D-lysergic acid activation and cell-free synthesis of D-lysergyl peptides in enzyme fractions from the ergot fungus Claviceps purpurea

The D-lysergic acid activating enzyme from the ergot fungus Claviceps purpurea was purified to near homogeneity. It has a native Mr of about 245,000 and in its denatured form is a single polypeptide chain of Mr 62,000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent on dihydrolysergic acid. Western blot analysis of SDS electropherograms of crude protein extracts from C. purpurea using monospecific antibodies directed against the D-lysergic acid activating enzyme revealed the immunostaining of one particular band which was identical with that of the D-lysergic acid activating enzyme. No significant immunoreactive band with higher molecular weight was seen, which precludes the possibility that the enzyme had arisen from the proteolysis of a high molecular weight ergot peptide synthetase. An ammonium sulfate fractionated enzyme fraction was prepared from C. purpurea strain C1 that catalyzed the incorporation of D-lysergic acid into two peptides which besides D-lysergic acid contained alanine, phenylalanine, and proline. Dihydrolysergic acid was efficiently incorporated into the corresponding dihydrolysergic acid containing analogues of the two compounds. Radiochemical analysis and degradation studies suggest that the two D-lysergic acid containing peptides most probably are N-[N-(D-lysergyl)-L-alanlyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-analyl]-L-phenylalanyl-D-proline lactam, respectively. N-[N-(D-Lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam is considered to be the immediate precursor of ergotamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural analysis of a peptide synthetase gene required for ergopeptine production in the endophytic fungus Neotyphodium lolii.

Lysergyl peptide synthetase 1 catalyzes the assembly of toxic ergopeptines from activated D-lysergic acid and three amino acids. The gene encoding this enzyme in the endophytic fungus Neotyphodium lolii was analyzed and compared to a homologous gene from the ergot fungus Claviceps purpurea. Each gene contained two introns, which were found in the same relative position within two modules of the gene. The 5' ends of the two genes were unusually divergent. Signature sequences determining substrate specificity were similar in adenylation domains that recognized identical amino acids but differed within the adenylation domain for the amino acid that varies between the major ergopeptines of the two fungi. Homologues were detected in several related endophytic fungi; the tall fescue endophyte Neotyphodium coenophialum contained a divergent, second copy of the gene. Our results provide new information on the structure and distribution of this important peptide synthetase involved in ergot alkaloid biosynthesis.     

Biotechnology and genetics of ergot alkaloids

Ergot alkaloids, i.e. ergoline-derived toxic metabolites, are produced by a wide range of fungi, predominantly by members of the grass-parasitizing family of the Clavicipitaceae. Naturally occurring alkaloids like the D-lysergic acid amides, produced by the "ergot fungus" Claviceps purpurea, have been used as medicinal agents for a long time. The pharmacological effects of the various ergot alkaloids and their derivatives are due to the structural similarity of the tetracyclic ring system to neurotransmitters such as noradrenaline, dopamine or serotonin. In addition to "classical" indications, e.g. migraine or blood pressure regulation, there is a wide spectrum of potential new applications of this interesting group of compounds. The biotechnology of ergot alkaloids has a long tradition, and efficient parasitic and submerse production processes have been developed; the biochemistry of the pathway and the physiology of production have been worked out in detail. The recent identification of a cluster of genes involved in ergot alkaloid biosynthesis in C. purpurea and the availability of molecular genetic techniques allow the development of strategies for rational drug design of ergoline-related drugs by enzyme engineering and by biocombinatorial approaches.     

Heterologous Expression of Lysergic Acid and Novel Ergot Alkaloids in Aspergillus fumigatus

