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Jonathan D. Walton - One of the best experts on this subject based on the ideXlab platform.
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Profiling of Amatoxins and Phallotoxins in the Genus Lepiota by Liquid Chromatography Combined with UV Absorbance and Mass Spectrometry
MDPI AG, 2014Co-Authors: Michael R. Sgambelluri, Sara Epis, Davide Sassera, Hong Luo, Evan R. Angelos, Jonathan D. WaltonAbstract:Species in the mushroom genus Lepiota can cause fatal mushroom poisonings due to their content of amatoxins such as α-amanitin. Previous studies of the toxin composition of poisonous Lepiota species relied on analytical methods of low sensitivity or resolution. Using liquid chromatography coupled to UV absorbance and mass spectrometry, we analyzed the spectrum of peptide toxins present in six Italian species of Lepiota, including multiple samples of three of them collected in different locations. Field taxonomic identifications were confirmed by sequencing of the internal transcribed spacer (ITS) regions. For comparison, we also analyzed specimens of Amanita phalloides from Italy and California, a specimen of A. virosa from Italy, and a laboratory-grown sample of Galerina marginata. α-Amanitin, β-amanitin, amanin, and amaninamide were detected in all samples of L. brunneoincarnata, and α-amanitin and γ-amanitin were detected in all samples of L. josserandii. Phallotoxins were not detected in either species. No amatoxins or Phallotoxins were detected in L. clypeolaria, L. cristata, L. echinacea, or L. magnispora. The Italian and California isolates of A. phalloides had similar profiles of amatoxins and Phallotoxins, although the California isolate contained more β-amanitin relative to α-amanitin. Amaninamide was detected only in A. virosa
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Colocalization of Amanitin and a Candidate Toxin-Processing Prolyl Oligopeptidase in Amanita Basidiocarps
Eukaryotic Cell, 2010Co-Authors: Heather E. Hallen-adams, John S. Scott-craig, Jonathan D. WaltonAbstract:Fungi in the basidiomycetous genus Amanita owe their high mammalian toxicity to the bicyclic octapeptide amatoxins such as α-amanitin. Amatoxins and the related Phallotoxins (such as the heptapeptide phalloidin) are encoded by members of the “MSDIN” gene family and are synthesized on ribosomes as short (34- to 35-amino-acid) proproteins. Antiamanitin antibodies and confocal microscopy were used to determine the cellular and subcellular localizations of amanitin accumulation in basidiocarps (mushrooms) of the Eastern North American destroying angel (Amanita bisporigera). Consistent with previous studies, amanitin is present throughout the basidiocarp (stipe, pileus, lamellae, trama, and universal veil), but it is present in only a subset of cells within these tissues. Restriction of amanitin to certain cells is especially marked in the hymenium. Several lines of evidence implicate a specific prolyl oligopeptidase, A. bisporigera POPB (AbPOPB), in the initial processing of the amanitin and Phallotoxin proproteins. The gene for AbPOPB is restricted taxonomically to the amatoxin-producing species of Amanita and is clustered in the genome with at least one expressed member of the MSDIN gene family. Immunologically, amanitin and AbPOPB show a high degree of colocalization, indicating that toxin biosynthesis and accumulation occur in the same cells and possibly in the same subcellular compartments.
