ArticleLiterature Review

Ribosomal Biosynthesis of the Cyclic Peptide Toxins of Amanita Mushrooms

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Abstract

Some species of mushrooms in the genus Amanita are extremely poisonous and frequently fatal to mammals including humans and dogs. Their extreme toxicity is due to amatoxins such as alpha- and beta-amanitin. Amanita mushrooms also biosynthesize a chemically related group of toxins, the phallotoxins, such as phalloidin. The amatoxins and phallotoxins (collectively known as the Amanita toxins) are bicyclic octa- and heptapeptides, respectively. Both contain an unusual Trp-Cys crossbridge known as tryptathionine. We have shown that, in Amanita bisporigera, the amatoxins and phallotoxins are synthesized as proproteins on ribosomes and not by nonribosomal peptide synthetases. The proproteins are 34-35 amino acids in length and have no predicted signal peptides. The genes for alpha-amanitin (AMA1) and phallacidin (PHA1) are members of a large family of related genes, characterized by highly conserved amino acid sequences flanking a hypervariable "toxin" region. The toxin regions are flanked by invariant proline (Pro) residues. An enzyme that could cleave the proprotein of phalloidin was purified from the phalloidin-producing lawn mushroom Conocybe apala. The enzyme is a serine protease in the prolyl oligopeptidase (POP) subfamily. The same enzyme cuts at both Pro residues to release the linear hepta- or octapeptide.

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... Amatoxine sind ribosomal synthetisierte toxische bicyclische Octapeptide, die hauptsächlich in mehreren Spezies der Gattung Amanita vorkommen, insbesondere in Amanita phalloides. [1][2][3][4] Sie inhibieren selektiv die eukaryotische RNA Polymerase II [a-Amanitin, K d = 10 À9 m], was letzten Endes zur Apoptose der Zielzelle führt. [4,5] Amatoxine wurden erstmals 1941 isoliert (Wieland und Hallermayer) und ihre Struktur in den 1950er und 1960er Jahren aufgeklärt (Wieland et al.). ...
... [1][2][3][4] Sie inhibieren selektiv die eukaryotische RNA Polymerase II [a-Amanitin, K d = 10 À9 m], was letzten Endes zur Apoptose der Zielzelle führt. [4,5] Amatoxine wurden erstmals 1941 isoliert (Wieland und Hallermayer) und ihre Struktur in den 1950er und 1960er Jahren aufgeklärt (Wieland et al.). [6,7] Alle Amatoxine besitzen acht l-konfigurierte Aminosäuren und eine Tryptathionin-Querverknüpfung zwischen den Seitenketten der Aminosäuren Tryptophan und Cystein, die ein (R)-konfiguriertes Sulfoxid besitzt. ...
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Eine neue Syntheseroute zum toxischen bicyclischen Octapeptid α‐Amanitin wird vorgestellt. Die Schlüsselschritte der konvergenten [5+1+2]‐Synthesestrategie sind die Bildung des Thioether‐Bausteins und der Zugang zu den enantiomerenreinen Aminosäuren l‐6‐Hydroxytryptophan und (3R,4R)‐l‐4,5‐Dihydroxyisoleucin im Multigramm‐Maßstab. Die Fragment‐basierende Methode führte zur ersten konvergenten α‐Amanitin‐Synthese, die vollständig in Flüssigphase durchgeführt wurde. Abstract Das toxische bicyclische Octapeptid α‐Amanitin kommt hauptsächlich in Spezies der Pilzgattung Amanita vor, wobei der Grüne Knollenblätterpilz (Amanita phalloides) der bekannteste Vertreter ist. Seine Toxizität beruht auf der Inhibition von RNA Polymerase II, insbesondere in Hepatocyten. Dies macht α‐Amanitin zu einem interessanten Wirkstoff für Antikörper‐Wirkstoff‐Konjugate in der Krebsforschung. Allerdings stellt die Isolierung von ausreichenden Mengen an α‐Amanitin aus Pilzen ein schwieriges Unterfangen dar, was sich limitierend auf die medizinalchemische Anwendung auswirkt. Ausgehend von der Synthese der nicht‐proteinogenen Aminosäure Dihydroxyisoleucin entwickelten wir eine robuste Totalsynthese für α‐Amanitin, die sich sowohl für eine Synthese im industriellen Maßstab als auch für zukünftige strukturelle Diversifizierungen eignet.
... Amatoxins are ribosomally synthesized toxic bicyclic octapeptides mainly found in different species of the fungus genus Amanita, most notably Amanita phalloides. [1][2][3][4] These compounds are selective inhibitors of eukaryotic RNA polymerase II [a-amanitin, K d = 10 À9 m], which ultimately leads to apoptosis. [4,5] The fungal toxins were first isolated by Heinrich Wieland and Rudolf Hallermayer in 1941 and their structure was elucidated in the 1950s and 1960s (Wieland et al.). ...
... [1][2][3][4] These compounds are selective inhibitors of eukaryotic RNA polymerase II [a-amanitin, K d = 10 À9 m], which ultimately leads to apoptosis. [4,5] The fungal toxins were first isolated by Heinrich Wieland and Rudolf Hallermayer in 1941 and their structure was elucidated in the 1950s and 1960s (Wieland et al.). [6,7] All amatoxins contain eight l-configured amino acids and a tryptathionine linkage between the side chains of tryptophan and cysteine, with an (R)-configured sulfoxide moiety. ...
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The toxic bicyclic octapeptide α-amanitin is mostly found in different species of the mushroom genus Amanita, with the death cap (Amanita phalloides) as one of the most prominent members. Due to its high selective inhibition of RNA polymerase II which is directly linked to its high toxicity, particularly to hepatocytes, α-amanitin received an increased attention as a toxin-component of antibody-drug conjugates (ADC) in cancer research. Furthermore, the isolation of α-amanitin from mushrooms as the sole source severely restricts compound supply as well as further investigations, as structure-activity relationship (SAR) studies. Based on a straightforward access to the non-proteinogenic amino acid dihydroxyisoleucine we herein present a robust total synthesis of α-amanitin providing options for production at larger scale as well as future structural diversifications.
... Amatoxins are also biosynthesized by mushrooms in other unrelated genera, such as Galerina, Lepiota, and Conocybe. They can cause liver failure and death by inhibiting ribonucleic acid (RNA) polymerase II [9]. Amanita mushrooms are also responsible for producing a structurally related group of toxins, called phallotoxins, which are orally inactive but toxic when injected. ...
... Amanita mushrooms are also responsible for producing a structurally related group of toxins, called phallotoxins, which are orally inactive but toxic when injected. Phallotoxins act by stabilizing F-actin, and have been utilized to stain the cytoskeleton ( Figure 2) [9]. Genome survey sequencing revealed that amatoxins and phallotoxins are biosynthesized via ribosomal pathways [8,10]. ...
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Natural products have played indispensable roles in drug development and biomedical research. Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a group of fast-expanding natural products attribute to genome mining efforts in recent years. Most RiPP natural products were discovered from bacteria, yet many eukaryotic cyclic peptides turned out to be of RiPP origin. This review article presents recent advances in the discovery of eukaryotic RiPP natural products, the elucidation of their biosynthetic pathways, and the molecular basis for their biosynthetic enzyme catalysis.
... In particular, the amatoxin α-amanitin is primarily responsible for the fatalities associated with Amanita (Berger and Guss, 2005a). In humans, a dose as low as~0.1 mg/kg can be fatal (Faulstich 1980;Wieland, 1986;Karlson-Stiber and Persson, 2003;Erden et al., 2013), and a single Amanita mushroom can contain~10-12 mg of α-amanitin (Mas, 2005;Walton et al., 2010). Studies of amatoxin toxicity in white mice calculated an LD 50 of~0.3-0.7 mg/kg (Wieland, 1983). ...
... These resistant fly stocks were originally collected in the 1960s in Taiwan (Ama-KTT), India (Ama-MI), and Malaysia (Ama-KLM). Because amatoxins are solely produced by mushrooms (Hallen et al., 2002;Hallen et al., 2007;Walton et al., 2010), it is quite astonishing that any strains of this mushroom-avoiding species, which is frugivorous and does not exhibit any mushroom-feeding behavior (Werner, 2017), have evolved resistance to the potent amatoxin, αamanitin. Their α-amanitin resistance was approximately two orders of magnitude higher than that of the susceptible reference strain Oregon-R (i.e., the resistant strains tolerated up to~10 mg of α-amanitin per gram of food, as compared to~0 ...
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Background: Evolutionary novelties, be they morphological or biochemical, fascinate both scientists and non-scientists alike. These types of adaptations can significantly impact the biodiversity of the organisms in which they occur. While much work has been invested in the evolution of novel morphological traits, substantially less is known about the evolution of biochemical adaptations. Methods: In this review, we present the results of literature searches relating to one such biochemical adaptation: α-amanitin tolerance/resistance in the genus Drosophila. Results: Amatoxins, including α-amanitin, are one of several toxin classes found in Amanita mushrooms. They act by binding to RNA polymerase II and inhibiting RNA transcription. Although these toxins are lethal to most eukaryotic organisms, 17 mushroom-feeding Drosophila species are tolerant of natural concentrations of amatoxins and can develop in toxic mushrooms. The use of toxic mushrooms allows these species to avoid infection by parasitic nematodes and lowers competition. Their amatoxin tolerance is not due to mutations that would inhibit α-amanitin from binding to RNA polymerase II. Furthermore, the mushroom-feeding flies are able to detoxify the other toxin classes that occur in their mushroom hosts. In addition, resistance has evolved independently in several D. melanogaster strains. Only one of the strains exhibits resistance due to mutations in the target of the toxin. Conclusions: Given our current understanding of the evolutionary relationships among the mushroom-feeding flies, it appears that amatoxin tolerance evolved multiple times. Furthermore, independent lines of evidence suggest that multiple mechanisms confer α-amanitin tolerance/resistance in Drosophila. © 2018 Higher Education Press and Springer-Verlag GmbH Germany, part of Springer Nature
... Hallen et al. (2007) found that POPase proteins are mainly present in Basidiomycetes and are predicted to be related to the biosynthesis of cyclic peptide toxins. Later, this hypothesis was proven via a physicochemical test by purification of the POPase protein from mushroom extracts, with C. apala as the starting material (Walton et al., 2010). In amanitin-producing species, two genes from POPase families, known as POPA and POPB in A. bisporigera and G. marginata, were cloned , and POPA was shown to be a ubiquitous protein in amanitin-producing and non-amanitin-producing species of Amanita, whereas POPB is restricted in taxonomic distribution to amanitin-producing species, as a pivotal POPase catalyzing two nonprocessive reactions in the biosynthesis of cyclic peptide toxins (Luo et al., 2012(Luo et al., , 2014. ...
