• Multisystem combined uranium resistance mechanisms and bioremediation potential of Stenotrophomonas bentonitica BII-R7: Transcriptomics and microscopic study

      Pinel-Cabello, M.; Jroundi, F.; López-Fernández, M.; Geffers, R.; Jarek, M.; Jauregui, R.; Link, A.; Vílchez-Vargas, R.; Merroun, M. L.; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Elsevier, 2021-02-05)
      The potential use of microorganisms in the bioremediation of U pollution has been extensively described. However, a lack of knowledge on molecular resistance mechanisms has become a challenge for the use of these technologies. We reported on the transcriptomic and microscopic response of Stenotrophomonas bentonitica BII-R7 exposed to 100 and 250 μM of U. Results showed that exposure to 100 μM displayed up-regulation of 185 and 148 genes during the lag and exponential phases, respectively, whereas 143 and 194 were down-regulated, out of 3786 genes (>1.5-fold change). Exposure to 250 μM of U showed up-regulation of 68 genes and down-regulation of 290 during the lag phase. Genes involved in cell wall and membrane protein synthesis, efflux systems and phosphatases were up-regulated under all conditions tested. Microscopic observations evidenced the formation of U-phosphate minerals at membrane and extracellular levels. Thus, a biphasic process is likely to occur: the increased cell wall would promote the biosorption of U to the cell surface and its precipitation as U-phosphate minerals enhanced by phosphatases. Transport systems would prevent U accumulation in the cytoplasm. These findings contribute to an understanding of how microbes cope with U toxicity, thus allowing for the development of efficient bioremediation strategies.
    • Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

      Schiefer, Andrea; Hübner, Marc P; Krome, Anna; Lämmer, Christine; Ehrens, Alexandra; Aden, Tilman; Koschel, Marianne; Neufeld, Helene; Chaverra-Muñoz, Lillibeth; Jansen, Rolf; et al. (PLOS, 2020-12-07)
      Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal-adult-worm killing-treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4-5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug.
    • Solubility and Stability Enhanced Oral Formulations for the Anti-Infective Corallopyronin A.

      Krome, Anna K; Becker, Tim; Kehraus, Stefan; Schiefer, Andrea; Steinebach, Christian; Aden, Tilman; Frohberger, Stefan J; López Mármol, Álvaro; Kapote, Dnyaneshwar; Jansen, Rolf; et al. (MDPI, 2020-11-18)
      Novel-antibiotics are urgently needed to combat an increase in morbidity and mortality due to resistant bacteria. The preclinical candidate corallopyronin A (CorA) is a potent antibiotic against Gram-positive and some Gram-negative pathogens for which a solid oral formulation was needed for further preclinical testing of the active pharmaceutical ingredient (API). The neat API CorA is poorly water-soluble and instable at room temperature, both crucial characteristics to be addressed and overcome for use as an oral antibiotic. Therefore, amorphous solid dispersion (ASD) was chosen as formulation principle. The formulations were prepared by spray-drying, comprising the water-soluble polymers povidone and copovidone. Stability (high-performance liquid chromatography, Fourier-transform-infrared spectroscopy, differential scanning calorimetry), dissolution (biphasic dissolution), and solubility (biphasic dissolution, Pion's T3 apparatus) properties were analyzed. Pharmacokinetic evaluations after intravenous and oral administration were conducted in BALB/c mice. The results demonstrated that the ASD formulation principle is a suitable stability- and solubility-enhancing oral formulation strategy for the API CorA to be used in preclinical and clinical trials and as a potential market product.
    • 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.
    • Unsaturated Fatty Acids Control Biofilm Formation of and Other Gram-Positive Bacteria.

      Yuyama, Kamila Tomoko; Rohde, Manfred; Molinari, Gabriella; Stadler, Marc; Abraham, Wolf-Rainer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-11-08)
      Infections involving biofilms are difficult to treat due to increased resistances against antibiotics and the immune system. Hence, there is an urgent demand for novel drugs against biofilm infections. During our search for novel biofilm inhibitors from fungi, we isolated linoleic acid from the ascomycete Hypoxylon fragiforme which showed biofilm inhibition of several bacteria at sub-MIC concentrations. Many fatty acids possess antimicrobial activities, but their minimum inhibitory concentrations (MIC) are high and reports on biofilm interferences are scarce. We demonstrated that not only linoleic acid but several unsaturated long-chain fatty acids inhibited biofilms at sub-MIC concentrations. The antibiofilm activity exerted by long-chain fatty acids was mainly against Gram-positive bacteria, especially against Staphylococcus aureus. Micrographs of treated S. aureus biofilms revealed a reduction in the extracellular polymeric substances, pointing to a possible mode of action of fatty acids on S. aureus biofilms. The fatty acids had a strong species specificity. Poly-unsaturated fatty acids had higher activities than saturated ones, but no obvious rule could be found for the optimal length and desaturation for maximal activity. As free fatty acids are non-toxic and ubiquitous in food, they may offer a novel tool, especially in combination with antibiotics, for the control of biofilm infections.
    • Simplicilones A and B Isolated from the Endophytic Fungus SPC3.

