David R. McMullin

Chaetoglobosins and azaphilones produced by Canadian strains of Chaetomium globosum isolated from the indoor environment

Chaetomium globosum is one of the most common species of fungi found growing on damp building materials in North America and Europe. At doses that could be experienced in a building with some mould damage, exposure to metabolites from other fungi results in inflammatory changes in vivo and in vitro. This research requires knowledge of the dominant toxins produced by fungal strains from the built environment and characterization of pure compounds for toxicity testing. We examined 25 strains of C. globosum isolated from the built environment in Canada. In varying amounts, these strains primarily produced chaetoglobosin A, C and F, chaetomugilin D, and chaetoviridin A. Spectroscopic data of the major isolated compounds are provided. Previous studies reported a number of metabolites from this species that we did not find. However, this appears to be due to misidentifications of the fungi they examined as well as problems with the analytical methods used. In addition, our data support the use of metabolite profiles for resolving the taxonomy of some economically important Chaetomium species.

Antimicrobial dihydrobenzofurans and xanthenes from a foliar endophyte of Pinus strobus

Foliar fungal endophytes of Pinus strobus (eastern white pine) were collected from different sites across south-eastern New Brunswick, Canada and screened for the production of bioactive metabolites. From one site, two fungal isolates representing a formerly unknown genus and species within the family Massarinaceae (Pleosporales, Dothideomycetes, Ascomycota) were resolved by phylogenetic analysis. These isolates produced crude organic extracts that were active against Microbotryum violaceum and Saccharomyces cerevisiae. From these strains, DAOM 242779 and 242780, four dihydrobenzofurans (1-4) and two xanthenes (5-6) were characterized. Structures were elucidated by HRMS, interpretation of NMR spectra and other spectroscopic techniques. All isolated metabolites displayed antimicrobial activity against the biotrophic fungal pathogen M. violaceum and Bacillus subtilis.

Isochromans and α‑Pyrones from Penicillium corylophilum

Seven new secondary metabolites, four isochromans (1-4) and three α-pyrones (5-7), were isolated from Penicillium corylophilum DAOM 242293 collected from a damp building in Halifax, Canada. Their structures were elucidated by HRESIMS, 1D and 2D NMR, chemical derivatization, ORD, UV, and comparison to the literature. Related isochromans have previously been reported from other Penicillium species; however, to our knowledge this is the first report of α-pyrones from P. corylophilum. Compounds 1-4 demonstrated antifungal activity against Saccharomyces cerevisiae at 100 μg mL(-1).

Ochratoxin A production by Penicillium thymicola.

Ochratoxin A (OTA) is a mycotoxin produced by some Aspergillus and Penicillium species that grow on economically important agricultural crops and food products. OTA is classified as Group 2B carcinogen and is potently nephrotoxic, which is the basis for its regulation in some jurisdictions. Using high resolution mass spectroscopy, OTA and ochratoxin B (OTB) were detected in liquid culture extracts of Penicillium thymicola DAOMC 180753 isolated from Canadian cheddar cheese. The genome of this strain was sequenced, assembled and annotated to probe for putative genes involved in OTA biosynthesis. Known OTA biosynthetic genes from Penicillium verrucosum or Penicillium nordicum, two related Penicillium species that produce OTA, were not found in P. thymicola. However, a gene cluster containing a polyketide synthase (PKS) and PKS-nonribosomal peptide synthase (NRPS) hybrid encoding genes were located in the P. thymicola genome that showed a high degree of similarity to OTA biosynthetic enzymes of Aspergillus carbonarius and Aspergillus ochraceus. This is the first report of ochratoxin from P. thymicola and a new record of the species in Canada.

Secondary metabolites from Penicillium corylophilum isolated from damp buildings

Indoor exposure to the spores and mycelial fragments of fungi that grow on damp building materials can result in increased non-atopic asthma and upper respiratory disease. The mechanism appears to involve exposure to low doses of fungal metabolites. Penicillium corylophilum is surprisingly common in damp buildings in USA, Canada and western Europe. We examined isolates of P. corylophilum geographically distributed across Canada in the first comprehensive study of secondary metabolites of this fungus. The sesquiterpene phomenone, the meroterpenoids citreohybridonol and andrastin A, koninginin A, E and G, three new alpha pyrones and four new isochromans were identified from extracts of culture filtrates. This is the first report of koninginins, meroterpenoids and alpha pyrones from P. corylophilum. These secondary metabolite data support the removal of P. corylophilum from Penicillium section Citrina and suggest that further taxonomic studies are required on this species.

Metabolites of Trichoderma species isolated from damp building materials

Buildings that have been flooded often have high concentrations of Trichoderma spores in the air while drying. Inhaled spores and spore and mycelial fragments contain large amounts of fungal glucan and natural products that contribute to the symptoms associated with indoor mould exposures. In this study, we considered both small molecules and peptaibol profiles of T. atroviride, T. koningiopsis, T. citrinoviride, and T. harzianum strains obtained from damp buildings in eastern Canada. Twenty-residue peptaibols and sorbicillin-derived metabolites (1-6) including a new structure, (R)-vertinolide (1), were characterized from T. citrinoviride. Trichoderma koningiopsis produced several koninginins (7-10), trikoningin KA V, and the 11-residue lipopeptaibols trikoningin KB I and trikoningin KB II. Trichoderma atroviride biosynthesized a mixture of 19-residue trichorzianine-like peptaibols, whereas T. harzianum produced 18-residue trichokindin-like peptaibols and the 11-residue harzianin HB I that was subsequently identified from the studied T. citrinoviride strain. Two α-pyrones, 6-pentyl-pyran-2-one (11) and an oxidized analog (12), were produced by both T. atroviride and T. harzianum. Aside from exposure to low molecular weight natural products, inhalation of Trichoderma spores and mycelial fragments may result in exposure to membrane-disrupting peptaibols. This investigation contributes to a more comprehensive understanding of the biologically active natural products produced by fungi commonly found in damp buildings.

New 1,3-benzodioxin-4-ones from Synnemapestaloides ericacearum sp. nov., a biosynthetic link to remarkable compounds within the Xylariales

Surveys of foliar endophytes from the Acadian forest region over the past three decades have identified numerous phylogenetically diverse fungi producing natural products toxic to forest pests and diseases. The life histories of some conifer endophytes can be restricted to plant foliage or may include saprotrophic phases on other plants tissues or even alternate hosts. Considering the potentially broad host preferences of conifer endophytes we explored fungi isolated from understory species and their metabolites as part of an ongoing investigation of fungal biodiversity from the Acadian forest. We report a hitherto unidentified Xylariomycetidae species isolated from symptomatic Labrador tea (Rhododendron groenlandicum) leaves and mountain laurel (Kalmia latifolia) collected in coastal southern New Brunswick, Canada. Morphological and phylogenetic evidence demonstrated the unknown species was a novel Synnemapestaloides (Sporocadaceae) species, described here as Syn. ericacearum. A preliminary screening assay indicated that the culture filtrate extract of the new species was potently antifungal towards the biotrophic pathogen Microbotryum violaceum, warranting an investigation of its natural products. Two natural products possessing a rare 1,3-benzodioxin-4-one scaffold, synnemadoxins A-B (1–2), and their postulated precursor, synnemadiacid A (3), were characterized as new structures and assessed for antimicrobial activity. All isolated compounds elicited in vitro inhibitory antifungal activity towards M. violaceum at 2.3 μg mL-1 and moderate antibiotic activity. Further, the characterization of synnemadoxins A-B provided a perspective on the biosynthesis of some related 1,3-benzodioxin-4-ones produced by other fungi within the Xylariales.

Toxigenic Foliar Endophytes from the Acadian Forest

This chapter describes the ecology of foliar endophytes of the Acadian Forest that dominates Canada’s Maritime Provinces extending into Eastern Quebec and Maine. Recent evidence has illuminated the ‘foraging ascomycete’ life habit of fungi that can be endophytic in conifer needles. These fungi can occupy several eco-niches other than the needles including as saprophytes in aquatic or terrestrial environments or as endophytes of understory species. Structurally diverse secondary antifungal and antiinsectan metabolites appear to mediate the exchange between plant and fungus. The plant provides nutrients and shelter, the fungus increases plant fitness by contributing to tolerance to herbivorous insects or needle pathogens. This work is enabled by the advent of affordable sequencing capability, a dedication to fieldwork and alpha taxonomy, and directed investigations of the metabolites produced by these interesting fungi.

Inflammation-associated gene expression in RAW 264.7 macrophages induced by toxins from fungi common on damp building materials

Most fungi that grow on damp building materials produce low molecular weight compounds, some of which are known to be toxic. In this study, we tested the hypothesis that exposure to some metabolites of fungi common on damp building materials would result in time-, dose-, and compound-specific responses in the production of various chemokines by RAW 264.7 cells. Cell cultures were exposed to a 10-7M or 10-8M metabolite dose for 2, 4, 8 or 24h. Metabolite concentrations used were based on those that might be expected in alveolar macrophages due to inhalation exposure from living or working in a damp building. Compared to controls, exposure provoked significant time-, dose- and compound-specific responses manifest as differentially elevated secretion of three of nine cytokines tested in culture supernatant of treated cells. The greatest number of cytokines produced in response to the metabolites tested were in andrastin A-treated cells (GM-CSF, TGFβ1, Tnf-α) followed by koninginin A (TGFβ1 and Tnf-α) and phomenone (GM-CSF, TGFβ1). Chaetoglobosin A, chaetomugilin D and walleminone exposures each resulted in significant time-specific production of Tnf-α only. This investigation adds to a body of evidence supporting the role of low molecular weight compounds from damp building materials as pathogen associated molecular patterns (PAMPs). Along with fungal glucan and chitin, these compounds contribute to the non-allergy based respiratory outcomes for people living and working in damp buildings.