... α-Amanitin, encoded by gene AMA, is the most representative toxin among lethal Amanita mushrooms Walton et al., 2010), whereas POPB is the key enzyme in the biosynthesis of amanitin (Luo et al., 2012). Some researchers have described the relevance of AeAMA1 expression and α-amanitin accumulation in A. exitialis, and suggested that the POPB gene was closely related to α-amanitin biosynthesis Luo et al., 2014), however, there have been no reports about the relations between POPB, AMA, and α-amanitin. ...
Article
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.
... Seven phallotoxins: phallacidin (191), phallacin (192), phallisacin (193), phallisin (194), phalloidin (195), phalloin (196), and prophallin (197), have been identified so far [150]. Phallotoxins were shown to be ribosomally synthesized and posttranslationally modified peptides (RiPPs) [151]. ...
... Amatoxins are synthesized as proproteins on ribosomes, and are considered as ribosomally synthesized and post-translationally modified peptides (RiPPs). The proproteins are 34-35 amino acids in length and do not have predicted signal peptides [151]. ...
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Cyclic peptides are cyclic compounds formed mainly by the amide bonds between either proteinogenic or non-proteinogenic amino acids. This review highlights the occurrence, structures and biological activities of fungal cyclic peptides (excluding cyclodipeptides, and peptides containing ester bonds in the core ring) reported until August 2017. About 293 cyclic peptides belonging to the groups of cyclic tri-, tetra-, penta-, hexa-, hepta-, octa-, nona-, deca-, undeca-, dodeca-, tetradeca-, and octadecapeptides as well as cyclic peptides containing ether bonds in the core ring have been isolated from fungi. They were mainly isolated from the genera Aspergillus, Penicillium, Fusarium, Acremonium and Amanita. Some of them were screened to have antimicrobial, antiviral, cytotoxic, phytotoxic, insecticidal, nematicidal, immunosuppressive and enzyme-inhibitory activities to show their potential applications. Some fungal cyclic peptides such as the echinocandins, pneumocandins and cyclosporin A have been developed as pharmaceuticals.
... Three different amino acid substitutions were found at the second (Cys/Trp), fourth (Val/Ala) and fifth (Phe/Thr) positions. Amatoxins as well as phallotoxins are the bicyclic peptides that normally contain the tryptathionine linkage (Trp-Cys cross-bridge) which is involved in the toxin biosynthesis [34]. The core amino acid sequence of A. brunneitoxicaria unknown peptide however, revealed the existence of a disulfide bond (Cys-Cys) instead of the tryptathionine linkage. ...
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A recent discovery of Amanita brunneitoxicaria revealed that this mushroom and A. exitialis are the main causative agents of fatal mushroom poisoning in Thailand. Poisonous amatoxins present are bicyclic octapeptides encoded by the MSDIN family and macrocyclized by prolyl oligopeptidase B enzyme. Diversity of the MSDIN toxin-associated genes in A. brunneitoxicaria however, remains unexplored. Therefore, this study aimed to characterize the MSDIN family members in A. brunneitoxicaria in comparison with A. exitialis based on whole genome sequencing, followed by gene annotation using bioinformatic tools. Identification of conserved core peptides of the MSDIN family revealed the presence of α-amanitin and β-amanitin as endogenous toxins in A. brunneitoxicaria. In addition to the major amatoxins, amanexitide and phallacidin were also found in A. exitialis. Phylogenetic analyses of the MSDIN family members showed that an unknown peptide present in A. brunneitoxicaria is clustered with phalloidin. On the basis of PCR detection, a modified forward primer was incorporated into the PCR reactions to obtain clear and distinctive DNA fragments of approximately 300 bp from three deadly poisonous mushrooms of A. brunneitoxicaria, A. exitialis and A. fuliginea in the section Phalloideae. Hence, they could be distinguished from other edible and less toxic species in the same genus. Such rapid and accurate identification of the target region of the MSDIN family from the lethal mushrooms is relevant as means to reduce mortality. HIGHLIGHTS Amanita brunneitoxicaria and A. exitialis are responsible for most cases of fatal mushroom poisoning in Thailand During wild mushroom foraging, A. brunneitoxicaria is often mistaken for the edible species of A. vaginata A. brunneitoxicaria and other lethal aminitas contain amatoxin-producing genes encoded by the MSDIN family PCR-based detection of the target region of the MSDIN family can be used to distinguish the lethal aminitas in the section Phalloideae This rapid detection is useful for timely and appropriate medical treatment of mushroom intoxication GRAPHICAL ABSTRACT
... Amanitin biosynthesis Amatoxins are ribosomally synthesized and post-translationally modified peptides (RiPPs) produced by select mushroom species that are responsible for severe mushroom poisonings [125]. Amatoxins consist of eight peptide residues that are cyclized and contain tryptathione, sulfoxide, and hydroxyl functionalities, and the biological target is RNA polymerase II [126]. ...
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Fungal specialized metabolites play an important role in the environment and have impacted human health and survival significantly. These specialized metabolites are often the end product of a series of sequential and collaborating biosynthetic enzymes that reside within different subcellular compartments. A wide variety of methods have been developed to understand fungal specialized metabolite biosynthesis in terms of the chemical conversions and the biosynthetic enzymes required, however there are far fewer studies elucidating the compartmentalization of the same enzymes. This review illustrates the biosynthesis of specialized metabolites where the localization of all, or some, of the biosynthetic enzymes have been determined and describes the methods used to identify the sub-cellular localization.
... Bacteriocins are unique amino acid sequences (peptides) produced by bacteria and some fungi and possess antimicrobial activities against various groups of microorganisms (Riley & Wertz 2002, Walton et al. 2010, Chikindas et al. 2017. Anaerobes from various samples like faecal, environmental and foods have been reported to produce bacteriocins. ...
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Anaerobic microorganisms occur in oxygen-free environments as they possess biomolecules and metabolic processes that have adapted to sustain them in the stressed environments. These biomolecules and metabolic processes are harnessed and employed in various biotechnological applications. Anaerobes are abundant in various ecological niches such as freshwater or marine sediments, subsurface aquifers, deep thermal vents etc. where, in the absence or limitation of oxygen, they utilise reduced ions such as nitrate, sulphate, ferric, carbonate and certain organic compounds like degrading organic matter, fatty acids or alcohols as electron acceptors during respiration/fermentation to produce more or fewer reduced products such as, alcohols, ammonia, organic acids, hydrogen and carbon dioxide as the oxidised product. Diversity of substrates that can be fermented and the wide range of products that can be formed make anaerobic processes useful in biotechnological applications. Their substrate versatility and flexibility, for example, they can utilize simple sugars as well as many of them can utilise complex renewable biomass make their biotechnological applications sustainable and environment friendly. Anaerobes have been employed in food, chemical and material industries for the production of food items, preservative, additives, chemical intermediates, solvents, catalysts and polymers. They find widespread usage in energy sector for generation of various kinds of biofuels that can mitigate our reliance on non-renewable sources for fuel production. An emerging aspect of anaerobic biotechnology is waste valorization that involves treatment of any kind of waste (industrial, municipal, agricultural, pharmaceutical), where organic fraction of the waste is converted into valuable products. Anaerobes play key role in maintaining the global cycles of carbon, nitrogen, and sulfur, and also facilitate bioremediation by breakdown of persistent compounds. Lately, role of anaerobes in medicine and health care sector is widely explored as they are found to produce several bacteriocins, antibiotics, immunomodulators, vitamins and anti-tumor compounds. Some of the anaerobes have also been employed as probiotics. In the present chapter, we discussed these and many more biotechnological applications of anaerobes in white, green, red and blue biotechnology. Keywords: Anaerobes, Fermentation, Anaerobic digestion, Bioreactors, Biotechnological applications
... Furthermore, the positive (eNH 2 þ ) and negative (eCOO -) ending groups are benefitted to the electrostatic connection between the L-Lysine molecules. The L-Lysine molecules can connect to each other via peptide bonding between the NH 2 and COOH groups [45]. Moreover, the Fe 2þ cations can be chelated by both amino and carboxyl groups [46,47]. ...
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LiFePO4/C powders were synthesized by solvothermal method using L-Lysine as a surfactant for controlling over the particle size and shape. The effects of L-Lysine contents on the phase, structure, microstructure, and electrochemical properties were studied by X-ray diffractometry, scanning electron microscopy, infrared spectroscopy, galvanic charge/discharge, and electrochemical impedance spectroscopy methods. As the amount of L-Lysine increased, the large sheet-like morphology was transformed to the spherical particulate and hollow-like structure. The LiFePO4/C powder obtained by 50 mmol L-Lysine showed the highest specific capacity of 96 mAh g⁻¹ at 1C rate and high capacity retention of 95.6% following 650 cycles because of the low antisite defects and particulate morphology of LiFePO4. Moreover, the high rate capability of the LiFePO4/C powders was attributed to their small spherical particles and hollow-like structure, leading to the high electrochemical kinetics as investigated by electrochemical impedance spectroscopy.
... It has been strongly indicated that a prolyl oligopeptidase (POP) plays an important role in the initial processing of MSDIN precursor peptides. Since the core toxin regions are flanked by two highly conserved proline (Pro) residues, this enzyme can cleave the C-terminus of Pro residues and release the peptide chain of the toxin to form a cyclopeptide [10]. It has been reported that there are two types of POP in amatoxin-producing mushrooms: POPA, which behaves like a conventional housekeeping protein that is present in all species, and POPB, which is the enzyme that actually catalyses the cutting and cyclization of precursor peptides [7,11,12]. ...
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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, β-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.
