Butyrate influences intracellular levels of adenine and adeninederivatives in the fungus Penicillium restrictum

Christoph Zutza, Yi Ming Chiangc, Bettina Faehnriche, Markus Bacherf,Roland Hellinger, Bernhard Kluger, Martin Wagner, Joseph Strauss, Kathrin Rychli

11 January 2017

Butyrate, a small fatty acid, has an important role in the colon of ruminants and mammalians includingthe inhibition of inflammation and the regulation of cell proliferation. There is also growing evidence thatbutyrate is influencing the histone structure in mammalian cells by inhibition of histone deacetylation.Butyrate shows furthermore an antimicrobial activity against fungi, yeast and bacteria, which is linkedto its toxicity at a high concentration. In fungi there are indications that butyrate induces the produc-tion of secondary metabolites potentially via inhibition of histone deacetylases. However, informationabout the influence of butyrate on growth, primary metabolite production and metabolism, besides lipidcatabolism, in fungi is scarce. We have identified the filamentous fungus Penicillium (P.) restrictum as asusceptible target for butyrate treatment in an antimicrobial activity screen. The antimicrobial activitywas detected only in the mycelium of the butyrate treated culture. We investigated the effect of butyrateranging from low (0.001 mM) to high (30 mM), potentially toxic, concentrations on biomass and antimi-crobial activity. Butyrate at high concentrations (3 and 30 mM) significantly reduced the fungal biomass.In contrast P. restrictum treated with 0.03 mM of butyrate showed the highest antimicrobial activity. Weisolated three antimicrobial active compounds, active against Staphylococcus aureus, from P. restrictumcellular extracts treated with butyrate: adenine, its derivate hypoxanthine and the nucleoside derivateadenosine. Production of all three compounds was increased at low butyrate concentrations. Further-more we found that butyrate influences the intracellular level of the adenine nucleoside derivate cAMP,an important signalling molecule in fungi and various organisms.In conclusion butyrate treatment increases the intracellular levels of adenine and its respective deriva-tives.

Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores

Christoph Zutz, Markus Bacher, Alexandra Parich, Bernhard Kluger, Agnieszka Gacek-Matthews, Rainer Schuhmacher, Martin Wagner, Kathrin Rychli and Joseph Strauss

13 April 2016

One of the biggest challenges in public health is the rising number of antibiotic resistant pathogens and the lack of novel antibiotics. In recent years there is a rising focus on fungi as sources of antimicrobial compounds due to their ability to
produce a large variety of bioactive compounds and the observation that virtually every fungus may still contain yet unknown so called “cryptic,” often silenced, compounds. These putative metabolites could include novel bioactive compounds. Considerable effort is spent on methods to induce production of these “cryptic” metabolites. One
approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi. We observed that the supernatant of the fungus Doratomyces (D.) microsporus treated with valproic acid (VPA) displayed antimicrobial activity against Staphylococcus (S.) aureus and two methicillin resistant clinical S. aureus isolates. VPA treatment resulted in enhanced production of seven antimicrobial compounds: cyclo- (L-proline-L-methionine) (cPM), p-hydroxybenzaldehyde, cyclo-(phenylalanine-proline) (cFP), indole-3-carboxylic acid, phenylacetic acid (PAA) and indole-3-acetic acid. The
production of the antimicrobial compound phenyllactic acid was exclusively detectable after VPA treatment. Furthermore three compounds, cPM, cFP, and PAA, were able to boost the antimicrobial activity of other antimicrobial compounds. cPM, for the first time isolated from fungi, and to a lesser extent PAA, are even able to decrease the minimal inhibitory concentration of ampicillin in MRSA strains. In conclusion we could show in this study that VPA treatment is a potent tool for induction of “cryptic” antimicrobial compound production in fungi, and that the induced compounds are not exclusively linked to the secondary metabolism. Furthermore this is the first discovery of the rare diketopiperazine cPM in fungi.  Additionally we could demonstrate that cPM and PAA boost antibiotic activity against antibiotic resistant strains, suggesting a possible application in combinatorial antibiotic treatment against resistant pathogens.

