, Ignacio; Rodriguez, Andre Luis; Lam, Carolyn Ming Chi; Kessler, Wolfgang; Microbial Drugs Group, Helmholtz Centre for Infection Research, Inhoffenstraße 7, 38124 Braunschweig, Germany. (2013-10-22)
      One of the major challenges in metabolic engineering for enhanced synthesis of value-added chemicals is to design and develop new strains which can be translated into well-controlled fermentation processes using bioreactors. The aim of this study was to assess the influence of various fed-batch strategies in the performance of metabolically-engineered Pseudomonas putida strains, ∆gcd and ∆gcd-pgl, for improving production of medium-chain-length poly-hydroxyalkanoates (mcl-PHAs) using glucose as the only carbon source. First we developed a fed-batch process which comprised an initial phase of biomass accumulation based on an exponential feeding carbon-limited strategy. For the mcl-PHA accumulation stage, three induction techniques were tested under nitrogen limitation. The substrate-pulse feeding was more efficient than the constant-feeding approach to promote the accumulation of the desirable product. Nonetheless, the most efficient approach for maximum PHA synthesis was the application of a dissolved-oxygen-stat feeding strategy (DO-stat), where P. putida ∆gcd mutant strain showed a final PHA content and specific PHA productivity of 67%, and 0.83 [g•L(-1)•h(-1)], respectively. To our knowledge this mcl-PHA titer is the highest value that has been ever reported using glucose as the solely carbon and energy source. Our results also highlighted the effect of different fed-batch strategies upon the extent of realization of the intended metabolic modification of the mutant strains.
    • Intragenomic polymorphisms in the ITS region of high-quality genomes of the Hypoxylaceae (Xylariales, Ascomycota)

      Stadler, Marc; Lambert, Christopher; Wibberg, Daniel; Kalinowski, Jörn; Cox, Russell J.; Kolařík, Miroslav; Kuhnert, Eric; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-03-01)
      The internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) has been established (and is generally accepted) as a primary “universal” genetic barcode for fungi for many years, but the actual value for taxonomy has been heavily disputed among mycologists. Recently, twelve draft genome sequences, mainly derived from type species of the family Hypoxylaceae (Xylariales, Ascomycota) and the ex-epitype strain of Xylaria hypoxylon have become available during the course of a large phylogenomic study that was primarily aimed at establishing a correlation between the existing multi-gene-based genealogy with a genome-based phylogeny and the discovery of novel biosynthetic gene clusters encoding for secondary metabolites. The genome sequences were obtained using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing, respectively, and resulted in high-quality sequences with an average N50 of 3.2 Mbp. While the main results will be published concurrently in a separate paper, the current case study was dedicated to the detection of ITS nrDNA copies in the genomes, in an attempt to explain certain incongruities and apparent mismatches between phenotypes and genotypes that had been observed during previous polyphasic studies. The results revealed that all of the studied strains had at least three copies of rDNA in their genomes, with Hypoxylon fragiforme having at least 19 copies of the ITS region, followed by Xylaria hypoxylon with at least 13 copies. Several of the genomes contained 2–3 copies that were nearly identical, but in some cases drastic differences, below 97% identity were observed. In one case, ascribable to the presence of a pseudogene, the deviations of the ITS sequences from the same genome resulted in only ca. 90% of overall homology. These results are discussed in the scope of the current trends to use ITS data for species recognition and segregation of fungi. We propose that additional genomes should be checked for such ITS polymorphisms to reassess the validity of this non-coding part of the fungal DNA for molecular identification.
    • Investigating the Function of Cryptic Cytochalasan Cytochrome P450 Monooxygenases Using Combinatorial Biosynthesis.

      Wang, Chongqing; Becker, Kevin; Pfütze, Sebastian; Kuhnert, Eric; Stadler, Marc; Cox, Russell J; Skellam, Elizabeth; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (ACS, 2019-10-23)
      Tailoring enzymes in cytochalasan biosynthesis are relatively promiscuous. Exploiting this property, we deduced the function of four cryptic cytochrome P450 monooxygenases via heterologous expression of six cytochrome P450-encoding genes, originating from Hypoxylon fragiforme and Pyricularia oryzae, in pyrichalasin H ΔP450 strains. Three cryptic cytochrome P450 enzymes (HffD, HffG, and CYP1) restored pyrichalasin H production in mutant strains, while CYP3 catalyzed a site-selective epoxidation leading to the isolation of three novel cytochalasans.
    • Isolation of a gene cluster from Armillaria gallica for the synthesis of armillyl orsellinate-type sesquiterpenoids.

