Amanita Exitialis Research Articles

Polymerase chain reaction-based methods for the rapid identification of Amanita exitialis

Amanita exitialis, a deadly mushroom found in eastern Asia, causes the highest death rates among all poisonous mushrooms in China. The aim of the present study was to develop an efficient, accurate, and user-friendly PCR-based method for identifying A. exitialis that could facilitate the prevention, diagnosis, and treatment of associated food poisoning. A. exitialis-specific primers and probes were designed based on the internal transcribed spacer region variations of 27 mushroom species. Specificity was confirmed using conventional and real-time PCR for 23 non-target mushroom species, including morphologically similar and closely related species. Compared to conventional PCR, real-time PCR was more sensitive (detectable DNA concentration: 1.36 × 10−2 ng/μL vs. 1.36 × 10−3) and efficient (analysis time: 1 h vs. 40 min). Furthermore, the real-time PCR results could be immediately visualized using amplification curve analysis. The results present two robust PCR-based methods for A. exitialis identification that can facilitate food safety.

The research of liver failure in Banna miniature pigs caused by amanita exitialis

Objective: To explore the characteristics of Banna miniature pig liver failure induced by amanita exitialis. Methods: From September to October 2020, a reverse high performance liquid chromatography (RP-HPLC) method was used to determine the toxin content of amanita exitialis solution, and 2.0 mg/kg amanita exitialis solution (α-amanitins+β-amanitins) was administered orally to Banna miniature pigs. Toxic symptoms, blood biochemical indexes and histopathological changes of liver, heart and kidney were observed at each time point. Results: All Banna miniature pigs died within 76 h of exposure, and different degrees of digestive tract symptoms such as nausea, vomiting and diarrhea appeared between 6 and 36 h. The biochemical indexes of alanine aminotransferase, aspartate aminotransferase, total bilirubin, lactate dehydrogenase, myoglobin, creatine kinase isoenzyme, blood urea nitrogen and creatinine increased significantly at 52 h after exposure, and the differences were statistically significant compared with 0 h (P<0.05). The bleeding of liver and heart was obvious under macroscopic and microscopic observation, hepatocyte necrosis, renal tubule epithelial cell swelling. Conclusion: Large dose of amanita exitialis can cause acute liver failure of Banna miniature pigs, which is in line with the pathophysiological characteristics of acute liver failure, and lays a foundation for further research on the toxic mechanism and detoxification drugs of amanita exitialis induced liver failure.

An outbreak of Amanita exitialis poisoning

Background The mushroom Amanita exitialis is reported to cause acute liver injury. It is found in Southern China, and has been previously associated with a high incidence of mortality. Methods We described a series of 10 patients with Amanita exitialis poisoning admitted to The Second Affiliated Hospital of the Chinese University of Hong Kong (Shenzhen) in April 2022. Patient demographics, clinical features, laboratory results, therapeutic interventions, and outcome data were collected. Results Among the 10 patients, 9 survived, while 1 died. Gastrointestinal symptoms were the first to appear (average latency period, 11 ± 4.2 h). Diarrhea was the most common clinical symptom (average duration, 4.4 days). Abdominal distention was an important sign, especially in severely-ill patients. Thrombocytopenia occurred on day 2 after mushroom ingestion and persisted for 3–4 days. Alanine aminotransferase and total bilirubin peaked on days 2–3. Conclusion Amanita exitialis poisoning is characterized by gastrointestinal symptoms and liver injury. In the patient who died, acute hepatic failure led to hepatic encephalopathy and cerebral edema. Abdominal distension accompanied by thrombocytopenia was common in critically ill patients in this outbreak.

Diversity, Dispersal and Mode of Reproduction of Amanita exitialis in Southern China.