Different lineages of fungi produce distinct classes of ergot alkaloids. Lysergic acid-derived ergot alkaloids produced by fungi in the Clavicipitaceae are particularly important in agriculture and medicine. The pathway to lysergic acid is partly elucidated, but the gene encoding the enzyme that oxidizes the intermediate agroclavine is unknown. We investigated two candidate agroclavine oxidase genes from the fungus Epichloë festucae var. lolii × Epichloë typhina isolate Lp1 (henceforth referred to as Epichloë sp. Lp1), which produces lysergic acid-derived ergot alkaloids. Candidate genes easH and cloA were expressed in a mutant strain of the mold Aspergillus fumigatus, which typically produces a subclass of ergot alkaloids not derived from agroclavine or lysergic acid. Candidate genes were coexpressed with the Epichloë sp. Lp1 allele of easA, which encodes an enzyme that catalyzed the synthesis of agroclavine from an A. fumigatus intermediate; the agroclavine then served as the substrate for the candidate agroclavine oxidases. Strains expressing easA and cloA from Epichloë sp. Lp1 produced lysergic acid from agroclavine, a process requiring a cumulative six-electron oxidation and a double-bond isomerization. Strains that accumulated excess agroclavine (as a result of Epichloë sp. Lp1 easA expression in the absence of cloA) metabolized it into two novel ergot alkaloids for which provisional structures were proposed on the basis of mass spectra and precursor feeding studies. Our data indicate that CloA catalyzes multiple reactions to produce lysergic acid from agroclavine and that combining genes from different ergot alkaloid pathways provides an effective strategy to engineer important pathway molecules and novel ergot alkaloids.     

Use of polymerase chain reaction for searching for producers of ergot alkaloids from among microscopic fungi]

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the genera Penicillium and Claviceps was evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerated primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as alpha-cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

Screening for Ergot Alkaloid Producers among Microscopic Fungi by Means of the Polymerase Chain Reaction

The potential of the polymerase chain reaction for the detection of ergot alkaloid producers among microscopic fungi of the generaPenicilliumand Clavicepswas evaluated. Twenty-three strains of various species of fungi with a previously studied capacity for alkaloid production were used. The internal fragment of the gene encoding 4-dimethylallyltryptophan synthase, the enzyme catalyzing the first step in the biosynthesis of ergot alkaloids, was amplified using degenerate primers. This approach revealed an about 1.2-kb specific DNA fragment in micromycetes synthesizing ergot alkaloids with complete tetracyclic ergoline system. Microorganisms that produce alkaloids with modified C or D ergoline rings, as well as -cyclopiazonic acid, did not yield the PCR fragment of the expected size. This fragment was also not found in fungi incapable of ergot alkaloid production.     

Distribution of ergot alkaloids and ricinoleic acid in different milling fractions

The sclerotia of the fungus Claviceps sp. are still a challenge for the milling industry. Ergot sclerotia are a constant contamination of the rye crop and have to be removed by modern milling technologies. Changing sizes and coloration of the sclerotia make it difficult to separate them from the grain. Ergot sclerotia are a problem when cleaning is insufficient and non-separated specimens or sclerotia fragments get into the milling stream and thus ergot alkaloids are distributed into the different cereal fractions. In model milling experiments, the residues of ergot in rye flour and the distribution of ergot into different milling fractions were investigated. Rye grains were mixed with whole ergot sclerotia and in another experiment with ergot powder and cleaned afterwards before milling. The ergot alkaloids ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristineand their related isomeric forms (-inine-forms), and additionally ricinoleic acid as a characteristic component of ergot, were quantified in the different milling fractions. From the first experiment, it can be shown that after harvesting even simple contact of sclerotia with bulk grains during ordinary handling or movement of bulk grain in the granary is sufficient to contaminate all the healthy or sound rye grains with ergot alkaloids. Thereby, the amount of ergot residue correlates with the amount of peripheral layers of rye grains in the flour. In an additional experiment without sclerotia specimens, bulk rye grains were loaded with powder of sclerotia. After subsequent cleaning, aconcentration of ergot alkaloids was detected, which was tenfold higher than the ergot alkaloidconcentration of the experiment with intact ergot sclerotia.     

