Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non-Natural Multiply Backbone N-Methylated Peptide Macrocycles in Yeast.

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.

Structural and Functional Analysis of Peptides Derived from KEX2-Processed Repeat Proteins in Agaricomycetes Using Reverse Genetics and Peptidomics.

Bioactivities of fungal peptides are of interest for basic research and therapeutic drug development. Some of these peptides are derived from "KEX2-processed repeat proteins" (KEPs), a recently defined class of precursor proteins that contain multiple peptide cores flanked by KEX2 protease cleavage sites. Genome mining has revealed that KEPs are widespread in the fungal kingdom. Their functions are largely unknown. Here, we present the first in-depth structural and functional analysis of KEPs in a basidiomycete. We bioinformatically identified KEP-encoding genes in the genome of the model agaricomycete Coprinopsis cinerea and established a detection protocol for the derived peptides by overexpressing the C. cinerea KEPs in the yeast Pichia pastoris. Using this protocol, which includes peptide extraction and mass spectrometry with data analysis using the search engine Mascot, we confirmed the presence of several KEP-derived peptides in C. cinerea, as well as in the edible mushrooms Lentinula edodes, Pleurotus ostreatus, and Pleurotus eryngii. While CRISPR-mediated knockout of C. cinerea kep genes did not result in any detectable phenotype, knockout of kex genes caused defects in mycelial growth and fruiting body formation. These results suggest that KEP-derived peptides may play a role in the interaction of C. cinerea with the biotic environment and that the KEP-processing KEX proteases target a variety of substrates in agaricomycetes, including some important for mycelial growth and differentiation. IMPORTANCE Two recent bioinformatics studies have demonstrated that KEX2-processed repeat proteins are widespread in the fungal kingdom. However, despite the prevalence of KEPs in fungal genomes, only few KEP-derived peptides have been detected and studied so far. Here, we present a protocol for the extraction and structural characterization of KEP-derived peptides from fungal culture supernatants and tissues. The protocol was successfully used to detect several linear and minimally modified KEP-derived peptides in the agaricomycetes C. cinerea, L. edodes, P. ostreatus, and P. eryngii. Our study establishes a new protocol for the targeted search of KEP-derived peptides in fungi, which will hopefully lead to the discovery of more of these interesting fungal peptides and allow a further characterization of KEPs.

Mycorrhiza-induced mycocypins of Laccaria bicolor are potent protease inhibitors with nematotoxic and collembola antifeedant activity.

Fungivory of mycorrhizal hyphae has a significant impact on fungal fitness and, by extension, on nutrient transfer between fungi and host plants in natural ecosystems. Mycorrhizal fungi have therefore evolved an arsenal of chemical compounds that are hypothesized to protect the hyphal tissues from being eaten, such as the protease inhibitors mycocypins. The genome of the ectomycorrhizal fungus Laccaria bicolor has an unusually high number of mycocypin-encoding genes. We have characterized the evolution of this class of proteins, identified those induced by symbiosis with a host plant and characterized the biochemical properties of two upregulated L. bicolor mycocypins. More than half of L. bicolor mycocypin-encoding genes are differentially expressed during symbiosis or fruiting body formation. We show that two L. bicolor mycocypins that are strongly induced during symbiosis are cysteine protease inhibitors and exhibit similar but distinct localization in fungal tissues at different developmental stages and during interaction with a host plant. Moreover, we show that these L. bicolor mycocypins have toxic and feeding deterrent effect on nematodes and collembolans, respectively. Therefore, L. bicolor mycocypins may be part of a mechanism by which this species deters grazing by different members of the soil food web.

Genome sequences of Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana and the identification of biosynthetic gene clusters for fungal peptide natural products.

