Isolation and Characterisation of Toxic Secondary Metabolites Produced by Black Mould

<p>Microbial secondary metabolites, commonly called natural products, have been crucial for the progression of modern medicine. Not essential for the basic functions of life, secondary metabolites are instead produced to provide a competitive advantage in the environment. The method of action is commonly toxicity to other species in their environment, thereby harming or killing the competition. These toxic properties have allowed them to be utilised as antimicrobial and antitumor agents, however this same toxicity is able to cause detrimental health effects in humans causing symptoms ranging from minor to life threatening. The black mould Stachybotrys chartarum is capable of producing very toxic secondary metabolites called macrocyclic trichothecenes. Satratoxin G (6) and H (7), are two of the most toxic naturally occurring compounds in the world. This has made S. chartarum a common target when adverse health has been associated with damp and mouldy dwellings. However, there is very little evidence for this link beyond its ubiquity and ability to produce the aforementioned highly toxic macrocyclic trichothecenes. This research investigates S. chartarum and the toxic secondary metabolites it produces, with special emphasis on satratoxin G and H. Different culturing methods and resulting morphology are assessed. The satratoxins were isolated from crude extracts and full characterisation by 1-D and 2-D NMR spectroscopy was done. This process revealed differences from the accepted literature, and spectra are reported herein to aid in future identification. The importance of genetics and the public health implications of mould contamination are also discussed.</p>

Isolation and Characterisation of Toxic Secondary Metabolites Produced by Black Mould

<p>Microbial secondary metabolites, commonly called natural products, have been crucial for the progression of modern medicine. Not essential for the basic functions of life, secondary metabolites are instead produced to provide a competitive advantage in the environment. The method of action is commonly toxicity to other species in their environment, thereby harming or killing the competition. These toxic properties have allowed them to be utilised as antimicrobial and antitumor agents, however this same toxicity is able to cause detrimental health effects in humans causing symptoms ranging from minor to life threatening. The black mould Stachybotrys chartarum is capable of producing very toxic secondary metabolites called macrocyclic trichothecenes. Satratoxin G (6) and H (7), are two of the most toxic naturally occurring compounds in the world. This has made S. chartarum a common target when adverse health has been associated with damp and mouldy dwellings. However, there is very little evidence for this link beyond its ubiquity and ability to produce the aforementioned highly toxic macrocyclic trichothecenes. This research investigates S. chartarum and the toxic secondary metabolites it produces, with special emphasis on satratoxin G and H. Different culturing methods and resulting morphology are assessed. The satratoxins were isolated from crude extracts and full characterisation by 1-D and 2-D NMR spectroscopy was done. This process revealed differences from the accepted literature, and spectra are reported herein to aid in future identification. The importance of genetics and the public health implications of mould contamination are also discussed.</p>

Differentiation of S. chartarum (Ehrenb.) S. Hughes Chemotypes A and S via FT-IR Spectroscopy

Stachybotrys (S.) chartarum is a cellulolytic mould with the ability to produce highly cytotoxic macrocyclic trichothecenes. Two chemotypes are defined according to their ability to produce either atranones or satratoxins. S. chartarum has been well known as the causative agent of the lethal disease stachybotryotoxicosis in horses. Further investigations revealed that this disease is strictly correlated with the presence of macrocyclic trichothecenes. Furthermore, their occurrence in water-damaged buildings has been linked to adverse health effects such as the sick building syndrome. As the chemotypes cannot be characterized via phenotypic criteria, different methods such as PCR, MALDI–TOF MS, LC–MS/MS, thin-layer chromatography and cytotoxicity assays have been used so far. Fourier-transform-infrared spectroscopy (FT-IR) is commonly used for the differentiation of bacteria and yeasts, but this technique is also applicable to filamentous fungi. Hence, this study aimed at evaluating to which extent a reliable differentiation of S. chartarum chemotypes A and S is possible. Besides, another objective was to verify if the recently introduced third genotype of S. chartarum can be identified. Therefore, 28 strains including the two chemotypes and the third genotype H were cultivated on malt extract agar (MEA) and potato dextrose agar in three biological replicates. Each sample was applied to FT-IR measurements on day 7, 14 and 21 of cultivation. In this study, we achieved a distinction of the chemotypes A and S via FT-IR spectroscopy after incubation for 7 days on MEA. In terms of genotype differentiation, the PCR detecting satratoxin- and atranone-gene clusters remained the only applicable method.