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Processing of the phalloidin proprotein by prolyl oligopeptidase from the mushroom conocybe albipes
Journal of Biological Chemistry, 2009Co-Authors: Heather E. Hallen-adams, Jonathan D. WaltonAbstract:Abstract The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (Phallotoxins). In Amanita bisporigera, α-amanitin and phallacidin are synthesized as 35- and 34-amino acid proproteins, respectively, in which the amino acid sequences found in the mature toxins are flanked by conserved amino acid sequences. The presence of invariant Pro residues immediately upstream of the toxin regions and as the last predicted amino acid in the toxin regions themselves suggests that a Pro-specific peptidase is responsible for the initial post-translational processing of the Amanita toxin proproteins. We purified an enzyme from the phalloidin-producing mushroom Conocybe albipes that cleaves a synthetic 22-mer phalloidin peptide to release the mature toxin peptide (AWLATCP). Mass spectrometric analysis of the purified protein combined with isolation and sequencing of the encoding gene indicates that the responsible processing enzyme is a member of the prolyl oligopeptidase (POP) subfamily of proteases (EC 3.4.21.26). The processing enzyme was able to use the chromogenic POP substrate benzyloxycarbonyl-Gly-Pro-p-nitroanilide and was inhibited by the specific POP inhibitor benzyloxycarbonyl-Pro-prolinal. Both Pro bonds in the proprotein are cleaved by the same enzyme, with the C-terminal Pro bond cleaved first or much faster than the N-terminal Pro bond. Transient accumulation of the N-terminal intermediate indicates that cleavage is not strongly processive. A synthetic peptide representing the phallacidin proprotein was also cleaved by the POP of C. albipes, but a precursor of amanitin (which is not made by C. albipes) was cleaved inefficiently.
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Gene family encoding the major toxins of lethal Amanita mushrooms
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Heather E. Hallen, John S. Scott-craig, Jonathan D. WaltonAbstract:Amatoxins, the lethal constituents of poisonous mushrooms in the genus Amanita, are bicyclic octapeptides. Two genes in A. bisporigera, AMA1 and PHA1, directly encode α-amanitin, an amatoxin, and the related bicyclic heptapeptide phallacidin, a Phallotoxin, indicating that these compounds are synthesized on ribosomes and not by nonribosomal peptide synthetases. α-Amanitin and phallacidin are synthesized as proproteins of 35 and 34 amino acids, respectively, from which they are predicted to be cleaved by a prolyl oligopeptidase. AMA1 and PHA1 are present in other toxic species of Amanita section Phalloidae but are absent from nontoxic species in other sections. The genomes of A. bisporigera and A. phalloides contain multiple sequences related to AMA1 and PHA1. The predicted protein products of this family of genes are characterized by a hypervariable “toxin” region capable of encoding a wide variety of peptides of 7–10 amino acids flanked by conserved sequences. Our results suggest that these fungi have a broad capacity to synthesize cyclic peptides on ribosomes.
Felix Carvalho - One of the best experts on this subject based on the ideXlab platform.
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amanita phalloides poisoning mechanisms of toxicity and treatment
Food and Chemical Toxicology, 2015Co-Authors: Juliana Garcia, Vera Marisa Costa, Alexandra T P Carvalho, Paula Baptista, Paula Guedes De Pinho, Maria De Lourdes Bastos, Felix CarvalhoAbstract:Amanita phalloides, also known as 'death cap', is one of the most poisonous mushrooms, being involved in the majority of human fatal cases of mushroom poisoning worldwide. This species contains three main groups of toxins: amatoxins, Phallotoxins, and virotoxins. From these, amatoxins, especially α-amanitin, are the main responsible for the toxic effects in humans. It is recognized that α-amanitin inhibits RNA polymerase II, causing protein deficit and ultimately cell death, although other mechanisms are thought to be involved. The liver is the main target organ of toxicity, but other organs are also affected, especially the kidneys. Intoxication symptoms usually appear after a latent period and may include gastrointestinal disorders followed by jaundice, seizures, and coma, culminating in death. Therapy consists in supportive measures, gastric decontamination, drug therapy and, ultimately, liver transplantation if clinical condition worsens. The discovery of an effective antidote is still a major unsolved issue. The present paper examines the clinical toxicology of A. phalloides, providing the currently available information on the mechanisms of toxicityinvolved and on the current knowledge on the treatment prescribed against this type of mushrooms. Antidotal perspectives will be raised as to set the pace to new and improved therapy against these mushrooms.
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determination of amatoxins and Phallotoxins in amanita phalloides mushrooms from northeastern portugal by hplc dad ms
Mycologia, 2015Co-Authors: Juliana Garcia, Alexandra T P Carvalho, Paula Baptista, Paula Guedes De Pinho, Maria De Lourdes Bastos, Ana Cristina Palmeira De Oliveira, Victor De Freitas, Eric Pereira, Felix CarvalhoAbstract:Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and Phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. α-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in Phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.