Amanita exitialis is a poisonous mushroom and has caused many deaths in southern China. In this study, we collected 118 fruiting bodies of A. exitialis from seven different sites in Guangdong Province in southern China and investigated their genetic relationships using 14 polymorphic molecular markers. These 14 markers grouped the 118 fruiting bodies into 20 multilocus genotypes. Among these 20 genotypes, eight were each found only once while the remaining 12 were each represented by two to 54 fruiting bodies. Interestingly, among the 12 shared genotypes, four were shared between/among local populations that were separated by as far as over 80 km, a result consistent with secondary homothallic reproduction and long-distance spore dispersal. Despite the observed gene flow, significant genetic differentiations were found among the local populations, primarily due to the over-representation of certain genotypes within individual local populations. STRUCTURE analyses revealed that the 118 fruiting bodies belonged to three genetic clusters, consistent with divergence within this species in this geographic region. Interestingly, we found an excess of heterozygous individuals at both the local and the total sample level, suggesting potential inbreeding depression and heterozygous advantage in these populations of A. exitialis. We discuss the implications of our results for understanding the life cycle, dispersal, and evolution of this poisonous mushroom.

Novel Cyclic Peptides from Lethal Amanita Mushrooms through a Genome-Guided Approach.

Most species in the genus Amanita are ectomycorrhizal fungi comprising both edible and poisonous mushrooms. Some species produce potent cyclic peptide toxins, such as α-amanitin, which places them among the deadliest organisms known to mankind. These toxins and related cyclic peptides are encoded by genes of the “MSDIN” family (named after the first five amino acid residues of the precursor peptides), and it is largely unknown to what extent these genes are expressed in the basidiocarps. In the present study, Amanita rimosa and Amanita exitialis were sequenced through the PacBio and Illumina techniques. Together with our two previously sequenced genomes, Amanita subjunquillea and Amanita pallidorosea, in total, 46 previously unknown MSDIN genes were discovered. The expression of over 80% of the MSDIN genes was demonstrated in A. subjunquillea. Through a combination of genomics and mass spectrometry, 12 MSDIN genes were shown to produce novel cyclic peptides. To further confirm the results, three of the cyclic peptides were chemically synthesized. The tandem mass spectrometry (MS/MS) spectra of the natural and the synthetic peptides shared a majority of the fragment ions, demonstrating an identical structure between each peptide pair. Collectively, the results suggested that the genome-guided approach is reliable for identifying novel cyclic peptides in Amanita species and that there is a large peptide reservoir in these mushrooms.

Mushroom Poisoning in Thailand: Incidence and Intoxication to Human Health

Mushroom poisoning is one of the public health issues related to food-borne diseases in Thailand and worldwide. This paper revealed the incidence of mushroom poisoning in Thailand between 2008 and 2017 and discussed relevant information involved. About 1,200 to 2,000 reported cases were reported each year with morbidity rates from 1.86 to 3.34 per 100,000 population and mortality rates from 0.12 to 1.12%. Overall, 15,680 patients of mushroom poisoning were reported in Thailand during the 10-year period from 2008 to 2017 with an average of 1,568 patients annually. The cases were reported mainly from northeast and north Thailand. Actually, the incidence of mushroom poisoning increased during rainy season between May and September. The highest risk was found among patients aged between 55 and 64 years and over 65 years followed by the group of 45-54 years. The poison risk to women was always greater than to men with ratios varying between 1.13:1 and 1.70:1 and the incidence was found mainly among farmers and general workers. Four groups of toxins have been found in Thailand including protoplasmic poisons, neurotoxins, gastrointestinal irritants and disulfiram-like toxins. The protoplasmic poisons are a major cause of death in most cases and are found among some mushrooms, such as Amanita exitialis, A. fuliginea, A. fuligineoides and A. gleocystidiosa. Gastrointestinal irritants are the most common toxins found in all regions of Thailand and found among some mushrooms, such as Chlorophyllum molybdites, Russula sp., Cantharocybe virosa, Entoloma sp., Lactarius sp.and Tricholoma sp. Commonly, consumption of poisonous mushrooms is usually a result from misidentification of some poisonous ones, closely similar to those of the commonly edible mushrooms. Therefore, educating the public to recognize poisonous mushrooms to avoid to harvesting them for consumption or sale is urgently needed.