      Anoumedem, Elodie Gisèle M; Mountessou, Bel Youssouf G; Kouam, Simeon F; Narmani, Abolfazl; Surup, Frank; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-29)
      Two new tetracyclic polyketides with a spirocenter, simplicilones A (1) and B (2) were isolated from the broth-culture of the endophytic fungus Simplicilliumsubtropicum (SPC3) in the course of our screening for new bioactive secondary metabolites. This endophytoic fungus is naturally harboured in the fresh bark of the Cameroonian medicinal plant Duguetia staudtii (Engl. and Diels) Chatrou. The planar structures of the simplicilones were elucidated by MS and 1D as well as 2D NMR spectroscopic techniques. The relative configuration was assigned by NOESY experiments in conjunction with coupling constants; subsequently, the absolute configurations were assigned by the modified Mosher's method. The compounds showed weak cytotoxic effects against the cell line KB3.1 (in vitro cytotoxicity (IC50) = 25 µg/mL for 1, 29 µg/mL for 2), but were inactive against the tested Gram-positive and Gram-negative bacteria as well as fungi.
    • Recent progress in biodiversity research on the Xylariales and their secondary metabolism.

      Becker, Kevin; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2020-10-23)
      The families Xylariaceae and Hypoxylaceae (Xylariales, Ascomycota) represent one of the most prolific lineages of secondary metabolite producers. Like many other fungal taxa, they exhibit their highest diversity in the tropics. The stromata as well as the mycelial cultures of these fungi (the latter of which are frequently being isolated as endophytes of seed plants) have given rise to the discovery of many unprecedented secondary metabolites. Some of those served as lead compounds for development of pharmaceuticals and agrochemicals. Recently, the endophytic Xylariales have also come in the focus of biological control, since some of their species show strong antagonistic effects against fungal and other pathogens. New compounds, including volatiles as well as nonvolatiles, are steadily being discovered from these ascomycetes, and polythetic taxonomy now allows for elucidation of the life cycle of the endophytes for the first time. Moreover, recently high-quality genome sequences of some strains have become available, which facilitates phylogenomic studies as well as the elucidation of the biosynthetic gene clusters (BGC) as a starting point for synthetic biotechnology approaches. In this review, we summarize recent findings, focusing on the publications of the past 3 years.
    • Erinacine C Activates Transcription from a Consensus ETS DNA Binding Site in Astrocytic Cells in Addition to NGF Induction.

      Rascher, Monique; Wittstein, Kathrin; Winter, Barbara; Rupcic, Zeljka; Wolf-Asseburg, Alexandra; Stadler, Marc; Köster, Reinhard W; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-10-14)
      Medicinal mushrooms of the genus Hericium are known to produce secondary metabolites with homeostatic properties for the central nervous system. We and others have recently demonstrated that among these metabolites cyathane diterpenoids and in particular erinacine C possess potent neurotrophin inducing properties in astrocytic cells. Yet, the signaling events downstream of erinacine C induced neurotrophin acitivity in neural-like adrenal phaeochromocytoma cells (PC12) cells have remained elusive. Similar, signaling events activated by erinacine C in astrocytic cells are unknown. Using a combination of genetic and pharmacological inhibitors we show that erinacine C induced neurotrophic activity mediates PC12 cell differentiation via the TrkA receptor and likely its associated PLCγ-, PI3K-, and MAPK/ERK pathways. Furthermore, a small library of transcriptional activation reporters revealed that erinacine C induces transcriptional activation mediated by DNA consensus binding sites of selected conserved transcription factor families. Among these, transcription is activated from an ETS consensus in a concentration dependent manner. Interestingly, induced ETS-consensus transcription occurs in parallel and independent of neurotrophin induction. This finding helps to explain the many pleiotropic functions of cyathane diterpenoids. Moreover, our studies provide genetic access to cyathane diterpenoid functions in astrocytic cells and help to mechanistically understand the action of cyathanes in glial cells.
    • Three novel species and a new record of Daldinia (Hypoxylaceae) from Thailand

