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The Ascomycete fungus Claviceps gigantea infects maize kernels and synthetizes several alkaloids, mostly dihydrolysergamides. There is limited information on the damage these toxins cause in mammals, despite reports from infested areas with 90% presence of the fungus sclerotia. With this background, it was decided to determine the biological activity of chemical compounds present in sclerotia of C. gigantea in rabbits 38 days after weaning. Sclerotia of C. gigantea were collected in fields with high incidence of the disease, ground and analysed for nutrients. Experimental diets were prepared with four treatments, where sclerotial powder was added, substituting for alfalfa flour in increasing proportions [C. gigantea/alfalfa flour (0:100, 5:95, 15:85 and 25:75)]. Total ergot alkaloid content was analysed by high-performance liquid chromatography. Male juvenile rabbits were utilised and distributed in completely randomised design with four replications. Initial weight was recorded in each animal, and experimental diet was offered. In this study, weight of animals, feed consumption and feed conversion were evaluated in individual animals. Blood samples were taken for haemograms, and finally euthanasia was practiced. The consumption of C. gigantea had a negative effect on body weight and feed consumption. The necropsies showed anomalies proportional to the consumption of feed contaminated with the fungus. [ABSTRACT FROM AUTHOR]

Bioactive ergot alkaloids produced by several species of fungi are important molecules in agriculture and medicine. Much of the ergot alkaloid pathway has been elucidated, but a few steps, including the gene controlling hydroxylation of festuclavine to fumigaclavine B, remain unsolved. Festuclavine is a key intermediate in the fumigaclavine branch of the ergot alkaloid pathway of the opportunistic pathogen Neosartorya fumigata and also in the dihydrolysergic acid-based ergot alkaloid pathway of certain Claviceps species. Based on several lines of evidence, the N. fumigata gene easM is a logical candidate to encode the festuclavine-hydroxylating enzyme. To test this hypothesis we disrupted easM function by replacing part of its coding sequences with a hygromycin resistance gene and transforming N. fumigata with this construct. High-pressure liquid chromatography analysis demonstrated that easM deletion mutants were blocked in the ergot alkaloid pathway at festuclavine, and downstream products were eliminated. An additional alkaloid, proposed to be a prenylated form of festuclavine on the basis of mass spectral data, also accumulated to higher concentrations in the easM knockout. Complementation with the wild-type allele of easM gene restored the ability of the fungus to produce downstream compounds. These results indicate that easM encodes an enzyme required for fumigaclavine B synthesis likely by hydroxylating festuclavine. The festuclavine-accumulating strain of N. fumigata may facilitate future investigations of the biosynthesis of dihydrolysergic acid derivatives, which are derived from festuclavine and are the basis for several important drugs. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus is an opportunistic human pathogen that synthesizes a group of mycotoxins via a branch of the ergot alkaloid pathway. This fungus is globally distributed, and genetic data indicate that isolates recombine freely over that range; however, previous work on ergot alkaloids has focused on a limited number of isolates. We hypothesized that A. fumigatus harbors variation in the chemotype of ergot alkaloids and genotype of the ergot alkaloid gene cluster. Analysis of 13 isolates by high performance liquid chromatography revealed four distinct ergot alkaloid profiles or chemotypes. Five isolates completed the A. fumigatus branch of the ergot alkaloid pathway to fumigaclavine C. Six independent isolates accumulated fumigaclavine A, the pathway intermediate immediately before fumigaclavine C. One isolate accumulated only the early pathway intermediates chanoclavine-I and chanoclavine-I aldehyde, and one isolate lacked ergot alkaloids altogether. A genetic basis for each of the observed chemotypes was obtained either by PCR analysis of the ergot alkaloid gene cluster or through sequencing of easL, the gene encoding the prenyl transferase that reverse prenylates fumigaclavine A to fumigaclavine C. Isolates also exhibited differences in pigmentation and sporulation. The ergot alkaloid chemotypes were widely distributed geographically and among substrate of origin. [ABSTRACT FROM AUTHOR]

Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes ( easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (Δ easC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The Δ easC mutant instead accumulated N-methyl-4-dimethylallyltryptophan ( N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, ). A Δ easE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the Δ easC mutant restored ergot alkaloid production. Complementation of either Δ easC or Δ easE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with HO but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I. [ABSTRACT FROM AUTHOR]

Ergot alkaloids are mycotoxins that affect the nervous and reproductive systems of exposed individuals through interactions with monoamine receptors. They have been studied more widely in ergot fungi and grass endophytes but also are found in Aspergillus fumigatus, an opportunistic human pathogen that reproduces and disseminates exclusively through conidia. The ergot alkaloids festuclavine and fumigaclavines A, B and C are present in or on conidia of A. fumigatus. Cultures of the fungus that are free of conidia are difficult to obtain, obscuring comparisons of conidia versus vegetative hyphae as sources of the ergot alkaloids. To create conidiation-deficient strains of A. fumigatus we manipulated the bristle A gene (br/A), which controls vesicle formation or budding growth necessary for conidiation in Aspergillus spp. Disruption of brlA in A. fumigatus, via homologous recombination, resulted in a nonconidiating mutant that produced bristle-like structures instead of conidiophores and conidia. Moreover the disrupted strain failed to produce ergot alkaloids as verified by HPLC analyses. Complementation with a wild-type allele restored conidiation and ergot alkaloid production. These results suggest that ergot alkaloids are not produced within the vegetative mycelium of the fungus and are associated directly with conidiation. [ABSTRACT FROM AUTHOR]

Several endophytic fungi of the genus Neotyphodium form symbiotic associations with Lolium spp. grasses and are renowned for production of bioactive alkaloids such as ergot alkaloids. Some of these endophytes make their grass partners less suitable as hosts for endoparasitic nematodes, including Pratylenchus spp. The potential for ergot alkaloids to affect nematode motility was investigated in vitro. Ergovaline, the ergot alkaloid pathway end product of several Neotyphodium spp., was the only ergot alkaloid tested that inhibited motility of Pratylenchus scribneri. The association of ergot alkaloids with nematode population suppression was examined in glasshouse experiments with strains of the perennial ryegrass endophyte Neotyphodium sp. isolate Lp1 (syn. Neotyphodium lolii × Epichloë typhina) that have been genetically modified to lack ergot alkaloids or to have an altered ergot alkaloid profile. Populations of P. scribneri were significantly smaller in pots of perennial ryegrass containing the wild-type, ergot alkaloid-producing endophyte than in pots of endophyte-free perennial ryegrass. Elimination of certain complex ergot alkaloids (ergovaline and lysergic acid amides) in one gene knockout strain, or complete elimination of ergot alkaloids in another, did not affect the ability of the endophyte to suppress populations of nematode. Presence and concentrations of ergot alkaloids in pseudostems were as expected based on presence and genotype of endophyte in each plant, but frequently were undetectable or in low concentration in roots. The data indicate that ergot alkaloids do not contribute significantly to the endophyte-associated suppression of Pratylenchus spp. [ABSTRACT FROM AUTHOR]

Abstract: Ergot alkaloids are a diverse family of indole-derived mycotoxins that collectively have activities against a variety of organisms including bacteria, nematodes, insects, and mammals. Different fungi accumulate different, often characteristic, profiles of ergot alkaloids rather than a single pathway end product. These ergot alkaloid profiles result from inefficiency in the pathway leading to accumulation of certain intermediates or diversion of intermediates into shunts along the pathway. The inefficiency generating these ergot alkaloid profiles may have been selected for as a means of accumulating a diversity of ergot alkaloids, potentially contributing in different ways to benefit the producing fungus. [Copyright &y& Elsevier]