More than just an eagle killer: The freshwater cyanobacterium Aetokthonos hydrillicola produces highly toxic dolastatin derivatives

Abstract

Cyanobacteria are infamous producers of toxins. While the toxic potential of planktonic cyanobacterial blooms is well documented, the ecosystem level effects of toxigenic benthic and epiphytic cyanobacteria are an understudied threat. The freshwater epiphytic cyanobacterium Aetokthonos hydrillicola has recently been shown to produce the "eagle killer" neurotoxin aetokthonotoxin (AETX) 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 AETX, prompting further investigation. Here, we describe the isolation and structure elucidation of the aetokthonostatins (AESTs), 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 AEST potently impacts microtubule dynamics and can bind to tubulin in a similar matter as dolastatin 10. We also show that AEST inhibits reproduction of the nematode Caenorhabditis elegans. Bioinformatic analysis revealed the AEST biosynthetic gene cluster encoding a nonribosomal peptide synthetase/polyketide synthase accompanied by a unique tailoring machinery. The biosynthetic activity of a specific N-terminal methyltransferase was confirmed by in vitro biochemical studies, establishing a mechanistic link between the gene cluster and its product.

Keywords: aetokthonostatin; biosynthesis; cyanotoxin; cytotoxicity; dolastatin.

Fig. 1.

A. hydrillicola produces dolastatin analogs. (A) Structures of AETX, AEST, MMAEST, DAAEST, as well as MMAE, MMAF and dolastatin 10. (B) AEST cluster in a GNPS feature-based MS/MS networking analysis of an HPLC-MS/MS analysis of an A. hydrillicola extract. Node size proportional to ion intensity, exact mass of [M+H]⁺ indicated next to the respective node. Red: AEST, orange: pentapeptide AEST derivatives like MMAEST, cyan: tetrapeptide AEST derivatives like DAAEST.

Fig. 2.

AEST is a cytotoxic tubulin binder. (A) Cytotoxicity of AEST (EC₅₀ 1 ± 0.2 nM), MMAEST (EC₅₀ 4 ± 0.2 nM), DAAEST (EC₅₀ 25 ± 1.4 nM), MMAE (EC₅₀ 3 ± 0.4 nM), and MMAF (EC₅₀ 18 ± 0.7 nM) on HeLa cells (n = 3). Data are represented as average ± SEM. (B) Decrease of the reproduction rate of C. elegans treated with AEST (EC₅₀ 0.8 ± 0.2 µM, n = 3). Data are represented as average ± SEM. (C) Predicted binding of AEST (Aph as 2S,3R) to tubulin; tubulin α-subunit of one heterodimer visualized as yellow cartoon, the β-subunit of its neighboring heterodimer in cyan. Interacting binding site residues represented as sticks colored the same way. Magenta: hydrogen bonds, orange: salt bridges, red: cation–π interactions. Cofactor GDP shown as white, transparent sticks. (D) Immunofluorescence microscopy of HeLa cells showing the effect of AEST and MMAF on the tubulin network (green); nuclei stained with DAPI (blue). Arrows indicate tubulin network depolymerization after AEST and MMAF treatment (disruption of thread-like organization of microtubules). Taxol treatment, used as technical control to show detectability of tubulin interactions, results in tubulin stabilization and bundle formation typical for taxol.

Fig. 3.

AESTs are synthesized via an NRPS/PKS pathway. (A) Organization of the AEST BGC compared to homologous putative BGCs of members of the dolastatin compound family found in publicly available metagenome-assembled genomes of environmental marine Symploca sp. The aes orthologs identified in Symploca sp. are labeled symA–K as they presumably do not code for AESTs but rather symplostatins. The order of symA–K genes is collinear with the proposed biosynthesis of the symplostatin backbone scaffold. (B) Scheme of the predicted biosynthetic assembly of AEST. The chronology of the tailoring methylation reactions catalyzed by AesE, H, and I (marked with asterisks) is unknown; the reactions are depicted at their first theoretically suitable substrate. The specific target moieties of AesH/I (the Dil/Dap/Aph OH groups) remain to be assigned experimentally. The proposed decarboxylation/reduction of the C-terminal AEST residue (gray box) was not fully explained by bioinformatic analysis. The N-terminal methylation (red arrow) of MMAEST was reconstituted by in vitro enzymatic activity of AesK. (C) HPLC-HRMS/MS analysis (extracted ion chromatograms) of AEST (red) in reaction mixtures of MMAEST (blue) incubated with heat-inactivated or fresh Strep-AesK (Top Left/Right) show that the inactivated enzyme had no effect on the substrate while the fresh Strep-AesK methylated MMAEST. MMAEST and AEST differ only in the degree of methylation of the N-terminal Ile (Bottom Left). Comparison of the MS/MS spectra of MMAEST and the product of the fresh enzyme reaction (Bottom Right) shows that AesK methylated the N terminus of MMAEST, producing AEST. Abbreviations: AEST, aetokthonostatin; Aph, aetophenine; BGC, biosynthetic gene cluster; Dap, Dolaproine; Dil, Dolaisoleuine; mal, malonyl-CoA; MMAEST, monomethylaetokthonostatin; N,N-di-Melle, N,N-dimethylisoleucine; OH-mal, hydroxymalonyl-CoA; SAM, S-adenosylmethionine; SDR, short-chain dehydrogenase/reductase.