Fungi Treated with Small Chemicals Exhibit Increased Antimicrobial Activity against Facultative Bacterial and Yeast Pathogens

Christoph Zutz, Dragana Bandian, Bernhard Neumayer, Franz Speringer, Markus Gorfer, Martin Wagner, Joseph Strauss and Kathrin Rychli

9 July 2014

For decades, fungi have been the main source for the discovery of novel antimicrobial drugs. Recent sequencing efforts revealed a still high number of so far unknown “cryptic” secondary metabolites. The production of these metabolites is presumably epigenetically silenced under standard laboratory conditions. In this study, we investigated the effect of six small mass chemicals, of which some are known to act as epigenetic modulators, on the production of antimicrobial compounds in 54 spore forming fungi. The antimicrobial effect of fungal samples was tested against clinically facultative pathogens and multiresistant clinical isolates. In total, 30 samples of treated fungi belonging to six different genera reduced significantly growth of different test organisms compared to the untreated fungal sample (growth log reduction 0.3–4.3). For instance, the pellet of Penicillium restrictum grown in the presence of butyrate revealed significant higher antimicrobial activity against Staphylococcus (S.) aureus andmultiresistant S. aureus strains and displayed no cytotoxicity against human cells, thusmaking it an ideal candidate for antimicrobial compound discovery. Our study shows that every presumable fungus, even well described fungi, has the potential to produce novel antimicrobial compounds and that our approach is capable of rapidly filling the pipeline for yet undiscovered antimicrobial substances.

A novel stable isotope labelling assisted workflow for improved untargeted LC–HRMS based metabolomics research

Christoph Bueschl, Bernhard Kluger, Marc Lemmens, Gerhard Adam, Gerlinde Wiesenberger, Valentina Maschietto, Adriano Marocco, Joseph Strauss, Stephan Bödi • Gerhard G. Thallinger, Rudolf Krska, Rainer Schuhmacher

4 December 2013

Many untargeted LC–ESI–HRMS based metabolomics studies are still hampered by the large proportion of non-biological sample derived signals included in the generated raw data. Here, a novel, powerful stable isotope labelling (SIL)-based metabolomics workflow is presented, which facilitates global metabolome extraction, improved metabolite annotation and metabolome wide internal standardisation (IS). The general concept is exemplified with two different cultivation variants, (1) co-cultivation of the plant pathogenic fungi Fusarium graminearum on nonlabelled and highly 13C enriched culture medium and (2) experimental cultivation under native conditions and use of globally U-13C labelled biological reference samples as exemplified with maize and wheat. Subsequent to LC– HRMS analysis of mixtures of labelled and non-labelled samples, two-dimensional data filtering of SIL specific isotopic patterns is performed to better extract truly biological derived signals together with the corresponding number of carbon atoms of each metabolite ion. Finally, feature pairs are convoluted to feature groups each representing a single metabolite. Moreover, the correction of unequal matrix effects in different sample types and the improvement of relative metabolite quantification with metabolome wide IS are demonstrated for the F. graminearum experiment. Data processing employing the presented workflow revealed about 300 SIL derived feature pairs corresponding to 87–135 metabolites in F. graminearum samples and around 800 feature pairs corresponding to roughly 350 metabolites in wheat samples. SIL assisted IS, by the use of globally U-13C labelled biological samples, reduced the median CV value from 7.1 to 3.6 % for technical replicates and from 15.1 to 10.8 % for biological replicates in the respective F. graminearum samples.

Small Chemical Chromatin Effectors Alter Secondary Metabolite Production in Aspergillus clavatus

Christoph Zutz, Agnieszka Gacek, Michael Sulyok, Martin Wagner, Joseph Strauss and Kathrin Rychli

7 October 2013

The filamentous fungus Aspergillus clavatus is known to produce a variety of secondary metabolites (SM) such as patulin, pseurotin A, and cytochalasin E. In fungi, the production of most SM is strongly influenced by environmental factors and nutrients. Furthermore, it has been shown that the regulation of SM gene clusters is largely based on modulation of a chromatin structure. Communication between fungi and bacteria also triggers chromatin-based induction of silent SM gene clusters. Consequently, chemical chromatin effectors known to inhibit histone deacetylases (HDACs) and DNA-methyltransferases (DNMTs) influence the SM profile of several fungi. In this study, we tested the effect of five different chemicals, which are known to affect chromatin structure, on SM production in A. clavatus using two growth media with a different organic nitrogen source. We found that production of patulin was completely inhibited and cytochalasin E levels strongly reduced, whereas growing A. clavatus in media containing soya-derived peptone led to substantially higher pseurotin A levels. The HDAC inhibitors valproic acid, trichostatin A and butyrate, as well as the DNMT inhibitor 5-azacytidine (AZA) and N-acetyl-D-glucosamine, which was used as a proxy for bacterial fungal co-cultivation, had profound influence on SM accumulation and transcription of the corresponding biosynthetic genes. However, the repressing effect of the soya-based nitrogen source on patulin production could not be bypassed by any of the small chemical
chromatin effectors. Interestingly, AZA influenced some SM cluster genes and SM production although no Aspergillus species has yet been shown to carry detectable DNA methylation.