Contribution of ergot alkaloids to suppression of a grass-feeding caterpillar assessed with gene knockout endophytes in perennial ryegrass

Neotyphodium and Epichloë species (Ascomycota: Clavicipitaceae) are fungal symbionts (endophytes) of grasses. Many of these endophytes produce alkaloids that enhance their hosts’ resistance to insects or are toxic to grazing mammals. The goals of eliminating from forage grasses factors such as ergot alkaloids that are responsible for livestock disorders, while retaining pasture sustainability, and of developing resistant turf grasses, require better understanding of how particular alkaloids affect insect herbivores. We used perennial ryegrass Lolium perenne L. (Poaceae) symbiotic with Neotyphodium lolii × Epichloë typhina isolate Lp1 (a natural interspecific hybrid), as well as with genetically modified strains of Lp1 with altered ergot alkaloid profiles, to test effects of ergot alkaloids on feeding, growth, and survival of the black cutworm, Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae), a generalist grass‐feeding caterpillar. Neonates or late instars were provided clippings from glasshouse‐grown plants in choice and rearing trials. Wild‐type endophytic grass showed strong antixenosis and antibiosis, especially to neonates. Plant‐endophyte symbiota from which complex ergot alkaloids (ergovaline and lysergic acid amides such as ergine) or all ergot alkaloids were eliminated by endophyte gene knockout retained significant resistance against neonates. However, this activity was reduced compared to that of wild‐type Lp1, providing the first direct genetic evidence that ergot alkaloids contribute to insect resistance of endophytic grasses. Similarity of larval response to the two mutants suggested that ergovaline and/or ergine account for the somewhat greater potency of wild‐type Lp1 compared to the knockouts, whereas simpler ergot alkaloids contribute little to that added resistance. All of the endophyte strains also produced peramine, which was probably their primary resistance component. This study suggests that ergot alkaloids can be eliminated from an endophyte of perennial ryegrass while retaining significant insect resistance.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Procedure for isolating the endophyte from tall fescue and screening isolates for ergot alkaloids.

A procedure was developed to isolate and determine ergot alkaloid production by Acremonium coenophialum, the endophytic fungus of tall fescue. The procedure established that macerated leaf sheath or pith from inflorescence stem placed either in a liquid medium or on a corn meal-malt extract agar medium produced isolated mycelium and characteristic conidia within a 3- to 3.5-week period. Once isolated, each fungus was placed in another liquid medium, M104T, where competent strains produced total ergot alkaloids ranging from 38 to 797 mg/liter. Several isolates were negative for ergot alkaloid synthesis. The production of ergot alkaloids by individual isolates was unstable; isolates rapidly degenerated in their ability to produce ergot alkaloids during subculture. However, the procedure as presented allows the assessment of an isolate for ergot alkaloid synthesis during its initial isolation.     

Biosynthesis of ergot alkaloids. Some new results on an old problem (Review)]

The biosynthetic pathway leading from L-tryptophan, mevalonic acid and methionine to the tetracyclic ergoline ring system of the ergot alkaloids in Claviceps species is reviewed. This pathway entails many mechanistically intriguing features. Recent studies are also discussed which reveal the stereochemical course of the isoprenylation of tryptophan and of the N-methylation of dimethylallyltryptophan (DMAT) and which shed some light on the likely steps leading from the open-chain precursors, N-methyl-DMAT to the tricyclic intermediate, chanoclavine-1. Finally, some plans are outlined to probe the evolutionary relationship of ergot alkaloid biosynthesis in fungi to that in higher plants of the family Convolvulaceae.     

Effects of different levels of ergot in concentrates on the growing and slaughtering performance of bulls and on carry-over into edible tissue

The aim of the present study was to examine long-term effects of low levels of ergot alkaloids on growing bulls. Natural grown ergot with a mean total alkaloid concentrations of 633 mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent alkaloids, was used. In a dose-response study 38 Holstein Friesian bulls were fed with three different doses of this ergot (0, 0.45 and 2.25 g/kg concentrate corresponding to an average total alkaloid concentration of the daily ration of 0, 69 and 421 microg/kg DM) over a period of approximately 230 days. Live weight, feed intake and health condition were monitored over the entire test period. The bulls were slaughtered at a live weight of approximately 550 kg. Carcass composition and quality were recorded and samples of liver, muscle, kidneys, fat, bile, urine and blood were analysed for ergot alkaloids. Liver enzyme activities and total bilirubin were measured in the blood. Statistically, no significant differences were detectable between the three feeding groups. Mean live weight gain over all groups was 1.41 kg/d with a mean dry matter intake of 7.35 kg/d. No carry over into tissues could be proved out of the experiment. To derive a no-effect level for beef cattle further research including higher ergot doses will be necessary.     