      Wongkanoun, Sarunyou; Becker, Kevin; Boonmee, Kanthawut; Srikitikulchai, Prasert; Boonyuen, Nattawut; Chainuwong, Boonchuai; Luangsa-ard, Jennifer; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Science and Business Media LLC, 2020-10-08)
      n an investigation of stromatic Xylariales in Thailand, several specimens of Daldinia were discovered. Three novel species (D. flavogranulata, D. phadaengensis, and D. chiangdaoensis) were recognized from a molecular phylogeny based on concatenated ITS, LSU, RPB2, and TUB2 sequence data, combined with morphological characters and secondary metabolite profiles based on high performance liquid chromatography coupled to diode array detection and mass spectrometry (HPLC-MS). The major components detected were cytochalasins (in D. flavogranulata and D. chiangdaoensis) and daldinin type azaphilones (in D. phadaengensis). In addition, D. brachysperma, which had hitherto only been reported from America, was found for the first time in Asia. Its phylogenetic affinities were studied, confirming previous suspicions from morphological comparisons that the species is closely related to D. eschscholtzii and D. bambusicola, both common in Thailand. Daldinia flavogranulata, one of the new taxa, was found to be closely related to the same taxa. The other two novel species, D. phadaengensis and D. chiangdaoensis, share characters with D. korfii and D. kretzschmarioides, respectively.
    • Seven New Cytotoxic and Antimicrobial Xanthoquinodins from Jugulospora vestita.

      Shao, Lulu; Marin-Felix, Yasmina; Surup, Frank; Stchigel, Alberto M; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-25)
      During the course of a screening for novel biologically active secondary metabolites produced by the Sordariomycetes (Ascomycota, Fungi), the ex-type strain of Jugulospora vestita was found to produce seven novel xanthone-anthraquinone heterodimers, xanthoquinodin A11 (1) and xanthoquinodins B10-15 (2-7), together with the already known compound xanthoquinodin B4 (8). The structures of the xanthoquinodins were determined by analysis of the nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric data. Moreover, the absolute configurations of these metabolites were established by analysis of the 1H-1H coupling constants, nuclear Overhauser effect spectroscopy (NOESY) correlations, and Electronic Circular Dichroism (ECD) spectroscopic data. Antifungal and antibacterial activities as well as cytotoxicity of all compounds were tested. Xanthoquinodin B11 showed fungicidal activities against Mucor hiemalis [minimum inhibitory concentration (MIC) 2.1 µg/mL], Rhodotorula glutinis (MIC 2.1 µg/mL), and Pichia anomala (MIC 8.3 µg/mL). All the compounds 1-8 displayed anti-Gram-positive bacteria activity (MIC 0.2-8.3 µg/mL). In addition, all these eight compounds showed cytotoxicity against KB 3.1, L929, A549, SK-OV-3, PC-3, A431, and MCF-7 mammalian cell lines. The six novel compounds (1-3, 5-7), together with xanthoquinodin B4, were also found in the screening of other strains belonging to Jugulospora rotula, revealing the potential chemotaxonomic significance of the compound class for the genus.
    • Cycloheximide-Producing Associated With and Fungus-Farming Ambrosia Beetles.

      Grubbs, Kirk J; Surup, Frank; Biedermann, Peter H W; McDonald, Bradon R; Klassen, Jonathan L; Carlson, Caitlin M; Clardy, Jon; Currie, Cameron R; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Frontiers, 2020-09-24)
      Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.
    • Amidochelocardin Overcomes Resistance Mechanisms Exerted on Tetracyclines and Natural Chelocardin.

      Hennessen, Fabienne; Miethke, Marcus; Zaburannyi, Nestor; Loose, Maria; Lukežič, Tadeja; Bernecker, Steffen; Hüttel, Stephan; Jansen, Rolf; Schmiedel, Judith; Fritzenwanker, Moritz; et al. (MDPI, 2020-09-18)
      The reassessment of known but neglected natural compounds is a vital strategy for providing novel lead structures urgently needed to overcome antimicrobial resistance. Scaffolds with resistance-breaking properties represent the most promising candidates for a successful translation into future therapeutics. Our study focuses on chelocardin, a member of the atypical tetracyclines, and its bioengineered derivative amidochelocardin, both showing broad-spectrum antibacterial activity within the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) panel. Further lead development of chelocardins requires extensive biological and chemical profiling to achieve favorable pharmaceutical properties and efficacy. This study shows that both molecules possess resistance-breaking properties enabling the escape from most common tetracycline resistance mechanisms. Further, we show that these compounds are potent candidates for treatment of urinary tract infections due to their in vitro activity against a large panel of multidrug-resistant uropathogenic clinical isolates. In addition, the mechanism of resistance to natural chelocardin was identified as relying on efflux processes, both in the chelocardin producer Amycolatopsis sulphurea and in the pathogen Klebsiella pneumoniae. Resistance development in Klebsiella led primarily to mutations in ramR, causing increased expression of the acrAB-tolC efflux pump. Most importantly, amidochelocardin overcomes this resistance mechanism, revealing not only the improved activity profile but also superior resistance-breaking properties of this novel antibacterial compound.
    • Molecular Phylogeny and Morphology of (=Lepteutypa ) (Amphisphaeriaceae).

