APPLIED AND ENVIRONMENTAL MICROBIOLOGY,0099-2240/99/$04.0010

Sept. 1999, p. 4207–4210 Vol. 65, No. 9

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Detection and Identification of Amanitins in the Wood-RottingFungi Galerina fasciculata and Galerina helvoliceps

SHINJIRO MURAOKA,1,2 NOBUKO FUKAMACHI,3 KIYOHISA MIZUMOTO,3

AND TAKAO SHINOZAWA1*

Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515,1 Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku,

Tokyo 108-8641,3 and Research and Development Division, Mori & Company, Ltd., Kiryu, Gunma 376-0051,2 Japan

Received 1 March 1999/Accepted 6 June 1999

More than 600 strains of wood-rotting fungi were screened for the detection of amanitins. Three strains ofGalerina fasciculata and 18 strains of Galerina helvoliceps contained amanitins. These strains contained mainlya- and b-amanitins in the native fruit bodies, while a- and g-amanitins were found in liquid-cultured mycelia.Purified amanitins were confirmed by their chromatographic profiles, spectra (UV, Fourier transform infrared,and atmospheric ionization mass), cytotoxicity for mammalian cell lines (3T3 and SiHa), and inhibitory effectson RNA polymerase II. The results revealed that the purified amanitin fractions from these species areidentical to authentic amanitins and suggest that these two species must be handled as poisonous mushrooms.

Amanitins belong to a family of bicyclic octapeptide myco-toxins that bind tightly to and inhibit eukaryotic DNA-depen-dent RNA polymerase II (4, 8, 9). Four types of amanitin (a,b, g, and ε) are characterized by differences in their side groups(16–18) and are used as inhibitors in eukaryotic gene transcrip-tion analysis. These compounds are extracted from poisonousmushrooms, especially Amanita phalloides (15). Many attemptsto obtain mycelial subcultures of Amanita species in artificialmedia have been unsuccessful because of their mycotrophy.For some species in the genera Lepiota and Galerina, identifi-cation of amanitins in fruit bodies has been reported (3, 7), andpreliminary work on the production of amanitins in liquidmedium with a strain of Galerina marginata has been reportedby Benedict et al. (2). By screening culturable strains possess-ing the ability to produce amanitins, two species were foundand identified as Galerina fasciculata and Galerina helvoliceps.In this paper, we report the isolation and identification ofamanitins from natural mushrooms and cultured mycelia ofthese species.

MATERIALS AND METHODS

Chemicals. Authentic a- and b-amanitins were purchased from Sigma Chem-ical Co. (St. Louis, Mo.). g-Amanitin was purified from a mycelial extract of G.fasciculata as described below and used as a standard based on its molar extinc-tion coefficient (15). Columns for high-pressure liquid chromatography (HPLC)were as follows: Superdex Peptide HR 10/30 (Amersham Pharmacia Biotech,Tokyo, Japan), Zorbax octyldecyl silane (ODS) (4.6 mm by 25 cm; Shimadzu-Dupont, Kyoto, Japan), Wakosil II 5C18 types RS, HG, and AR (4.6 mm by 25cm; Wako Pure Chemical, Osaka, Japan), mBondasphere 5 m C18-100Å (19 mmby 15 cm; Waters, Tokyo, Japan), and Discovery RP-Amide C16 5mm (4.6 mmby 25 cm; Sigma). HeLa cell nuclear extract for in vitro transcription was pre-pared as described by Manley et al. (10). [a-32P]UTP (400 Ci/mmol, 6 mCi/ml)was from Amersham Pharmacia Biotech. All other reagents were of the highestgrade available from commercial sources.