Effect of Biliary Drainage on the Toxicity and Toxicokinetics of Amanita exitialis in Beagles.

Amatoxin poisoning induces delayed-onset acute liver failure, which are responsible for more than 90% of deaths in mushroom poisoning. It has been postulated from animal and human studies that biliary drainage interrupting enterohepatic amatoxin circulation may affect amatoxin poisoning. Dogs were randomly divided into four groups of six animals each. In 20 mg/kg and 60 mg/kg with biliary drainage groups, after accepting bile drainage operation, beagles were fed Amanita exitialis powder (20 or 60 mg/kg) in starch capsules. In control and bile drainage groups, the beagle dogs were fed with empty capsules. They were assessed for toxicity signs, biochemical and pathological changes, and peptide toxins in plasma, urine and bile. The data were directly compared with those from our published studies on Amanita exitialis-exposed beagles without biliary drainage. Amatoxins were rapidly absorbed and eliminated from plasma after Amanita exitialis ingestion. Amatoxins in 0–1-day urine accounted for more than 90% of the total urine excretion, and amatoxins in bile accounted for less than 20% of the total urine and bile excretion. The dogs with biliary drainage showed less severe toxicity signs and biochemical and pathological changes and much lower internal exposure than dogs without biliary drainage. Biliary drainage caused a more than 70% reduction in intestinal amatoxin absorption and could reduce amatoxin absorption from the gastrointestinal tract.

Molecular cloning and the expression pattern of AePOPB involved in the α-amanitin biosynthesis in Amanita exitialis fruiting bodies

Amanita exitialis Zhu L. Yang & T. H. Li is the species responsible for the largest number of mushroom-associated human poisonings and fatalities in South China due to its lethal cyclic peptide toxins. Prolyl oligopeptidase B (POPB) is considered a key enzyme in the production of the highly toxic cyclic peptide α-amanitin. However, the POPB gene of A. exitialis has not been studied. In the present study we cloned and sequenced the full-length A. exitialis POPB (AePOPB) gene. The aim was to verify the gene structure and functions of AePOPB. The full-length sequence of AePOPB is 3144 bp, including 18 exons encoding 730 aa, and the advanced structure is very similar to that of the previously reported POPB in Galerina marginata (GmPOPB). The amino acid sequence of AePOPB is highly homologous with those from other amanitin-producing lethal mushrooms, implying that AePOPB may have a similar role in the biosynthesis of cyclic peptide toxins. Expression levels of AePOPB were detectable in all parts and developmental stages of the fruiting bodies, and AePOPB was expressed more strongly at early development stages (early and late elongation stages). At early and late elongation stages, the expression peaks occurred in the stipe, whereas at early and late mature stages, the expression peaks occurred in the pileus. The expression patterns of AePOPB in different stages and different parts of the fruiting bodies were highly consistent with those of Aeα-AMA, which is required for α-amanitin accumulation. These results indicate that AePOPB should be involved in the α-amanitin biosynthesis in A. exitialis.

Experimental study on acute hepatotoxicity in beagles induced by Amanita exitialis

