Hack Sung Jung, Department of Biological Sciences, College of Natural Sciences, Seoul National University, Kwanak-gu, Seoul 151-747, Korea (e-mail: firstname.lastname@example.org).
Aims: To identify a new fungal strain, Hypocrea sp. F000527 producing a trichothecene metabolite, harzianum A, and to evaluate its cytotoxicity to tumour cell lines.
Methods and Results: A fungal strain, F000527, with cytotoxic activity was identified as a new Hypocrea strain based on morphological characteristics and internal transcribed spacers rDNA sequence data. Harzianum A was isolated from wheat bran culture by 50% acetone extraction, silica gel chromatography, Sephadex LH-20 chromatography and HPLC. The chemical structures were determined by ESI- or HRFAB-MS and 1H and 13C-NMR analyses. Harzianum A showed cytotoxicity to HT1080 and HeLa cell lines with IC50 value of 0·65 and 5·07 μg ml−1 respectively.
Conclusions: Harzianum A with a chemical formula of C23H28O6 was isolated from a new Hypocrea strain and showed moderate to strong cytotoxicity to human cancer cell lines.
Significance and Impact of the Study: This is the first report of the production of cytotoxic harzianum A by a new Hypocrea strain.
Mycotoxins, by-products of fungal metabolism, have been implicated as causative agents of adverse health effects in humans and animals that have consumed fungus-infected agricultural products (Ciegler 1975; Ciegler and Bennett 1980). Various genera of toxigenic fungi are capable of producing diverse mycotoxins such as aflatoxins, rubratoxins, ochratoxins, fumonisins and trichothecenes (Ciegler 1975; Ciegler and Bennett 1980). The trichothecenes are a very large family of chemically related toxins produced by various species of Cephalosporium, Fusarium, Myrothecium, Stachybotrys, Trichoderma and Verticimonosporium (Ueno 1989). They are markedly stable under different environmental conditions. The distinguishing chemical feature of trichothecenes is the presence of a trichothecene ring, which contains a double bond at C-9 and C-10, and an epoxide group at C-12 (Wannemacher and Wiener 1997). This family of mycotoxins causes multiorgan effects including emesis and diarrhoea, weight loss, nervous disorders, cardiovascular alterations, immunodepression, haemostatic derangements, skin toxicity, decreased reproductive capacity and bone marrow damage (Ueno 1989; Wannemacher et al. 1991). This family of mycotoxins are also known to be potent inhibitors of protein and/or DNA synthesis and can impact on actively dividing tissues, with the immune system being particularly susceptible (Thompson and Wannemacher 1984; Islam et al. 2002).
Trichoderma and Gliocladium are known as anamorphs of Hypocrea and characterized by their ability to produce a wide range of secondary metabolites or enzymes with diverse biological actions (Papavizas 1985; Hermosa et al. 2000). Especially, serious outbreaks of green mould caused by Hypocrea have occurred world widely on commercial mushrooms (Seaby 1987; Castle et al. 1998). Green mould disease is characterized by an aggressive mycelium causing a soft decay and a rapid infestation of economically important crops including mushrooms (Castle et al. 1998; Dodd et al. 2002). Recently, a number of species of Hypocrea have been re-evaluated or newly found to be teleomorphs of Trichoderma (Dodd et al. 2002, 2003). However, only harzianum A was once found in T. harzianum (Corley et al. 1994) without descriptions of fungal morphology and phylogenetic analysis, but there has been no precedent for harzianum in Hypocrea or other Trichoderma species.
In this paper, we report the identification of a new harzianum toxin-producing fungal strain, Hypocrea sp. F000527, chemical elucidation of a trichothecene compound, harzianum A produced by the strain, and its cytotoxicity to human cell lines.
