17ß-Estradiol promotes metastasis in triple-negative breast cancer through the Calpain/YAP/ß-catenin signaling axis.

ß-catenin is an important regulator of malignant progression. 17ß-Estradiol (E2), an important sex hormone in women, promotes the growth and metastasis of triple-negative breast cancer (TNBC). However, whether ß-catenin is involved in E2-induced metastasis of TNBC remains unknown. In this study, we show that E2 induces the proliferation, migration, invasion, and metastasis of TNBC cells. E2 induces ß-catenin protein expression and nuclear translocation, thereby regulating the expression of target genes such as Cyclin D1 and MMP-9. The inhibition of ß-catenin reversed the E2-induced cell malignant behaviors. Additionally, E2 activated Calpain by increasing intracellular Ca2+ levels and reducing calpastatin levels. When Calpain was inhibited, E2 did not induce the proliferation, migration, invasion, or metastasis of TNBC cells. In addition, E2 promoted translocation of YAP into the nucleus by inhibiting its phosphorylation. Calpain inhibition reversed the E2-induced YAP dephosphorylation. Inhibition of YAP transcriptional activity reversed the effects of E2 on the proliferation, migration, invasion, and ß-catenin of TNBC cells. In conclusion, we demonstrated that E2 induced metastasis-related behaviors in TNBC cells and this effect was mediated through the Calpain/YAP/ß-catenin signaling pathway.

Rare NRPS Gene Cluster for Desferriferrichrome Biosynthesis Controls the Conflict between Trap Formation and Nematicidal Activity in Arthrobotrys oligospora.

The formation of the trapping device induced by nematodes has been assumed as an indicator for a switch from saprophytic to predacious lifestyles for nematode-trapping fungi. However, fungal nematocidal activity is not completely synonymous with fungal trap formation. We found that the predominant nematode-trapping fungus Arthrobotrys oligospora harbored a rare NRPS (Ao415) gene cluster that was mainly distributed in nematode-trapping fungi. The gene Ao415 putatively encodes a protein with a unique domain organization, distinct from other NRPSs in other fungi. Mutation of the two key biosynthetic genes Ao415 and Ao414 combined with nontarget metabolic analysis revealed that the Ao415 gene cluster was responsible for the biosynthesis of a hydroxamate siderophore, desferriferrichrome (1). Lack of desferriferrichrome (1) and its hydroxamate precursor (3) could lead to significantly increased Fe3+ content, which induced fungal trap formation without a nematode inducer. Furthermore, the addition of Fe3+ strongly improved fungal trap formation but deleteriously caused broken traps. The addition of 1 significantly attenuated trap formation but enhanced fungal nematicidal activity. Our findings indicate that iron is a key factor for trap formation and provide a new insight into the underlying mechanism of siderophores in nematode-trapping fungi.

Exploration of the profiles of steroidal saponins from Rhizoma Paridis and their metabolites in rats by UPLC-Q-TOF-MS/MS.

INTRODUCTION: Steroidal saponins characterised by intricate chemical structures are the main active components of a well-known traditional Chinese medicine (TCM) Rhizoma Paridis. The metabolic profiles of steroidal saponins in vivo remain largely unexplored, despite their renowned antitumor, immunostimulating, and haemostatic activity. OBJECTIVE: To perform a comprehensive analysis of the chemical constituents of Rhizoma Paridis total saponins (RPTS) and their metabolites in rats after oral administration. METHOD: The chemical constituents of RPTS and their metabolites were analysed using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS). RESULTS: A reliable UPLC-Q-TOF-MS/MS method was established, and a total of 142 compounds were identified in RPTS. Specifically, diosgenin-type saponins showed the diagnostic ions at m/z 415.32, 397.31, 283.25, 271.21, and 253.20, whereas pennogenin-type saponins exhibited the diagnostic ions at m/z 413.31, 395.30, and 251.20. Based on the characteristic fragments and standard substances, 15 specific metabolites were further identified in the faeces, urine, plasma, and bile of rats. The metabolic pathways of RPTS, including phase I reactions (de-glycosylation and oxidation) and phase II reactions (glucuronidation), were explored and summarised, and the enrichment of metabolites was characterised by multivariate statistical analysis. CONCLUSION: The intricate RPTS could be transformed into relatively simple metabolites in rats through de-glycosylation, which provides a reference for further metabolic studies and screening of active ingredients for TCM.