In recent years, a variety of fungal cyclic peptides with interesting bioactivities have been discovered. For many of these peptides, the biosynthetic pathways are unknown and their elucidation often holds surprises. The cyclic and backbone N-methylated omphalotins from Omphalotus olearius were recently shown to constitute a novel class (borosins) of ribosomally synthesized and posttranslationally modified peptides, members of which are produced by many fungi, including species of the genus Rhizopogon. Other recently discovered fungal peptide macrocycles include the mariannamides from Mariannaea elegans and the backbone N-methylated verrucamides and broomeanamides from Myrothecium verrucaria and Sphaerostilbella broomeana, respectively. Here, we present draft genome sequences of four fungal species Rhizopogon roseolus, Mariannaea elegans, Myrothecium verrucaria, and Sphaerostilbella broomeana. We screened these genomes for precursor proteins or gene clusters involved in the mariannamide, verrucamide, and broomeanamide biosynthesis including a general screen for borosin-producing precursor proteins. While our genomic screen for potential ribosomally synthesized and posttranslationally modified peptide precursor proteins of mariannamides, verrucamides, broomeanamides, and borosins remained unsuccessful, antiSMASH predicted nonribosomal peptide synthase gene clusters that may be responsible for the biosynthesis of mariannamides, verrucamides, and broomeanamides. In M. verrucaria, our antiSMASH search led to a putative NRPS gene cluster with a predicted peptide product of 20 amino acids, including multiple nonproteinogenic isovalines. This cluster likely encodes a member of the peptaibols, an antimicrobial class of peptides previously isolated primarily from the Genus Trichoderma. The nonribosomal peptide synthase gene clusters discovered in our screenings are promising candidates for future research.

Structure-function relationship of a novel fucoside-binding fruiting body lectin from Coprinopsis cinerea exhibiting nematotoxic activity.

Lectins are non-immunoglobulin-type proteins that bind to specific carbohydrate epitopes and play important roles in intra- and inter-organismic interactions. Here, we describe a novel fucose-specific lectin, termed CML1, which we identified from fruiting body extracts of Coprinopsis cinerea. For further characterization, the coding sequence for CML1 was cloned and heterologously expressed in Escherichia coli. Feeding of CML1-producing bacteria inhibited larval development of the bacterivorous nematode Caenorhabditis tropicalis, but not of C. elegans. The crystal structure of the recombinant protein in its apo-form and in complex with H type I or Lewis A blood group antigens was determined by X-ray crystallography. The protein folds as a sandwich of 2 antiparallel ß-sheets and forms hexamers resulting from a trimer of dimers. The hexameric arrangement was confirmed by small-angle X-ray scattering (SAXS). One carbohydrate-binding site per protomer was found at the dimer interface with both protomers contributing to ligand binding, resulting in a hexavalent lectin. In terms of lectin activity of recombinant CML1, substitution of the carbohydrate-interacting residues His54, Asn55, Trp94, and Arg114 by Ala abolished carbohydrate-binding and nematotoxicity. Although no similarities to any characterized lectin were found, sequence alignments identified many non-characterized agaricomycete proteins. These results suggest that CML1 is the founding member of a novel family of fucoside-binding lectins involved in the defense of agaricomycete fruiting bodies against predation by fungivorous nematodes.

Incubation media modify silver nanoparticle toxicity for whitefish (Coregonus lavaretus) and roach (Rutilus rutilus) embryos.

Toxicological studies were performed to examine silver nanoparticle (AgNP, size: 14.4 ± 2.5 nm) transformation within three different test media and consequent effects on embryos of whitefish (Coregonus lavaretus) and roach (Rutilus rutilus). The test media, namely ASTM very hard water, ISO standard dilution medium, and natural lake water differed predominantly in ionic strength. Total silver was determined using inductively coupled plasma mass spectrometry (ICP-MS), while AgNPs were characterized by transmission electron microscopy and single particle ICP-MS. Silver species distributions were estimated via thermodynamic speciation calculations. Data demonstrated that increased AgNP dissolution accompanied by decreasing ionic strength of the test medium did not occur as noted in other studies. Further, other physicochemical parameters including AgNP size and metallic species distribution did not markedly affect AgNP-induced toxicity. Irrespective of the test medium, C. lavaretus were more sensitive to AgNP exposure (median lethal concentration after 8 weeks: 0.51-0.73 mg/L) compared to R. rutilus, where adverse effects were only observed at 5 mg/L in natural lake water. In addition, AgNP-induced toxicity was lower in the two standard test media compared to natural lake water. Currently, there are no apparent studies assessing simultaneously the sensitivity of C. lavaretus and R. rutilus to AgNP exposure. Therefore, the aim of this study was to (1) investigate AgNP-induced toxicity in C. lavaretus and R. rutilus cohabiting in the same aquatic environment and (2) the role played by test media in the observed effects of AgNPs on these aquatic species.