Collection and isolation of amanitin-containing basidiomycetes. Fruiting bod-ies of wood-rotting fungi were collected between July 1995 and October 1997from decayed wood in forests in the middle latitudes of Japan. The mushroomswere identified by referring to studies by Ammirati et al. (1), Imazeki and Hongo(6), and Singer (11). After HPLC analysis as described by Enjalbert et al. (5), thestrains found to contain amanitins were subcultured. The liquid medium (HSV)

for amanitin production contained the following, per liter: 1 g of yeast extract(Difco, Detroit, Mich.), 2 g of glucose, 0.1 g of NH4Cl, 0.1 g of KCl, 0.1 g ofCaSO4 z 1/2H2O, 1 mg of thiamine z HCl, and 0.1 mg of biotin (medium pH withHCl, 5.2). Agar medium (HSVA) for subculture contained 2% agar in HSV.Vegetative mycelial stocks were prepared by culturing aseptic fragments offruiting bodies on HSVA plates. Fungal colonies were transferred and reisolateduntil pure cultures were obtained. The stocks were subcultured every 6 monthsand deposited at The Mushroom Research Institute of Japan (Kiryu, Gunma,Japan).

Screening of amanitin-producing strains. Isolated strains were cultured in 30ml of HSV (in a 100-ml Erlenmeyer flask by rotary shaking at 150 rpm) at 25°Cfor 30 days in the dark. The mycelia were collected by centrifugation at 3,000 3g for 20 min, washed twice with distilled water, lyophilized, and weighed. Theextraction and determination of amanitin content were carried out according tothe methods described by Enjalbert et al. (5). For the analytical reversed-phaseHPLC, a 4.6- by 250-mm Zorbax ODS column was used.

Large-scale cultivation. To confirm the amanitins, mainly two strains, G.fasciculata GF-060 and G. helvoliceps GH-343, were used for large-scale culti-vation. The mycelia cultivated for 10 days in 30 ml of HSV medium (in a 100-mlErlenmeyer flask) as described above were dispersed aseptically with a homog-enizer (Biomixer SBM-1; Nihonseiki, Tokyo, Japan) for 10 s at 30,000 rpm. Thedispersed mycelia (30 ml) were mixed in the same medium (400 ml) in a 1-literErlenmeyer flask, and the mixture was further cultivated under the same condi-tions for 30 days.

Purification of amanitins. (i) Step 1: preparation of cell extract. Washed andlyophilized mycelia from 430 ml of culture broth were suspended in 500 ml ofmethanol containing 0.083% (vol/vol) HCl. The suspension was treated with ahomogenizer (at 30,000 rpm for 5 min at 4°C). The supernatant fluid wasobtained by centrifugation at 15,000 3 g for 15 min at 4°C, and the precipitateswere extracted three times under the same conditions. The combined superna-tant fluids were concentrated and dried in a rotary evaporator at 35°C. The cellextract was dissolved in 10 ml of distilled water and defatted with an equalvolume of diethyl ether.

(ii) Step 2: solid-phase extraction by C18 cartridge. The extract from step 1 wasdiluted to 10 ml with distilled water and applied to a Sep-Pak Vac tC18 cartridge(200 mg; Waters) previously equilibrated with distilled water. The cartridge waswashed with 10 ml of distilled water, and then the amanitins were eluted with40% (vol/vol) methanol. The amanitin fractions in 10 ml of 40% (vol/vol) meth-anol were dried in vacuo at 35°C.

(iii) Step 3: size exclusion HPLC. The extract from step 2 was dissolved inwater, passed through a membrane filter (pore size, 0.45 mm; Millipore, Tokyo,Japan), and applied to size exclusion HPLC (LC-10AT; Shimadzu) under thefollowing conditions: column, Superdex Peptide HR 10/30; elution buffer, 100mM ammonium acetate containing 35% (vol/vol) acetonitrile (pH 6.5 with aceticacid); flow rate, 1.0 ml/min at 25°C; and monitoring with a photodiode arraydetector (MD-910; Jasco Corp., Tokyo, Japan) from 210 to 500 nm. The fractioncorresponding to the amanitins (eluted at 14.6 to 16.3 min) was collected andconcentrated in vacuo at 35°C.