... In-vitro genetic code reprogramming, easily achievable with FIT-Laz, offers an alternative route to similar hybrids, many of which are inaccessible by existing methods. We demonstrated that thiopeptide-NRP hybrids (macrocycles containing hydroxyacids, D-, β-, Nmethylated-, and α,α-disubstituted amino acids), thiopeptide-RiPP hybrids (N-methylation and D-amino acids are found in borosins 66 , lanthipeptides 49 , proteusins 67 , phallotoxins 68 , and many other RiPPs families 7 ), and thiopeptides with unnatural amino acids (Phe(F 5 ) and cLeu) can be routinely accessed with FIT-Laz. Such noncanonical hybrid architectures can further expand the range of available molecular complexity for biotechnology and drug discovery. ...
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Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we establish a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis reveals remarkable substrate tolerance of the biosynthetic enzymes and leads to the development of the minimal lactazole scaffold, a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz opens access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and 18 amino acid-long tail regions, as well as to hybrid thiopeptides containing non-proteinogenic amino acids. This work suggests that the minimal lactazole scaffold is amenable to extensive bioengineering and opens possibilities to explore untapped chemical space of thiopeptides. Lactazole A is a thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, the authors show a platform for in vitro biosynthesis of lactazole A via a combination of a flexible in vitro translation system with recombinantly produced lactazole biosynthetic enzymes.
... In another route, the amino acids are ordered without direct ribosome involvement, via non-ribosomal peptide synthetases (Finking and Marahiel, 2004). Accumulated evidence suggests that ribosome-dependent biosynthesis of CPs were widespread in animals and fungi (Walton et al., 2010;Luo et al., 2012;Cai et al., 2016). Moreover, ribosome-dependent biosynthesis of CPs also have been reported in plant. ...
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Plant cyclic peptides (CPs) are a large group of small molecule metabolites found in a wide variety of plants, including traditional Chinese medicinal plants. However, the majority of plant CPs have not been studied for their biosynthetic mechanisms, including heterophyllin B (HB), which is one of the characteristic chemical components of Pseudostellaria heterophylla. Here, we screened the precursor gene (prePhHB) of HB in P. heterophylla and functionally identified its correctness in vivo and in vitro. First, we developed a new method to screen the precursors of HB from 16 candidate linear peptides. According to transcriptome sequencing data, we cloned the genes that encoded the HB precursor peptides and confirmed that the prePhHB-encoded precursor peptide could enzymatically synthesize HB. Next, we generated the transgenic tobacco that expressed prePhHB, and the results showed that HB was detected in transgenic tobacco. Moreover, we revealed that prePhHB gene expression is positively correlated with HB accumulation in P. heterophylla. Mutations in the prePhHB gene may influence the accumulation of HB in P. heterophylla. These results suggest that HB is ribosomally synthesized and posttranslationally modified peptide (RiPP) derived from the precursor gene prePhHB-encoded precursor peptide, and the core peptide sequence of HB is IFGGLPPP in P. heterophylla. This study developed a new idea for the rapid identification of Caryophyllaceae-type CP precursor peptides via RNA-sequencing data mining.
... Amatoxins and phallotoxins are structurally similar peptides synthesized by several species of the basidiomycete genera Amanita, Galerina, Lepiota, and Conocybe (Hallen et al. 2007;Luo et al. 2009;Walton et al. 2010;Luo et al. 2012;Sgambelluri et al. 2014). Amatoxins, exemplified by α-amanitin, are highly toxic to insects, nematodes, and mammals and many of the amatoxin-synthesizing fungi, such as the death cap (Amanita phalloides), are infamous for causing fatal mushroom poisonings. ...
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Bioactive peptide natural products are an important source of therapeutics. Prominent examples are the antibiotic penicillin and the immunosuppressant cyclosporine which are both produced by fungi and have revolutionized modern medicine. Peptide biosynthesis can occur either non-ribosomally via large enzymes referred to as non-ribosomal peptide synthetases (NRPS) or ribosomally. Ribosomal peptides are synthesized as part of a larger precursor peptide where they are posttranslationally modified and subsequently proteolytically released. Such peptide natural products are referred to as ribosomally synthesized and posttranslationally modified peptides (RiPPs). Their biosynthetic pathways have recently received a lot of attention, both from a basic and applied research point of view, due to the discoveries of several novel posttranslational modifications of the peptide backbone. Some of these modifications were so far only known from NRPSs and significantly increase the chemical space covered by this class of peptide natural products. Latter feature, in combination with the promiscuity of the modifying enzymes and the genetic encoding of the peptide sequence, makes RiPP biosynthetic pathways attractive for synthetic biology approaches to identify novel peptide therapeutics via screening of de novo generated peptide libraries and, thus, exploit bioactive peptide natural products beyond their direct use as therapeutics. This review focuses on the recent discovery and characterization of novel RiPP biosynthetic pathways in fungi and their possible application for the development of novel peptide therapeutics.
... During evolution, fungi have developed several defense lines to protect themselves against these predators. These defense lines can be categorized into three groups; physical barriers [4,5], secondary metabolites [6,7] and peptides/proteins [8,9]. Amongst defense proteins, biotin-binding proteins [10], protease inhibitors [11], pore-forming proteins [12], ribotoxins [13], and lectins [14] have been characterized. ...
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Background Fungi are an attractive source of nutrients for predators. As part of their defense, some fungi are able to induce the production of anti-predator protein toxins in response to predation. A previous study on the interaction of the model mushroom Coprinopsis cinerea by the fungivorous nematode Aphelenchus avenae on agar plates has shown that the this fungal defense response is most pronounced in the part of the mycelium that is in direct contact with the nematode. Hence, we hypothesized that, for a comprehensive characterization of this defense response, an experimental setup that maximizes the zone of direct interaction between the fungal mycelium and the nematode, was needed. Results In this study, we conducted a transcriptome analysis of C. cinerea vegetative mycelium upon challenge with A. avenae using a tailor-made microfluidic device. The device was designed such that the interaction between the fungus and the nematode was confined to a specific area and that the mycelium could be retrieved from this area for analysis. We took samples from the confrontation area after different time periods and extracted and sequenced the poly(A)⁺ RNA thereof. The identification of 1229 differentially expressed genes (DEGs) shows that this setup profoundly improved sensitivity over co-cultivation on agar plates where only 37 DEGs had been identified. The product of one of the most highly upregulated genes shows structural homology to bacterial pore-forming toxins, and revealed strong toxicity to various bacterivorous nematodes. In addition, bacteria associated with the fungivorous nematode A. avenae were profiled with 16S rRNA deep sequencing. Similar to the bacterivorous and plant-feeding nematodes, Proteobacteria and Bacteroidetes were the most dominant phyla in A. avenae. Conclusions The use of a novel experimental setup for the investigation of the defense response of a fungal mycelium to predation by fungivorous nematodes resulted in the identification of a comprehensive set of DEGs and the discovery of a novel type of fungal defense protein against nematodes. The bacteria found to be associated with the fungivorous nematode are a possible explanation for the induction of some antibacterial defense proteins upon nematode challenge. Electronic supplementary material The online version of this article (10.1186/s12864-019-5607-3) contains supplementary material, which is available to authorized users.
... The research showed that A. rimosa can produce abundant toxic peptides and related peptides, which will lay a strong foundation for the toxic peptide gene expression and exploitation of new cyclopeptide resources. Keywords Amanita rimosa, Transcriptome, MSDIN, Phylogenetic analysis 裂皮鹅膏(Amanita rimosa P. Zhang & Zhu L. Yang)是 2010 年发现的中国特有剧毒蘑菇 (Zhang et al., 2010),在我国江西、湖南、浙江、广东和海南等南方地区分布较广 。近年来,由裂皮鹅膏 引起的中毒事件频频发生,据不完全统计,2015~2017 年我国裂皮鹅膏中毒事件发生 6 起,中毒 32 人,死 亡 21 人。最严重的是在 2015 年 7 月浙江温州和 2016 年 5 月广东东莞发生的两起中毒事件,一共 11 个人 误食裂皮鹅膏,经抢救无效后全部死亡,死亡率达到了 100% (Tang et al., 2016;熊佳慧和刘猛, 2017)。裂 皮鹅膏含有丰富的鹅膏肽类毒素, 每克干重子实体中鹅膏毒肽(amatoxins)和鬼笔毒肽(phallotoxins)总含量可 达 14.19 mg,是目前所有已知剧毒鹅膏中最毒的种类 (Tang et al., 2016)。 目前已报道的鹅膏肽类毒素有 22 种,均为 6~8 个氨基酸组成的小环肽 (Wieland, 1986)。这些鹅膏肽类 毒素是由毒环肽基因编码的,最先由核糖体合成的前体肽序列包含 32~39 个氨基酸,氨基酸序列上、下游 区域是相对保守的,其中上游为 10 个氨基酸,下游为 15~17 个氨基酸,中间是 6~10 个氨基酸组成的毒肽 高变区,该区的第一个氨基酸与末尾的氨基酸均为脯氨酸,由于所有前体肽序列上游的前 5 个氨基酸多为 保守的"MSDIN"氨基酸序列,因此这些前体肽被称为 MSDIN 家族 (Hallen et al., 2007)。MSDIN 基因家族除 了编码毒素多肽,也编码其他多肽,如 antamanide、cycloamanide 以及许多未知结构和功能的多肽 (Hallen et al., 2007;Walton and Luo, 2010;Li et al., 2014) ...
... Both of these toxins contain a characteristic Trp-Cys (tryptathionine) cross bridge 46 , as well as hydroxylated Trp, Pro, and Ile residues. In addition, phallotoxins can also contain hydroxylated Asp, and Leu residues 41 . Amanita bisporigera and other members of the Amanita species are capable of synthesizing both amatoxins and phallotoxins. ...
Article
Circular peptides have long been sought after as scaffolds for drug design as they demonstrate protein-like properties in the context of small, constrained peptides. Traditional routes towards the production of cyclic peptides rely on synthesis or semi-synthetic methods, which restrict their use as platforms for the production of large, structurally diverse chemical libraries. Here, we discuss the biosynthetic routes towards the N-C macrocyclization of linear peptide precursors, specifically, those transformations that are catalyzed by peptidases. While canonical peptidases catalyze the proteolysis of linear peptides, the biosynthetic macrocyclases couple proteolytic cleavage with cyclization to produce a macrocyclic compounds. In this Perspective, we explore the different structural features that impart on each of these biosynthetic proteases the distinct ability carry out macrocyclization, and focus on their potential use in biotechnology.