Isabelle Morel - One of the best experts on this subject based on the ideXlab platform.
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Amatoxins (a- and b-Amanitin) and Phallotoxin (Phalloidin) Analyses in Urines Using High-Resolution Accurate Mass LC–MS Technology
2016Co-Authors: Thomas Gicquel, Sylvie Lepage, Manon Fradin, Olivier Tribut, Isabelle MorelAbstract:Mycotoxin intoxications can result from the consumption of amatox-ins like a- and b-amanitin or of Phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatox-ins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography–high-resolution-mass spectrometry, LC–HR-MS), Q ExactiveTM (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 3 2.1 mm, 2.6 mm) using a gradient of 10 mM ammonium acetate buffer containing 0.1 % (v/v) formic acid and of acetonitrile with 0.1 % (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for a-amanitin, b-amanitin, phalloidin and IS of 1.9
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Amatoxins (α- and β-Amanitin) and Phallotoxin (Phalloidin) analyses in urines using high-resolution accurate mass LC-MS technology
Journal of Analytical Toxicology, 2014Co-Authors: Thomas Gicquel, Sylvie Lepage, Manon Fradin, Olivier Tribut, Bénédicte Duretz, Isabelle MorelAbstract:Mycotoxin intoxications can result from the consumption of amatoxins like α- and β-amanitin or of Phallotoxin, present in several toxic mushrooms like Amanita phalloides. To identify and quantify amatoxins and phallotoidin in biological matrixes, we developed a method using liquid chromatography coupled with an ultra-high-resolution and accurate mass instrument (liquid chromatography-high-resolution-mass spectrometry, LC-HR-MS), Q Exactive™ (Thermo Fisher). The method includes a simple solid-phase extraction of urine samples spiked with flurazepam as internal standard (IS), using Bond Elut Agilent Certify cartridges (C18, 200 mg, 3 mL). LC separation was performed on a C18 Accucore column (100 × 2.1 mm, 2.6 µm) using a gradient of 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid and of acetonitrile with 0.1% (v/v) formic acid. Separation of analytes was obtained in 7 min, with respective retention times for α-amanitin, β-amanitin, phalloidin and IS of 1.9, 1.7, 3.5 and 3.8 min, respectively. Quantitation on the LC-HR-MS system was performed by extracting the exact mass value of each protonated species using a 5-p.p.m. mass window, which was 919.3614, 920.3455, 789.3257 and 388.1586 for α-amanitin, β-amanitin, phalloidin and IS, respectively. Calibration curves were obtained by spiking drug-free urine at 1-100 ng/mL. Mean correlation coefficients, r(2), were above 0.99 for each amatoxins and phalloidin. According to currently accepted validation procedures, the method was tested for selectivity, calibration, accuracy, matrix effect, precision and recovery. Authentic urine samples from 43 patients suffering from a suspected intoxication with mushrooms were analyzed by LC-HR-MS, and the results were compared with ELISA competitive immunoassay. The LC-HR-MS presented large benefits over immunoassay of being specific, faster and more sensitive, making it suitable for daily emergency toxicological analysis
Juliana Garcia - One of the best experts on this subject based on the ideXlab platform.
