Journal: Environment international

Article Title: Identification of the bacterial metabolite aerugine as potential trigger of human dopaminergic neurodegeneration

doi: 10.1016/j.envint.2023.108229

Figure Lengend Snippet: Purification of neurotoxic small molecules from Streptomyces bacterial strains. A Workflow of metabolite identification from bacterial extracts. Subfigures that provide activity data on the respective purification stages are indicated. Information on the purification procedures is given in the subfigure paragraphs below. B-E The neurotoxicity of extracts, fractions and pure compounds was tested on human dopaminergic neurons. After 6 days of differentiation (d6), cultures of LUHMES neurons were exposed to bacterial fractions. The cell viability was assessed by calcein-AM & H-33342 staining, automated fluorescence microscopy and quantification by an image processing algorithm. B Cultures of Streptomyces venezuelae and S. lividans were extracted with ethyl acetate (EA) or dichloromethane (DCM). The crude extracts were fractionated via Sep-Pak C18 and eluted using a stepwise gradient with methanol and water (MeOH = 60% or 100%). Dried and DMSO-reconstituted fractions were tested for effects on neuronal viability. Data from at least two fully independent runs. Viability data was obtained after 24 h of exposure. C The EA/60% MeOH extract of S. venezuelae was further fractionated by reverse phase HPLC (C18 column, MeOH gradient). The collected fractions are indicated in the absorption spectrum (a part of the spectrum is shown, the full spectrum is given in ; the MeOH gradient started at 50%). Fractions were dried, taken up in DMSO and added to neurons for 48 h (final dilution 1:1000). Viability data are means ± SEM (n = 3). D Fraction 14 of the previous step was further purified by several reverse phase HPLC (C-18) runs in acetonitrile/water eluent. Absorption peaks were collected in fractions as depicted (red numbers in the spectrum). They were tested for their effect on neuronal viability (24 h, at 1:1000 and 1:10000 dilutions). E Two single substances (aerugine and aeruginol) of fraction 4 of the previous purification step were separated and purified to homogeneity. The chemical structures (elucidation details in and spectral data 1) are displayed next to their respective peaks in the elution trail diagram. Note the additional double bond in the thiazolidine ring of aeruginol. The two compounds were assessed in two separate experiments for their effect on neuronal viability after incubation times of 24 h and 48 h. All statistical analyses were performed using a one-way ANOVA with Dunnett’s multiple comparisons test (** = p < 0.005, *** = p < 0.0001).

Article Snippet: Automated flash chromatography with 1:1 hexane:EA yielded a semi-pure product, which was further purified with semipreparative HPLC with a normal phase Si-column with Si Reprosil 100 (250 × 10 mm, 5 μm, Dr. Maisch).

Techniques: Purification, Activity Assay, Staining, Fluorescence, Microscopy, Incubation