      Engels, Benedikt; Heinig, Uwe; McElroy, Christopher; Meusinger, Reinhard; Grothe, Torsten; Stadler, Marc; Jennewein, Stefan; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer, 2020-11-16)
      Melleolides and armillyl orsellinates are protoilludene-type aryl esters that are synthesized exclusively by parasitic fungi of the globally distributed genus Armillaria (Agaricomycetes, Physalacriaceae). Several of these compounds show potent antimicrobial and cytotoxic activities, making them promising leads for the development of new antibiotics or drugs for the treatment of cancer. We recently cloned and characterized the Armillaria gallica gene Pro1 encoding protoilludene synthase, a sesquiterpene cyclase catalyzing the pathway-committing step to all protoilludene-type aryl esters. Fungal enzymes representing secondary metabolic pathways are sometimes encoded by gene clusters, so we hypothesized that the missing steps in the pathway to melleolides and armillyl orsellinates might be identified by cloning the genes surrounding Pro1. Here we report the isolation of an A. gallica gene cluster encoding protoilludene synthase and four cytochrome P450 monooxygenases. Heterologous expression and functional analysis resulted in the identification of protoilludene-8α-hydroxylase, which catalyzes the first committed step in the armillyl orsellinate pathway. This confirms that ∆-6-protoilludene is a precursor for the synthesis of both melleolides and armillyl orsellinates, but the two pathways already branch at the level of the first oxygenation step. Our results provide insight into the synthesis of these valuable natural products and pave the way for their production by metabolic engineering. KEY POINTS: • Protoilludene-type aryl esters are bioactive metabolites produced by Armillaria spp. • The pathway-committing step to these compounds is catalyzed by protoilludene synthase. • We characterized CYP-type enzymes in the cluster and identified novel intermediates.
    • KdmB, a Jumonji Histone H3 Demethylase, Regulates Genome-Wide H3K4 Trimethylation and Is Required for Normal Induction of Secondary Metabolism in Aspergillus nidulans.

      Gacek-Matthews, Agnieszka; Berger, Harald; Sasaki, Takahiko; Wittstein, Kathrin; Gruber, Clemens; Lewis, Zachary A; Strauss, Joseph; Helmholtzzentrum für Infektionsforschung, Inhoffenstrasse 7, 38124 Braunschweig (2016-08)
      Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) in which the corresponding genes-usually physically linked in co-regulated clusters-are silenced under optimal physiological conditions (nutrient-rich) but are activated when nutrients are limiting. The exact molecular mechanisms by which HPTMs influence silencing and activation, however, are still to be better understood. Here we show by a combined approach of quantitative mass spectrometry (LC-MS/MS), genome-wide chromatin immunoprecipitation (ChIP-seq) and transcriptional network analysis (RNA-seq) that the core regions of silent A. nidulans SM clusters generally carry low levels of all tested chromatin modifications and that heterochromatic marks flank most of these SM clusters. During secondary metabolism, histone marks typically associated with transcriptional activity such as H3 trimethylated at lysine-4 (H3K4me3) are established in some, but not all gene clusters even upon full activation. KdmB, a Jarid1-family histone H3 lysine demethylase predicted to comprise a BRIGHT domain, a zinc-finger and two PHD domains in addition to the catalytic Jumonji domain, targets and demethylates H3K4me3 in vivo and mediates transcriptional downregulation. Deletion of kdmB leads to increased transcription of about ~1750 genes across nutrient-rich (primary metabolism) and nutrient-limiting (secondary metabolism) conditions. Unexpectedly, an equally high number of genes exhibited reduced expression in the kdmB deletion strain and notably, this group was significantly enriched for genes with known or predicted functions in secondary metabolite biosynthesis. Taken together, this study extends our general knowledge about multi-domain KDM5 histone demethylases and provides new details on the chromatin-level regulation of fungal secondary metabolite production.
    • Kenalactams A-E, Polyene Macrolactams Isolated from Nocardiopsis CG3.