Objective To establish acute hepatotoxic model induced by Amanita exitialis and to study the characteristics of acute toxic liver failure induced by mushrooms containing peptide toxins, in hope for providing some help to experimental research on poisoning induced by mushrooms containing peptide toxins. Methods UPLC-MS/MS(Ultra performance liquid chromatography- tandem mass spectrometry) method was used to detect peptide toxins in Amanita exitialis. To establish acute toxic liver hepatic failure model, the beagles were fed with 60 mg/kg of lyophilized powder of Amanita exitialis fungus which encapsulated in starch capsules. Toxic sighs were observed, coagulation function, hepatic and renal function, liver histopathological morphology, peptide toxin concentration in plasma and urine were detected during the experiment. Results Total peptide toxins in Amanita exitialis was (3 482.6 ± 124.94) mg/kg. All the beagles had toxic signs including vomiting and diarrhea in 12-48 h after ingestion. On 24 h after ingestion, the beagles’ ALT, AST, TBIL, ALP, PT and APTT levels increased obviously. On 36 h after ingestion, the beagles’ ALT, AST, PT and APTT values reached their peaks (ALT: 283.2±112.9 Kallmann unit; AST: 223.9±93.8 Kallmann units; PT: 132.9±152.6 s; APTT: 131.4±153.9 s). On 48 h after ingestion, the beagles’ TBIL and ALP levels reached their peaks (TBIL: 23.3±14.6 mol/L; ALP: 274.5±115.5 U/L). The beagles’ TBIL, TP and APTT returned to normal 1 week after ingestion, their ALT, AST and ALP levels returned to normal 3 weeks after ingestion. Three dogs died during 24-72 h after ingestion. Liver histopathological morphology study showed hemorrhagic necrosis of hepatocytes. Peptide toxins can be detected in plasma within 24 h after ingestion. Peptide toxins can be detected in urine within 96 h after ingestion. Conclusion Amanita peptide toxins can cause hemorrhagic necrosis of liver cells and lead to acute liver failure. This model is consistent with clinical pathophysiological process of acute toxic liver failure induced by mushrooms containing peptide toxins, and it can be applied to the study of diagnosis and treatment of poisoning induced by mushrooms containing peptide toxins. Key words: Mushroom poisoning; Amanita exitialis; Amanita peptide toxins; Beagles; Hepatotoxicity; Hemorrhagic necrosis of liver cells; Acute hepatic failure; Toxin detection; Model

Molecular cloning of α-amanitin and characterization of its expression pattern in different parts and development stages of Amanita exitialis fruitbody

Amanita exitialis is a lethal mushroom in East Asia. α-Amanitin is one kind of the leading peptide toxins responsible for mushroom poisoning, which are mainly from the fatal amanitae. In the study, the full-length cDNA sequences of α-amanitin (α-AMA) and phallacidin (PHA) from A. exitialis were cloned and analyzed; in addition, the expression patterns of α-AMA in three parts as well as four developmental stages of the fruitbody were first characterized by quantitative real-time PCR. Results demonstrate that α-AMA and PHA were highly homologous to the toxin genes reported from A. bisporigera; the α-AMA could be expressed in all parts and stages of the fruitbody, from late elongation stage to late mature stage, α-AMA showed the highest expression level in the pileus, followed by the stipe and volva in sequence, while at the early elongation stage, the highest expression level was found in the stipe, followed by the pileus and volva. Further analysis revealed that the higher expression level of α-AMA was closely associated with the development stages, and the expression of α-AMA was accumulated in the more vigorous growth parts and stages, from which could be inferred that α-AMA might play an important role in the development of A. exitialis.

Illumina-based de novo transcriptome sequencing and analysis of Amanita exitialis basidiocarps

Amanita exitialis is a lethal mushroom that was first discovered in Guangdong Province, China. The high content of amanitin in its basidiocarps makes it lethal to humans. To comprehensively characterize the A. exitialis transcriptome and analyze the Amanita toxins as well as their related gene family, transcriptome sequencing of A. exitialis was performed using Illumina HiSeq 2000 technology. A total of 25,563,688 clean reads were collected and assembled into 62,137 cDNA contigs with an average length of 481bp and N50 length of 788bp. A total of 27,826 proteins and 39,661 unigenes were identified among the assembled contigs. All of the unigenes were classified into 166 functional categories for understanding the gene functions and regulation pathways. The genes contributing to toxic peptide biosynthesis were analyzed. From this set, eleven gene sequences encoding the toxins or related cyclic peptides were discovered in the transcriptome. Three of these sequences matched the peptide toxins α-amanitin, β-amanitin, and phallacidin, while others matched amanexitide and seven matched unknown peptides. All of the genes encoding peptide toxins were confirmed by polymerase chain reaction (PCR) in A. exitialis, and the phylogenetic relationships among these proprotein sequences were discussed. The gene polymorphism and degeneracy of the toxin encoding sequences were found and analyzed. This study provides the first primary transcriptome of A. exitialis, which provided comprehensive gene expression information on the lethal amanitas at the transcriptional level, and could lay a strong foundation for functional genomics studies in those fungi.