... The younger fruit body of mushrooms are containing lower toxin. Conversely, the well-developed fungus have higher toxin concentrations (5). Phallotoxins are not absorbed by the digestive tract in mammals, but can cause toxicity by injection (3,4). ...
Article
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Amanita phalloides is one of the most poisonous mushrooms that contains three groups of toxins: amatoxins, virotoxins and phallotoxins. Gastrointestinal symptoms are the first clinical manifestations. The toxin can cause liver necrosis. This poisoning management includes supportive care, gastric decontamination, chemotherapy and liver transplantation.
... GmPOPB-S577A (the higher resolution of the pair) bound to the full-length substrate (35mer) belongs to space group P2 1 with four monomers in the asymmetric unit. For ease of discussion, we split the 35mer into four regions (Fig. 1b), the 10 residue leader (residues 1-10), the 8 residue core (11)(12)(13)(14)(15)(16)(17)(18), 6 residue linker (19)(20)(21)(22)(23)(24), and the 11 residue recognition tail (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35). The refined model (PDB:5N4C) includes residues 6-225 and 228-727 of the protein and residues 3-35 of the peptide (Fig. 1c). ...
Article
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Peptide macrocycles are promising therapeutic molecules because they are protease resistant , structurally rigid, membrane permeable, and capable of modulating protein-protein interactions. Here, we report the characterization of the dual function macrocyclase-peptidase enzyme involved in the biosynthesis of the highly toxic amanitin toxin family of macrocycles. The enzyme first removes 10 residues from the N-terminus of a 35-residue substrate. Conformational trapping of the 25 amino-acid peptide forces the enzyme to release this intermediate rather than proceed to macrocyclization. The enzyme rebinds the 25 amino-acid peptide in a different conformation and catalyzes macrocyclization of the N-terminal eight residues. Structures of the enzyme bound to both substrates and biophysical analysis characterize the different binding modes rationalizing the mechanism. Using these insights simpler substrates with only five C-terminal residues were designed, allowing the enzyme to be more effectively exploited in biotechnology.
... Examples of nonribosomal peptide synthetase-produced SMs include the beta-lactam antibiotics such as penicillin and cephalosporin and the immunosuppressant cyclosporin (115). Amatoxins, the deadly RNA transcription inhibitors produced by death angel mushrooms such as Amanita bisporigera, are produced by cleaving 35 amino acid proteins into octopeptides, which are then cyclized by a mechanism that is not fully understood (116). Polyketide synthetases, the most abundant SM-producing enzymes in fungi, polymerize fatty acid subunits rather than amino acid subunits like the nonribosomal peptide synthetases (115). ...
Article
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Six Key Traits of Fungi: Their Evolutionary Origins and Genetic Bases, Page 1 of 2 Abstract The fungal lineage is one of the three large eukaryotic lineages that dominate terrestrial ecosystems. They share a common ancestor with animals in the eukaryotic supergroup Opisthokonta and have a deeper common ancestry with plants, yet several phenotypes such as morphological, physiological, or nutritional traits make them unique among all living organisms. This article provides an overview of some of the most important fungal traits, how they evolve, and what major genes and gene families contribute to their development. The traits highlighted here represent just a sample of the characteristics that have evolved in fungi, including polarized multicellular growth, fruiting body development, dimorphism, secondary metabolism, wood decay, and mycorrhizae. However, a great deal of other important traits also underlie the evolution of the taxonomically and phenotypically hyperdiverse fungal kingdom, which could fill up a volume on its own. After reviewing the evolution of these six well-studied traits in fungi, we discuss how the recurrent evolution of phenotypic similarity, that is, convergent evolution in the broad sense, has shaped their phylogenetic distribution in extant species.
... 327 A similar thioether linkage is formed during the biosynthesis of the potent fungal toxins α-amanitin (Scheme 40) and phalloidin. 328 These macrocyclic RiPPs are produced by members of the phylum Basidomycota and harbor a tryptathionine moiety originating from core peptide-encoded Cys and Trp. While the biosynthetic gene clusters for each of these natural products are known and the enzymes responsible for core peptide macrocyclization have been characterized, it is still unclear how the tryptathionine cross-bridge is formed. ...
Article
Sulfur plays a critical role for the development and maintenance of life on earth, which is reflected by the wealth of primary metabolites, macromolecules, and cofactors bearing this element. Whereas a large body of knowledge has existed for sulfur trafficking in primary metabolism, the secondary metabolism involving sulfur has long been neglected. Yet, diverse sulfur functionalities have a major impact on the biological activities of natural products. Recent research at the genetic, biochemical, and chemical levels has unearthed a broad range of enzymes, sulfur shuttles, and chemical mechanisms for generating carbon-sulfur bonds. This Review will give the first systematic overview on enzymes catalyzing the formation of organosulfur natural products.
... A prolyl oligopeptidase (cutting at Pro residue), a serine protease was isolated from the lawn mushroom Conocybe apala. 62 The cytotoxic components in the sclerotium of Lignosus rhinocerotis (tiger milk mushroom) were identified as serine proteases (31 and 36 kDa). 63 Wild Ascomycete mushroom Helvella lacunose has serine protease helvellisin (33.5-kDa). ...
Article
Proteolytic activity is fundamental to survival, so it is not surprising that all living organisms have proteases, especially seine protease. This enzyme in its numerous isoforms and homologues, constitutes the quintessential offence and defence factors, in the form of surface proteins, secreted molecules, gut digestive enzymes, venom in specialised glands or plant latex, among other manifestations. Occurring as trypsin, chymotrypsin, elastase, collagenase, thrombin, subtilisin etc., it mediates a diverse array of functions, including pathological roles as inflammatory, coagulatory to haemorrhagic. This review emphasizes that despite the superficial differences in mechanisms, most health issues, be they infectious, allergic, metabolic, or neural have a common conduit. This enzyme, in its various glycosylated forms leads to signal misinterpretations, wreaking havoc. However, organisms are endowed with serine protease inhibitors which might restrain this ubiquitous yet deleterious enzyme. Hence, serine proteases-driven pathogenesis and antagonising role of inhibitors is the focal point of this critical review.
... Phallotoxins and virotoxins induce polymerization of G-actin, promoting an anomalous stabilization of F-actin filaments (Wieland 1977). Beyond their importance as toxins, these natural products from Amanita species are significant as the first ribosomally synthesized and post-translationally modified peptides (RiPPs) identified from fungi (Hallen et al. 2007, Walton et al. 2010. RiPPs are a recently recognised class of secondary metabolites that have been gaining significant attention in recent years (Arnison et al. 2013). ...
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Fungi are often inconspicuous in nature and this means it is all too easy to overlook their importance. Often referred to as the “Forgotten Kingdom”, fungi are key components of life on this planet. The phylum Basidiomycota, considered to contain the most complex and evolutionarily advanced members of this Kingdom, includes some of the most iconic fungal species such as the gilled mushrooms, puffballs and bracket fungi. Basidiomycetes inhabit a wide range of ecological niches, carrying out vital ecosystem roles, particularly in carbon cycling and as symbiotic partners with a range of other organisms. Specifically in the context of human use, the basidiomycetes are a highly valuable food source and are increasingly medicinally important. In this review, seven main categories, or ‘roles’, for basidiomycetes have been suggested by the authors: as model species, edible species, toxic species, medicinal basidiomycetes, symbionts, decomposers and pathogens, and two species have been chosen as representatives of each category. Although this is in no way an exhaustive discussion of the importance of basidiomycetes, this review aims to give a broad overview of the importance of these organisms, exploring the various ways they can be exploited to the benefit of human society.
... Paradoxically, mushroom toxin resistance is even found in some mushroomavoiding fruit flies, such as certain stocks of the genetic model organism D. melanogaster 14,15 . Because α-amanitin is solely produced by mushrooms [49][50][51] , these flies should never encounter this mushroom toxin in nature. In the 1960s, the first three α-amanitin-resistant Furthermore, the addition of α-amanitin to the larval food increases female fecundity, but also affects larva-to-adult development and longevity of the resistant fly stocks. ...
Article
Insect pesticide resistance has become a costly problem in the US. To make things more problematic, resistance to one pesticide is often associated with cross-resistance to other toxins, including pesticides that have not yet even been developed. In this study, we investigated a possible type of cross-resistance in the model fruit fly Drosophila melanogaster, some stocks of which are resistant to the very potent mushroom toxin α-amanitin. Because α-amanitin is solely produced by mushrooms, and because D. melanogaster does not feed on mushrooms in nature, the fruit flies should not be resistant to this toxin. In order to understand how this mushroom toxin resistance evolved, we first examined the physiological aspects of α-amanitin resistance in three D. melanogaster stocks, which were isolated in Asia half a century ago: Ama-KTT, Ama-MI, and Ama-KLM. We showed that all three fly stocks have not lost their α-amanitin resistance over time, even though they were maintained in the absence of selective pressure over the past ~1,200 generations. When we reared these flies on sub-lethal α-amanitin concentrations in our laboratory, the females unexpectedly doubled their fecundity. This effect could have dramatic ecological consequences by enabling D. melanogaster to invade the toxic mushroom niche. As a result of this potential niche switch, other mushroom-feeding flies could be driven to extinction. However, we also noted signs of developmental retardation and a shortened life span of the flies in response to increasing α-amanitin concentrations in the food, suggesting that the flies are not yet well adapted to the toxin. We next elucidated the molecular mechanisms causing α-amanitin resistance by performing a whole genome microarray study. Our data suggest that 1) cuticular proteins block α-amanitin from entering cells, 2) phase I and phase II detoxification enzymes modify α-amanitin to prepare it for excretion, 3) intracellular lipid particles sequester α-amanitin in the cytoplasm, and 4) peptidases cleave α-amanitin. Because the most highly up-regulated genes in our microarray study were Cytochrome P450/phase I detoxification genes that are known to detoxify pesticides, we speculate that the use of pesticides was the primary cause for the observed cross-resistance to the mushroom toxin α-amanitin.
