Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy

H. verticillata samples collected from numerous watersheds from 2014 to 2020 were screened for the presence of A. hydrillicola. After isolation of A. hydrillicola from environmental samples and adaptation to laboratory conditions, the cyanobacterium was cultivated in BG11 medium with or without the addition of potassium bromide. H. verticillata leaves colonized with A. hydrillicola were analyzed using fluorescence microscopy as well as AP-MALDI-MSI (9-AA as matrix in negative-ionization mode; lateral resolution, 10 μm). Environmental bromide and bromine concentrations in H. verticillata, sediment, and water samples were analyzed by x-ray fluorescence spectroscopy and ion chromatography. The structure of AETX was elucidated by NMR spectroscopy, high-resolution tandem mass spectrometry, infrared spectroscopy, and x-ray crystallography after isolation of the compound using flash chromatography, semipreparative HPLC, and recrystallization. The genome of A. hydrillicola was amplified from single filaments using multiple displacement amplification and then sequenced using the Illumina MiSeq platform. The putative AETX biosynthetic gene cluster was identified by BLASTp searches for bacterial halogenases against the A. hydrillicola genome. The halogenase AetF was heterologously expressed in E. coli and then purified. Biochemical assays to characterize its activity used tryptophans and indoles as substrates. Reaction products were structurally characterized by HPLC-MS and NMR spectroscopy. A. hydrillicola–H. verticillata extract fractions and pure AETX were tested for activity on C. dubia, D. rerio, C. elegans, and G. gallus. Bioassays on D. rerio and G. gallus were performed in accordance with the National Insitutes of Health Guide for the Care and Use of Laboratory Animals and followed protocol A2017 11-007-Y1-A0, which was reviewed, approved, and overseen by the University of Georgia Institutional Animal Care and Use Committee. VM occurrence in treated birds was confirmed by analysis of the white matter of their optic lobe using light microscopy and transmission electron microscopy. Tissues of deceased wild birds were extracted and analyzed for AETX by HPLC-MS. A full description of the materials and methods used in this study is provided in the supplementary materials. Portions of this dataset are inaccessible because: Data belongs to coauthors at Martin-Luther-University Halle-Wittenberg, Halle (Saale), University of Georgia, Czech Academy of Sciences, and Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP), They can be accessed through the following means: NMR and MS raw data are available at Figshare (https://doi.org/10.6084/m9.figshare.6025748.v1). 

X-ray data and models are available at the Cambridge Crystallographic Data Centre under accession no. CCDC-2018827. (https://doi.org/10.5517/ccdc.csd.cc25rrg5)

The whole-genome assemblies (Whole Genome Shotgun projects) of two A. hydrillicola strains, CCALA 1050 and Thurmond2011, have been deposited at DDBJ/ENA/GenBank under the accession nos. JAALHA000000000 and JAAKGC000000000, respectively. The versions described in this paper are JAALHA010000000 and JAAKGC010000000. 

The sequence of the putative AETX biosynthetic gene cluster can be found at DDBJ/ENA/GenBank under the accession no. MT225528. 

All other data are available in the main text or the supplementary materials of the journal article. Format: data are in several formats, see accessed information for more information. 

This dataset is associated with the following publication:
Breinlinger, S., T. Phillips, B. Haram, J. Mareš, J. Martínez Yerena, P. Hrouzek, R. Sobotka, W. Henderson, P. Schmieder, S.M. Williams, J.D. Lauderdale, H.D. Wilde, W. Gerrin, A. Kust, J. Washington, C. Wagner, M. Liebeke, H. Enke, T. Niedermeyer, and S. Wilde. Hunting the eagle killer: A cyanobacterial neurotoxin causes vacuolar myelinopathy.   SCIENCE. American Association for the Advancement of Science (AAAS), Washington, DC, USA, 371(6536): eaax9050, (2021).

Tags: aquatic-vegetation, bald-eagles, novel-epiphytic-cyanobacterial, vacuolar-myelinopathy
Organization: U.S. Environmental Protection Agency