Large-Scale Metabolomics Reveals a Complex Response of Aspergillus nidulans to Epigenetic Perturbation

Abstract

The microbial world offers a rich source of bioactive compounds for those able to sift through it. Technologies capable of quantitatively detecting natural products while simultaneously identifying known compounds would expedite the search for new pharmaceutical leads. Prior efforts have targeted histone deacetylases in fungi to globally activate the production of new secondary metabolites, yet no study has directly assessed its effects with minimal bias at the metabolomic level. Using untargeted metabolomics, we monitored changes in >1000 small molecules secreted from the model fungus, Aspergillus nidulans, following genetic or chemical reductions in histone deacetylase activity (HDACi). Through quantitative, differential analyses, we found nearly equal numbers of compounds were up- and down-regulated by >100 fold. We detected products from both known and unknown biosynthetic pathways and discovered that A. nidulans is capable of producing fellutamides, proteasome inhibitors whose expression was induced by ~100 fold or greater upon HDACi. This work adds momentum to an ‘omics’-driven resurgence in natural products research, where direct detection replaces bioactivity as the primary screen for new pharmacophores.

Multicomponent Analysis of the Differential Induction of Secondary Metabolite Profiles in Fungal Endophytes

“…We decided to use mass spectrometry analyses, as metabolomics is currently getting stronger foundation on LC/MS data. In recent studies, Volcano-plots have been applied to these data with success in describing and obtaining conclusions on the biosynthesis of fellutamides by A. nidulans, hence supporting the use of HDAC inhibitors and said techniques for the discovery of cryptic secondary metabolites [12,13].…”

“…Among the metabolites with increased production, we could identify the presence of curvicollide A/B (m/z 432; C 26 In general, some of these most outlier ion masses were induced independently of the production method applied, being four of them the most statistically significative: m/z 171, 185-186, 225-226-227 and 243-244-245. These four sets of ions were studied then by UHPLC/HRMS-MS and their molecular formulae identified as C 9 H 6 N 4 (9), C 10 H 8 N 4 (10), C 13 H 14 N 4 (11) and C 14 H 16 N 2 O 2 (12). Interestingly the first three outlier molecules presented a molecular formula closely related to that of hydralazine (C 8 H 8 N 4 , 2), and could not be found also in public nor commercial natural products databases.…”

“…HRMS/NMR results indicated that m/z 171, m/z 185-186, and m/z 227 ions corresponded to biotransformation products (9-11) of hydralazine (2) ocurring in the fermentation broth. On the other hand, the molecular formula C 14 H 16 N 2 O 2 , deduced from the ions m/z 243-244-245, that could be associated to 19 possible molecules in the natural products databases, was finally purified and identified as the diketopiperazine natural product cyclo(phenylalanyl-prolyl) (12) by HRMS and NMR data ( Figure 6 and Supplementary Materials).…”

“…Figure 6. Most differential metabolites detected by the Volcano-plots methodology described, from the biotransformation (9-11), or the elicitor induction (12), of the epigenetic modifier hydralazine (2) on the fermentation broth of the endophyte fungus CF-285353.…”

“…Recently Albright et al [12] proved global changes in Aspergillus nidulans metabolome upon treatment with epigenetic modifiers in response to an upregulated reduced expression of rpdA by using volcano-plots statistical analyses [13]. This study suggested that, although HDAC inhibition generally leads to an up-regulation of the Aspergillus nidulans biosynthetic machinery as evidenced by transcriptomics, the response at the level of the secondary metabolome is more complex than just a global increase in the abundance of the secondary metabolites already produced by the strain.…”