Exploring Secondary Metabolite Profiles of Stachybotrys spp. by LC-MS/MS

Abstract

The genus Stachybotrys produces a broad diversity of secondary metabolites, including macrocyclic trichothecenes, atranones, and phenylspirodrimanes. Although the class of the phenylspirodrimanes is the major one and consists of a multitude of metabolites bearing various structural modifications, few investigations have been carried out. Thus, the presented study deals with the quantitative determination of several secondary metabolites produced by distinct Stachybotrys species for comparison of their metabolite profiles. For that purpose, 15 of the primarily produced secondary metabolites were isolated from fungal cultures and structurally characterized in order to be used as analytical standards for the development of an LC-MS/MS multimethod. The developed method was applied to the analysis of micro-scale extracts from 5 different Stachybotrys strains, which were cultured on different media. In that process, spontaneous dialdehyde/lactone isomerization was observed for some of the isolated secondary metabolites, and novel stachybotrychromenes were quantitatively investigated for the first time. The metabolite profiles of Stachybotrys species are considerably influenced by time of growth and substrate availability, as well as the individual biosynthetic potential of the respective species. Regarding the reported adverse effects associated with Stachybotrys growth in building environments, combinatory effects of the investigated secondary metabolites should be addressed and the role of the phenylspirodrimanes re-evaluated in future research.

Keywords: LC-MS/MS; Stachybotrys spp.; biosynthetic production; metabolite profiles; phenylspirodrimanes; satratoxins; stachybotrychromenes.

Figure 1

Chemical structures of isolated secondary metabolites from Stachybotrys spp. (the used color for each structure is reflected in the chromatograms and all diagrams shown in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10).

Figure 2

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) chromatograms of isolated secondary metabolites in the positive (a) and negative ionization mode (b) of a calibration standard with respective selected reaction monitoring (SRM) transitions (details of chromatographic methods are described in Section 5.3); isomerization of dial-containing metabolites to the corresponding lactone forms (c) (enlarged view).

Figure 3

Extracted ion chromatograms with respective SRM) transitions of a micro-scale extract from S. chartarum CT S ATCC 34916 after 7 days of growth at 25 °C on potato dextrose agar (PDA) in the dark (1:100 dilution), negative (a) and positive ionization mode (b) (details of chromatographic methods are described in Section 5.3). The figure is split in several panes, due to the differences in the intensities of the observed metabolites.

Figure 4

Relative secondary metabolite profiles of S. chlorohalonata CBS 109238 on potato dextrose agar (PDA) after 3 days (a), 5 days (b), 7 days (c), and 21 days (d) of cultivation at 25 °C in the dark. * ∑ of metabolites < LOD/LOQ (see Figure 1 for chemical structures and abbreviations).

Figure 5

Relative secondary metabolite profiles of S. chartarum CT S IBT 40293 on potato dextrose agar (PDA) after 3 days (a), 5 days (b), 7 days (c), and 21 days (d) of cultivation at 25 °C in the dark. * ∑ of metabolites < LOD/LOQ (see Figure 1 for chemical structures and abbreviations).

Figure 6

Absolute production of 10 phenylspirodrimanes (PSDs) (µg/cm²) by S. chlorohalonata over time of growth on PDA) (3–21 days), n = 3 (see Figure 1 for chemical structures and abbreviations).

Figure 7

Absolute concentrations of 10 PSDs (µg/cm²) by S. chartarum CT S over time of growth on PDA (3–21 days), n = 3 (see Figure 1 for chemical structures and abbreviations).

Figure 8

Relative secondary metabolite profiles of S. chartarum CT S ATCC 34916 on malt extract agar (MEA) after 7 days (a), 14 days (b) and after 21 days (c) of cultivation at 25 °C in the dark. * ∑ of metabolites < LOD/LOQ (see Figure 1 for chemical structures and abbreviations).

Figure 9

Relative secondary metabolite profiles of S. chartarum CT A IBT 40288 (a) and S. chartarum CT A DSM 63425 (b) on Czapek Yeast autolysate agar (CYA) after 21 days of cultivation at 25 °C in the dark (see Figure 1 for chemical structures and abbreviations).

Figure 10

Relative secondary metabolites of 5 different Stachybotrys strains after 14 days of cultivation on synthetic-nutrient-poor agar (SNA) at 25 °C in the dark. S. chlorohalonata CBS 109283 (a), S. chartarum CT A DSM 63425 (b), S. chartarum CT A IBT 40288 (c), S. chartarum CT S IBT 40293 (d) and S. chartarum CT S ATCC 34916 (e). * ∑ of metabolites < LOD/LOQ (see Figure 1 for chemical structures and abbreviations).