Genes and evolutionary fates of the amanitin biosynthesis pathway in poisonous mushrooms.

The deadly toxin α-amanitin is a bicyclic octapeptide biosynthesized on ribosomes. A phylogenetically disjunct group of mushrooms in Agaricales (Amanita, Lepiota, and Galerina) synthesizes α-amanitin. This distribution of the toxin biosynthetic pathway is possibly related to the horizontal transfer of metabolic gene clusters among taxonomically unrelated mushrooms with overlapping habitats. Here, our work confirms that two biosynthetic genes, P450-29 and FMO1, are oxygenases important for amanitin biosynthesis. Phylogenetic and genetic analyses of these genes strongly support their origin through horizontal transfer, as is the case for the previously characterized biosynthetic genes MSDIN and POPB. Our analysis of multiple genomes showed that the evolution of the α-amanitin biosynthetic pathways in the poisonous agarics in the Amanita, Lepiota, and Galerina clades entailed distinct evolutionary pathways including gene family expansion, biosynthetic genes, and genomic rearrangements. Unrelated poisonous fungi produce the same deadly amanitin toxins using variations of the same pathway. Furthermore, the evolution of the amanitin biosynthetic pathway(s) in Amanita species generates a much wider range of toxic cyclic peptides. The results reported here expand our understanding of the genetics, diversity, and evolution of the toxin biosynthetic pathway in fungi.

[Investigation of a family cluster poisoning incident caused by Galerina sulciceps mushroom].

Mushroom poisoning is the most important cause of death in food-borne poisoning in China, mainly caused by amanitin, which is caused by rapid progression, complex mechanism and latency. Early identification, diagnosis and treatment are important to improve the prognosis of fatal mushroom poisoning. This article analyzes the clinical characteristics, identification process and treatment of 14 patients with amanitin-containing Galerina sulciceps mushroom poisoning in a family, so as to improve the identification ability of the first physician in recognizing and managing early-stage mushroom poisoning, and to increase the cure rate through early bundle therapy of mushroom poisoning.

Diversity of MSDIN family members in amanitin-producing mushrooms and the phylogeny of the MSDIN and prolyl oligopeptidase genes.

BACKGROUND: Amanitin-producing mushrooms, mainly distributed in the genera Amanita, Galerina and Lepiota, possess MSDIN gene family for the biosynthesis of many cyclopeptides catalysed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be an efficient way to mine MSDIN and POP genes in these lethal mushrooms. Thus far, only A. palloides and A. bisporigera from North America and A. exitialis and A. rimosa from Asia have been studied based on transcriptome analysis. However, the MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied; hence, the transcriptomes of these speices deserve to be analysed. RESULTS: In this study, the MSDIN and POP genes from ten Amanita species, two Galerina species and Lepiota venenata were studied and the phylogenetic relationships of their MSDIN and POP genes were analysed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, ß-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that (1) MSDIN genes generally clustered depending on the taxonomy of the genus, while Amanita MSDIN genes clustered depending on the chemical substance; and (2) the POPA genes of Amanita, Galerina and Lepiota clustered and were separated into three different groups, but the POPB genes of the three distinct genera were clustered in a highly supported monophyletic group. CONCLUSIONS: These results indicate that lethal Amanita species have the genetic capacity to produce numerous cyclopeptides, most of which are unknown, while lethal Galerina and Lepiota species seem to only have the genetic capacity to produce α-amanitin. Additionally, the POPB phylogeny of Amanita, Galerina and Lepiota conflicts with the taxonomic status of the three genera, suggesting that underlying horizontal gene transfer has occurred among these three genera.

Uptake of certain heavy metals from contaminated soil by mushroom--Galerina vittiformis.

Remediation of soil contaminated with heavy metals has received considerable attention in recent years. In this study, the heavy metal uptake potential of the mushroom, Galerina vittiformis, was studied in soil artificially contaminated with Cu (II), Cd (II), Cr (VI), Pb (II) and Zn (II) at concentrations of 50 and 100mg/kg. G. vittiformis was found to be effective in removing the metals from soil within 30 days. The bioaccumulation factor (BAF) for both mycelia and fruiting bodies with respect to these heavy metals at 50mg/kg concentrations were found to be greater than one, indicating hyper accumulating nature by the mushroom. The metal removal rates by G. vittiformis was analyzed using different kinetic rate constants and found to follow the second order kinetic rate equation except for Cd (II), which followed the first order rate kinetics.

Wild mushroom poisoning: A case study of amatoxin-containing mushrooms and implications for public health.