Melanin precursors mediated adaption to temperature changes in fungus and animal via inhibition of lipid-mediated ferroptosis.

The discovery of biological activities of natural products plays a vital part in drug development. The mechanism by which organisms respond to temperature changes via biosynthesis of natural products remained largely cryptic. A thermophilic fungus under cold stress turned black and accumulated a polyketide metabolite 1 and lipid mass. Deficiency in 1 caused melanin loss and accumulated extra lipid mass, unexpectedly leading to seriously damaged mitochondria diagnostic for ferroptosis. Further analysis revealed that lipid mass induced by cold stress intensively increased ferroptosis risk and 1 functioned as cell wall reinforcer against mass lipid accumulation and as reactive oxygen species scavenger against lipid peroxidation. We also found that melanin in mice lowered lipid level but enhanced animal resistance to cold stress. Treatment with melanin precursors significantly increased mouse cell survival rate under cold stress. Our results unveiled a metabolite-lipid-ferroptosis-cold relationship, which provided mechanistic insights into the functions of most common metabolites and into diseases related to cold stress. These findings opened a perspective for developing anti-cold and anti-ferroptosis therapeutics and agents.

Arthrocolins Synergizing with Fluconazole Inhibit Fluconazole-Resistant Candida albicans by Increasing Riboflavin Metabolism and Causing Mitochondrial Dysfunction and Autophagy.

Our previous study reported that seminaturally occurring arthrocolins A to C with unprecedented carbon skeletons could restore the antifungal activity of fluconazole against fluconazole-resistant Candida albicans. Here, we showed that arthrocolins synergized with fluconazole, reducing the fluconazole minimum and dramatically augmenting the survivals of 293T human cells and nematode Caenorhabditis elegans infected with fluconazole-resistant C. albicans. Mechanistically, fluconazole can induce fungal membrane permeability to arthrocolins, leading to the intracellular arthrocolins that were critical to the antifungal activity of the combination therapy by inducing abnormal cell membranes and mitochondrial dysfunctions in the fungus. Transcriptomics and reverse transcription-quantitative PCR (qRT-PCR) analysis indicated that the intracellular arthrocolins induced the strongest upregulated genes that were involved in membrane transports while the downregulated genes were responsible for fungal pathogenesis. Moreover, riboflavin metabolism and proteasomes were the most upregulated pathways, which were accompanied by inhibition of protein biosynthesis and increased levels of reactive oxygen species (ROS), lipids, and autophagy. Our results suggested that arthrocolins should be a novel class of synergistic antifungal compounds by inducing mitochondrial dysfunctions in combination with fluconazole and provided a new perspective for the design of new bioactive antifungal compounds with potential pharmacological properties. IMPORTANCE The prevalence of antifungal-resistant Candida albicans, which is a common human fungal pathogen causing life-threatening systemic infections, has become a challenge in the treatment of fungal infections. Arthrocolins are a new type of xanthene obtained from Escherichia coli fed with a key fungal precursor toluquinol. Different from those artificially synthesized xanthenes used as important medications, arthrocolins can synergize with fluconazole against fluconazole-resistant Candida albicans. Fluconazole can induce the fungal permeability of arthrocolins into fungal cells, and then the intracellular arthrocolins exerted detrimental effects on the fungus by inducing fungal mitochondrial dysfunctions, leading to dramatically reduced fungal pathogenicity. Importantly, the combination of arthrocolins and fluconazole are effective against C. albicans in two models, including human cell line 293T and nematode Caenorhabditis elegans. Arthrocolins should be a novel class of antifungal compounds with potential pharmacological properties.

Tryptophan-centered metabolic alterations coincides with lipid-mediated fungal response to cold stress.