      Messaoudi, Omar; Sudarman, Enge; Bendahou, Mourad; Jansen, Rolf; Stadler, Marc; Wink, Joachim; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (American Cemical Society (ACS), 2019-05-24)
      In our screening program for new biologically active secondary metabolites, a new strain, Nocardiopsis CG3 (DSM 106572), isolated from the saltpan of Kenadsa, was found to produce five new polyene macrolactams, the kenalactams A-E (1-5). Their structures were elucidated by spectral methods (NMR and HRESIMS), and the absolute configuration was derived by chemical derivatization (Mosher's method). Through a feeding experiment, alanine was proven to be the nitrogen-bearing starter unit involved in biosynthesis of the polyketide kenalactam A (1). Kenalactam E (5) was cytotoxic against human prostate cancer PC-3 cells with an IC50 value of 2.1 μM.
    • Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

      Blockus, Sebastian; Sake, Svenja M; Wetzke, Martin; Grethe, Christina; Graalmann, Theresa; Pils, Marina; Le Goffic, Ronan; Galloux, Marie; Prochnow, Hans; Rox, Katharina; et al. (Elsevier, 2020-03-18)
      Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
    • Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

      Prochnow, Hans; Rox, Katharina; Birudukota, N V Suryanarayana; Weichert, Loreen; Hotop, Sven-Kevin; Klahn, Philipp; Mohr, Kathrin; Franz, Sergej; Banda, Dominic H; Blockus, Sebastian; et al. (ASM, 2019-10-30)
      To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.
    • Lanyamycin, a macrolide antibiotic from Sorangium cellulosum, strain Soce 481 (Myxobacteria)

      Mulwa, Lucky S.; Jansen, Rolf; Praditya, Dimas F.; Mohr, Kathrin I.; Okanya, Patrick W.; Wink, Joachim; Steinmann, Eike; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Beilstein-Institut, 2018-06-26)
      Lanyamycin (1/2), a secondary metabolite occurring as two epimers, was isolated from the myxobacterium Sorangium cellulosum, strain Soce 481. The structures of both epimers were elucidated from HRESIMS and 1D and 2D NMR data and the relative configuration of their macrolactone ring was assigned based on NOE and vicinal 1H NMR coupling constants and by calculation of a 3D model. Lanyamycin inhibited HCV infection into mammalian liver cells with an IC50 value of 11.8 µM, and exhibited a moderate cytotoxic activity against the mouse fibroblast cell line L929 and the human nasopharyngeal cell line KB3 with IC50 values of 3.1 and 1.5 μM, respectively, and also suppressed the growth of the Gram-positive bacterium Micrococcus luteus.
    • Large Scale Production and Downstream Processing of Labyrinthopeptins from the Actinobacterium .

      Rupcic, Zeljka; Hüttel, Stephan; Bernecker, Steffen; Kanaki, Sae; Stadler, Marc; Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2018-06-05)
      A method was established for the production of 1.2-fold and 4.2-fold increased amounts of the antiviral and central nervous system-active lantipeptides, labyrinthopeptins A1 and A2, respectively, isolated from the actinobacterium
    • Lentinulactam, a hirsutane sesquiterpene with an unprecedented lactam modification

      Helaly, Soleiman E.; Richter, Christian; Thongbai, Benjarong; Hyde, Kevin D.; Stadler, Marc; Helmholtz Centre for infection research, Inhoffenstr. 7, 38124 Braunschweig, Germany. (2016-12)
    • Microfungi associated with Clematis (Ranunculaceae) with an integrated approach to delimiting species boundaries

