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Antifungal Agents from Pseudallescheria boydii SNB-CN73 Isolated from a Nasutitermes sp. Termite Charlotte Nirma,†,‡ Véronique Eparvier,*,† and Didier Stien*,† †
CNRS, Institut de Chimie des Substances Naturelles, UPR 2301, 1 Avenue de la Terrasse, 91198 Gif-sur-Yvette, France UMR Ecofog, Institut Pasteur de la Guyane, Université des Antilles et de la Guyane, 23 Avenue Pasteur, 97306 Cayenne, France
‡
S Supporting Information *
ABSTRACT: Defense mutualisms between social insects and microorganisms have been described in the literature. The present article describes the discovery of a Pseudallescheria boydii strain isolated from Nasutitermes sp. The microbial symbiont produces two antifungal metabolites: tyroscherin and N-methyltyroscherin, a compound not previously described in the literature. Methylation of tyroscherin has confirmed the structure of N-methyltyroscherin. Both compounds are effective antifungal agents with favorable selectivity indices for Candida albicans and Trichophyton rubrum.
I
microbial strain growing outside the insect was collected, purified, and identified as Pseudallescheria boydii by genetic analysis. An EtOAc extract of the culture was highly active against human pathogenic fungi and moderately active against pathogenic bacteria. A bioguided fractionation allowed us to isolate two antifungal compounds (Figure 1).
nfectious diseases, particularly those affecting skin and mucosal surfaces, are a serious problem worldwide, especially in tropical and subtropical developing countries. An important class of skin pathogens are fungi, among which dermatophytes and yeasts are the most common.1 Furthermore, since the end of the 1990s, the number of HIV-infected and immunocompromised patientswho frequently develop opportunistic systemic and superficial mycoses such as candidiases, aspergilloses, and filamentous fungal infections has dramatically increased.2−4 Today, the drugs available to treat these diseases are limited by a number of drawbacks, such as low potency, poor solubility, emergence of resistant strains, and toxicity.5,6 These factors demonstrate the urgent need for novel antifungal drugs with greater efficacy. Social insect colonies may host large numbers of individuals in a constrained environment. Promiscuity, frequent interactions, and genetic homogeneity are factors that in principle favor the transmission of infectious pathogens. The literature demonstrates that social insects have evolved behavioral and physiological defense systems to prevent colonies from spreading infectious pathogens and have also developed mutually beneficial associations with microbes that provide colonies with antimicrobial agents.7−14 However, microbes associated with insect colonies have not been well studied. Owing to the extreme diversity of insects and microbes,15 insect colonies represent an alternative and attractive natural source of bioactive compounds. Excluding studies related to trophobioses,16,17 beneficial associations of termites with microbial symbionts have not been studied. Therefore, we wanted to collect microbes associated with termites in French Guiana, evaluate the antifungal and antibacterial activities of microbial extracts, and identify the compounds responsible for their antimicrobial activity. An Amazonian Nasutitermes sp. termite worker was surface sterilized and placed into a Petri dish. After one week, a © XXXX American Chemical Society and American Society of Pharmacognosy
Figure 1. Tyroscherin and N-methyltyroscherin isolated from Pseudallescheria boydii.