Coma,Hyperthermia and Bleeding Associated with Massive LSD Overdose: A Report of Eight Cases

Eight patients were seen within 15 minutes of intranasal self-administration of large amounts of pure D-lysergic acid diethylamide (LSD) tartrate powder. Emesis and collapse occurred along with signs of sympathetic overactivity, hyperthermia, coma and respiratory arrest. Mild generalized bleeding occurred in several patients and evidence of platelet dysfunction was present in all. Serum and gastric concentrations of LSD tartrate ranged from 2.1 to 26 nanograms per ml and 1,000 to 7,000 μg per 100 ml, respectively. With supportive care, all patients recovered. Massive LSD overdose in man is life-threatening and produces striking and distinctive manifestations.     

Effect of Intravenous Administration of D-Lysergic Acid Diethylamide on Initiation of Protein Synthesis in a Cell-Free System Derived from Brain

An initiating cell-free protein synthesis system derived from brain was utilized to demonstrate that the intravenous injection of D-lysergic acid diethylamide (LSD) to rabbits resulted in a lesion at the initiation stage of brain protein synthesis. Three inhibitors of initiation, edeine, poly(I), and aurintricarboxylic acid were used to demonstrate a reduction in initiation-dependent amino acid incorporation in the brain cell-free system. One hour after LSD injection, there was also a measurable decrease in the formation of 40S and 80S initiation complexes in vitro, using either [35S]methionine or [35S]Met-tRNAf. Analysis of the methionine pool size after LSD administration indicated there was no change in methionine levels. Analysis of the formation of initiation complexes in the brain cell-free protein synthesis system prepared 6 h after LSD administration indicated that there was a return to control levels at this time. The effects of LSD on steps in the initiation process are thus reversible.     

Dietary ergot alkaloids as a possible cause of tail necrosis in rabbits

This study describes the association between tail necrosis in rabbits and mycotoxins in rabbit feed. Clinical cases of tail necrosis were observed in 14 out of 103 rabbits kept in an outdoor group housing, fed with hay and a commercial pelleted feed. The observed clinical symptoms, alopecia, erosions, crusts and necrosis were restricted to the tail area and exclusively occurred in young rabbits aged 113?±?20 days. Dermatological examination suggested that ischemia had caused necrosis. Analysis of blood samples showed an elevated level of creatine kinase. No weight loss occurred in affected rabbits. Trauma caused by injuries or technopathic lesions was also excluded. Histopathologically, the lesions were characterized by acute muscle fibre degeneration and chronic active dermatitis with granulation tissue formation. Necropsy of one rabbit revealed hepatocellular degeneration and necrosis as remarkable findings. Feed analysis for ergot alkaloids by enzyme immunoassays yielded a mean and maximum ergot alkaloid content of 410?±?250 μg/kg and 1,700 μg/kg, respectively. Faeces of affected rabbits contained ergot alkaloids at levels up to 200 μg/kg. The mean and maximum dietary intake of total ergot alkaloids were 17 and 71 μg/kg bodyweight, respectively. Fusarium toxins (trichothecenes, zearalenone, fumonisins) were also found in the feed, but at levels which did not explain the observed effects. The results indicate that ergot alkaloids may have been the cause of tail necrosis, which is supported by literature data showing that rabbits are especially sensitive towards these toxins.     

Effects of different levels of ergot in concentrates on the growing and slaughtering performance of bulls and on carry-over into edible tissue

Abstract

The aim of the present study was to examine long-term effects of low levels of ergot alkaloids on growing bulls. Natural grown ergot with a mean total alkaloid concentrations of 633 mg/kg, and ergotamine (25%), ergocristine (15%) and ergosine (13%) as the most prominent alkaloids, was used. In a dose-response study 38 Holstein Friesian bulls were fed with three different doses of this ergot (0, 0.45 and 2.25 g/kg concentrate corresponding to an average total alkaloid concentration of the daily ration of 0, 69 and 421 µg/kg DM) over a period of approximately 230 days. Live weight, feed intake and health condition were monitored over the entire test period. The bulls were slaughtered at a live weight of approximately 550 kg. Carcass composition and quality were recorded and samples of liver, muscle, kidneys, fat, bile, urine and blood were analysed for ergot alkaloids. Liver enzyme activities and total bilirubin were measured in the blood. Statistically, no significant differences were detectable between the three feeding groups. Mean live weight gain over all groups was 1.41 kg/d with a mean dry matter intake of 7.35 kg/d. No carry over into tissues could be proved out of the experiment. To derive a no-effect level for beef cattle further research including higher ergot doses will be necessary.     