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amanita phalloides poisoning mechanisms of toxicity and treatment
Food and Chemical Toxicology, 2015Co-Authors: Juliana Garcia, Vera Marisa Costa, Alexandra T P Carvalho, Paula Baptista, Paula Guedes De Pinho, Maria De Lourdes Bastos, Felix CarvalhoAbstract:Amanita phalloides, also known as 'death cap', is one of the most poisonous mushrooms, being involved in the majority of human fatal cases of mushroom poisoning worldwide. This species contains three main groups of toxins: amatoxins, Phallotoxins, and virotoxins. From these, amatoxins, especially α-amanitin, are the main responsible for the toxic effects in humans. It is recognized that α-amanitin inhibits RNA polymerase II, causing protein deficit and ultimately cell death, although other mechanisms are thought to be involved. The liver is the main target organ of toxicity, but other organs are also affected, especially the kidneys. Intoxication symptoms usually appear after a latent period and may include gastrointestinal disorders followed by jaundice, seizures, and coma, culminating in death. Therapy consists in supportive measures, gastric decontamination, drug therapy and, ultimately, liver transplantation if clinical condition worsens. The discovery of an effective antidote is still a major unsolved issue. The present paper examines the clinical toxicology of A. phalloides, providing the currently available information on the mechanisms of toxicityinvolved and on the current knowledge on the treatment prescribed against this type of mushrooms. Antidotal perspectives will be raised as to set the pace to new and improved therapy against these mushrooms.
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determination of amatoxins and Phallotoxins in amanita phalloides mushrooms from northeastern portugal by hplc dad ms
Mycologia, 2015Co-Authors: Juliana Garcia, Alexandra T P Carvalho, Paula Baptista, Paula Guedes De Pinho, Maria De Lourdes Bastos, Ana Cristina Palmeira De Oliveira, Victor De Freitas, Eric Pereira, Felix CarvalhoAbstract:Amanita phalloides is a toxic mushroom responsible for the majority of deaths occurring after mushrooms ingestion, mainly due to amatoxins. In the present study the contents and distribution of the major amatoxins and Phallotoxins in different tissues of A. phalloides from two different sites of Portugal were analyzed by liquid chromatography (LC) coupled to diode array (DAD) and mass spectrometry (MS) detection. The main toxins were separated by LC and its chemical structures confirmed by MS. α-Amanitin contents in caps, stipe and volva tissues were quantified by RP-HPLC. The results show that caps have the highest content of amatoxins, whereas the volva was richest in Phallotoxins. Moreover variability in the toxins composition from different geographic sites was also observed. This study provides for the first time the content of toxins in A. phalloides from Portugal.
Zeki Severoglu - One of the best experts on this subject based on the ideXlab platform.
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amanitin and Phallotoxin concentration in amanita phalloides var alba mushroom
Toxicon, 2013Co-Authors: Ertugrul Kaya, Recep Bayram, Kursat Oguz Yaykasli, Ayhan Saritas, Selim Karahan, Serdar Colakoglu, Ismail Yilmaz, Zeynep Aydin Sinirlioglu, Zeki SeverogluAbstract:Although rarely seen, Amanita phalloides var. alba, a variety of A. phalloides type mushrooms, causes mushroom poisoning resulting in death. Since it is frequently confused with some edible mushrooms due to its white colored cap and macroscopic appearance, it becomes important in toxicological terms. Knowledge of the toxin amount contained in this mushroom type is invaluable in the treatment of cases involving poisoning. In this study, we examined the toxin levels of various parts of the A. phalloides var. alba mushroom growing Duzce region of Turkey. Toxin analyses were carried out for A. phalloides var. alba, which were collected from the forests Duzce region of Turkey in 2011, as a whole and also separately in its spore, pileus, gills, stipe and volva parts. The alpha amanitin, beta amanitin, gamma amanitin, phalloidin and phallacidine analyses of the mushrooms were carried out using the RP-HPLC method. A genetic analysis of the mushroom showed that it had similar genetic characteristics as A. phalloides and was a variety of it. The lowest toxins quantity was detected in spores, volva and stipe among all parts of the mushroom. The maximum amount of amatoxins was measured in the gills. The pileus also contained a high amount of amatoxins. Generally, amatoxins and Phallotoxin concentrations were lower as compared to A. phalloides, but interestingly all toxins other than gamma toxin were higher in the spores of A. phalloides var. alba. The amount of toxin in all of its parts had sufficient concentrations to cause death. With this study, the amatoxin and Phallotoxin concentrations in A. phalloides var. alba mushroom and in its parts have been revealed in detail for the first time.