... However, ribosomally synthesized and posttranslationally modified peptides (RiPPs), a rapidly growing class of natural products (17), have rarely been found in Ascomycetes. It has been known since 2007 that amatoxins and phallotoxins are RiPPs produced by strains of another fungal phylum, Basidiomycetes (which, together with Ascomycetes, compose the subkingdom Dikarya) (17)(18)(19). It also was recently reported that the ustiloxins produced by Aspergillus flavus and Ustilaginoidea virens belong to the RiPP class (20,21). ...
... However, ribosomally synthesized and posttranslationally modified peptides (RiPPs), a rapidly growing class of natural products (17), have rarely been found in Ascomycetes. It has been known since 2007 that amatoxins and phallotoxins are RiPPs produced by strains of another fungal phylum, Basidiomycetes (which, together with Ascomycetes, compose the subkingdom Dikarya) (17)(18)(19). It also was recently reported that the ustiloxins produced by Aspergillus flavus and Ustilaginoidea virens belong to the RiPP class (20,21). ...
Article
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Significance Ascomycetes are a prolific source of natural products that are of great significance for human health, yet production of ribosomally synthesized and posttranslationally modified peptides (RiPPs), a ubiquitous class of natural products, have rarely been reported in this fungal phylum. Here we show that phomopsins, a family of antimitotic mycotoxins, have a ribosomal origin and demonstrate the widespread presence of a fungal RiPP pathway for cyclic peptides that we term dikaritins. The framework described herein provides a foundation for mining for additional dikaritin members and investigating the biological activities and biosynthetic chemistry of this family of fungal natural products.
... Interactions between organisms can be either beneficial or detrimental for the partners involved. In order to avoid the detrimental effects caused by antagonistic organisms, in particular multicellular organisms have evolved sophisticated defense strategies, comprising mechanisms to recognize the presence of other organisms, and to distinguish between self and nonself (Zhang et al. 2010;Zipfel 2008), as well as the production of defense molecules, such as proteins (Bleuler-Martinez et al. 2011;Gallo and Hooper 2012;Vandenborre et al. 2011), RNAs (Liu et al. 2012), peptides (Walton et al. 2010), and secondary metabolites (Engel et al. 2002;Rohlfs and Churchill 2011;Spiteller 2008). It has been hypothesized that such defense systems originally evolved to prevent the fusion of somatic conspecifics that were genetically different (Muller and Muller 2003;Srivastava et al. 2010). ...
Article
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The dung of herbivores, the natural habitat of the model mushroom Coprinopsis cinerea, is a nutrient-rich but also very competitive environment for a saprophytic fungus. We showed previously that C. cinerea expresses constitutive, tissue-specific armories against antagonists such as animal predators and bacterial competitors. In order to dissect the inducible armories against such antagonists, we sequenced the poly(A)-positive transcriptome of C. cinerea vegetative mycelium upon challenge with fungivorous and bacterivorous nematodes, Gram-negative and Gram-positive bacteria and mechanical damage. As a response to the fungivorous nematode Aphelenchus avenae, C. cinerea was found to specifically induce the transcription of several genes encoding previously characterized nematotoxic lectins. In addition, a previously not characterized gene encoding a cytoplasmic protein with several predicted Ricin B-fold domains, was found to be strongly up-regulated under this condition. Functional analysis of the recombinant protein revealed a high toxicity towards the bacterivorous nematode Caenorhabditis elegans. Challenge of the mycelium with A. avenae also lead to the induction of several genes encoding putative antibacterial proteins. Some of these genes were also induced upon challenge of the mycelium with the bacteria Escherichia coli and Bacillus subtilis. These results suggest that fungi have the ability to induce specific innate defense responses similar to plants and animals.
... Fungal Genetics & Biology R.D. Johnson et al., gigA However, here we present evidence that multiple epichloëcyclins may be present in an endophyte-infected grass plant, and that they are post-translationally modified products of the gigA gene synthesised by ribosomal peptide synthesis (RiPS). RiPS was first described for the antibiotic nicin in Streptococcus lactis (Buchman et al., 1988) and is common in bacteria and plants (Velasquez and van der Donk, 2011; Arnison et al., 2013), with only a few examples previously reported for fungi (Hallen et al., 2007; Walton et al., 2010; Luo et al., 2012; Tsukui et al., 2014; ). The GigA cyclic peptides from fungal endophytes of grasses therefore represent a further example of a RiPS pathway but with seemingly greater chemical diversity being produced from a single gene. ...
Article
Fungal endophytes belonging to the genus Epichloë form associations with temperate grasses belonging to the sub-family Poöideae that range from mutualistic through to pathogenic. We previously identified a novel endophyte gene (designated gigA for grass induced gene) that is one of the most abundantly expressed fungal transcripts in endophyte-infected grasses and which is distributed and highly expressed in a wide range of Epichloë grass associations. Molecular and biochemical analyses indicate that gigA encodes a small secreted protein containing an imperfect 27 amino acid repeat that includes a kexin protease cleavage site. Kexin processing of GigA liberates within the plant multiple related products, named here as epichloëcyclins, which we have demonstrated by MS/MS to be cyclic peptidic in nature. Gene deletion of gigA leads to the elimination of all epichloëcyclins with no conspicuous phenotypic impact on the host grass, suggesting a possible bioactive role. This is a further example of a ribosomal peptide synthetic (RiPS) pathway operating within the Ascomycetes, and is the first description of such a pathway from a mutualistic symbiotic fungus from this Phylum.
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Amatoxins are strong inhibitors of RNA polymerase II, and cause cell death. Because of their cytotoxicity, they are candidates for anti-cancer drugs, and understanding their structure-activity relationship is crucial. Amatoxins have a rigid bicyclic scaffold which consists of a cyclic octapeptide bridged by cysteine and tryptophan side chain forming a tryptathionine bridge. Here, we show the influence of the N-methylation on the amatoxin scaffold by studying Gly5Sar-amanullin with MD simulations and NMR experiments. Since we have shown recently that the amatoxin scaffold allows for two isomeric forms (ansamers), we studied both isomers of Gly5Sar-amanullin. We found that both isomers of Gly5Sar-amanullin form two long-living conformations, which is unusual for amatoxins, and that they are differently affected by the N-methylation. The natural Gly5Sar-amanullin forfeits the hydrogen bonds to Gly5 due to the N-methylation, which is expected from existing crystal structures for alpha-amanitin. Our results however indicate that this does not cause more flexibility due to a shift in the hydrogen bond pattern. In the unnatural isomer, we observe an interesting cis-trans-isomerisation of the backbone angles in Trp4 and Gly7, which is enabled by the N-methylation. We expect that our perspective on the effect of N-methylation in amatoxins could be a starting point for further SAR-studies which are urgently needed for the design of better anti-cancer agents.
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Recombinant peptide production in Escherichia coli is often accomplished through cloning and expression of a fusion protein. The fusion protein partner generally has two requirements: (a) it contains an affinity tag to assist with purification and (b) it can be cleaved off to leave only the desired peptide sequence behind. Common soluble fusion partners include small ubiquitin‐like modifier protein (SUMO), maltose‐binding protein (MBP), glutathione S‐transferase (GST), or intein proteins. However, heterologously expressed peptides can suffer from proteolytic degradation or instability. This degradation can pose a major issue for applications requiring a large amount of purified peptide, such as NMR structural assignments or biochemical assays. Improving peptide yield by testing various expression and isolation conditions requires a significant amount of effort and may not lead to improved results. Here, we cloned and expressed four different peptides as SUMO fusion proteins. These peptides (lactococcin A, leucocin A, faerocin MK, neopetrosiamide A) were truncated during expression and isolation as SUMO fusions, resulting in low yields of purified peptide. To prevent this degradation and improve yield, we designed a new expression system to create a “sandwiched” fusion protein of the form: His6‐SUMO‐peptide‐intein (SPI). These sandwiched peptides were more stable and protected against degradation, resulting in improved yields (up to 17‐fold) under a set of standard expression and isolation procedures. This SPI expression system uses only two commercially available vectors and standard protein purification techniques, and therefore may offer an economical and facile route to improve yields for peptides that undergo degradation.
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The increasing length and complexity of peptide drug candidates foster the development of novel strategies for their manufacture, which should include sustainable and efficient technologies. In this context, including enzymatic catalysis in the production of peptide molecules has gained interest. Here, several enzymes from ribosomally synthesized and post-translationally modified peptides biosynthesis pathways are reviewed, with attention to their capacity to introduce stability-promoting structural features on peptides, providing an initial framework towards their use in therapeutic peptide production processes.
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Covering up to June 2021Ribosomally synthesized and post-translationally modified peptides (RiPPs) from fungi are an underexplored class of natural products, despite their propensity for diverse bioactivities and unique structural features. Surveys of fungal genomes for biosynthetic gene clusters encoding RiPPs have been limited in their scope due to our incomplete understanding of fungal RiPP biosynthesis. Through recent discoveries, along with earlier research, a clearer picture has been emerging of the biosynthetic principles that underpin fungal RiPP pathways. In this Highlight, we trace the approaches that have been used for discovering currently known fungal RiPPs and show that all of them can be assigned to one of three distinct families based on hallmarks of their biosynthesis, which are in turn imprinted on their corresponding gene clusters. We hope that our systematic exposition of fungal RiPP structural and gene cluster features will facilitate more comprehensive approaches to genome mining efforts in the future.
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Wild mushrooms are a vital source of income and nutrition for many poor communities and of value to recreational foragers. Literature relating to the edibility of mushroom species continues to expand, driven by an increasing demand for wild mushrooms, a wider interest in foraging, and the study of traditional foods. Although numerous case reports have been published on edible mushrooms, doubt and confusion persist regarding which species are safe and suitable to consume. Case reports often differ, and the evidence supporting the stated properties of mushrooms can be incomplete or ambiguous. The need for greater clarity on edible species is further underlined by increases in mushroom-related poisonings. We propose a system for categorizing mushroom species and assigning a final edibility status. Using this system, we reviewed 2,786 mushroom species from 99 countries, accessing 9,783 case reports, fromover 1,100 sources.We identified 2,189 edible species, of which 2,006 can be consumed safely, and a further 183 species which required some form of pretreatment prior to safe consumption or were associated with allergic reactions by some. We identified 471 species of uncertain edibility because of missing or incomplete evidence of consumption, and 76 unconfirmed species because of unresolved, differing opinions on edibility and toxicity. This is the most comprehensive list of edible mushrooms available to date, demonstrating the huge number of mushrooms species consumed. Our review highlights the need for further information on uncertain and clash species, and the need to present evidence in a clear, unambiguous, and consistent manner.