An ergot alkaloid biosynthesis gene and clustered hypothetical genes from Aspergillus fumigatus

The ergot alkaloids are a family of indole-derived mycotoxins with a variety of significant biological activities. Aspergillus fumigatus, a common airborne fungus and opportunistic human pathogen, and several fungi in the relatively distant taxon Clavicipitaceae (clavicipitaceous fungi) produce different sets of ergot alkaloids. The ergot alkaloids of these divergent fungi share a four-member ergoline ring but differ in the number, type, and position of the side chains. Several genes required for ergot alkaloid production are known in the clavicipitaceous fungi, and these genes are clustered in the genome of the ergot fungus Claviceps purpurea. We investigated whether the ergot alkaloids of A. fumigatus have a common biosynthetic and genetic origin with those of the clavicipitaceous fungi. A homolog of dmaW, the gene controlling the determinant step in the ergot alkaloid pathway of clavicipitaceous fungi, was identified in the A. fumigatus genome. Knockout of dmaW eliminated all known ergot alkaloids from A. fumigatus, and complementation of the mutation restored ergot alkaloid production. Clustered with dmaW in the A. fumigatus genome are sequences corresponding to five genes previously proposed to encode steps in the ergot alkaloid pathway of C. purpurea, as well as additional sequences whose deduced protein products are consistent with their involvement in the ergot alkaloid pathway. The corresponding genes have similarities in their nucleotide sequences, but the orientations and positions within the cluster of several of these genes differ. The data indicate that the ergot alkaloid biosynthetic capabilities in A. fumigatus and the clavicipitaceous fungi had a common origin.     

Zum Einfluss von Mutterkorn im Futter auf Absetzferkel und Masthähnchen

Two preliminary tests were conducted with piglets and chicken to study the influence of feeding ergot, which was specified concerning alkaloid content and pattern, on growth performance and health parameters. Treatments were 5 ergot concentrations in the diets (0; 0.05; 0.1; 0.2; 0.4%) which were fed to 5×16 piglets for 35 days and 5×28 chicken for 21 days. A content of 0.4% ergot in the diet decreased feed intake and growth performance of piglets significantly. Contents of 0.1% and 0.2% ergot in the diet showed a tendency towards improved weight gain as compared to controls. Feed to gain ratio was found to be the lowest in the group with 0.2% ergot in the diet. In the study conducted with chicken significant effects of ergot feeding on growth performance were not detected. With increased ergot concentrations in the diet the weight of hearts decreased, which proved to be significant only at a level of 0.2 % ergot in the diet. Moderate to severe inflammations were found in the proximal duodenum of the ergot fed groups, which, however, were not dosedependent. Further studies with ergot from other sources differing in alkaloid content and pattern are necessary to evaluate toxic alkaloid levels for the various animal species or categories.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus Penicillium citrinum

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to absorb autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to absorb ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12- day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.     

Transport of ergot alkaloids and quinocitrinins in the producing fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>

The decrease in the concentration of alkaloids in the culture liquid of Penicillium citrinum grown to the early stationary phase was found to be due to the uptake of quinocitrinins and ergot alkaloids by fungal cells. The ability of the fungal mycelium to take up autogenous quinocitrinins does not depend on the mycelium age, whereas its ability to take up ergot alkaloids is higher in the young than in the 12-day-old mycelium. The uptake of exogenously added ergot alkaloids by the fungal mycelium is accompanied by excretion of intracellular quinocitrinins. The addition of quinocitrinins to the medium was found to exert different effects in different growth stages. Namely, the uptake of exogenously added quinocitrinins by the actively growing young mycelium inhibits the excretion of ergot alkaloids, but the excretion of ergot alkaloids by the 12-day-old mycelium occurs throughout the cultivation period. The excretion of both ergot alkaloids and quinocitrinins does not require energy.