Heterologous Production and Functional Characterization of Ageritin, a Novel Type of Ribotoxin Highly Expressed during Fruiting of the Edible Mushroom Agrocybe aegerita.

Fungi produce various defense proteins against antagonists, including ribotoxins. These toxins cleave a single phosphodiester bond within the universally conserved sarcin-ricin loop of ribosomes and inhibit protein biosynthesis. Here, we report on the structure and function of ageritin, a previously reported ribotoxin from the edible mushroom Agrocybe aegerita The amino acid sequence of ageritin was derived from cDNA isolated from the dikaryon A. aegerita AAE-3 and lacks, according to in silico prediction, a signal peptide for classical secretion, predicting a cytoplasmic localization of the protein. The calculated molecular weight of the protein is slightly higher than the one reported for native ageritin. The A. aegerita ageritin-encoding gene, AaeAGT1, is highly induced during fruiting, and toxicity assays with AaeAGT1 heterologously expressed in Escherichia coli showed a strong toxicity against Aedes aegypti larvae yet not against nematodes. The activity of recombinant A. aegerita ageritin toward rabbit ribosomes was confirmed in vitro Mutagenesis studies revealed a correlation between in vivo and in vitro activities, indicating that entomotoxicity is mediated by ribonucleolytic cleavage. The strong larvicidal activity of ageritin makes this protein a promising candidate for novel biopesticide development.IMPORTANCE Our results suggest a pronounced organismal specificity of a protein toxin with a very conserved intracellular molecular target. The molecular details of the toxin-target interaction will provide important insight into the mechanism of action of protein toxins and the ribosome. This insight might be exploited to develop novel bioinsecticides.

Discovery of novel fungal RiPP biosynthetic pathways and their application for the development of peptide therapeutics.

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.

Temporary deafferentation evoked by cutaneous anesthesia: behavioral and electrophysiological findings in healthy subjects.

Motor function and motor excitability can be modulated by changes of somatosensory input. Here, we performed a randomized single-blind trial to investigate behavioral and neurophysiological changes during temporary deafferentation of left upper arm and forearm in 31 right-handed healthy adults. Lidocaine cream was used to anesthetize the skin from wrist to shoulder, sparing the hand. As control condition, on a different day, a neutral cream was applied to the same skin area. The sequence (first Lidocaine, then placebo or vice versa) was randomized. Behavioral measures included the Grating Orientation Task, the Von Frey hair testing and the Nine-hole-peg-test. Transcranial magnetic stimulation was used to investigate short-interval intracortical inhibition, stimulus response curves, motor evoked potential amplitudes during pre-innervation and the cortical silent period (CSP). Recordings were obtained from left first dorsal interosseous muscle and from left flexor carpi radialis muscle. During deafferentation, the threshold of touch measured at the forearm was significantly worse. Other behavioral treatment-related changes were not found. The CSP showed a significant interaction between treatment and time in first dorsal interosseous muscle. CSP duration was longer during Lidocaine application and shorter during placebo exposure. We conclude that, in healthy subjects, temporary cutaneous deafferentation of upper and lower arm may have minor effects on motor inhibition, but not on sensory or motor function for the adjacent non-anesthetized hand.