Wild mushroom poisoning is a global public health concern, with mushrooms containing amatoxins being the main cause of fatalities. Mushrooms from the genus Amanita and Galerina contain amatoxins. Here we present a case of wild mushroom poisoning that affected three individuals, resulting in two fatalities. Within 10-15 hours after consumption, they experienced symptoms of gastroenteritis such as vomiting, abdominal pain, and diarrhea. One individual sought medical attention promptly and recovered, while the other two sought medical help nearly two or three days after the onset of symptoms, by which time their conditions had already worsened and led to their deaths. The mushrooms were identified belonging to genus Galerina, and laboratory test revealed variations in toxin levels among mushrooms collected from different parts of the decaying stump. The higher levels of α-amanitin, ß-amanitin, and γ-amanitin were detected near the base of the tree stump, but trace levels of α-amanitin were found near the top of the stump, while ß-amanitin and γ-amanitin were undetectable. This case emphasizes the importance of seeking immediate medical attention when experiencing delayed-onset gastrointestinal symptoms, as it may indicate more severe mushroom poisoning, particularly amatoxin poisoning. Timely and appropriate treatment is equally important. Additionally, consuming different units of the mushrooms in the same incident can lead to varying prognoses due to differences in toxin levels.

An outbreak of Galerina sulciceps-like (Galerina cf. sulciceps) mushroom poisoning.

OBJECTIVE: Amatoxin-containing mushroom poisoning is a significant threat to public health worldwide. We report a mass poisoning of Galerina sulciceps-like mushrooms (Galerina cf. sulciceps) in Luzhou, Sichuan Province, China, aiming to offer insights for future prevention and treatment strategies. METHODS: We performed a retrospective survey of mass mushroom poisoning patients admitted to our hospital. The demographic data, clinical presentations, laboratory findings, therapeutic measures and prognostic information were collected and analyzed. We used the 2020 Chinese consensus on the clinical diagnosis and treatment of amatoxin-containing mushroom poisoning to assess the severity of poisoning. Mushrooms were examined through morphological analysis, molecular biology identification, and toxin detection. RESULTS: Our patient cohort consisted of nine males and six females, with mean (±SD) age of 34.9 ± 13.0 years. Gastrointestinal symptoms were the first to manifest, with mean (±SD) latency period of 13.4 ± 3.9 h. The majority of patients (86.7%) experienced nausea, vomiting, and diarrhea. Liver dysfunction was noted in 66.7% of patients, and thrombocytopenia was present in 26.7% of patients. In terms of the severity of poisoning, there were 10 mild cases and 5 severe cases. The mushrooms were provisionally labeled as Galerina cf. sulciceps, containing the toxins α-amanitin, ß-amanitin, and γ-amanitin. All patients eventually recovered. DISCUSSION: We report what appears to be a new type of mushroom that is morphologically and phylogenetically similar to the known Galerina sulciceps, but further study is required to determine if it represents a distinct species. CONCLUSION: This poisoning event was caused by unintentional ingestion of Galerina cf. sulciceps, an amatoxin-containing mushroom. Early symptoms are primarily gastrointestinal, with acute liver damage and coagulopathy being the main toxic effects. Thrombocytopenia is also prominent, particularly in severe cases. Accurate assessment and prompt, individualized, and intensive treatment are crucial for managing patients with acute Galerina cf. sulciceps poisoning effectively.

Toxin components and toxicological importance of Galerina marginata from Turkey.

Amatoxins, most of which are hepatotoxic, can cause fatal intoxication. While mushrooms in the amatoxin-containing Galerina genus are rare, they can poison humans and animals worldwide. Few studies have profiled the toxicity of Galerina marginata. In addition, many studies indicate that macrofungi can have different characteristics in different regions. In this study, the quantities of toxins present in G. marginata from different provinces in Turkey were analysed using reversed-phase high-performance liquid chromatography with ultraviolet detection (RP-HPLC-UV) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). G. marginata samples were collected from three different regions of Turkey. The taxonomic categorization of mushrooms was based on their micro- and macroscopic characteristics. The presence of toxins α-amanitin (AA), ß-amanitin (BA), γ-amanitin (GA), phalloidin (PHD) and phallacidin (PHC) quantities were measured using RP-HPLC-UV and then were confirmed using LC-ESI-MS/MS. BA levels were higher than AA levels in G. marginata mushrooms collected from all three regions. Moreover, the levels of GA were below the detection limit and no phallotoxins were detected. This is the first study to identify and test the toxicity of G. marginata collected from three different regions of Turkey using RP-HPLC-UV. This is also the first study to confirm the UV absorption of amatoxins in G. marginata using LC-ESI-MS/MS, which is a far more sensitive process. More studies evaluating the toxicity of G. marginata in other geographic regions of the world are needed.

Investigation and analysis of Galerina sulciceps poisoning in a canteen.