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Wild mushrooms are a vital source of income and nutrition for many poor com-munities and of value to recreational foragers. Literature relating to the edibilityof mushroom species continues to expand, driven by an increasing demand forwild mushrooms, a wider interest in foraging, and the study of traditional foods.Although numerous case reports have been published on edible mushrooms,doubt and confusion persist regarding which species are safe and suitable to con-sume. Case reports often differ, and the evidence supporting the stated proper-ties of mushrooms can be incomplete or ambiguous. The need for greater clarityon edible species is further underlined by increases in mushroom-related poi-sonings. We propose a system for categorizing mushroom species and assigninga final edibility status. Using this system, we reviewed 2,786 mushroom speciesfrom 99 countries, accessing 9,783 case reports, from over 1,100 sources. We iden-tified 2,189 edible species, of which 2,006 can be consumed safely, and a further183 species which required some form of pretreatment prior to safe consumptionor were associated with allergic reactions by some. We identified 471 species ofuncertain edibility because of missing or incomplete evidence of consumption,and 76 unconfirmed species because of unresolved, differing opinions on edibil-ity and toxicity. This is the most comprehensive list of edible mushrooms avail-able to date, demonstrating the huge number of mushrooms species consumed.Our review highlights the need for further information on uncertain and clashspecies, and the need to present evidence in a clear, unambiguous, and consistent manner.
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The paper has now been published and is open access. https://doi.org/10.1111/1541-4337.12708 A full list of global species has been added this entry (also available as supplementary information for the review article). Wild mushrooms are a vital source of income and nutrition for many poor communities and of value to recreational foragers. Literature relating to the edibility of mushroom species continues to expand, driven by an increasing demand for wild mushrooms, a wider interest in foraging, and the study of traditional foods. Although numerous case reports have been published on edible mushrooms, doubt and confusion persist regarding which species are safe and suitable to consume. Case reports often differ, and the evidence supporting the stated properties of mushrooms can be incomplete or ambiguous. The need for greater clarity on edible species is further underlined by increases in mushroom-related poisonings. We propose a system for categorizing mushroom species and assigning a final edibility status. Using this system, we reviewed 2,786 mushroom species from 99 countries, accessing 9,783 case reports, from over 1,100 sources. We identified 2,189 edible species, of which 2,006 can be consumed safely, and a further 183 species which required some form of pretreatment prior to safe consumption or were associated with allergic reactions by some. We identified 471 species of uncertain edibility because of missing or incomplete evidence of consumption, and 76 unconfirmed species because of unresolved, differing opinions on edibility and toxicity. This is the most comprehensive list of edible mushrooms available to date, demonstrating the huge number of mushrooms species consumed. Our review highlights the need for further information on uncertain and clash species, and the need to present evidence in a clear, unambiguous, and consistent manner.
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Objective: To assess the value of Ploton silver staining and phalloidin-iFlour 488 staining in observation of the morphology of osteocyte dendrites of mice at different developmental stages. Methods: The humerus and femurs were harvested from mice at 0 (P0), 5 (P5), 15 (P15), 21 (P21), 28 (P28), and 35 days (P35) after birth to prepare cryo-sections and paraffin sections. HE staining of P35 mouse femur sections served as a reference for observing osteocytes in the trabecular bone and cortical bone. The humeral sections at different developmental stages were stained with Ploton silver staining to observe the morphology of osteocytes and canaliculi, and the canalicular lengths in the cortical and trabecular bones of the humerus of the mice in each developmental stage were recorded. The cryo-sections of the humerus from P10 and P15 mice were stained with phalloidin iFlour-488 to observe the morphology of osteocytes and measurement of the length of osteocyte dendrites in the cortical bone. Results: In the trabecular bone of the humerus of P0-P15 mice, Ploton silver staining only visualized the outline of the osteocytes, and the morphology of the canaliculi was poorly defined. In P21 or older mice, Ploton silver staining revealed the morphology of the trabecular bone osteocytes and the canaliculi, which were neatly arranged and whose lengths increased significantly with age (P21 vs P28, P < 0.05; P21 vs P35, P < 0.05). In the humeral cortical bone of P15 mice, the morphology of the osteocytes and canalicular could be observed with Ploton silver staining, and the length of the regularly arranged canaliculi of the osteocytes increased significantly with age (P15 vs P21, P < 0.005; P15 vs P28, P < 0.0001; P15 vs P35, P < 0.0001). Phalloidin iFlour-488 staining was capable of visualizing the complete morphology of the osteocytes at P10 and P15; the osteocyte dendrites elongated progressively with age (P10 vs P15, P < 0.01) to form connections with the surrounding osteocytes. Conclusions: Mouse osteocyte dendrites elongate progressively and their arrangement gradually becomes regular with age. Ploton silver staining can clearly visualize the morphology of the osteocytes and the canaliculi in adult mice but not in mice in early stages of development. Phalloidin iFlour-488 staining for labeling the cytoskeleton can be applied for mouse osteocytes at all developmental stages and allows morphological observation of mouse osteocytes in early developmental stages.
Article
The interest in cyclic peptide research has increased in the last decade. These compounds are resistant to enzymatic degradation and possess a more rigid scaffold than their linear analogues. This paper presents the results of combined potentiometric and spectroscopic studies on the coordination properties of hetero-site cyclopeptide (CDP) with the c(HHKHHPHDHKHP) sequence. Thermodynamic stability constants for ligands and complex forms have been determined in a system with a double excess of copper ions, i.e. with a molar ratio to ligand L:Cu(II) of 1:2. The interaction was characterised by UV-Vis, CD, EPR and ESI-MS methods over a wide pH range. CDP is a good chelator for divalent metal ions, it creates mono-, di- and trinuclear complexes with copper(II) ions over a wide range of pH. It was found that promotion of di-copper(II) complexes is connected with a higher flexibility of the cyclopeptide chain.
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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 gened to encode 98 non-duplicated cyclopeptides, including α- amanitin, β-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.
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Background Amanitin-producing mushrooms, mainly distributed in the genera Amanita , Galerina and Lepiota , possess MSDIN gene family for the biosynthesis of many cyclopeptides catalyzed by prolyl oligopeptidase (POP). Recently, transcriptome sequencing has proven to be a efficient way to mine MSDIN and POP genes in these lethal mushrooms. Until now, only A . palloides and A. bisporigera from North America and A . exitialis from Asia have been studied based on transcriptome analysis. However, MSDIN and POP genes of many amanitin-producing mushrooms in China remain unstudied, and 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 analyzed. Through transcriptome sequencing and PCR cloning, 19 POP genes and 151 MSDIN genes predicted to encode 98 non-duplicated cyclopeptides, including α-amanitin, β-amanitin, phallacidin, phalloidin and 94 unknown peptides, were found in these species. Phylogenetic analysis showed that toxin peptide genes were clustered depending on the chemical substance within genus while depending on the taxonomy between genus and that the POPA genes of Amanita , Galerina and Lepiota were clustered and separated in three different groups, but the POPB genes of the three distinct genera were clustered in a highly monophyletic group. Conclusions These results above 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 horizontal gene transfer might occur among the three genera.
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Lactazole A is a cryptic thiopeptide from Streptomyces lactacystinaeus, encoded by a compact 9.8 kb biosynthetic gene cluster. Here, we established a platform for in vitro biosynthesis of lactazole A, referred to as the FIT-Laz system, via a combination of the flexible in vitro translation (FIT) system with recombinantly produced lactazole biosynthetic enzymes. Systematic dissection of lactazole biosynthesis revealed remarkable substrate tolerance of the biosynthetic enzymes, and led to the development of the "minimal lactazole scaffold", a construct requiring only 6 post-translational modifications for macrocyclization. Efficient assembly of such minimal thiopeptides with FIT-Laz enabled access to diverse lactazole analogs with 10 consecutive mutations, 14- to 62-membered macrocycles, and up to 18 amino acid-long tail regions. Moreover, utilizing genetic code reprogramming, we demonstrated synthesis of pseudo-natural lactazoles containing 4 non-proteinogenic amino acids. This work opens possibilities in exploring novel sequence space of pseudo-natural thiopeptides.
Chapter
Antibody–drug conjugates (ADCs) represent one of the most promising and exciting areas of anticancer drug discovery. Five ADCs are now approved in the US and EU [i.e., ado-trastuzumab emtansine (Kadcyla™), brentuximab vedotin (Adcetris™), inotuzumab ozogamicin (Besponsa™), gemtuzumab ozogamicin (Mylotarg™) and moxetumomab pasudotox-tdfk (Lumoxiti®)] and over 70 others are in various stages of clinical development, with impressive interim results being reported for many. The technology is based on the concept of delivering a cytotoxic payload selectively to cancer cells by attaching it to an antibody targeted to antigens on the cell surfaces. This approach has several advantages including the ability to select patients as likely responders based on the presence of antigen on the surface of their cancer cells and a wider therapeutic index, given that ADC targeting enables a more efficient delivery of cytotoxic agents to cancer cells than can be achieved by conventional chemotherapy, thus minimising systemic toxicity. Although there are many examples of antibodies that have been developed for this purpose, along with numerous linker technologies used to attach the cytotoxic agent to the antibody, there is presently a relatively small number of payload molecules in clinical use. The purpose of this book is to describe the variety of payloads used to date, along with a discussion of their advantages and disadvantages and to provide information on novel payloads at the research stage that may be used clinically in the future.
