Mostafa E.
Rateb
ab,
Irene
Hallyburton
c,
Wael E.
Houssen
ad,
Alan T.
Bull
e,
Michael
Goodfellow
f,
Rakesh
Santhanam
f,
Marcel
Jaspars
a and
Rainer
Ebel
*a
aMarine Biodiscovery Centre, Department of Chemistry, University of Aberdeen, Meston Walk, AB24 3UE, Scotland, UK. E-mail: r.ebel@abdn.ac.uk; Fax: +44 1224 272921; Tel: +44 1224 272930
bPharmacognosy Department, Faculty of Pharmacy, Beni-Suef University, Salah Salem St., 62111, Beni-Suef, Egypt
cDrug Discovery Unit, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee, Sir James Black Centre, Dundee, DD1 5EH, UK
dInstitute of Medical Sciences, University of Aberdeen, Ashgrove Road West, Aberdeen AB25 2ZD, Scotland, UK
eSchool of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK
fSchool of Biology, University of Newcastle, Newcastle upon Tyne NE1 7RU, UK
First published on 10th June 2013
An established culture of Aspergillus fumigatus MBC-F1-10 proved to be very receptive to external stimuli and reacted with the production of secondary metabolites which were undetectable when the fungus was grown under standard conditions. Firstly, co-cultivation with the type strain of Streptomyces bullii, an isolate from hyper-arid Atacama desert soil, led to the isolation of ergosterol 1, seven metabolites belonging to the diketopiperazine alkaloids; brevianamide F 2, spirotryprostatin A 3, 6-methoxy spirotryprostatin B 4, fumitremorgin C and its 12,13-dihydroxy derivative (5–6), fumitremorgin B 7, and verruculogen 8, in addition to 11-O-methylpseurotin A 9 and its new isomer 11-O-methylpseurotin A210. In an independent experiment, addition of N-butyryl-DL-homoserine lactone to the culture medium led to the production of emestrins A and B (11–12). Neither microbe produced these compounds when cultured alone. The structures of all compounds were elucidated using NMR spectroscopic techniques and mass spectrometric analysis. The isolated compounds were tested for their potential antibacterial and antiprotozoal activities.
Most previous co-culture experimental studies reported an increase in biological activity without the identification of the secondary metabolites responsible for this effect, confirmed the induction of antibiotic biosynthesis but not the induction of specific metabolic pathways or increased yields of previously described metabolites.7,8 However, the induction of a previously unexpressed biosynthetic pathway leading to the production of new secondary metabolites in response to co-culture is less often described in the literature. Fenical and co-workers reported that the competing co-culture of the marine-derived fungus Pestalotia sp. with a marine α-proteobacterium (strain CNJ-328) led to the production of the new antibiotic, pestalone9 and subsequently, culturing the same bacterium with the marine-derived fungus Libertella sp. resulted in the discovery of four potent cytotoxic pimarane diterpenoids, libertellenones A–D.4 They also isolated two new antibacterial cyclic depsipeptides, emericellamides A–B through the co-culture of the marine-derived fungus Emericella sp. and the obligate marine actinomycete Salinispora arenicola.10 More recently, a new antibiotic-antitumor metabolite, glionitrin A was isolated during the co-culture of a mine drainage-derived Aspergillus fumigatus and a mine drainage-derived Sphingomonas isolate strain KMK-001,11 while co-culturing A. fumigatus with Streptomyces peucetius led to the production of the new formyl xanthocillin analogue fumiformamide.12 Interaction of the two fungi Penicillium pinophilum and Trichoderma harzianum led to the discovery of a new prenylated polyketide, secopenicillide C,13 while co-cultivation of Pseudomonas aeruginosa and Candida albicans resulted in induction of a bacterial gene cluster required for phenazine biosynthesis.14 This listing does not aim at complete coverage, and the reader is referred to excellent recent review articles covering bacterial-fungal interactions15 as well as the control of secondary metabolite biosynthetic pathways in the genus Aspergillus.16
In this communication, we report on another example of the induction of a presumed fungal biosynthetic pathway in response to microbial co-culture. The addition of the type strain Streptomyces bullii,17 an isolate from hyper-arid Atacama Desert soil, to an established culture of A. fumigatus MBC-F1-10 has led to the isolation of ergosterol (1), seven metabolites belonging to the diketopiperazine (DKP) class of alkaloids; brevianamide F (2), spirotryprostatin A (3), 6-methoxy spirotryprostatin B (4), fumitremorgin C and its 12,13-dihydroxy derivative (5–6), fumitremorgin B (7), and verruculogen (8), in addition to two metabolites from the rare class of pseurotins, 11-O-methylpseurotin A (9) and its new isomer 11-O-methylpseurotin A2 (10). None of these compounds were observed in pure independent cultures of the fungus or the bacterium and all of them are considered to be of fungal origin due to their chemical nature.