Materials and Methods
Fungal isolation, culture and identification
The fungal strain, F000527 used in this study, was isolated from a soil sample collected at Daejeon, Korea. The strain was cultured on potato dextrose agar (PDA; Difco Laboratories, Detroit, MI, USA) at 25°C and identified through the investigation of electron microscopic morphological features of spores, spore masses, phialides, conidiophores and hyphae. For the scanning electron microscopy, samples were fixed in 2·5% paraformaldehyde–glutaraldehyde mixture buffer with 0·1 mol l−1 phosphate (pH 7·2) for 2 h, postfixed in 1% osmium tetroxide in the same buffer for 1 h, dehydrated in ethanol and substituted by isoamyl acetate. Then it was dried at the critical point in CO2. Finally, the sample was sputtered with gold in a sputter coater (SC502; Polaron, West Sussex, UK) and observed using a scanning electron microscope (SEM515; Phillips, Eindhoven, the Netherlands). The fungal strain was cultured on wheat bran medium in order to evaluate its toxigenic potential. Wheat bran (200 g) as a solid medium was adjusted to about 45–50% moisture content in 500 ml Erlenmeyer flasks and autoclaved at 120°C at 15 psi for 20 min. The cultures were incubated at 28°C for 2 weeks prior to harvesting.
Fungal DNA extraction, amplification, sequencing and phylogenetic analyses
Total genomic DNAs were extracted from mycelia cultured on PDA plates covered with cellophane using AccuPrep® Genomic DNA Extraction Kit (Bioneer, Daejeon, Korea). From extracted genomic DNA, the internal transcribed spacers (ITS) 1 and 2 including 5·8S and 28S of nuclear rDNA were amplified with ITS1 and LR5 (1·5 kb) primers (White et al. 1990) using Quick PCR Premix containing Taq DNA polymerase, dNTPs, reaction buffer and tracking dye (GENENMED, Seoul, Korea). Each PCR reaction was conducted for 30 thermal cycles according to following conditions; 1 min at 95°C for denaturation, 1 min at 52°C for primer annealing, 1 min at 72°C for extension and 10 min at 72°C for terminal extension. Amplified PCR products were detected on 0·75% agarose gel through electrophoresis. Checked amplicons were purified with AccuPrep® PCR Purification Kit (Bioneer, Daejeon, Korea). The purified PCR products were sequenced with ABI3700 Automated DNA Sequencer (Applied Biosystems, Foster, CA, USA). For sequencing ITS and 28S region, primer pairs ITS4 and LR3 (White et al. 1990) were used. Sequences generated in this study were aligned with sequences retrieved from GenBank using CLUSTAL × ver. 1·83 (Thompson et al. 1997) with gap opening penalty 10·0 and gap extension penalty 0·02. Using PHYDIT program ver. 3·2 (Chun 1995), ambiguous and uninformative variable sites were excluded and a sequence data set was submitted to subsequent phylogenetic analyses. Parsimony analysis for most parsimonious (MP) tree was performed by PAUP 4·0b10 (Swofford 2002) using tree bisection reconnection branch swapping with MAXTREES unrestricted. All gaps were treated as missing data. Phylogenetic inference was performed by the neighbour-joining (NJ) method with the NEIGHBOR program of the PHYLIP package (Saitou and Nei 1987). Verticillium catenulatum (AY555963) was used as an outgroup species for the phylogenetic trees of Figs 2 and 3. The strain was frozen in sterile 20% glycerol and deposited in KRIBB (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea) and CBS (Centraalbureau voor Schimmelcultures, the Netherlands) under accession number of CBS 113214.
Extraction and isolation of harzianum
Wheat bran cultures were extracted with 50% acetone at room temperature twice. Acetone was evaporated and the residue was partitioned between H2O and ethyl acetate. The ethyl acetate fraction was chromatographed on a silica gel column (6 × 40 cm) eluting with a step gradient of CH2Cl2-MeOH (100 : 1, 50 : 1, 30 : 1, 20 : 1, 10 : 1, 5 : 1, 1 : 1, 100% MeOH, each 1·5 l) to give 11 fractions (Fr1 to Fr11). Active fraction Fr7 was subjected to Sephadex LH-20 (3·5 × 100 cm) chromatography eluted with MeOH to give four fractions (Fr7-1 to Fr7-4). Active subfraction Fr7-2 (156 mg) was subjected to semi-preparative HPLC (ODS-H80, 150 × 10 mm, YMC, Tokyo, Japan; CH3CN-H2O =30: 70 v/v, 0·05% trifluoroacetic acid in both solvent; flow rate, 3 ml min−1). Fractions eluted from 140 to 149 min yielded 52·4 mg of pure compound.