      Phukhamsakda, Chayanard; McKenzie, Eric H. C.; Phillips, Alan J. L.; Gareth Jones, E. B.; Jayarama Bhat, D.; Stadler, Marc; Bhunjun, Chitrabhanu S.; Wanasinghe, Dhanushka N.; Thongbai, Benjarong; Camporesi, Erio; et al. (Springer Science and Business Media LLC, 2020-07-07)
      The cosmopolitan plant genus Clematis contains many climbing species that can be found worldwide. The genus occurs in the wild and is grown commercially for horticulture. Microfungi on Clematis were collected from Belgium, China, Italy, Thailand and the UK. They are characterized by morphology and analyses of gene sequence data using an integrated species concept to validate identifications. The study revealed two new families, 12 new genera, 50 new species, 26 new host records with one dimorphic character report, and ten species are transferred to other genera. The new families revealed by multigene phylogeny are Longiostiolaceae and Pseudomassarinaceae in Pleosporales (Dothideomycetes). New genera are Anthodidymella (Didymellaceae), Anthosulcatispora and Parasulcatispora (Sulcatisporaceae), Fusiformispora (Amniculicolaceae), Longispora (Phaeosphaeriaceae), Neobyssosphaeria (Melanommataceae), Neoleptosporella (Chaetosphaeriales, genera incertae sedis), Neostictis (Stictidaceae), Pseudohelminthosporium (Neomassarinaceae), Pseudomassarina (Pseudomassarinaceae), Sclerenchymomyces (Leptosphaeriaceae) and Xenoplectosphaerella (Plectosphaerellaceae). The newly described species are Alloleptosphaeria clematidis, Anthodidymella ranunculacearum, Anthosulcatispora subglobosa, Aquadictyospora clematidis, Brunneofusispora clematidis, Chaetosphaeronema clematidicola, C. clematidis, Chromolaenicola clematidis, Diaporthe clematidina, Dictyocheirospora clematidis, Distoseptispora clematidis, Floricola clematidis, Fusiformispora clematidis, Hermatomyces clematidis, Leptospora clematidis, Longispora clematidis, Massariosphaeria clematidis, Melomastia clematidis, M. fulvicomae, Neobyssosphaeria clematidis, Neoleptosporella clematidis, Neoroussoella clematidis, N. fulvicomae, Neostictis nigricans, Neovaginatispora clematidis, Parasulcatispora clematidis, Parathyridaria clematidis, P. serratifoliae, P. virginianae, Periconia verrucose, Phomatospora uniseriata, Pleopunctum clematidis, Pseudocapulatispora clematidis, Pseudocoleophoma clematidis, Pseudohelminthosporium clematidis, Pseudolophiostoma chiangraiense, P. clematidis, Pseudomassarina clematidis, Ramusculicola clematidis, Sarocladium clematidis, Sclerenchymomyces clematidis, Sigarispora clematidicola, S. clematidis, S. montanae, Sordaria clematidis, Stemphylium clematidis, Wojnowiciella clematidis, Xenodidymella clematidis, Xenomassariosphaeria clematidis and Xenoplectosphaerella clematidis. The following fungi are recorded on Clematis species for the first time: Angustimassarina rosarum, Dendryphion europaeum, Dermatiopleospora mariae, Diaporthe ravennica, D. rudis, Dichotomopilus ramosissimum, Dictyocheirospora xishuangbannaensis, Didymosphaeria rubi-ulmifolii, Fitzroyomyces cyperacearum, Fusarium celtidicola, Leptospora thailandica, Memnoniella oblongispora, Neodidymelliopsis longicolla, Neoeutypella baoshanensis, Neoroussoella heveae, Nigrograna chromolaenae, N. obliqua, Pestalotiopsis verruculosa, Pseudoberkleasmium chiangmaiense, Pseudoophiobolus rosae, Pseudoroussoella chromolaenae, P. elaeicola, Ramusculicola thailandica, Stemphylium vesicarium and Torula chromolaenae. The new combinations are Anthodidymella clematidis (≡ Didymella clematidis), A. vitalbina (≡ Didymella vitalbina), Anthosulcatispora brunnea (≡ Neobambusicola brunnea), Fuscohypha kunmingensis (≡ Plectosphaerella kunmingensis), Magnibotryascoma rubriostiolata (≡ Teichospora rubriostiolata), Pararoussoella mangrovei (≡ Roussoella mangrovei), Pseudoneoconiothyrium euonymi (≡ Roussoella euonymi), Sclerenchymomyces jonesii (≡ Neoleptosphaeria jonesii), Stemphylium rosae (≡ Pleospora rosae), and S. rosae-caninae (≡ Pleospora rosae-caninae). The microfungi on Clematis is distributed in several classes of Ascomycota. The analyses are based on morphological examination of specimens, coupled with phylogenetic sequence data. To the best of our knowledge, the consolidated species concept approach is recommended in validating species.