Moreover, the addition of the known quorum sensing metabolite, N-butyryl-DL-homoserine lactone (HSL), to the culture media of the fungus led to the isolation of emestrins A–B (11–12) which were not traced in the pure fungal culture. The isolated compounds were tested for their potential antibacterial and antiprotozoal activities. Diseases caused by parasitic protozoa affect many people around the world, notably, Chagas' disease (American trypanosomiasis), sleeping sickness (human African trypanosomiasis), and leishmaniasis. The treatment for these diseases mainly depends on chemotherapy.18 Although several antiparasitic drugs are available, most have toxic side effects, and furthermore development of drug resistance is commonly observed. Therefore, searching for new and effective drugs against neglected tropical diseases (NTD) is needed. Since DKPs with diverse skeletons were reported to have efficient trypanocidal activity, especially disulfide-containing DKPs like gliotoxin which showed nanomolar activity against the whole cell parasite,19 DKPs isolated from Aspergillus fumigatus MBC-F1-10 were tested for their potential antiparasitic activity against Trypanosoma brucei brucei and Leishmania donovani. They were also tested against normal human MRC5 cells (diploid foetal lung fibroblasts) as a counter-screen for indications of toxicity and non-selective inhibition.
Quite unexpectedly, chemical analysis of the fungus A. fumigatus MBC-F1-10 cultivated on malt extract medium (Fig. 1a) resulted in a sparse metabolite profile, with hardly any dominating peaks visible in the LC-MS chromatogram. As a control, the S. bullii strain was also cultivated alone on ISP2 medium (Fig. 1b), resulting in a similar finding. However, deliberately co-cultivating both microorganisms together, i.e. addition of a fresh culture of S. bullii strain C2 to an established three day old culture of A. fumigatus MBC-F1-10, resulted in a very complex LC-MS profile, which was confirmed in two subsequent analogous experiments (Fig. 1c, only one representative HPLC chart depicted). Seven metabolites belonging to the DKP alkaloid family were isolated, specifically brevianamide F24 (2), spirotryprostatin A25 (3), 6-methoxyspirotryprostatin B26 (4), fumitremorgin C27 (5) and its 12,13-dihydroxy derivative28 (6), fumitremorgin B29 (7), and verruculogen30 (8), in addition to two metabolites from the rare class of pseurotins, 11-O-methylpseurotin A31 (9) and its new isomer 11-O-methylpseurotin A2 (10). All of these compounds were consistently detected in all three experiments, and based on their structures and their known fungal origin it is reasonable to assume that they were produced by the fungus and not by the bacterium during the fungal bacterial co-culture.
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Fig. 1 HPLC profiles of different bacterial/fungal co-culture approaches. (a) bacteria alone; (b) fungus alone; (c) fungal-bacterial co-cultivation; (d) fungus with bacteria-free ISP2 medium; (e) fungus with bacterial MeOH extract; (f) fungus with dead bacterial cells; (g) fungus with HSL. |
In order to gain insight into the possible mechanism of induction of the biosynthesis of these fungal metabolites by the streptomycete, an additional series of experiments was performed. To an established fungal culture cultured by itself and obtained as described above, was added a treated culture of the streptomycete cultured on its own, as follows: A bacteria-free medium only consisting of ISP2 medium (to rule out induction of media components) (Fig. 1d); an extract of the bacterial biomass obtained with MeOH (Fig. 1e), and a full bacterial culture broth, but inactivated by autoclaving (Fig. 1f). In all three cases, induction of the fungal metabolites 2–10 was not observed. However, addition of N-butyryl-DL-homoserine lactone, a known bacterial quorum sensing compound (Fig. 1g), to the established fungal culture led to induction of the known cyclic DKPs emestrin A32 (11) and B33 (12). It is worth noting that neither 11 nor 12 could be detected in the co-cultivation experiment represented in Fig. 1c, while none of the fungal metabolites 2–10 were observed in this experiment.