Chapter
Mushrooms, the macrofungi, are enigmatic in their composition, which renders some of them umami-flavored edible, some medicinal, some hallucinogenic/psychoactive, while some lethal. In current times, appreciation and demand of the medicinal mushrooms is rising, with the validation of their efficacy as antioxidant, anti-inflammatory, immunomodulatory, anticancer, endocrine restorative, etc. Containing a rich repertoire of bioactive components as β-glucan, phenolics, peptides, and sterols, they have attracted the attention of researchers. Ganoderma lucidum, Hericium erinaceus, Grifola frondosa, Lentinus edodes, Inonotus obliquus, and Cordyceps sinensis are some of the most popular medicinal mushrooms. The immunomodulation, anti-inflammation, and anticancer attributes of the mycochemicals mostly rely on the inhibition of mTOR translational pathway, cell apoptosis, G1 phase cell cycle arrest, downregulation of cyclins A and B1, upregulation of p21 and p27, and induction of cytokines (TNF-α, IL-2, and IFN- γ), among others. Further attention can facilitate the identification of components of biological interest from mushrooms. In this regard, this chapter discusses the biological effects of some major mushroom mycochemicals.
Article
Mushroom consumption is a global tradition that is still gaining popularity. However, foraging for wild mushrooms and accidental ingestion of toxic mushrooms can result in serious illness and even death. The early diagnosis and treatment of mushroom poisoning are quite difficult, as the symptoms are similar to those caused by common diseases. Chemically, mushroom poisoning is related to very powerful toxins, suggesting that the isolation and identification of toxins has great research value, especially in determining the lethal components of toxic mushrooms. In contrast, most of these toxins have remarkable physiological properties that could promote advances in chemistry, biochemistry, physiology, and pharmacology. Although more than 100 toxins have been elucidated, there are a number of lethal mushrooms that have not been fully investigated. This review provides information on the chemistry (including chemical structures, total synthesis and biosynthesis), and the toxicology of these toxins, hoping to inspire further research in this area.
Chapter
In this chapter, the occurrence of these compounds within the kingdom Eumycota (true fungi), the diversity of their chemical building blocks and structures, their ecological roles, as well as their different biological activities will be described. Finally the importance of cyclic peptides and depsipeptides as drugs and lead compounds for agricultural and pharmaceutical applications will be discussed.
Article
Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol–water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.
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Background: Amino acids are the essential building blocks of proteins and, therefore, living organisms. While the focus often lies on the canonical or proteinogenic amino acids, there is also a large number of non-canonical amino acids to explore. Some of them are part of toxins or antibiotics in fungi, bacteria or animals (e.g. sponges). Some others operate at the translational level like an "undercover agent". Scope of review: Here we give an overview of natural aliphatic amino acids, up to a side chain length of five carbons, without rings and with an unmodified backbone, and have a closer look on each of them. Some of them are dehydro amino acids with double or even triple bonds. Moreover, we outline mathematical methods for enumerating the complete list of all potential aliphatic amino acids of a given chain length, which should be of interest for synthetic biology. Major conclusions: Most non-proteinogenic amino acids are found within fungi, with particularly many produced by Amanita species as defence chemicals. Several are incorporated into peptide antibiotics. Some of the amino acids occur due to broad substrate specificity of the branched-chain amino acid synthesis pathways. A large variety of amino acids were also found in the Murchison meteorite. General significance: Non-proteinogenic amino acids are of interest for numerous medical applications: discovery of new antibiotics, support in designing synthetic antibiotics, improvement of protein and peptide pharmaceuticals by avoiding incorporation of non-canonical amino acids, study of toxic cyanobacteria and other applications.
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Aerobic microorganisms have been sources of medicinal agents for several decades and an impressive variety of drugs have been isolated from their cultures, studied and formulated to treat or prevent diseases. On the other hand, anaerobes, which are believed to be the oldest life forms on earth and evolved remarkably diverse physiological functions, have largely been neglected as sources of bioactive compounds. However, results obtained from the limited research done so far show that anaerobes are capable of producing a range of interesting bioactive compounds that can promote human health. In fact, some of these bioactive compounds are found to be novel in their structure and/or mode of action.
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Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.
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Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins, but engenders the resultant cyclic proteins with exceptional stability. The mechanisms involved are poorly understood and this review attempts to summarize what is known of the events that lead to cyclization. Cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the theta defensins, found in rhesus macaques, and the retrocyclins. Two types of cyclic proteins have been found in plants, the sunflower trypsin inhibitor and the larger, more prolific, group known as cyclotides. The cyclotides from Oldenlandia affinis, the plant in which these cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. Cysteine proteases are commonly associated with transpeptidation reactions, which, for suitable substrates can lead to cyclization events. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack by the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide. © 2010 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 94: 573–583, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
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The peptide toxins of poisonous Amanita mushrooms are bicyclic octapeptides (amatoxins) or heptapeptides (phallotoxins). In Amanita bisporigera, alpha-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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The staphylococcal agr locus encodes a quorum sensing (QS) system that controls the expression of virulence and other accessory genes by a classical two-component signaling module. Like QS modalities in other Gram-positive bacteria, agr encodes an autoactivating peptide (AIP) that is the inducing ligand for AgrC, the agr signal receptor. Unlike other such systems, agr variants have arisen that show strong cross-inhibition in heterologous combinations, with important evolutionary implications. Also unlike other systems, the effector of global gene regulation in the agr system is a major regulatory RNA, RNAIII. In this review, we describe the functions of the agr system's elements, show how they interact to bring about the regulatory response, and discuss the role of QS in staphylococcal pathobiology. We conclude with the suggestion that agr autoactivation, unlike classical enzyme induction, can occur under suboptimal conditions and can distinguish self from non-self by inducing an exclusive and coordinated population wide response.
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The structure of RNA polymerase II in a complex with the inhibitor alpha-amanitin has been determined by x-ray crystallography. The structure of the complex indicates the likely basis of inhibition and gives unexpected insight into the transcription mechanism.
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The 500 different species of venomous cone snails (genus Conus) use small, highly structured peptides (conotoxins) for interacting with prey, predators, and competitors. These peptides are produced by translating mRNA from many genes belonging to only a few gene superfamilies. Each translation product is processed to yield a great diversity of different mature toxin peptides (approximately 50,000-100,000), most of which are 12-30 aa in length with two to three disulfide crosslinks. In vitro, forming the biologically relevant disulfide configuration is often problematic, suggesting that in vivo mechanisms for efficiently folding the diversity of conotoxins have been evolved by the cone snails. We demonstrate here that the correct folding of a Conus peptide is facilitated by a posttranslationally modified amino acid, gamma-carboxyglutamate. In addition, we show that multiple isoforms of protein disulfide isomerase are major soluble proteins in Conus venom duct extracts. The results provide evidence for the type of adaptations required before cone snails could systematically explore the specialized biochemical world of "microproteins" that other organisms have not been able to systematically access. Almost certainly, additional specialized adaptations for efficient microprotein folding are required.
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Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron spp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium. • heterocycle • tunicate • ascidian • genome sequencing • lantibiotic
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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 microg g(-1) (dry matter) of alpha-Amanitin and 60.0 +/- 20.7 microg g(-1) (dry matter) of beta-Amanitin, respectively, a yield which is about 10% of that produced by fruiting bodies.
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We have isolated a protein-disulfide isomerase (PDI) from Oldenlandia affinis (OaPDI), a coffee family (Rubiaceae) plant that accumulates knotted circular proteins called cyclotides. The novel plant PDI appears to be involved in the biosynthesis of cyclotides, since it co-expresses and interacts with the cyclotide precursor protein Oak1. OaPDI exhibits similar isomerase activity but greater chaperone activity than human PDI. Since domain c of OaPDI is predicted to have a neutral pI, we conclude that this domain does not have to be acidic in nature for PDI to be a functional chaperone. Its redox potential of -157 ± 4 mV supports a role as a functional oxidoreductase in the plant. The mechanism of enzyme-assisted folding of plant cyclotides was investigated by comparing the folding of kalata B1 derivatives in the presence and absence of OaPDI. OaPDI dramatically enhanced the correct oxidative folding of kalata B1 at physiological pH. A detailed investigation of folding intermediates suggested that disulfide isomerization is an important role of the new plant PDI and is an essential step in the production of insecticidal cyclotides.
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Microcin J25 (MccJ25) is a gene-encoded lasso peptide secreted by Escherichia coli which exerts a potent antibacterial activity by blocking RNA polymerase. Here we demonstrate that McjB and McjC, encoded by genes in the MccJ25 gene cluster, catalyze the maturation of MccJ25. Requirement for both McjB and McjC was shown by gene inactivation and complementation assays. Furthermore, the conversion of the linear precursor McjA into mature MccJ25 was obtained in vitro in the presence of McjB and McjC, all proteins being produced by recombinant expression in E. coli. Analysis of the amino acid sequences revealed that McjB could possess proteolytic activity, whereas McjC would be the ATP/Mg(2+)-dependent enzyme responsible for the formation of the Gly1-Glu8 amide bond. Finally, we show that putative lasso peptides are widespread among Proteobacteria and Actinobacteria.
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Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.
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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. • amanitin • cyclic peptide • phalloidin • phallotoxin • amatoxin
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More than 100 cyclic peptides harboring heterocyclized residues are known from marine ascidians, sponges and different genera of cyanobacteria. Here, we report an assembly line responsible for the biosynthesis of these diverse peptides, now called cyanobactins, both in symbiotic and free-living cyanobacteria. By comparing five new cyanobactin biosynthetic clusters, we produced the prenylated antitumor preclinical candidate trunkamide in Escherichia coli culture using genetic engineering.
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On water agar, Conocybe lactea (Lange) Metrod produces droplets of toxin on conspicuous secretory cells. Both free-living rhabditoid nematodes and fungus-feeding nematodes (Aphelenchoides sp.) are immobilized when they contact the toxin droplets. Prolonged and (or) repeated exposure to the toxin results in death of the nematodes, which may take place at some distance from the contact point. Unlike Pleurotus ostreatus (Jacq.:Fr.) Kummer, the hyphae of C. lactea do not locate and colonize immobilized nematodes and (or) consume them as a nutrient source. It is considered that the toxin droplets are for protection of the hyphal system and function as antifeedants to repel or kill fungus-feeding nematodes and possibly other fungus-feeding soil microfauna. No differences were observed in nematode response between dikaryotic and monokaryotic cultures, although the latter produced a spermatial-like arthrosporic anamorph. Panaeolina foenisecii (Pers.:Fr.) R. Maire also produces secretory cells with fluid droplets. These droplets, however, are not as toxic to nematodes as those of C. lactea. Keywords: secretory cells, nematotoxin, antifeedants, lawn mushroom.
