Brefeldin A, a Cytotoxin Produced by Paecilomyces Sp. and Aspergillus Clavatus Isolated from Taxus Mairei and Torreya Grandis
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FEMS Immunology and Medical Microbiology 34 (2002) 51^57 www.fems-microbiology.org Brefeldin A, a cytotoxin produced by Paecilomyces sp. and Aspergillus clavatus isolated from Taxus mairei and Torreya grandis Jianfeng Wang a;b;Ã, Yaojian Huang a, Meijuan Fang b, Yongjie Zhang a, Zhonghui Zheng a, Yufen Zhao b, Wenjin Su a;c a The Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, P.O. Box 958, Xiamen 361005, PR China b Bioorganic Phosphorus Chemistry Lab., Department of Chemistry, Xiamen University, Xiamen 361005, PR China c School of Biotechnology, Jimei University, Xiamen 361021, PR China Received 24 April 2002; received in revised form 29 May 2002; accepted 30 May 2002 First published online 3 August 2002 Abstract Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in liquid culture. Nuclear magnetic resonance techniques, infrared spectrometry, electrospray ionization mass spectroscopy and X-ray analysis identified brefeldin A, a bioactive metabolite produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora and Phyllosticta. This is the first report of the isolation of the cytotoxin from Paecilomyces sp. and A. clavatus. The relevance of brefeldin A to the association between these fungi and their host plants is discussed. ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Brefeldin A; Endophytic fungi; Paecilomyces sp.; Aspergillus clavatus; Taxus mairei; Torreya grandis 1. Introduction showed antitumor and antifungal activity, respectively, demonstrating that endophytic fungi are to be a promising Fungi are fundamental to the health and prosperity of source of bene¢cial bioactive products [5]. every terrestrial ecosystem and are essential to the sustain- Paecilomyces and Aspergillus, belonging to fungi imper- ability of biodiversity. Endophytic fungi, which live inside fecti, were commonly found as endophytic fungi in our plants as an inconspicuous embroidery of thread-like ¢la- investigation. However, the former fungus was encoun- ments, provide yet another dimension to the fungal sup- tered more frequently than the latter [5]. The genus Pae- port system. The role of endophytic fungi in plant^herbi- cilomyces includes several species that are able to produce vore interactions and their production of extremely a wide array of bioactive secondary metabolites of di¡er- bene¢cial compounds (suchas taxol) havereceived partic- ent chemical classes and with di¡erent biological activities, ular attention in recent years [1^4]. suchas antimicrobial activity, cytotoxic activity and im- We have isolated hundreds of endophytic fungi from munostimulating activity [6]. Aspergillus clavatus has usu- Taxus mairei, Cephalataxus fortunei and Torreya grandis, ally been found as a saprophytic fungus, producing myco- which are traditional Chinese pharmaceutical plants [5]. toxins suchas patulin and cytochalasinE [7]. Further 14.5 and 52.3% of endophytic fungi fermentation broths investigation of Paecilomyces sp. and A. clavatus will not only be a way of ¢nding useful bioactive compounds, but also provide us withmuchmore bene¢cial ecological in- formation. The present paper describes the isolation and chemical identi¢cation of a metabolite from three endophytic fungi of T. mairei and T. grandis. The systematic identi¢cation of these endophytic fungi and the relevance of the cyto- * Corresponding author. Tel./Fax: +86 (592) 2185780. toxin to their association with their host plants are also E-mail address: [email protected] (J. Wang). discussed. 0928-8244 / 02 / $22.00 ß 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S0928-8244(02)00346-2 FEMSIM 1426 21-8-02 52 J. Wang et al. / FEMSImmunology and Medical Microbiology 34 (2002) 51^57 2. Materials and methods Chinese Academy of Sciences. All the cell lines were cul- tured in RPMI1640 medium supplemented with10% heat- 2.1. Identi¢cation of endophytic fungi inactivated fetal bovine serum, 100 U ml31 penicillin, 80 U ml31 kanamycin and 100 U ml31 streptomycin. Cultures Endophytic fungi strains W-001, H-036 and H-037 were were maintained in a humidi¢ed incubator at 37‡C in an studied in this work. They were isolated from bark sam- atmosphere of 5% CO2. The cytotoxic e¡ect of the fermen- ples of T. mairei and T. grandis, which were gathered at tation brothwas tested using a protocol (theMTT assay) Wuyi Mountain nature conservation area, Fujian prov- adapted from that described by Mosmann [11]. The opti- ince, southeast China, at an altitude of about 900 m. Fun- cal density of the wells was measured with a microplate gal identi¢cation was