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Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores.

Zutz C, Bacher M, Parich A, Kluger B, Gacek-Matthews A, Schuhmacher R, Wagner M, Rychli K, Strauss J - Front Microbiol (2016)

Bottom Line: One approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Milk Hygiene, University of Veterinary Medicine ViennaVienna, Austria; Research Platform Bioactive Microbial Metabolites, Bioresources and Technologies Campus in TullnTulln an der Donau, Austria.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Antimicrobial effect of the supernatant of valproic acid treated D. microsporus on S. aureus. Effect of the supernatant of untreated (-VPA) and VPA treated (+VPA) D. microsporus on growth [OD600, (A)] and log10 CFU reduction (B) and percentage of dead cells (C) of S. aureus measured after 0, 6, and 18 h of incubation at 37°C. Control comprises of S. aureus cells grown without fungal extract. Data are presented as mean values ± standard deviations of three biological replicates performed in triplicate. Asterisks indicate statistically significant difference compared to the control (p < 0.05) according to student’s t-test.
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Figure 1: Antimicrobial effect of the supernatant of valproic acid treated D. microsporus on S. aureus. Effect of the supernatant of untreated (-VPA) and VPA treated (+VPA) D. microsporus on growth [OD600, (A)] and log10 CFU reduction (B) and percentage of dead cells (C) of S. aureus measured after 0, 6, and 18 h of incubation at 37°C. Control comprises of S. aureus cells grown without fungal extract. Data are presented as mean values ± standard deviations of three biological replicates performed in triplicate. Asterisks indicate statistically significant difference compared to the control (p < 0.05) according to student’s t-test.

Mentions: The supernatants of the VPA treated D. microsporus cultures showed a strong antimicrobial activity against S. aureus (Figure 1A) and the MRSA strains B337919 and B335466 whereas the untreated fungal extracts displayed no antimicrobial activity. The active fungal extracts showed a log10 growth reduction of 3.7–4 log units for S. aureus after 6 h of incubation (Figure 1B). Furthermore a log reduction of 3.3–3.6 log units against both MRSA strains was observed. Weak activity was observed against E. coli, ESBL E. coli and K. pneumoniae (Table 2). The cell viability assay (LIVE/DEAD) revealed that the VPA treated fungal extracts led to 30% of dead cells after 6 h of incubation which indicated a bacteriostatic activity of the fungal culture (Figure 1C). To further characterize the antibacterial compound(s) in the extracts we determined proteinase K stability. The antimicrobial activity after incubation with 1 × 105 μg/l for 24 h and with 1 × 106 μg/l proteinase K at 37°C for 1 week (data not shown) (Supplementary Figure S1) was unchanged. This indicated that the active compound(s) were not proteins or peptides susceptible to proteinase K digestion. Heat stability of the fungal extracts was assessed after incubation at 96°C for 1 h. No significant reduction of bioactivity was observed (data not shown).


Valproic Acid Induces Antimicrobial Compound Production in Doratomyces microspores.

Zutz C, Bacher M, Parich A, Kluger B, Gacek-Matthews A, Schuhmacher R, Wagner M, Rychli K, Strauss J - Front Microbiol (2016)

Antimicrobial effect of the supernatant of valproic acid treated D. microsporus on S. aureus. Effect of the supernatant of untreated (-VPA) and VPA treated (+VPA) D. microsporus on growth [OD600, (A)] and log10 CFU reduction (B) and percentage of dead cells (C) of S. aureus measured after 0, 6, and 18 h of incubation at 37°C. Control comprises of S. aureus cells grown without fungal extract. Data are presented as mean values ± standard deviations of three biological replicates performed in triplicate. Asterisks indicate statistically significant difference compared to the control (p < 0.05) according to student’s t-test.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829596&req=5

Figure 1: Antimicrobial effect of the supernatant of valproic acid treated D. microsporus on S. aureus. Effect of the supernatant of untreated (-VPA) and VPA treated (+VPA) D. microsporus on growth [OD600, (A)] and log10 CFU reduction (B) and percentage of dead cells (C) of S. aureus measured after 0, 6, and 18 h of incubation at 37°C. Control comprises of S. aureus cells grown without fungal extract. Data are presented as mean values ± standard deviations of three biological replicates performed in triplicate. Asterisks indicate statistically significant difference compared to the control (p < 0.05) according to student’s t-test.
Mentions: The supernatants of the VPA treated D. microsporus cultures showed a strong antimicrobial activity against S. aureus (Figure 1A) and the MRSA strains B337919 and B335466 whereas the untreated fungal extracts displayed no antimicrobial activity. The active fungal extracts showed a log10 growth reduction of 3.7–4 log units for S. aureus after 6 h of incubation (Figure 1B). Furthermore a log reduction of 3.3–3.6 log units against both MRSA strains was observed. Weak activity was observed against E. coli, ESBL E. coli and K. pneumoniae (Table 2). The cell viability assay (LIVE/DEAD) revealed that the VPA treated fungal extracts led to 30% of dead cells after 6 h of incubation which indicated a bacteriostatic activity of the fungal culture (Figure 1C). To further characterize the antibacterial compound(s) in the extracts we determined proteinase K stability. The antimicrobial activity after incubation with 1 × 105 μg/l for 24 h and with 1 × 106 μg/l proteinase K at 37°C for 1 week (data not shown) (Supplementary Figure S1) was unchanged. This indicated that the active compound(s) were not proteins or peptides susceptible to proteinase K digestion. Heat stability of the fungal extracts was assessed after incubation at 96°C for 1 h. No significant reduction of bioactivity was observed (data not shown).

Bottom Line: One approach is the use of small molecule effectors, potentially influencing chromatin landscape in fungi.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Milk Hygiene, University of Veterinary Medicine ViennaVienna, Austria; Research Platform Bioactive Microbial Metabolites, Bioresources and Technologies Campus in TullnTulln an der Donau, Austria.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus