Verruculogen

Background:The role of Aspergillus fumigatus mycotoxins in the colonization of the respiratory tract by conidia has not been studied extensively, even though patients at risk from invasive aspergillosis frequently exhibit respiratory epithelium damage. In a previous study, we found that filtrates of A. fumigatus cultures can specifically alter the electrophysiological properties of human nasal epithelial cells (HNEC) compared to those of non pathogenic moulds.Results:We fractionated the organic phase of filtrate from 3-day old A. fumigatus cultures using high-performance liquid chromatography. The different fractions were tested for their ability to modify the electrophysiological properties of HNEC in an in vitro primary culture model. The fraction collected between 20 and 30 min mimicked the effects of the whole filtrate, i.e. decrease of transepithelial resistance and increase of potential differences, and contained secondary metabolites such as helvolic acid, fumagillin, and verruculogen. Only verruculogen (10(-8) M) had effects similar to the whole filtrate. We verified that verruculogen was produced by a collection of 67 human, animal, plant and environmental A. fumigatus isolates. Using MS-MS analysis, we found that verruculogen was associated with both mycelium and conidia extracts.Conclusion:Verruculogen is a secondary metabolite that modifies the electrophysiological properties of HNEC. The role of these modifications in the colonization and invasion of the respiratory epithelium by A. fumigatus on first contact with the epithelium remains to be determined.

Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin-type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O-prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His(8)-FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K(M) values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM, respectively. Average turnover number (k(cat)) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s(-1). FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13-dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.

Verruculogen is a tremorgenic mycotoxin and contains an endoperoxide bond. In this study, we describe the cloning, overexpression and purification of a non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase FtmOx1 from Aspergillus fumigatus, which catalyses the conversion of fumitremorgin B to verruculogen by inserting an endoperoxide bond between two prenyl moieties. Incubation with (18)O(2)-enriched atmosphere demonstrated that both oxygen atoms of the endoperoxide bond are derived from one molecule of O(2). FtmOx1 is the first endoperoxide-forming non-heme Fe(ii) and alpha-ketoglutarate-dependent dioxygenase reported so far. A mechanism of FtmOx1-catalysed verruculogen formation is postulated and discussed.