Tryptophan and its derived metabolites have been assumed to play important roles in the development and survival of organisms. However, the links of tryptophan and its derived metabolites to temperature change remained largely cryptic. Here we presented that a class of prenyl indole alkaloids biosynthesized from tryptophan dramatically accumulated in thermophilic fungus Thermomyces dupontii under cold stress, in which lipid droplets were also highly accumulated and whose conidiophores were highly build-up. Concurrently, disruption of the key NRPS gene involved in the biosynthesis of prenyl indole alkaloids, resulted in decreased lipid and shrunken mitochondria but enlarged vacuoles. Moreover, the Fe3+ and superoxide levels in ΔNRPS were significantly increased but the reactive oxygen species lipid peroxidation and autophagy levels decreased. Metabolomics study revealed that most enriched metabolites in ΔNRPS were mainly composed of tryptophan degraded metabolites including well known ROS scavenger kynurenamines, and lipid-inhibitors, anthranilic acid and indoleacetic acid, and free radical reaction suppressor free fatty acids. Transcriptomic analysis suggested that the key gene involved in tryptophan metabolism, coinciding with the lipid metabolic processes and ion transports were most up-regulated in ΔNRPS under stress. Our results confirmed a lipid-mediated fungal response to cold stress and unveiled a link of tryptophan-based metabolic reprogramming to the fungal cold adaption.

Characterization and discrimination of two varieties of eggplants using multi-element and metabolomics profiles coupled with chemometrics analysis.

The main purpose of the present study was to clarify the differences present in the multi-element and metabolite profiles between two varieties of eggplants. A total of 54 elements were identified by inductively coupled plasma mass spectrometry, 16 elements were significantly different between peel (n = 3) and flesh (n = 3). Besides, untargeted metabolomics combined with chemometrics was used to discriminate the peel (n = 4) and flesh (n = 4) of the two varieties of eggplants. A total of 178 metabolites were screened out with criteria of p < 0.05, fold change > 1.5 or < 0.67 and variable importance in projection score > 1 for the PLS-DA model. Maltitol and d-proline were the most important discriminants of the two varieties of eggplants. Kaempferol-3-O-rutinoside was the most important identification components between peel and flesh of the two varieties of eggplant. Results showed that the two varieties of eggplants could be distinguished based on their multi-element and metabolite profiles, which may provide new directions for eggplant function research.

The Multifaceted Gene 275 Embedded in the PKS-PTS Gene Cluster Was Involved in the Regulation of Arthrobotrisin Biosynthesis, TCA Cycle, and Septa Formation in Nematode-Trapping Fungus Arthrobotrys oligospora.

The predominant nematode-trapping fungus Arthrobotrys oligospora harbors a unique polyketide synthase-prenyltransferase (PKS-PTS) gene cluster AOL_s00215g responsible for the biosynthesis of sesquiterpenyl epoxy-cyclohexenoids (SECs) that are involved in the regulation of fungal growth, adhesive trap formation, antibacterial activity, and soil colonization. However, the function of one rare gene (AOL_s00215g275 (275)) embedded in the cluster has remained cryptic. Here, we constructed two mutants with the disruption of 275 and the overexpression of 275, respectively, and compared their fungal growth, morphology, resistance to chemical stress, nematicidal activity, transcriptomic and metabolic profiles, and infrastructures, together with binding affinity analysis. Both mutants displayed distinct differences in their TCA cycles, SEC biosynthesis, and endocytosis, combined with abnormal mitochondria, vacuoles, septa formation, and decreased nematicidal activity. Our results suggest that gene 275 might function as a separator and as an integrated gene with multiple potential functions related to three distinct genes encoding the retinoic acid induced-1, cortactin, and vacuolar iron transporter 1 proteins in this nematode-trapping fungus. Our unexpected findings provide insight into the intriguing organization and functions of a rare non-biosynthetic gene in a biosynthetic gene cluster.

Modulating Activity Evaluation of Gut Microbiota with Versatile Toluquinol.

Gut microbiota have important implications for health by affecting the metabolism of diet and drugs. However, the specific microbial mediators and their mechanisms in modulating specific key intermediate metabolites from fungal origins still remain largely unclear. Toluquinol, as a key versatile precursor metabolite, is commonly distributed in many fungi, including Penicillium species and their strains for food production. The common 17 gut microbes were cultivated and fed with and without toluquinol. Metabolic analysis revealed that four strains, including the predominant Enterococcus species, could metabolize toluquinol and produce different metabolites. Chemical investigation on large-scale cultures led to isolation of four targeted metabolites and their structures were characterized with NMR, MS, and X-ray diffraction analysis, as four toluquinol derivatives (1-4) through O1/O4-acetyl and C5/C6-methylsulfonyl substitutions, respectively. The four metabolites were first synthesized in living organisms. Further experiments suggested that the rare methylsulfonyl groups in 3-4 were donated from solvent DMSO through Fenton's reaction. Metabolite 1 displayed the strongest inhibitory effect on cancer cells A549, A2780, and G401 with IC50 values at 0.224, 0.204, and 0.597 µM, respectively, while metabolite 3 displayed no effect. Our results suggest that the dominant Enterococcus species could modulate potential precursors of fungal origin and change their biological activity.