Determination of the chemical structure of harzianum
Preparative HPLC was carried out on J'sphere ODS-H80 (150 × 20 mm, YMC). Foetal bovine serum, media and supplement materials for cell culture were purchased from Gibco-BRL (Gaithersberg, MD, USA). Optical rotation of the compound was measured on a JASCO DIP-370 polarimeter. Melting point was measured on an Electrothermal 9100 instrument (Dubuque, IA, USA) without correction. UV spectra were obtained on a Milton Roy 3000 spectrometer (Ivyland, PA, USA). 1H NMR, 13C NMR, DEPT, HMQC, HMBC and NOESY spectra were recorded on Bruker DMX 600 NMR spectrometer (Karlsruhe, Germany) with CDCl3 as a solvent. HRFAB-MS was obtained on a JMS-HX110A/HX110A Tandem Mass spectrometer (JEOL, Tokyo, Japan).
Cytotoxicity assay of harzianum A
A cytotoxicity assay was carried out according to Denizot and Lang (1986). Cell lines used in this study were human fibrosarcoma (HT1080), HeLa, and human breast cancer (MCF-7). Each cell (concentration of 1 × 104) was seeded in each well containing 100 μl DMEM. Subsequently, various concentrations of samples were added. The cells were incubated for 48 h at 37°C in an atmosphere containing 5% (v/v) CO2, then 10 μl FBS-free medium containing MTT solution (5 mg ml−1) was added to the wells. After 4 h of incubation at 37°C, the medium was discarded and the formazan blue which formed in the cells was dissolved by adding 100 μl DMSO. Optical density was measured at 570 nm using a microplate reader (Molecular Devices, Menlo Park, CA, USA). Doxorubicin was used as a positive control.
Identification of the fungal strain F000527
On solid medium, fungal strain F000527 manifested rapid growth, of a white colour with a downy appearance and, with increasing maturity, turned green to deep green, sometimes forming concentric ring-like zones on the agar surface (Fig. 1). The strain F000527 had pyramidal pustular structure of conidiophores with paired branches and distinctive ellipsoidal conidia tending to coalesce to some extent from separate heads into larger round to ovoid masses (Fig. 1). Branches of conidiophores arising at more or less a right angle were observed. Phialides were short and wide, and solitary or in groups. Chlamydospores were absent in culture. Teleomorphic forms of asci and ascospores were not found in culture. The morphology of the strain resembled Trichoderma species. NJ and MP trees were constructed by using the ITS1 and ITS2 sequences, and almost the same topologies were obtained in both methods (Figs 2 and 3). When the 18S rDNA ITS sequence of the fungus was compared with 24 isolates of Hypocrea and Trichoderma species retrieved from GenBank, the strain had 100% and 98% sequence homologies to two unidentified Trichoderma species (AY154921 and AY154943) and H. lutea (AB027384) respectively. The new clade of F000527 and two unidentified Trichodermas, along with Trichoderma sp. Ir.29 (AY154920), formed a sister relationship with the clade that contains H. lutea group (Figs 2 and 3). However, the strain was distinguished from H. virens and H. koningii which were phylogenetically distant. It was also distinguished from other Trichoderma species in terms of phylogeny. Thus, the clade under investigation was believed to be a new Hypocrea group which is phylogenetically different from H. lutea. In addition, gene tree data from the sequence analysis showed that the strain is also different from T. harzianum [anamorph of H. lixii (AY222351)] previously known as a harzianum A producer.
Structure elucidation of harzianum
Signals in the 1H-NMR and 13C-NMR spectra of the compound showed the expected resonances for a trichothecene nucleus: a vinyl methyl at δH 1·72; an olefinic proton at δH 5·42; a 1,1-disubstituted epoxide AB pattern at δH 2·86 and δH 3·16; two oxygen-bearing methine signals at δH 3·87 and δH 3·64; and two methyl signals at δH 0·73 and δH 0·97. A signal at δH 5·66, which correlated to C-4 at δC 75·27 in HMQC, showed long-range correlation with a carbonyl at δC 165·66 in HMBC spectrum, indicating an ester linkage at this site. The UV spectrum (λmax = 305, logε 4·33) was indicative of a triene moiety. The coupling constants of olefinic protons suggested geometric arrangements between each pair of the triene moiety. These were supported by observation of the NOE enhancement. Consequently, compound A was concluded to be cis-trans-trans stereochemistry for 2′, 4′, 6′-octatrienedioic acid esterified on the 4β hydroxyl group of trichodermol. Signals in the 1H-NMR and 13C-NMR spectra of the compound matched a pattern of harzianum A with almost same chemical shifts (Corley et al. 1994; Table 1). Its molecular formula was determined to be C23H28O6 from HRFAB-MS. Harzianum A was obtained as colourless oil [α]: 71·25° (c. 1·00, CHCl3); UV: λmax(CHCl3) = 205 (4·17), 306 (logɛ 4·36) nm; ESI-MS m/z 399·4 [M-H]+ (calculated for C23H28O6). The compound A was identified as a trichothecene derivative, harzianum A.