Compound 1 was obtained as a yellow solid. Its molecular formula (C21H35NO2) was determined by HRESIMS, which suggested five degrees of unsaturation. The NMR data (Table 1) and other spectroscopic properties of compound 1 were found to be identical to those of tyroscherin previously isolated by Watanabe from a Pseudallescheria sp.,18 the structure of which was revised by the same author, who also established its absolute configuration by synthesis. 19 Tyroscherin was originally reported to inhibit insulin-like growth factor-1 (IGF-1)-induced and fetal bovine serum (FBS)-induced growth of MCF-7 human breast cancer cells with IC50 values of 30 nM and 6.2 μM, respectively.18 Overall, the NMR data of compound 2 were similar to those of tyroscherin (1) (Table 1). The main differences for compound 2 include the 1H and 13C chemical shifts in the Received: February 28, 2013
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Table 1. NMR Spectroscopic Data (1H 500 MHz, 13C 125 MHz, CD3OD) for Tyroscherin (1) and N-Methyltyroscherin (2) Formates N-methyltyroscherin (2)
tyroscherin (1) position
δC, type
1
32.4, CH2
2 3 4 5
66.6, 68.7, 33.0, 29.8,
CH CH CH2 CH2
6 7 8 9
128.3, 138.6, 35.6, 45.5,
CH CH CH CH2
10 11 12 13 14 1′ 2′/6′ 3′/5′ 4′ NMe HCO2− a
33.0, CH 31.1, CH2 11.6, CH3 22.1, CH3 19.4, CH3 127.7, C 131.1, CH 116.7, CH 157.9, C 32.3, CH3 n.d.a
2.93, 2.87, 3.35, 3.85, 1.54, 2.21, 2.00, 5.35, 5.22, 2.16, 1.24, 1.00, 1.33, 1.30, 1.14, 0.86, 0.92, 0.84,
δH (J in Hz)
δC, type
dd (14.8, 6.9) dd (14.8, 7.6) m m m m m dt (15.2, 6.6) dd (15.2, 8.2) m ddd (13.2, 9.5, 4.4) ddd (13.2, 8.8, 5.4) m m m t (7.2) d (6.6) d (6.3)
28.5, CH2
7.12, d (8.5) 6.78, d (8.5) 2.63, s 8.56, s
70.9, 68.4, 34.1, 28.5,
CH CH CH2 CH2
126.8, 137.2, 34.3, 44.1,
CH CH CH CH2
31.6, CH 29.7, CH2 10.3, CH3 20.8, CH3 18.1, CH3 127.1, C 129.7, CH 115.3, CH 156.3, C 40.7, CH3 n.d.a
3.15, 3.01, 3.55, 4.04, 1.51, 2.20, 1.98, 5.35, 5.22, 2.18, 1.27, 1.03, 1.34, 1.30, 1.18, 0.90, 0.96, 0.86,
δH (J in Hz)
COSY
dd (15.1, 6.9) dd (15.1, 6.6) m m m m m dt (15.4, 6.3) dd (15.1, 7.9) m ddd (13.8, 9.1, 4.1) ddd (13.5, 8.5, 5.0) m m m t (7.2) d (6.6) d (6.3)
2 1, 2, 3, 4,
7.20, d (8.2) 6.82, d (8.2)
HMBC 2, 1′, 2′/6′
3 4 5 6, 7
NMe 5 5, 3 4, 6, 7
5, 7 5, 6 9, 13 8
4, 5, 7, 8 5, 6 8, 10, 11, 14
14 12
14 9, 10, 12, 14
11 8 10
10, 11 7, 8, 9 9, 10, 11
3′/5′ 2′/6′
4′, 2′/6′ 1′, 4′, 3′/5′
2.90, s 8.57, s
2
Not detected.
C-1−C-3 system and the 6H singlet signal at δ 2.90 that corresponds to the 3H singlet signal at δ 2.63 in compound 1. Therefore, it was clear that compound 2 was dimethylated on the nitrogen atom at C-2. Also of note, compound 2 was obtained as its ammonium formate form.20 The same or an analogous compound has been cited before under the name JM971A in a patent without any stereochemical information or spectroscopic data associated with it.21 We therefore sought to convert tyroscherin (1) into 2 with MeI/N,N-diisopropylethylamine in order to ascertain the relative and absolute configurations of the four stereocenters in 2. Starting material (1) and its expected N-methylated analogue were recovered from the reaction mixture by HPLC. Analytical data (NMR, specific rotation, and CD) of the N-methylated analogue were identical to those of compound 2. Compound 2 was therefore named N-methyltyroscherin, and its absolute configuration was 2S, 3R, 8R, 10R. The antimicrobial activities of tyroscherin (1) and Nmethyltyroscherin (2) were determined for the following strains: Candida albicans, C. parapsilosis, the filamentous fungi Aspergillus f umigatus and Trichophyton rubrum, the Gramnegative bacteria Escherichia coli, and the Gram-positive bacteria Staphylococcus aureus. Minimum inhibitory concentrations (MIC) and selectivity indexes (SI) are reported in Table 2. Selectivity indexes were calculated based upon cytotoxicity measurements for a skin cancer cell line, which are relevant in the context of topical application of antimicrobial agents. Overall, the antimicrobial activities of compounds 1 and 2 were moderate to good, depending on the strain tested. In the case of C. parapsilosis, A. f umigatus, and both bacteria E. coli and S. aureus, the antimicrobial activities did not exceed the cytotoxicity. However, both compound 1 and to a lesser extent