based on the morphology of the reader (M-3550, Bio-Rad) at 595 nm with655 nm as refer- fungal culture, the mechanism of spore production and ence. Growth inhibition rate was calculated by the follow- the characteristics of the spores [8^10]. ing equation: ODcontrol well3ODtreated well 2.2. Scanning electron microscopy Inhibition rate ¼ ¼ U100% ODcontrol well For scanning electron microscopy, the materials were ID50 and IC50 were respectively de¢ned as the dilution of ¢xed in 2.5% (v/v) glutaraldehyde in 0.1 M phosphate fermentation broths and concentration of compounds that bu¡er (pH 7.0) for 2 h and then step dehydrated (15 resulted in (at least) a 50% inhibition of growth rate. min each) with 50, 70, 90, 100% (v/v) ethanol, isoamyl acetate/ethanol (1:1 v/v) and ¢nally 100% isoamyl acetate. 2.5. Spectroscopic measurements Then the samples were critical-point dried, gold coated with a sputter coater and observed and photographed X-ray crystallographic data were collected on an Enraf- witha HitachiS520 scanning electron microscope. Nonius CAD-4 di¡ractometer equipped witha graphite monochromator MoK K radiation (V = 0.7013). A total 2.3. Fermentation and compound isolation of 1742 independent re£ections were collected in the range of 1 6 2a 6 26‡ by the g^2a scan technique at room tem- Eachfungus was cultured in potato^dextrose liquid perature, and the structure was solved by direct methods (PDA) medium for 7 days at 25‡C, 120 rpm. Crude fer- that yielded the positions of all non-hydrogen atoms, and mentation broth was blended thoroughly and centrifuged which were re¢ned with anisotropic thermal parameters. at 4000 rpm for 5 min. The supernatant was passed All hydrogen atoms were generated assuming idealized ge- through a ¢ltration membrane (0.22 Wm, Minipore) before ometry (C^H bond lengths ¢xed at 0.96 Aî ), assigned ap- its bioactivity was assayed. 1 l of homogenized broth was propriate isotropic thermal parameters, and allowed to extracted three times with ethyl acetate. The combined ride on their parent atoms. The computation was per- organic extract was evaporated under reduced pressure formed on a PIII-600 compatible PC withtheSHELXL97 yielding a crude semi-solid. The crude extract was sepa- for full-matrix least-square re¢nement. rated by silica gel column chromatography applying gra- Nuclear magnetic resonance spectroscopy was undertak- dient elution from petroleum ether to ethyl acetate to en on a Brucker 500 instrument using deuterated ethyl methanol to yield 50 fractions, which were analyzed by ester. The mass spectra were taken on a Brucker Esquire thin-layer chromatography (TLC) and combined to eight 3000 Plus mass spectrometer. The infrared ray spectrum fractions. The spots on silica plates were visualized by was taken on a Nicolet Avatar FT-IR360 IR spectrometer. exposure to UV radiation and/or by spraying with1% vanillin in H2SO4^methanol (4:1, v/v), followed by heating at 110‡C for 10 min. These eight fractions were screened for their ability to inhibit the growth of KB cells (see Section 2.4). Fraction 5 was found to be most active and was further puri¢ed by re-crystallization to yield ‘com- pound 1’ as colorless prism crystals. The compound was Table 1 homogeneous by TLC in solvent (A) benzene/ethyl ace- Cytotoxicity of the endophytic fungi and brefeldin A tate/diethylamine 7:2:1 (v/v) (R 0.23), solvent (B) chloro- f HL-60 KB Hela MCF-7 Spc-A-1 form/methanol 7:1 (v/v) (Rf 0.51) and solvent (C) chloro- form/methanol 7:3 (v/v) (R 0.68). ID50 f W-001 1:300 1:1000 1:5000 1:4000 1:1000 H-036 1:1500 1:3000 1:3000 1:2000 1:3000 2.4. Cell culture and cytotoxicity study H-037 1:200 1:200 1:1000 1:200 1:4000 31 IC50 (ng ml ) Human tumor cell lines HL60, KB, Hela, SPC-A-1 and Taxol 1.2 0.16 1.8 5.0 0.8 MCF-7 were obtained from the Cell Line Bank of the Brefeldin A 10.0 9.0 1.8 2.0 1.0 FEMSIM 1426 21-8-02 J. Wang et al. / FEMSImmunology and Medical Microbiology 34 (2002) 51^57 53 Fig. 1. The morphological observation of Paecilomyces sp. strain H-036. A: eleistothecia; B: ascospore inside the eleistothecium; C: conidiophore in single branch; D: conidiophore in three level branches; E: conidio-chain; F: conidia. 3. Results numerous sexual fruiting bodies, which indicated that it was an eleistothecia. Each eleistothecium was about 50^ 3.1. Identi¢cation of endophytic fungi 200 Wm in diameter witha thickwall and smoothwavy surface (Fig. 1A). The eleistothecia were engorged with Three endophytic fungi were studied in this work. ascospores, which were approximately 2.5 Wm in diameter, Strains W-001 from T. mairei and H-036 and H-037 hyaline, cylindrical and with spinous protuberances on from T. grandis were chosen for study because they pos- the surface (Fig.