Toxin Production by Stachybotrys chartarum Genotype S on Different Culture Media

Stachybotrys (S.) chartarum had been linked to severe health problems in humans and animals, which occur after exposure to the toxic secondary metabolites of this mold. S. chartarum had been isolated from different environmental sources, ranging from culinary herbs and improperly stored fodder to damp building materials. To access the pathogenic potential of isolates, it is essential to analyze them under defined conditions that allow for the production of their toxic metabolites. All Stachybotrys species are assumed to produce the immunosuppressive phenylspirodrimanes, but the highly cytotoxic macrocyclic trichothecenes are exclusively generated by the genotype S of S. chartarum. In this study, we have analyzed four genotype S strains initially isolated from three different habitats. We grew them on five commonly used media (malt-extract-agar, glucose-yeast-peptone-agar, potato-dextrose-agar, cellulose-agar, Sabouraud-dextrose-agar) to identify conditions that promote mycotoxin production. Using LC-MS/MS, we have quantified stachybotrylactam and all S-type specific macrocyclic trichothecenes (satratoxin G, H, F, roridin E, L-2, verrucarin J). All five media supported a comparable fungal growth and sporulation at 25 °C in the dark. The highest concentrations of macrocyclic trichothecenes were detected on potato-dextrose-agar or cellulose-agar. Malt-extract-agar let to an intermediate and glucose-yeast-peptone-agar and Sabouraud-dextrose-agar to a poor mycotoxin production. These data demonstrate that the mycotoxin production clearly depends on the composition of the respective medium. Our findings provide a starting point for further studies in order to identify individual components that either support or repress the production of mycotoxins in S. chartarum.

Recent Advances on Macrocyclic Trichothecenes, Their Bioactivities and Biosynthetic Pathway

Macrocyclic trichothecenes are an important group of trichothecenes bearing a large ring. Despite the fact that many of trichothecenes are of concern in agriculture, food contamination, health care and building protection, the macrocyclic ones are becoming the research hotspot because of their diversity in structure and biologic activity. Several researchers have declared that macrocyclic trichothecenes have great potential to be developed as antitumor agents, due to the plenty of their compounds and bioactivities. In this review we summarize the newly discovered macrocyclic trichothecenes and their bioactivities over the last decade, as well as identifications of genes tri17 and tri18 involved in the trichothecene biosynthesis and putative biosynthetic pathway. According to the search results in database and phylogenetic trees generated in the review, the species of the genera Podostroma and Monosporascus would probably be great sources for producing macrocyclic trichothecenes. Moreover, we propose that the macrocyclic trichothecene roridin E could be formed via acylation or esterification of the long side chain linked with C-4 to the hydroxyl group at C-15, and vice versa. More assays and evidences are needed to support this hypothesis, which would promote the verification of the proposed pathway.

Aerosolization of Mycotoxins after Growth of Toxinogenic Fungi on Wallpaper

ABSTRACT Many fungi can develop on building material in indoor environments if the moisture level is high enough. Among species that are frequently observed, some are known to be potent mycotoxin producers. This presence of toxinogenic fungi in indoor environments raises the question of the possible exposure of occupants to these toxic compounds by inhalation after aerosolization. This study investigated mycotoxin production by Penicillium brevicompactum, Aspergillus versicolor, and Stachybotrys chartarum during their growth on wallpaper and the possible subsequent aerosolization of produced mycotoxins from contaminated substrates. We demonstrated that mycophenolic acid, sterigmatocystin, and macrocyclic trichothecenes (sum of 4 major compounds) could be produced at levels of 1.8, 112.1, and 27.8 mg/m2, respectively, on wallpaper. Moreover, part of the produced toxins could be aerosolized from the substrate. The propensity for aerosolization differed according to the fungal species. Thus, particles were aerosolized from wallpaper contaminated with P. brevicompactum when an air velocity of just 0.3 m/s was applied, whereas S. chartarum required an air velocity of 5.9 m/s. A. versicolor was intermediate, since aerosolization occurred under an air velocity of 2 m/s. Quantification of the toxic content revealed that toxic load was mostly associated with particles of size ≥3 μm, which may correspond to spores. However, some macrocyclic trichothecenes (especially satratoxin H and verrucarin J) can also be found on smaller particles that can deeply penetrate the respiratory tract upon inhalation. These elements are important for risk assessment related to moldy environments. IMPORTANCE The possible colonization of building material by toxinogenic fungi in cases of moistening raises the question of the subsequent exposure of occupants to aerosolized mycotoxins. In this study, we demonstrated that three different toxinogenic species produce mycotoxins during their development on wallpaper. These toxins can subsequently be aerosolized, at least partly, from moldy material. This transfer to air requires air velocities that can be encountered under real-life conditions in buildings. Most of the aerosolized toxic load is found in particles whose size corresponds to spores or mycelium fragments. However, some toxins were also found on particles smaller than spores that are easily respirable and can deeply penetrate the human respiratory tract. All of these data are important for risk assessment related to fungal contamination of indoor environments.