      Samarakoon, Milan C; Maharachchikumbura, Sajeewa S N; Liu, Jian-Kui Jack; Hyde, Kevin D; Promputtha, Itthayakorn; Stadler, Marc; HZI, Helmholtz Zentrum für Infektionsforschung, GmbH, Inhoffenstr. 7, 38124 Braunschweig, Germany. (MDPI, 2020-09-17)
      Amphisphaeriaceous taxa (fungi) are saprobes on decaying wood in terrestrial, mangrove, and freshwater habitats. The generic boundaries of the family have traditionally been based on morphology, and the delimitation of genera has always been challenging. Amphisphaeria species have clypeate ascomata and 1-septate ascospores and a coelomycetous asexual morph. Lepteutypa is different from Amphisphaeria in having eutypoid stromata and more than 1-septate ascospores. These main characters have been used for segregation of Lepteutypa from Amphisphaeria for a long time. However, the above characters are overlapping among Amphisphaeria and Lepteutypa species. Therefore, here we synonymized Lepteutypa under Amphisphaeria based on holomorphic morphology and multigene phylogeny. Further, our cluster analysis reveals the relationship between seven morphological traits among Amphisphaeria/Lepteutypa species and suggests those morphologies are not specific to either genus. Three new species (i.e., Amphisphaeria camelliae, A. curvaticonidia, and A. micheliae) are introduced based on morphology and LSU-ITS-RPB2-TUB2 phylogenies. Furthermore, the monotypic genus Trochilispora, which had been accepted in Amphisphaeriaceae, is revisited and synonymized under Hymenopleella and placed in Sporocadaceae.
    • Re-Evaluation of the Order Sordariales: Delimitation of Lasiosphaeriaceae s. str., and Introduction of the New Families Diplogelasinosporaceae, Naviculisporaceae, and Schizotheciaceae.

      Marin-Felix, Yasmina; Miller, Andrew N; Cano-Lira, José F; Guarro, Josep; García, D; Stadler, Marc; Huhndorf, Sabine M; Stchigel, Alberto M; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-17)
      The order Sordariales includes the polyphyletic family Lasiosphaeriaceae, which comprises approximately 30 genera characterized by its paraphysate ascomata, asci with apical apparati, and mostly two-celled ascospores, which have a dark apical cell and a hyaline lower cell, frequently ornamented with mucilaginous appendages[...].
    • Biosynthesis of oxygenated brasilane terpene glycosides involves a promiscuous N-acetylglucosamine transferase.

      Feng, Jin; Surup, Frank; Hauser, Maurice; Miller, Anna; Wennrich, Jan-Peer; Stadler, Marc; Cox, Russell J; Kuhnert, Eric; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Royal Sciety of Chemistry, 2020-09-16)
      Investigation of the metabolome of the ascomycete Annulohypoxylon truncatum led to the identification of novel oxygenated brasilane glycosides and the revision of the stereochemistry of the brasilane A octahydro-1H-indene core scaffold to trans. The bra biosynthetic gene cluster containing five genes (braA-braE) was identified and verified by heterologous expression experiments in Aspergillus oryzae demonstrating that BraC is a multifunctional P450 monooxygenase. In vitro studies of BraB revealed it to be a very rare fungal UDP-GlcNAc dependent N-acetylglucosamine transferase. UDP-glucose is also accepted as a donor, and a broad acceptor substrate tolerance for various primary and secondary alcohols was observed.
    • Phylogenetic Assignment of the Fungicolous (Ascomycota, Xylariales) and Investigation of its Secondary Metabolites.