(iv) Step 4: preparative reversed-phase HPLC. Purification of each amanitinfrom the step 3 fraction was carried out by binary gradient HPLC (LC-6A;Shimadzu): column, mBondasphere 5m C18-100 Å; solvent A, 20 mM ammoniumacetate containing 5% (vol/vol) methanol (pH 5.0 with acetic acid); solvent B, 20

* Corresponding author. Mailing address: Department of Biologicaland Chemical Engineering, Faculty of Engineering, Gunma Univer-sity, Kiryu, Gunma 376-8515, Japan. Phone: 81-277-30-1431. Fax: 81-277-30-1431. E-mail: shinozawa@bce.gunma-u.ac.jp.

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mM ammonium acetate containing 80% (vol/vol) methanol (pH 5.85 with aceticacid). Separation was carried out at a flow rate of 10 ml/min at 35°C with thefollowing three eluents: 20% solvent B for 15 min, a linear gradient of 20 to 99%solvent B for 15 min, and 99% solvent B for 10 min. Detection and UV spectralanalysis of the eluents were performed with a photodiode array detector at 210to 500 nm. The fractions, corresponding to b-amanitin eluted at 16.5 min,a-amanitin eluted at 18.5 min, and g-amanitin eluted at 25.2 min, were collectedseparately and lyophilized. The purified fractions from the Galerina species(defined as the b-, a-, and g-amanitin fractions) were used in the followingexperiments.

Spectrometric analyses. Fourier transform infrared (FT-IR) spectra were ob-tained with a KBr pellet on an FT-IR spectrophotometer (model 1600; Perkin-Elmer, Yokohama, Japan). UV spectra in water were obtained with a spectro-

photometer (U-3210; Hitachi, Tokyo, Japan). b-, a-, and g-amanitin fractionswere dissolved in solvent (2.5 mM ammonium acetate in 50% [vol/vol] metha-nol), and their mass spectra were analyzed on a Perkin-Elmer API-100 byatmospheric ionization (API) in positive-ion mode.

Determination of melting point. The melting point was determined in a glasscapillary with a melting point apparatus (MFB595; Gallenkamp, Sussex, UnitedKingdom).

Cytotoxicity test. Two mammalian cell lines (3T3 [laboratory stock] and SiHa[gift of T. Kanda, National Institute of Health, Tokyo, Japan]) (12) were used toassay cytotoxicity. Cells were grown in 96-well plates (no. 3860; Iwaki Glass,Chiba, Japan) on Dulbecco’s modified Eagle’s medium (Iwaki Glass) supple-mented with 10% fetal bovine serum (Iwaki Glass) at 37°C in 5% CO2, with themedium changed every 3 days. Authentic a-amanitin and the a-amanitin fraction

FIG. 1. Fruiting bodies of amanitin-containing mushrooms G. helvoliceps (A) and G. fasciculata (B). Bar 5 1 cm.