Compounds 1–9, 11, and 12 were identified based on their accurate mass analysis and comparison of their MS and NMR spectral data with those reported in the literature. The HRESIMS data of 10 established its molecular formula as C23H27NO8, the same as that of 11-O-methylpseurotin A (9). Both compounds displayed virtually identical UV and IR spectra. The analysis of the 1H and 13C NMR data of 10 (Table 1) indicated the presence of a monosubstituted benzene ring, a disubstituted cis double bond (J = 10.9 Hz), one aliphatic and one allylic methyl group, two O-methyl groups, and three carbonyls including one α,β-unsaturated ketone and one amide. The 1H- and 13C NMR data of both compounds were reminiscent of each other with the exception of slight variations in chemical shifts at four positions (Table 1, ESI†). The analysis of COSY and HMBC spectra (Table 1, Fig. 2) revealed that the planar structure of compound 10 was identical to that of 9, thus indicating that they were stereoisomers. Analysis of the 1D NMR data (Table S1, ESI†) suggested that both compounds should differ with regard to the relative configuration of the pyrrolidinone ring, and a comparison of the 13C NMR data of this substructure with that reported for the structurally related compounds, pseurotin A34 and its diastereomer pseurotin A2,35 revealed that 10 possessed the same relative stereochemistry as pseurotin A2, based on almost identical 13C chemical shift values (Table S1, ESI†). Similar optical rotation values for 10 and pseurotin A2, the absolute configuration of which was established on the basis of X-ray crystallographic analysis and the use of circular dichroism (CD) spectroscopy,35 indicated that both compounds should also have the same absolute configuration.
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Fig. 2 Selected COSY (![]() ![]() |
Position | δ H, mult. (J in Hz)a | δ C, multa | δ H, multb (J in Hz) | δ C, multb | HMBCc |
---|---|---|---|---|---|
a In CDCl3. b In DMSO-d6. c From H to C. | |||||
2 | 184.4, C | 186.8, C | |||
3 | 113.3, C | 111.4, C | |||
4 | 198.1, C | 199.8, C | |||
5 | 88.5, C | 88.2, C | |||
6 | 167.9, C | 167.5, C | |||
8 | 95.4, C | 97.0, C | |||
9 | 4.78, s | 76.5, CH | 4.62 (d, 6.2) | 75.6, CH | 4, 6, 8 |
10 | 4.56 (d, 6.4) | 70.3, CH | 4.48 (t, 5.4) | 70.4, CH | 2, 11, 12 |
11 | 4.28 (dd, 6.5, 9.1) | 77.6, CH | 4.17 (dd, 5.5, 9.3) | 77.1, CH | 2, 10, 13 |
12 | 5.27 (t, 10.7) | 124.7, CH | 5.31 (t, 10.9) | 125.6, CH | 14 |
13 | 5.82, m | 140.3, CH | 5.62, m | 136.9, CH | 11 |
14 | 2.10, m | 21.4, CH2 | 2.04, m | 20.7, CH2 | 12, 13, 15 |
1.97, m | |||||
15 | 0.98 (t, 7.5) | 14.3, CH3 | 0.87 (t, 7.6) | 14.1, CH3 | 13, 14 |
16 | 1.77, s | 5.8, CH3 | 1.64, s | 5.6, CH3 | 2, 3, 4 |
17 | 193.3, C | 194.7, C | |||
18 | 134.0, C | 134.8, C | |||
19/23 | 8.21 (d, 7.9) | 129.9, CH | 8.11 (d, 7.7) | 129.6, CH | 17, 21 |
20/22 | 7.47 (t, 7.8) | 128.7, CH | 7.52 (t, 7.8) | 128.3, CH | 18 |
21 | 7.60 (t, 7.2) | 134.0, CH | 7.63 (t, 7.6) | 133.1, CH | |
8-OMe | 3.29, s | 51.6, CH3 | 3.12, s | 51.4, CH3 | 8 |
11-OMe | 3.25, s | 56.7, CH3 | 3.15, s | 55.7, CH3 | 11 |
9-OH | 6.09 (d, 6.2) | ||||
10-OH | 5.75 (d, 5.8) | ||||
NH-7 | 7.40, br s | 9.89, s | 5 |
Molecular mechanics simulations as well as a comparison of their NOESY spectra were carried out for both 9 (featuring a 9α-OH) and 10 (displaying a 9β-OH, Fig. 2). The proximity between H-9 and 8-OMe was confirmed by a strong NOE correlation from H-9 to 8-OMe in 10, indicating that the 9-OH was in the β-configuration in 10. This fact was also corroborated by the absence of an NOE between H-9 and 8-OMe in 9 when 9-OH was in the α-configuration. On this basis 10 was identified as the previously unreported 9-epimer of 11-O-methylpseurotin A for which the name 11-O-methylpseurotin A2 is suggested.
