More than just an Eagle Killer: The freshwater cyanobacteriumAetokthonos hydrillicolaproduces highly toxic dolastatin derivatives

Abstract

AbstractCyanobacteria are infamous producers of toxins. While the toxic potential of planktic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacteriumAetokthonos hydrillicolahas recently been shown to produce the “eagle killer” neurotoxin aetokthonotoxin causing the fatal neurological disease Vacuolar Myelinopathy. The disease affects a wide array of wildlife in the southeastern United States, most notably waterfowl and birds of prey, including the bald eagle. In an assay for cytotoxicity, we found the crude extract of the cyanobacterium to be much more potent than pure aetokthonotoxin, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins, linear peptides belonging to the dolastatin compound family, featuring a unique modification of the C-terminal phenylalanine derived moiety. Using immunofluorescence microscopy and molecular modeling, we confirmed that aetokthonostatin acts as a potent tubulin binder. We also show that aetokthonostatin inhibits reproduction of the nematodeC. elegans, resulting in increased population lethality of the combined action of the two toxins produced byA. hydrillicola. Bioinformatic analysis revealed the aetokthonostatin biosynthetic gene cluster encoding a non-ribosomal peptide synthe-tase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed byin vitrobiochemical studies, establishing a mechanistic link between the gene cluster and its product.Significance StatementCyanotoxins have adverse effects on ecosystems. Our understanding of their potential risk has recently been expanded by the discovery of aetokthonotoxin, produced by the cyanobacteriumAetokthonos hydrillicolagrowing on invasive plants. Via trophic transfer, it acts as a neurotoxin causing mortality in animals including top predators like Bald Eagles. Closer examination ofA. hydrillicolarevealed that it also produces highly toxic dolastatin derivatives.A. hydrillicolais the first cultured cyanobacterium producing dolastatin derivatives, allowing us to uncover biosynthetic gene clusters of this compound family. In contrast to all other known dolastatin-producers, which are marine cyanobacteria,A. hydrillicolathrives in freshwater reservoirs, making it a potential threat also for human health. Monitoring of the cyanobacterium and its toxins is strongly recommended.