Induction of cytoplasmic leakage from Rhizoctonia solani hyphae by Gliocladium virens and partial characterization of a leakage factor

The biocontrol fungus Gliocladium virens (Gl‐21)was grown on various solid and liquid substrates. Aqueous extracts of wheat bran and peanut hull meal (PHM)as well as spent glucose tartrate broth (GTB), Czapek‐Dox broth (CDB) and potato dextrose broth (PDB) upon which Gl‐21 was grown caused leakage of carbohydrates and electrolytes from hyphae of the soilborne plant pathogen Rhizoctonia solani. In addition, the mycelial weight of R. solani was reduced. Most of the leakage factor was formed in the substrates within six days of incubation. Acidification of the bran and the PHM media before inoculation stimulated production of the leakage factor by G. virens. Size‐fractionation experiments indicated that a combination of factors produced by G. virens induced leakage from R. solani. Although the < 1 k Da fraction from the water‐extracted bran culture contained significant leakage activity, it was less than in the non‐fractionated bran culture. The > 1 k Da fraction from bran culture did not induce leakage activity. When the < 1 k Da and the > 1 k Da fractions were recombined, leakage activity was restored to a level similar to that of the non‐fractionated preparation. Gliotoxin was detected in culture filtrates from G. virens grown on bran and PHM media. Gliotoxin preparations induced leakage of carbohydrates and electrolytes from R. solani and caused a concomitant reduction in mycelial weight, which suggests that it is a leakage factor.     

Development of a continuous cell line, PBLE, from an American eel peripheral blood leukocyte preparation

Summary A continuous cell line, PBLE, was developed from the adherent cells in a culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. The cells were grown in Leibovitz's L-15 basal medium supplemented with 20% fetal bovine serum (FBS). Under normal culture conditions at 18° C, the morphology of PBLE was fibroblast-like. The cultures have been subcultured over 80 times and have been cryopreserved successfully. These cells have a diploid karyotype of 38 chromosomes, survived temperatures from 5 to 36° C, and proliferated at temperatures from 5° C to at least 30° C. PBLE underwent apoptosis in response to gliotoxin, but did not show a respiratory burst. Results suggest that PBLE may have arisen from a circulating mesenchymal stem cell. PBLE was susceptible to Chum salmon reovirus (CSV) and supported CSV replication. Therefore this cell line should be useful in studying eel specific virus-host interactions.     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Plasmodium falciparum: the fungal metabolite gliotoxin inhibits proteasome proteolytic activity and exerts a plasmodicidal effect on P. falciparum

The in vitro antimalarial activity of the fungal metabolite gliotoxin (GTX) was evaluated, and its mechanism of action was studied. GTX showed plasmodicidal activity against both Plasmodium falciparum chloroquine-resistant strain K-1 and chloroquine-susceptible strain FCR-3. GTX cytotoxicity was significantly lower against a normal liver cell line (Chang Liver cells). The intracellular reduced glutathione level of parasitized and of normal red blood cells was not affected by GTX treatment. However, GTX decreased the chymotrypsin-like activity of parasite proteasomes in a time-dependent manner. The results of this study indicate that GTX possesses plasmodicidal activity and that this effect is due to inhibition of parasite proteasome activity, suggesting that GTX may constitute a useful antimalarial therapy.     

Clinical isolates of yeast produce a gliotoxin-like substance

Candida infections are major causes of morbidity in compromised human hosts, but our understanding of the virulence of Candida remains incomplete. The possibility that toxic fungal metabolites belonging to the chemical class epipolythiodioxopiperzine (ETP), which are reported to possess immunomodulating and antiphagocytic properties may be produced by Candida species was investigated. Reversed phase HPLC analysis of flash evaporated chloroform extracts of 7 day cultures of clinical Candida isolates grown in Minimal Essential Medium (MEM) with 5% fetal calf serum revealed the presence of a compound which eluted at the same time as the ETP, gliotoxin. Of 50 strains of yeast tested, 32 produced this gliotoxin-like material. This material was tested for other properties of ETP type toxins including the presence of mercaptans (Ellman reaction), ultraviolet absorbance spectrum and antibacterial activity against Micrococcus lutea. These tests revealed gliotoxin-like material from yeast cultures to be similar to commercially supplied gliotoxin. This represents the first report of the presence of ETP-like compounds in yeast and raises the possibility that ETP's may contribute to the virulence of the organism.     

胶霉毒素的研究进展

胶霉毒素(gliotoxin,GT)是一个分子量为326 Da的小分子化合物,其骨架是由非核糖体肽合成酶Gli P催化苯丙氨酸和丝氨酸缩合成的环二肽,属于表聚硫代哌嗪二酮类化合物,是一种重要的真菌次级代谢产物。体内外研究已经表明,GT对动植物产生多种效应,不仅具有免疫抑制功能和诱导细胞凋亡作用,在生物防治方面也具有潜在的应用价值。本文将对有关GT生物合成、诱导宿主效应机制及其潜在应用价值进行综述。     

Glutathione intensifies gliotoxin-induced cytotoxicity in human neuroblastoma SH-SY5Y cells