Since DKP production was induced through a microbial co-culture, which can be considered as a simulated competitive environment, antibacterial activity was expected for these compounds. However, none of the isolated compounds showed significant antibacterial activity when screened against Staphylococcus aureus and Escherichia coli (data not shown). This result does not exclude that these compounds were produced by the fungus to combat the existing bacteria in the culture environment, but could also be due to the compounds acting as quorum sensing inhibitors which do not inevitably kill or inhibit the growth of a microbial strain but instead modulate its phenotype, for instance via virulence attenuation.36,37 Meanwhile, assays for trypanocidal and leishmanicidal activities were performed on the isolated compounds (Table 2). All tested compounds proved active against these two parasites except brevianamide F (2) and 11-O-methylpseurotin A (9). Fumitremorgin B (7) and verruculogen (8) were the most potent growth inhibitors on both parasites exhibiting EC50 values of 0.2 μM on Trypanosoma brucei brucei and 3–4 μM on Leishmania donovani, followed by 12,13-dihydroxyfumitremorgin C (6) which exhibited an EC50 value of 7.4 μM on Trypanosoma brucei brucei. Although these compounds proved moderate to potent inhibitors of these parasites, they were not selective and showed pronounced toxicity on MRC5 cells (normal human lung fibroblasts) which suppressed further investigation and precluded their value as a lead scaffold. These results were in agreement with the literature where the fused pentacyclic DKPs showed pronounced trypanocidal activity when compared with other DKPs with a different skeleton.18 It could be concluded that the 12,13-diol moiety in DKP derivatives plays an important role in potentiating the antiparasitic effect of the fused pentacyclic DKPs. 6-methoxyspirostatin B (4) showed a moderate leishmanicidal activity with an EC50 value of 24.7 μM, but displayed selectivity in not being toxic toward MRC5 cells.
Aspergillus fumigatus strain MBC-F1-10 was isolated from the S. bullii culture, purified on malt extract agar (15.0 g malt extract, 15.0 g agar, distilled water 1000 mL adjusted to pH 7.2) and identified by sequence comparison of its amplified ITS region (GenBank accession number JN153038) with published sequences obtained from GenBank as described previously.38 The fungal strain was inoculated in 10 × 100 mL malt extract broth (15.0 g malt extract, distilled water 1000 mL adjusted to pH 7.2 in the presence of 50 g Diaion HP-20 resin) for 5 days in a rotary incubator at 28 °C with shaking at 150 rpm.
A set of experiments (a–g) were performed on these 2 cultures, each experiment was performed in triplicate on small scale cultures (1 L) and monitored by HPLC while experiments c and g were repeated on a larger scale basis with 3 L ISP2 medium. The time frame for experiment (c) was established during 11 independent experiments, in which the addition of the bacterial to the fungal culture was changed by 12 h each, ranging from 0 h to 144 h, and maximum induction of metabolites was observed after 72 h (data not shown). (a) Bacteria alone: 1 mL S. bullii culture was transferred aseptically to 100 mL fresh ISP2 broth and re-incubated using the same conditions for 5 days. (b) Fungus alone: 1 mL fungal culture was transferred aseptically to 100 mL fresh malt extract broth and re-incubated using the same conditions for 5 days. (c) Bacterial fungal interaction: 1 mL S. bullii culture was transferred aseptically to 100 mL three day-established fungal culture in ISP2 broth and re-incubated using the same conditions for 5 days. (d) Fungus with bacteria-free ISP2 broth: 1 mL fresh ISP2 broth was transferred aseptically to 100 mL fungal culture in malt extract broth and re-incubated using the same conditions for 5 days. (e) Fungus with bacterial extract: 0.5 mL biomass extract [(bacterial biomass obtained from 100 mL bacterial culture for 3 days was extracted with MeOH; the resulting extract was concentrated and the residue dissolved in 1 mL MeOH:H2O (1:
1)] was transferred aseptically to 100 mL fungal culture in ISP2 broth and re-incubated using the same conditions for 5 days. (f) Fungus with dead bacterial cells: 1 mL autoclaved bacterial broth was transferred aseptically to 100 mL fungal culture in ISP2 broth and re-incubated using the same conditions for 5 days. (g) Fungus with HSL: 1 mg L−1N-butyryl-DL-homoserine lactone was added aseptically to the fresh fungal culture in ISP2 broth and incubated using the same conditions for 7 days.
Footnote |
† Electronic supplementary information (ESI) available: NMR spectra of 9–10 including 1H and 13C in CDCl3 and DMSO-d6, COSY, HSQC, HMBC and NOESY in CDCl3. See DOI: 10.1039/c3ra42378f |
This journal is © The Royal Society of Chemistry 2013 |