TABLE 1. Galerina strains containing amanitins in Japan

Strain Date of collection(mo/day/yr) Location

Amanitin content (mg/g of freshwt)a

a b

G. helvolicepsGH-323 09/08/1996 Azegata, Nikko, Tochigi 138.41 97.51GH-324 09/08/1996 Azegata, Nikko, Tochigi 125.98 81.78GH-326 09/08/1996 Azegata, Nikko, Tochigi 89.21 38.49GH-327 09/08/1996 Azegata, Nikko, Tochigi 145.21 99.68GH-340 09/15/1996 Koutoku, Nikko, Tochigi 127.91 81.97GH-343 09/15/1996 Azegata, Nikko, Tochigi 391.06 165.16GH-344 09/15/1996 Azegata, Nikko, Tochigi 122.34 120.24GH-345 09/15/1996 Azegata, Nikko, Tochigi 52.92 17.71GH-349 09/15/1996 Azegata, Nikko, Tochigi 64.29 28.80GH-353 09/15/1996 Kuriyama, Tochigi 89.47 37.65GH-355 09/15/1996 Kuriyama, Tochigi 233.66 97.81GH-364 09/23/1996 Mizorogi, Akagi, Gunma 243.05 87.11GH-368 09/23/1996 Mizorogi, Akagi, Gunma 210.17 74.93GH-380 09/29/1996 Tanbara, Numata, Gunma 172.72 55.04GH-386 10/04/1996 Ashiwada, Yamanashi 128.45 112.14GH-403 10/20/1996 Nagaoka, Niigata 41.73 10.21GH-408 09/21/1997 Azegata, Nikko, Tochigi 38.48 9.33GH-409 10/10/1997 Hotaka, Tone, Gunma 41.53 13.45

G. fasciculataGF-060 09/27/1995 Kawamata, Tochigi 112.84 147.12GF-329 09/08/1996 Azegata, Nikko, Tochigi 100.29 122.04GF-405 11/10/1996 Shitada, Niigata 255.59 Tr

a Amanitins in natural fruiting bodies were measured by reversed-phase HPLC analysis.

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were diluted to the appropriate concentration with Dulbecco’s modified Eagle’smedium containing 10% fetal bovine serum. After incubation for 7 days with orwithout a-amanitin, cell growth was measured with an MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay kit (Sigma) at A570 toA620. After the calculation, each 50% lethal dose (LD50) was estimated from thedose-response curve.

Inhibitory effect on RNA polymerases II and III. The specific inhibition ofRNA polymerase II activity was measured by transcribing specific templates withclass II and class III promoters. Two DNAs, plasmid pSmaF, containing theSmaI-F fragment of adenovirus type 2 major late promoter (template for RNApolymerase II), and pAdSalIC, containing the VA1 RNA gene of adenovirustype 5 (for RNA polymerase III), provided by H. Handa and I. Saito (TheInstitute of Medical Science, University of Tokyo), respectively, were used as thetemplates (14) for in vitro transcription in a reconstituted system of HeLa cellcomponents (10). Authentic a-amanitin or the a-amanitin fraction was added toeach reaction mixture at the appropriate concentration. The transcription reac-tions contained 0.5 ml of [a-32P]UTP (400 Ci/mmol, 6 mCi/ml); reactions werecarried out according to methods described by Manley et al. (10). Transcriptswere analyzed in denaturing 4% polyacrylamide gel containing 7 M urea. Auto-radiographs of the transcripts were obtained by exposing the gel to an X-ray filmwith an intensifying screen overnight at 270°C.

RESULTS AND DISCUSSION

Screening of amanitin-containing basidiomycetes. Strainscontaining amanitin in their fruiting bodies are listed in Table1. They were identified as G. helvoliceps and G. fasciculata. Ingeneral, these two species share similar habitats but are dis-tinguished by fruiting body size (Fig. 1) and cystidia shape.Amanitin content in the fruiting body of each strain is also

shown in Table 1. The reasons for the various amanitin contentlevels might be geographical differences, seasonal conditions,moisture content, or the diversity of inheritance. According tothe LD50 of a-amanitin in humans (13), the ingestion of 5 to10 g of these fresh mushrooms would be fatal for an adult.When these strains were cultured in HSV medium, a-amanitinand small amounts of g-amanitin accumulated intracellularly,while little b-amanitin was observed (Fig. 2C). On the otherhand, in the fruiting bodies or mycelia from cultivation on solidHSVA, mainly a- and b-amanitins were observed, with onlytrace amounts of g-amanitin detected (Fig. 2A and B). Traceamounts of extracellular amanitins were detected at everystage of the culture period (data not shown). These resultssuggest that the production patterns of amanitins are affectedby culture conditions, such as liquid or solid medium.