Gliotoxin is a fungal second metabolite produced by diverse species that can be found in compost, stored crops, moist animal feed and sawdust. The role of glutathione in gliotoxin-induced toxicity was studied in order to elucidate the toxic mechanisms leading to neurite degeneration and cell death in differentiated human neuroblastoma (SH-SY5Y) cells. After 72 h of exposure to gliotoxin, moderate cytotoxicity was induced at 0.1 μmol/L, which was more severe at higher concentrations. A reduction in the number of neurites per cell was also observed. By decreasing the level of intracellular glutathione with l-buthionine-sulfoxamine (BSO) a specific inhibitor of glutathione synthesis, the cytotoxic effect of gliotoxin was significantly attenuated. The gliotoxin-induced cytotoxicity was also slightly reduced by the antioxidant vitamin C. However, the neurite degenerative effect was not altered by BSO, or by vitamin C. A concentration-dependent increase in the ratio between oxidized and reduced forms of glutathione, as well as the total intracellular glutathione levels, was noted after exposure to gliotoxin. The increase of glutathione was also reflected in western blot analyses showing a tendency for the regulatory subunit of γ-glutamylcysteine synthetase to be upregulated. In addition, the activity of glutathione reductase was slightly increased in gliotoxin-exposed cells. These results indicate that glutathione promotes gliotoxin-induced cytotoxicity, probably by reducing the ETP (epipolythiodioxopiperazine) disulfide bridge to the dithiol form.     

Gliotoxin enhances radiotherapy via inhibition of radiation-induced GADD45a, p38, and NFkappaB activation

The purpose of the study was to elucidate the mechanism underlying the enhancement of radiosensitivity to 60Co gamma-irradiation in human hepatoma cell line HepG2 pretreated with gliotoxin. Enhancement of radiotherapy by gliotoxin was investigated in vitro with human hepatoma HepG2 cell line. Apoptosis related proteins were evaluated by Western blotting. Annexin V/PI and reactive oxygen species (ROS) were quantified by Flow Cytometric (FACS) analysis. Gliotoxin (200 ng/ml) combined with radiation (4 Gy) treated cells induced apoptosis. Cells treated with gliotoxin (200 ng/ml) prior to irradiation at 4 Gy induced the expression of bax and nitric oxide (NO). The gliotoxin-irradiated cells also increased caspase-3 activation and ROS. Gadd45a, p38, and nuclear factor kappa B (NFkappaB) activated in irradiated cells was inhibited by Gliotoxin. Specific inhibitors of p38 kinase, SB203580, significantly inhibited NFkappaB activation and increased the cytotoxicity effect in cells exposed to gliotoxin combined with irradiation. However, SB203580 did not suppress the activation of Gadd45a in irradiated cells. Gliotoxin inhibited anti-apoptotic signal pathway involving the activation of Gadd45a-p38-NFkappaB mediated survival pathway that prevent radiation-induced cell death. Therefore, gliotoxin, blocking inflammation pathway and enhancing irradiation-induced apoptosis, is a promising agent to increase the radiotherapy of tumor cells.     

三个木霉菌株防病促生效应

【背景】木霉(Trichoderma sp.)是重要的生防微生物资源,对不同的木霉菌株进行生物学特性比较,可为获得功能较广的复配型生防菌剂提供参考信息。【目的】通过对绿木霉(Trichoderma virens) T23、哈茨木霉(Trichoderma harzianum) T22、G30的防病效果及对作物的促生潜力等生物学特性进行比较分析,明确不同菌株的生物功能差异。【方法】用平板拮抗法比较分析菌株T23、T22、G30对植物病原真菌的拮抗效果;用乙酸乙酯萃取法对菌株T23、T22、G30培养液中的胶毒素进行提取并通过高效液相色谱法(high-performance liquid chromatography, HPLC)检测胶毒素;用打孔法比较分析培养液萃取物对植物病原真菌的拮抗效果;通过温室接种验证菌株T23、T22、G30的防病效果;分别以磷酸钙、卵磷脂为唯一磷源检测菌株T23、T22、G30对难溶性磷的转化利用潜力,用电感耦合等离子发射光谱法(inductively coupled plasma-atomic emission spectrometry, ICP-AES)检...     

Cytotoxic Substances from Aspergillus fumigatus in Oxygenated or Poorly Oxygenated Environment

Aspergillus fumigatus often causes serious health problems. The airway of the human body, the most common initial site of damage, is always exposed to an oxygenated condition, and the oxygen concentration may play a critical role in the virulence of A. fumigatus. In this study, oxygen content, fungal growth, the production of cytotoxic substance(s) in the fungal culture, and their relationship were investigated. Two clinical strains of A. fumigatus were cultured under certain oxygen contents (10, 14 and 20%), and cytotoxicity of their culture filtrates on murine macrophages and their fungal growth were evaluated. The components of these filtrates were analyzed by gas chromatography-mass spectrometry. All culture filtrates contained gliotoxin and showed potent cytotoxicity on macrophages at very low concentration. The amount of gliotoxin in the culture filtrate prepared at 10% oxygen was markedly less, but diminutions in fungal growth and cytotoxicity of this culture filtrate were negligible. These results suggest that a well-oxygenated condition is suitable for the production of gliotoxin by A. fumigatus. A significant role of cytotoxic substances(s) other than gliotoxin is also suggested.