Table 2. Antimicrobial Activities of 1 and 2 SIb
MIC (μg/mL) strain Candida albicans (ATCC 10231) Candida parapsilosis (ATCC 22019) Aspergillus fumigatus (SNBAF1) Trichophyton rubrum (SNBTR1) Escherichia coli (ATCC 25922) Staphylococcus aureus (ATCC 29213) IC50 KB cells MDA435 cells
8.6 20.0
positive controla
1
2
1
2
2
4
4
10.0
6.1
16
32
4
1.3
0.8
16
32
0.5
1.3
0.8
2
4
0.25
10.0
6.1
16 8
32 16
8 0.5
1.3 2.5
0.8 1.5
(μg/mL) 16.0 24.3
a
Fluconazole (Candida sp.), itraconazole (filamentous fungi), gentamycin (E. coli), oxacillin (S. aureus). bSelectivity Index (SI = IC50/MIC) based on IC50 measured in skin cancer cells MDA435.
compound 2 showed promising antifungal potential against C. albicans and T. rubrum, with MIC values equivalent to those of reference antifungals and selectivity indexes of 10 (1) and 6 (2) for both strains. In conclusion, this article provides the first literature description of N-methyltyroscherin (2). Additionally, we demonstrate that tyroscherin (1) and its methylated analogue 2 possess antimicrobial activity. This finding illustrates how termite symbionts may inspire the discovery of antifungal agents. As these two compounds were obtained from a P. boydii B
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Table 1; HRESIMS m/z 348.2892 [M + H]+ (calcd for C22H38NO2, 348.2903). Methylation of Tyroscherin. Tyroscherin formate (2.0 mg, 5.3 μmol) and N,N-diisopropylethylamine (2.1 μL, 13.2 μmol) were mixed in an HPLC vial with a conical bottom insert and a Teflon cap. After adding a solution of methyl iodide in THF (20 μL, 13 M, 6 μmol), the vial was sealed and maintained for 2 h at rt and 1 h at 50 °C. In the end, the solvent was evaporated, and the crude reaction mixture was separated by prep-HPLC, yielding N-methyltyroscherin [0.7 mg, 38%, [α]25D −15 (c 0.31; MeOH)] along with recovered starting material (0.8 mg). Antimicrobial Assays. The ATCC strains were purchased, and the clinical isolates were kindly provided by Prof. Philippe Loiseau, Université Paris Sud. These strains were identified by Philippe Loiseau and Christian Bories, with molecular analyses conducted by BACTUP. ITS sequences were deposited in the NCBI GenBank database under the registry numbers KC692746 (Trichophyton rubrum SNB-TR1) and KC692747 (Aspergillus f umigatus SNB-AF1). Extracts, fractions, and pure compounds were tested according to the reference protocol of the European Committee on Antimicrobial Susceptibility Testing.22,23 The MIC value was obtained after 18 h for yeasts, 5 days for T. rubrum, and 24 h for the other pathogens. Cytotoxicity Assays. Cytotoxicity assays were conducted with KB (nasopharyngeal epidermoid carcinoma) and MDA435 (melanoma, previously described as mammary gland carcinoma) cell lines according to the procedure described by Tempête et al.24
strain isolated from a surface-sterilized termite worker, it is reasonable to hypothesize that this microbe provided its host with an effective antifungal therapy. Whether this strain is shared by the whole colony is a question that remains to be addressed.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured on an Anton Paar MCP 300 polarimeter in a 100 mm long 350 μL cell. UV spectra were recorded using a Perkin-Elmer Lambda 5 spectrophotometer. CD spectra were measured at 25 °C on a JASCO J-810 spectropolarimeter. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 MHz spectrometer or a Bruker 600 MHz spectrometer equipped with a 1 mm inverse detection probe. Chemical shifts (δ) are reported as ppm based on TMS signal. HRESIMS measurements were performed using a Waters Acquity UPLC system with column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with an electrospray interface (ESI). Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection equipped with an 80 g silica column. For UV-based experiments, effluents were monitored at 254 and 280 nm. TLCs were conducted on 60 A F254 Merck plates and visualized using UV and phosphomolybdic acid. Analytical and preparative HPLCs were conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector, and a prepELSII detector electrospray nebulizer. Columns used for these experiments included a Phenomenex Luna C18 5 μm 4.6 × 250 mm analytical column and Phenomenex Luna C18 5 μm 21.2 × 250 mm preparative column. The flow rate was set to 1 or 17 mL/min, respectively, using a linear gradient of H2O mixed with an increasing proportion of CH3CN. Both solvents were modified with 0.1% formic acid. All solvents were HPLC grade. Potato dextrose agar (PDA) was purchased from Fluka Analytical. Molecular analyses were performed externally by BACTUP, France. Collection and Identification of Pseudallescheria boydii SNBCN73. A termite worker was collected from an aerial termite nest located in Rémire-Montjoly, French Guiana, in July 2011. The worker was surface-sterilized by successive soakings in 70% EtOH (2 min), 5% NaOCl (2 min), and sterile water rinse. The termite was subsequently placed in a Petri dish containing a solid PDA medium. After 1 week at 25 °C, the first fungal hyphae to emerge from the insect were sampled and transferred into another Petri dish. The microbial colony consisted of a pure fungus, which was saved in triplicate at −80 °C in H2O− glycerol (50/50). A sample submitted for amplification and nuclear ribosomal internal transcribed spacer region ITS4 sequencing allowed for strain identification by NCBI sequence comparison. The sequence has been registered in the NCBI GenBank database (http://www.ncbi. nlm.nih.gov) under registry number KC684883. Culture, Extraction, and Isolation. The P. boydii strain was cultivated on PDA at 26 °C for 15 days, initially on a small scale and then on 150 14-cm Petri dishes. The fungus and culture medium were then transferred into a large container and macerated with EtOAc for 24 h. The organic solvent was then collected by filtration, washed with H2O in a separatory funnel, and evaporated, yielding 2.46 g of the extract. A portion of the extract (1.23 g) was purified by flash chromatography with a linear gradient of hexane−EtOAc followed by another gradient of EtOAc−MeOH. Twelve fractions were gathered based on their TLC profiles. The antifungal activity was found to be concentrated in fraction XII. Upon further fractionation with prepHPLC, tyroscherin (1, 5.4 mg, 0.44%) and N-methyltyroscherin (2, 3.1 mg, 0.25%) were isolated. Tyroscherin formate (1): yellow solid; [α]25D −49 (c 0.54, MeOH) [lit. [α]25D −21 (c 0.35, MeOH)];19 1H (500 MHz) and 13C (125 MHz) NMR data (CD3OD) in Table 1; HRESIMS m/z 334.2729 [M + H]+ (calcd for C21H36NO2, 334.2746). N-Methyltyroscherin formate (2): yellow solid; [α]25D −12 (c 0.31; MeOH); UV (MeOH) λmax (log ε) 225.0 (2.37), 280.0 (0.98) nm; 1H (500 MHz) and 13C (125 MHz) NMR data (CD3OD) in
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR spectra of tyroscherin (1) and Nmethyltyroscherin (2) formates as well as CD spectra of 1, 2, and synthetic N-methyltyroscherin are available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +33 1 69 82 36 10 (D.S.), +33 1 69 82 36 79 (V.E.). Fax: +33 1 69 82 37 84. E-mail:
[email protected] (D.S.),
[email protected] (V.E.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has benefited from an “Investissement d’Avenir” grant managed by Agence Nationale de la Recherche (CEBA, ref ANR-10-LABX-0025). The authors are very grateful to P. Loiseau for providing wild strains of pathogenic fungi.