Purification and confirmation of amanitins. The purifica-tion and confirmation of the amanitins were carried out asdescribed in Materials and Methods. The purity of each aman-itin fraction was confirmed, and each produced a single peakon analytical reversed-phase HPLC with various columns andelution conditions. The purities of the final products of boththe a- and b-amanitin fractions, calculated from the peak areasin analytical reversed-phase HPLC, were more than 99% (datanot shown). Elution profiles of the amanitin fractions fromGalerina strains were also compared in six types of reversed-phase chromatography (Zorbax ODS; Wakosil II 5C18 typesRS, HG, and AR; mBondasphere 5m C18-100 Å and DiscoveryRP-Amide C16 5mm). All profiles of a- and b-amanitin frac-tions obtained in these chromatographies were identical tothose of authentic a- and b-amanitin, respectively (data notshown). Physical and biological properties were also com-pared. Estimation of the molecular weight by API mass spec-trometry (919), determination of the melting point (decompo-sition at 254°C), and calculation of the LD50s for 3T3 (0.35 mgml21) and SiHa (0.32 mg ml21) cells showed no differencesbetween the authentic a-amanitin and the a-amanitin fraction.The UV and FT-IR spectra of the amanitin fractions wereidentical to those of the authentic amanitins (data not shown).a- and b-amanitin fractions from mycelial extracts from otherGalerina strains, as listed in Table 1, grown on HSVA or HSVmedium were also confirmed by the same spectrometric anal-yses. All g-amanitin fractions from the listed strains were con-firmed by reference to the various spectrometric analyses and

FIG. 2. Typical chromatograms of intracellular amanitins of G. helvolicepsGH-343, obtained by analytical reversed-phase HPLC. (A) Naturally occurringfruiting body; (B) mycelia cultured on solid (HSVA) medium; (C) myceliacultured on liquid (HSV) medium. Separation conditions are described in Ma-terials and Methods.

FIG. 3. Inhibitory effects of authentic a-amanitin and the a-amanitin fractionfrom G. fasciculata GF-060 on RNA polymerases II and III in an in vitrotranscription system. Two DNAs, pSmaF for polymerase II and pAdSalIC forpolymerase III, were used as templates for the reaction. The conditions for invitro transcription analysis are described in Materials and Methods.

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chromatographic studies described by Wieland (15). Becauseamanitins are well known as inhibitors of mammalian RNApolymerase II, the inhibitory activities of authentic a-amanitinand the a-amanitin fraction were tested (Fig. 3). Both a-aman-itins inhibited the transcription of RNA polymerase II (majorproduct from pSmaF, 536 bases) at a low concentration (0.4mg/ml), while neither a-amanitin preparation inhibited thetranscription of RNA polymerase III (major product frompAdSalIC, 156 bases), even at a high concentration (1.6 to 2.0mg/ml). These observations illustrate one important character-istic of amanitins.

In conclusion, it was found that G. fasciculata and G. helvo-liceps produce a-, b-, and g-amanitins in cultured mycelia, inaddition to in their fruit bodies. In the past, these Galerinaspecies were considered to be poisonous mushrooms if in-gested; however, the toxic components were not clarified. Inthis report, endogenous amanitins in these species were iden-tified. Therefore, these two species must be handled as poi-sonous mushrooms. The availability of two fermentation styles(solid and liquid medium culture) for these strains makes itpossible to obtain sufficient amounts of each amanitin for fur-ther studies. Moreover, these amanitin-producing strains willcontribute to the elucidation of the amanitin biosynthesis path-ways.

ACKNOWLEDGMENTS

We express our thanks to G. Kawai of Mori & Company, Ltd., andT. Toyomasu of The Mushroom Research Institute of Japan for theirhelpful suggestions and to T. Kanda of the National Institute of Healthfor his gift of SiHa cells. We also thank M. Abe and H. Ozaki ofGunma University for the FT-IR and API mass spectra measurements,respectively, and Y. Nakabayashi for taxonomical advice.

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