Table 1. 1H and 13C NMR spectral data of harzianum A (CDCl3) [δ values, (J) in Hz]
3·87 d (5·4)
2·59 dd (15·6, 7·5)
2·08 ddd (15·6, 5·2, 3·6)
5·66 dd (7·8, 3·3)
5·42 d (4·2)
3·64 d (5·4)
3·16 d (3·9)
2·86 d (3·9)
5·87 d (11·1)
6·66 t (11·4)
7·95 dd (14·7, 11·7)
6·57 dd (15·0, 11·4)
7·51 dd (15·3, 11·4)
6·02 d (15·3)
Cytotoxicity of harzianum A
Harzianum A was tested for their cytotoxicity to HeLa, MCF-7 and HT1080 cell lines. The results (IC50 values) are summarized in Table 2. Harzianum A showed cytotoxicity to a human fibrosarcoma cell line (HT1080) with IC50 value of 0·65 μg ml−1, HeLa cell line with IC50 value of 5·07 μg ml−1, and a human breast cancer cell line (MCF-7) with IC50 value of 10·13 μg ml−1.
Table 2. Cytotoxicity of harzianum A to tumour cell lines
Data are mean ± SD (IC50, μg ml−1) from two separate experiments.
5·07 ± 0·18
10·13 ± 0·33
0·65 ± 0·03
0·24 ± 0·02
9·68 ± 0·21
0·48 ± 0·01
This study reports the production of cytotoxic harzianum A from a new Hypocrea strain. In spite of serious outbreaks of green mould caused by Hypocrea (Seaby 1987; Castle et al. 1998), a survey of Hypocrea distribution, the natural occurrence of Hypocrea-derived mycotoxins and associated toxicities when isolated from contaminated agricultural products have not yet been undertaken. However, it seems quite possible for Hypocrea or Trichoderma and related species to exploit various natural and commercial niches such as mushroom production facilities, woodlands, varying crops, soils, producing trichothecene derivatives and mediating green mould disease.
In the course of our preliminary screening for NF-κB modulators, harzianum A metabolite has been isolated from Hypocrea. This is the first report on such a screening from a new Hypocrea species and its cytotoxicity to human cancer cell lines. Corley et al. (1994) reported that harzianum A from T. harzianum showed no cytotoxicity when assayed with baby hamster kidney cells at concentrations up to 1 μg ml−1, no antimicrobial activity when assayed with E. coli, B. subtilis, M. luteus or S. aureus at 1 mg ml−1, and only modest antifungal activity when tested with C. albicans and S. cerevisiae at 100 μg ml−1. However, our study showed that harzianum A was apparently cytotoxic to several human cancer cell lines, especially to a fibrosarcoma cell line.
In conclusion, the morphology along with the sequence data of ITS rDNA proved that the strain has a taxon of its own. Our study suggested that there is a possibility that harzianum A and related trichothecenes could be produced by Hypocrea or Trichoderma which as hazardous mycotoxins may widely contaminate agricultural crops, byproducts and wastes, giving rise to serious consequences. More studies on other harzianum derivatives produced by the new strain are now under way. Agricultural products always have economical importance, and hence further investigation of the distribution of trichothecene producing Hypocrea should be envisaged.
This work was supported by grant no. R01-2002-000-00304-0 from the Basic Research Program of the Korea Science and Engineering Foundation and by grant (MG02-0101-002-1-0-0) from Microbial Genomics and Application Center of 21st Century Frontier R and D Program funded by the Ministry of Science and Technology of the Korean Government. This research was also supported by the Brain Korea 21 Research Fellowship from the Ministry of Education and Human Resources Development.