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REFERENCES
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(12) Currie, C. R.; Poulsen, M.; Mendenhall, J.; Boomsma, J. J.; Billen, J. Science 2006, 311, 81−83. (13) Oh, D.-C.; Poulsen, M.; Currie, C. R.; Clardy, J. Nat. Chem. Biol. 2009, 5, 391−393. (14) Kaltenpoth, M.; Go, W.; Herzner, G.; Strohm, E. Curr. Biol. 2005, 15, 475−479. (15) Purvis, A.; Hector, A. Nature 2000, 405, 212−219. (16) Bignell, D. E.; Oskarsson, H.; Anderson, J. M. Appl. Environ. Microbiol. 1979, 37, 339−342. (17) Grassé, P.-P. Termitologia: Anatomie, Physiologie, Biologie, Systématique des Termites; Masson: Paris, 1982; p 676. (18) Hayakawa, Y.; Yamashita, T.; Mori, T.; Nagai, K.; Shin-Ya, K.; Watanabe, H. J. Antibiot. 2004, 57, 634−638. (19) Ishigami, K.; Katsuta, R.; Shibata, C.; Hayakawa, Y.; Watanabe, H.; Kitahara, T. Tetrahedron 2009, 65, 3629−3638. (20) Compounds 1 and 2 were also isolated in the free amine form with 0.1% NH4OH in the HPLC solvent instead of HCO2H. Protonation with 0.1% HCO2H in CH3CN and evaporation confirmed identification and attribution of the broad singlet at δ 8.56 and 8.57 in the formates (Table 1). Tyroscherin (free amine form): 1H NMR (500 MHz, CD3OD) δ 0.84 (d, 3H, J = 6.4), 0.86 (t, 3H, J = 7.6), 0.92 (d, 3H, J = 6.7), 1.00 (ddd, 1H, J = 13.4, 8.9, and 4.9), 1.14 (m, 1H), 1.24 (ddd, 1H, J = 13.4, 9.5, and 4.6), 1.26−1.37 (m, 2H), 1.48−1.61 (m, 2H), 2.01 (m, 1H), 2.14−2.24 (m, 2H), 2.38 (s, 3H), 2.67 (d, 2H, J = 7.0), 2.77 (m, 1H), 3.67 (dt, 1H, J = 9.5 and 3.1), 5.23 (dd, 1H, J = 15.3 and 8.2), 5.38 (dt, 1H, J = 15.3 and 6.7), 6.73 (br d, 2H, J = 8.3), 7.05 (br d, 1H, J = 8.3). N-Methyltyroscherin (free amine form): 1H NMR (500 MHz, CD3OD) δ 0.83 (d, 3H, J = 6.4), 0.86 (t, 3H, J = 7.3), 0.93 (d, 3H, J = 6.7), 0.99 (ddd, 1H, J = 13.4, 9.2, and 5.2), 1.14 (m, 1H), 1.24 (ddd, 1H, J = 13.4, 9.5, and 4.6), 1.25−1.37 (m, 2H), 1.42 (m, 1H), 1.51 (m, 1H), 1.97 (m, 1H), 2.09−2.19 (m, 2H), 2.34 (s, 6H), 2.73−2.83 (m, 3H), 3.76 (dt, 1H, J = 8.9 and 3.7), 5.19 (dd, 1H, J = 15.3 and 8.2), 5.35 (dt, 1H, J = 15.3 and 6.4), 6.70 (br d, 2H, J = 8.3), 7.08 (br d, 1H, J = 8.3). (21) Cuomo, V.; Gruner, J.; Müller, J. Ger. Offen. Patent 3,333,553, 1984. (22) EUCAST.. Clin. Microbiol. Infect. 2008, 14, 398−405. (23) Rodrigues, A. M. S.; Theodoro, P. N. E. T.; Basset, C.; Silva, M. R. R.; Beauchêne, J.; Espindola, L. S.; Stien, D. J. Nat. Prod. 2010, 73, 1706−1707. (24) Tempête, C.; Werner, G.; Favre, F. Eur. J. Med. Chem. 1995, 30, 647−650.
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