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Sunflower trypsin inhibitor-1 (SFTI-1) is a 14 amino acid cyclic peptide from sunflower seeds, which possesses exceptionally potent trypsin-inhibitory activity, and has promise as a stable peptide-based drug template. Within its compact structure, SFTI-1 combines a head-to-tail cyclized backbone and a disulfide bond. In this study, we synthesized a range of acyclic and disulfide-deficient analogs of SFTI-1 to investigate enzyme-assisted cyclization of the peptide backbone and proteolytic degradation that occurs as a result of incubation with trypsin. Electrospray and matrix-assisted laser desorption ionization mass spectrometry allowed the characterization of a range of novel degradation products and elucidation of the time-course for cyclization and/or proteolysis. Trypsin displayed the ability to resynthesize the scissile bond(s) and hence cyclize two of the linear permutants, whereas irreversible degradation was observed for another two permutants. An interesting ring contraction mediated by trypsin was observed, supporting a role for protease catalyzed splicing as a way of increasing the combinatorial diversity of cyclic peptides in nature. Disulfide-deficient mutants were degraded within minutes, emphasizing the critical role of the cysteine bridge in maintaining proteolytic stability of SFTI-1. Overall, the study provides additional support for the proposal that naturally occurring cyclic peptides like SFTI-1 are biosynthesized by proteolytic enzymes effectively catalyzing the reverse of their normal reaction to make, rather than break peptide bonds.
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Depudecin, an eleven-carbon linear polyketide made by the pathogenic fungus Alternaria brassicicola, is an inhibitor of histone deacetylase (HDAC). A chemically unrelated HDAC inhibitor, HC toxin, was earlier shown to be a major virulence factor in the interaction between Cochliobolus carbonum and its host, maize. In order to test whether depudecin is also a virulence factor for A. brassicicola, we identified the genes for depudecin biosynthesis and created depudecin-minus mutants. The depudecin gene cluster contains six genes (DEP1 to DEP6), which are predicted to encode a polyketide synthase (AbPKS9 or DEP5), a transcription factor (DEP6), two monooxygenases (DEP2 and DEP4), a transporter of the major facilitator superfamily (DEP3), and one protein of unknown function (DEP1). The involvement in depudecin production of DEP2, DEP4, DEP5, and DEP6 was demonstrated by targeted gene disruption. DEP6 is required for expression of DEP1 through DEP5 but not the immediate flanking genes, thus defining a coregulated depudecin biosynthetic cluster. The genes flanking the depudecin gene cluster but not the cluster itself are conserved in the same order in the related fungi Stagonospora nodorum and Pyrenophora tritici-repentis. Depudecin-minus mutants have a small (10%) but statistically significant reduction in virulence on cabbage (Brassica oleracea) but not on Arabidopsis. The role of depudecin in virulence is, therefore, less dramatic than that of HC toxin.
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Ribosomally synthesized bacterial natural products rival the nonribosomal peptides in their structural and functional diversity. The last decade has seen substantial progress in the identification and characterization of biosynthetic pathways leading to ribosomal peptide natural products with new and unusual structural motifs. In some of these cases, the motifs are similar to those found in nonribosomal peptides, and many are constructed by convergent or even paralogous enzymes. Here, we summarize the major structural and biosynthetic categories of ribosomally synthesized bacterial natural products and, where applicable, compare them to their homologs from nonribosomal biosynthesis.
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Small N-C terminally cyclic ribosomal peptides are common in nature, yet the mechanisms underlying their biosyntheses remain largely unknown. We recently identified candidate peptide cyclase genes in the metagenomes of marine animals. Here we report that PatG, a protease discovered in this analysis, cleaves variables, short peptides out of a precursor protein, and cyclizes these peptides in vivo and in vitro.
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The results of 50 years of effort in the chemistry of Amanita toxins are reviewed. The phallotoxins, fast acting components, but not responsible for fatal intoxications after ingestion, are bicyclic heptapeptides. They combine with F-actin, stabilizing this protein against several destabilizing influences. The virotoxins likewise fast acting are monocyclic heptapeptides. The amatoxins which are the real toxins lead to death within several days by inhibiting the enzymatic synthesis of m-RNA. They are bicyclic octapeptides. The structures of all of these compounds are described, as well as conformations, chemical reactions and modification, syntheses and correlations between structures and biological activities.
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Proline is unique among the 20 amino acids due to its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins and confers particular biological properties upon a wide range of physiologically important biomolecules. In order to adequately deal with such peptides, nature has developed a group of enzymes that recognise this residue specifically. These peptidases cover practically all situations where a proline residue might occur in a potential substrate. In this paper we endeavour to discuss these enzymes, particularly those responsible for peptide or protein hydrolysis at proline sites. We have detailed their discovery, biochemical attributes and substrate specificities and have provided information as to the methodology used to detect and manipulate their activities. We have also described the roles, or potential roles that these enzymes may play physiologically and the consequences of their dysfunction in varied disease states.
Article
Strains of the filamentous fungus Cochliobolus carbonum that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (Zea mays L.). Production of HC-toxin is under the control of a complex locus, TOX2, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of C. carbonum. One of these genes, TOXE, was earlier shown to be required for the expression of the other TOX2 genes. TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper. Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the "tox-box") without dyad symmetry that is present in the promoters of all of the known TOX2 genes. Both the basic region and the ankyrin repeats are involved in DNA binding. A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast. The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi. TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by C. carbonum.
Article
Circular proteins are a recently discovered phenomenon. They presumably evolved to confer advantages over ancestral linear proteins while maintaining the intrinsic biological functions of those proteins. In general, these advantages include a reduced sensitivity to proteolytic cleavage and enhanced stability. In one remarkable family of circular proteins, the cyclotides, the cyclic backbone is additionally braced by a knotted arrangement of disulfide bonds that confers additional stability and topological complexity upon the family. This article describes the discovery, structure, function and biosynthesis of the currently known circular proteins. The discovery of naturally occurring circular proteins in the past few years has been complemented by new chemical and biochemical methods to make synthetic circular proteins; these are also briefly described.
Article
Conocybe lactea was examined as part of a larger study on the distribution of amatoxins and phallotoxins in fungi, and the taxonomic relationships between these fungi. As amatoxins are present in the congener C. filaris, the locally abundant C. lactea was examined using HPLC and mass spectroscopy. Amatoxins were not found in C. lactea, but the related phallotoxins were present in small quantities making it the first fungus outside of the genus Amanita in which phallotoxins have been detected. Despite the presence of a related toxin, C. lactea was found not to be taxonomically close to C. filaris. Phylogenetic analyses using nuclear ribosomal RNA genes indicated that North American specimens of C. lactea were conspecific with North American specimens of C. crispa in Conocybe sect. Candidae. European C. crispa was a distinct taxon. The implications of the use of the name C. albipes for these taxa are discussed. Nucleotide data confirmed placement of the sequestrate taxon Gastrocybe lateritia in sect. Candidae, but as a distinct taxon. It is hypothesized that the unique sequestrate morphology of G. lateritia may be caused by a bacterial infection.
Article
Spider venoms are a rich source of novel pharmacologically and agrochemically interesting compounds that have received increased attention from pharmacologists and biochemists in recent years. The application of technologies derived from genomics and proteomics have led to the discovery of the enormous molecular diversity of those venoms, which consist mainly of peptides and proteins. The molecular diversity of spider peptides has been revealed by mass spectrometry and appears to be based on a limited set of structural scaffolds. Genetic analysis has led to a further understanding of the molecular evolution mechanisms presiding over the generation of these combinatorial peptide libraries. Gene duplication and focal hypermutation, which has been described in cone snails, appear to be common mechanisms to venomous mollusks and spiders. Post-translational modifications, fine structural variations and new molecular scaffolds are other potential mechanisms of toxin diversification, leading to the pharmacologically complex cocktails used for predation and defense.
Article
Cyclotides are mini-proteins with a macrocyclic peptide backbone and cystine-knot arrangement of disulfide bonds that makes them exceptionally stable to chemical, thermal or enzymatic degradation. They have a diverse range of bioactivities and are amenable to chemical synthesis, making them accessible as molecular templates for protein engineering and drug design applications. In the last two years, methods have been developed for the production of cyclotides using inteins in bacterial expression systems and using plant cell cultures, adding to established methods based on solid-phase peptide synthesis. The availability of an enhanced armory of synthetic methods promises to expand the potential range of cyclotide-based applications.
Article
The current need for antibiotics with novel target molecules has coincided with advances in technical approaches for the structural and functional analysis of the lantibiotics, which are ribosomally synthesized peptides produced by gram-positive bacteria. These peptides have antibiotic or morphogenetic activity and are structurally defined by the presence of unusual amino acids introduced by posttranslational modification. Lantibiotics are complex polycyclic molecules formed by the dehydration of select Ser and Thr residues and the intramolecular addition of Cys thiols to the resulting unsaturated amino acids to form lanthionine and methyllanthionine bridges, respectively. Importantly, the structural and functional diversity of the lantibiotics is much broader than previously imagined. Here we discuss this growing collection of molecules and introduce some recently discovered peptides, review advances in enzymology and protein engineering, and discuss the regulatory networks that govern the synthesis of the lantibiotics by the producing organisms.
Article
Cystidia in fruit bodies are taxonomically important characters. However, little is known about their ecological functions. The defensive role of cystidia against the collembolans Ceratophysella denisana and Mitchellania horrida was examined in fruit bodies of Russula bella and Strobilurus ohshimae. Cystidium-destruction experiments demonstrated that R. bella and S. ohshimae cystidia decrease the number of collembola found on gills, although the effects were not significant for R. bella against C. denisana. Furthermore, R. bella cystidia increased collembolan mortality in the laboratory, and in the field, collembola were found dead on parts of the fruit body of this species where cystidia were abundant. In the cystidium-destruction experiment, approximately one-third of collembola appeared to avoid R. bella. Therefore, deadly cystidia may be selected for in R. bella to avoid collembolan attack. Laboratory feeding experiments revealed that collembola can extensively damage R. bella and S. ohshimae basidiospores by feeding. These results suggest that the cystidia of R. bella and S. ohshimae may protect basidiospores from collembolan predation.
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