      Becker, Kevin; Lambert, Christopher; Wieschhaus, Jörg; Stadler, Marc; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-11)
      The ascomycete Hypoxylon invadens was described in 2014 as a fungicolous species growing on a member of its own genus, H.fragiforme, which is considered a rare lifestyle in the Hypoxylaceae. This renders H.invadens an interesting target in our efforts to find new bioactive secondary metabolites from members of the Xylariales. So far, only volatile organic compounds have been reported from H.invadens, but no investigation of non-volatile compounds had been conducted. Furthermore, a phylogenetic assignment following recent trends in fungal taxonomy via a multiple sequence alignment seemed practical. A culture of H.invadens was thus subjected to submerged cultivation to investigate the produced secondary metabolites, followed by isolation via preparative chromatography and subsequent structure elucidation by means of nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS). This approach led to the identification of the known flaviolin (1) and 3,3-biflaviolin (2) as the main components, which had never been reported from the order Xylariales before. Assessment of their antimicrobial and cytotoxic effects via a panel of commonly used microorganisms and cell lines in our laboratory did not yield any effects of relevance. Concurrently, genomic DNA from the fungus was used to construct a multigene phylogeny using ribosomal sequence information from the internal transcribed spacer region (ITS), the 28S large subunit of ribosomal DNA (LSU), and proteinogenic nucleotide sequences from the second largest subunit of the DNA-directed RNA polymerase II (RPB2) and β-tubulin (TUB2) genes. A placement in a newly formed clade with H.trugodes was strongly supported in a maximum-likelihood (ML) phylogeny using sequences derived from well characterized strains, but the exact position of said clade remains unclear. Both, the chemical and the phylogenetic results suggest further inquiries into the lifestyle of this unique fungus to get a better understanding of both, its ecological role and function of its produced secondary metabolites hitherto unique to the Xylariales.
    • A dynamic portal for a community-driven, continuously updated classification of Fungi and fungus-like organisms: outlineoffungi.org

      Wijayawardene, NN; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Mushroom Research Foundation, 2020-09-10)
    • Phylogeny- and morphology-based recognition of new species in the spider-parasitic genus (Hypocreales, Cordycipitaceae) from Thailand.

      Kuephadungphan, Wilawan; Tasanathai, Kanoksri; Petcharad, Booppa; Khonsanit, Artit; Stadler, Marc; Luangsa-Ard, J Jennifer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (PenSoft publisher, 2020-09-02)
      Thailand is known to be a part of what is called the Indo-Burma biodiversity hotspot, hosting a vast array of organisms across its diverse ecosystems. This is reflected by the increasing number of new species described over time, especially fungi. However, a very few fungal species from the specialized spider-parasitic genus Gibellula have ever been reported from this region. A survey of invertebrate-pathogenic fungi in Thailand over several decades has led to the discovery of a number of fungal specimens with affinities to this genus. Integration of morphological traits into multi-locus phylogenetic analysis uncovered four new species: G. cebrennini, G. fusiformispora, G. pigmentosinum, and G. scorpioides. All these appear to be exclusively linked with torrubiella-like sexual morphs with the presence of granulomanus-like asexual morph in G. pigmentosinum and G. cebrennini. A remarkably high host specificity of these new species towards their spider hosts was revealed, and for the first time, evidence is presented for manipulation of host behavior in G. scorpioides.
    • Phylogenetic and chemotaxonomic studies confirm the affinities of Stromatoneurospora phoenix to the Coprophilous xylariaceae

      Becker, Kevin; Wongkanoun, Sarunyou; Wessel, Anna Charleen; Bills, Gerald F.; Stadler, Marc; Luangsa-ard, J. Jennifer; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2020-09-01)
      The genus Stromatoneurospora was erected in 1973 by Jong and Davis to accommodate the pyrophilic pyrenomycete Sphaeria phoenix and has traditionally been placed in the family Xylariaceae based on morphological features. However, no living culture of this genus has so far been available in the public domain. Molecular data were restricted to an internal transcribed spacer (ITS) sequence that only confirmed the familial position, and was generated from a strain that is not deposited in a public culture collection. We have recently collected fresh material and were able to culture this fungus from Thailand. The secondary metabolites of this strains were analysed after fermentation in multiple media. The the prominent components of these fermentation were purified, using preparative chromatography. Aside from two new eremophilane sesquiterpenoids named phoenixilanes A-B (1-2), four other components that are known from species of the xylariaceous genera Xylaria and Poronia were identified by spectral methods (nuclear magnetic resonance spectroscopy and high resolution mass spectrometry). Notably, (-)-(R)-6-hydroxy-3-methyl-4-dihydroisocoumarin-5-carboxylic acid (6) has not been reported as a natural product before. Moreover, DNA sequences of Stromatoneurospora phoenix clustered with members of the genera Poronia and Podosordaria in a multi-locus molecular phylogeny. These results confirmed that the genus belongs to the same evolutionary lineage as the coprophilic Xylariaceae. The results also suggest that this lineage has evolved independently from the plant-inhabiting saprotrophs and endophytes that are closely related to the genus Xylaria. These findings are discussed in relation to some theories about the endophytic vs. the pyrophilic/coprophilic fungal life style.
    • 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.