Hepatoprotective Effects of Aqueous Extract from Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Higher Basidiomycetes) on α-Amanitin - Induced Liver Injury in Mice

The Lingzhi or Reishi mushroom Ganoderma lucidum is a well-known traditional medicinal mushroom that has been shown to have obvious hepatoprotective effects. The aim of this study was to evaluate the hepatoprotective effects of G. lucidum aqueous extracts (GLEs) on liver injury induced by α-amanitin (α-AMA) in mice and to analyze the possible hepatoprotective mechanisms related to radical scavenging activity. Mice were treated with α-AMA prepared from Amanita exitialis and then administrated with GLE after the α-AMA injection. The hepatoprotective activity of the GLE was compared with the reference drug silibinin (SIL). α-AMA induced a significant elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities and provoked a significant reduction of superoxide dismutase (SOD) and catalase (CAT) activities and a significant increment of malondialdehyde (MDA) content in liver homogenate. Treatment with GLE or SIL significantly decreased serum ALT and AST levels, significantly increased SOD and CAT activities, and decreased MDA content in liver compared with the α-AMA control group. The histopathological examination of liver sections was consistent with that of biochemical parameters. The results demonstrated that GLE induces hepatoprotective effects on acute liver injury induced by α-AMA; these protective effects may be related in part to the antioxidant properties of GLE.

Determination of amatoxins in different tissues and development stages of Amanita exitialis

Mushroom poisoning is the main cause of human death by food poisoning in China. Most lethal mushrooms belong to the Amanita genus, whose amatoxins are responsible for the death of humans. Amanita exitialis is a lethal white mushroom commonly found in Guangdong Province, China. In this study the contents and distribution of the major amatoxins in different tissues and development stages of A. exitialis were systematically analysed. The amatoxin contents and distribution in six different mushroom tissues of A. exitialis were analysed by reverse phase high-performance liquid chromatography. The highest concentrations of amatoxins were found in the gills and pileus, followed by the stipe and annulus, with the lowest concentrations in the volva and spores. Further analysis of mushrooms in different development stages showed that the amatoxin content was relatively high and steady during early development, reached its peak when the fruit body was in the vigorous growth stage and then decreased sharply when the mushroom entered its mature stage. Furthermore, the α-amanitin/β-amanitin ratio varied significantly in different tissues but remained constant within a specific tissue throughout development. The contents and distribution of amatoxins in different tissues and development stages of A. exitialis are markedly different. The distribution of α-amanitin and β-amanitin varies in different tissues but remains constant throughout development.

Production and characterization of Amanitin toxins from a pure culture of Amanita exitialis

Amanita exitialis Zhu L. Yang and T.H. Li is a lethal mushroom species recently isolated in Guangdong Province, China. In this report, a pure culture of this species was obtained for the first time. To confirm the identity of the pure culture, the internal transcribed spacer regions of the nuclear ribosomal DNA of the pure culture and of a typical fruiting body of the species were sequenced and compared. Further, amatoxins produced by pure cultures were analyzed and characterized by high-performance liquid chromatography and mass spectrometry analysis. The results showed that the pure cultures produced 728.3 ± 43.8 μg g −1 (dry matter) of α-Amanitin and 60.0 ± 20.7 μg g −1 (dry matter) of β-Amanitin, respectively, a yield which is about 10% of that produced by fruiting bodies.