INTRODUCTION: Guizhou Province in China has an abundant resource of wild mushrooms, including numerous poisonous species which contain various toxins. The mortality rate from wild mushroom poisoning has been high in this area in recent years. Galerina sulciceps is a dangerously toxic mushroom which can be fatal if ingested. METHODS: we report on an epidemiological investigation of G. sulciceps poisoning which occurred in Duyun City of Guizhou Province. The characteristics of this species, its toxicity, observed clinical features, laboratory data, treatment modality, and prognosis were investigated in order to provide a reference point for the prevention and treatment of this kind of mushroom poisoning. RESULTS: Thirteen employees showed toxic symptoms after ingesting wild mushrooms the previous day in a company canteen. Clinical manifestation varied from gastroenteritis to hepatic and renal dysfunction. Most of the 13 patients presented with nausea, vomiting, abdominal pain, diarrhea, and elevated levels of biochemical indices of hepatic and renal function, during which transaminase concentration peaked within 48-72 h. At 48 hours post-ingestion, all patients had hemodialysis, in addition to supportive care for hepatic and renal injury with oral Silibinin and Shenshuaining. All acute renal injury had resolved by day 10, and liver transaminases had trended toward normal in all patients and they were discharged. At follow-up in 30 days, both liver and renal function had completely recovered in all. CONCLUSION: This poisoning occurs as a result of unintentional consumption of G. sulciceps, which is relatively rare in mushroom poisonings. All patients recovered fully after timely diagnosis and treatment. To prevent wild mushroom poisoning, the best preventive measure is to educate the public not to gather and eat any unidentified wild mushrooms.

The MSDIN family in amanitin-producing mushrooms and evolution of the prolyl oligopeptidase genes.

The biosynthetic pathway for amanitins and related cyclic peptides in deadly Amanita (Amanitaceae) mushrooms represents the first known ribosomal cyclic peptide pathway in the Fungi. Amanitins are found outside of the genus in distantly related agarics Galerina (Strophariaceae) and Lepiota (Agaricaceae). A long-standing question in the field persists: why is this pathway present in these phylogenetically disjunct agarics? Two deadly mushrooms, A. pallidorosea and A. subjunquillea, were deep sequenced, and sequences of biosynthetic genes encoding MSDINs (cyclic peptide precursor) and prolyl oligopeptidases (POPA and POPB) were obtained. The two Amanita species yielded 29 and 18 MSDINs, respectively. In addition, two MSDIN sequences were cloned from L. brunneoincarnata basidiomes. The toxin MSDIN genes encoding amatoxins or phallotoxins from the three genera were compared, and a phylogenetic tree constructed. Prolyl oligopeptidase B (POPB), a key enzyme in the biosynthetic pathway, was used in phylogenetic reconstruction to infer the evolutionary history of the genes. Phylogenies of POPB and POPA based on both coding and amino acid sequences showed very different results: while POPA genes clearly reflected the phylogeny of the host species, POPB did not; strikingly, it formed a well-supported monophyletic clade, despite that the species belong to different genera in disjunct families. POPA, a known house-keeping gene, was shown to be restricted in a branch containing only Amanita species and the phylogeny resembled that of those Amanita species. Phylogenetic analyses of MSDIN and POPB genes showed tight coordination and disjunct distribution. A POPB gene tree was compared with a corresponding species tree, and distances and substitution rates were compared. The result suggested POPB genes have significant smaller distances and rates than the house-keeping rpb2, discounting massive gene loss. Under this assumption, the incongruency between the gene tree and species tree was shown with strong support. Additionally, k-mer analyses consistently cluster Galerina and Amanita POPB genes, while Lepiota POPB is distinct. Our result suggests that horizontal gene transfer (HGT), at least between Amanita and Galerina, was involved in the acquisition of POPB genes, which may shed light on the evolution of the α-amanitin biosynthetic pathway.

Production of (15)N-labeled α-amanitin in Galerina marginata.

α-Amanitin is the major causal constituent of deadly Amanita mushrooms that account for the majority of fatal mushroom poisonings worldwide. It is also an important biochemical tool for the study of its target, RNA polymerase II. The commercial supply of this bicyclic peptide comes from Amanita phalloides, the death cap mushroom, which is collected from the wild. Isotopically labeled amanitin could be useful for clinical and forensic applications, but α-amanitin has not been chemically synthesized and A. phalloides cannot be cultured on artificial medium. Using Galerina marginata, an unrelated saprotrophic mushroom that grows and produces α-amanitin in culture, we describe a method for producing (15)N-labeled α-amanitin using growth media containing (15)N as sole nitrogen source. A key to success was preparing (15)N-enriched yeast extract via a novel method designated "glass bead-assisted maturation." In the presence of the labeled yeast extract and (15)N-NH4Cl, α-amanitin was produced with >97% isotope enrichment. The labeled product was confirmed by HPLC, high-resolution mass spectrometry, and NMR.