Association of Intrauterine Microbes with Endometrial Factors in Intrauterine Adhesion Formation and after Medicine Treatment.

Intrauterine adhesions (IUAs) have caused serious harm to women's reproductive health. Although emerging evidence has linked intrauterine microbiome to gynecological diseases, the association of intrauterine microbiome with IUA, remains unknown. We performed metagenome-wide association, metabolomics, and transcriptomics studies on IUA and non-IUA uteri of adult rats to identify IUA-associated microbial species, which affected uterine metabolites and endometrial transcriptions. A rat model was used with one side of the duplex uterus undergoing IUA and the other remaining as a non-IUA control. Both 16S rRNA sequencing and metagenome-wide association analysis revealed that instead of Mycoplasmopsis specie in genital tract, murine lung pathogen Mycoplasmopsispulmonis markedly increased in IUA samples and displayed a distinct positive interaction with the host immune system. Moreover, most of the IUA-enriched 58 metabolites positively correlate with M.pulmonis, which inversely correlates with a mitotic progression inhibitor named 3-hydroxycapric acid. A comparison of metabolic profiles of intrauterine flushing fluids from human patients with IUA, endometritis, and fallopian tube obstruction suggested that rat IUA shared much similarity to human IUA. The endometrial gene Tenascin-N, which is responsible for extracellular matrix of wounds, was highly up-regulated, while the key genes encoding parvalbumin, trophectoderm Dkkl1 and telomerase involved in leydig cells, trophectoderm cells, activated T cells and monocytes were dramatically down-regulated in rat IUA endometria. Treatment for rat IUA with estrogen (E2), oxytetracycline (OTC), and a traditional Chinese patent medicine GongXueNing (GXN) did not reduce the incidence of IUA, though inflammatory factor IL-6 was dramatically down-regulated (96-86%) with all three. Instead, in both the E2 and OTC treated groups, IUA became worse with a highly up-regulated B cell receptor signaling pathway, which may be associated with the significantly increased proportions of Ulvibacter or Staphylococcus. Our results suggest an association between intrauterine microbiota alterations, certain uterine metabolites, characteristic changes in endometrial transcription, and IUA and the possibility to intervene in IUA formation by targeting the causal factors, microbial infection, and Tenascin-like proteins.

Acetylation of Sesquiterpenyl Epoxy-Cyclohexenoids Regulates Fungal Growth, Stress Resistance, Endocytosis, and Pathogenicity of Nematode-Trapping Fungus Arthrobotrys oligospora via Metabolism and Transcription.

Sesquiterpenyl epoxy-cyclohexenoids (SECs) that depend on a polyketide synthase-terpenoid synthase (PKS-TPS) pathway are widely distributed in plant pathogenic fungi. However, the biosynthesis and function of the acetylated SECs still remained cryptic. Here, we identified that AOL_s00215g 273 (273) was responsible for the acetylation of SECs in Arthrobotrys oligospora via the construction of Δ273, in which the acetylated SECs were absent and major antibacterial nonacetylated SECs accumulated. Mutant Δ273 displayed increased trap formation, and nematicidal and antibacterial activities but decreased fungal growth and soil colonization. Glutamine, a key precursor for NH3 as a trap inducer, was highly accumulated, and biologically active phenylpropanoids and antibiotics were highly enriched in Δ273. The decreased endocytosis and increased autophagosomes, with the most upregulated genes involved in maintaining DNA and transcriptional stability and pathways related to coronavirus disease and exosome, suggested that lack of 273 might result in increased virus infection and the acetylation of SECs played a key role in fungal diverse antagonistic ability.

Polyketide Synthase-Terpenoid Synthase Hybrid Pathway Regulation of Trap Formation through Ammonia Metabolism Controls Soil Colonization of Predominant Nematode-Trapping Fungus.

Polyketide synthase-terpenoid synthase (PKS-TPS) hybrid pathways for biosynthesis of unique sesquiterpenyl epoxy-cyclohexenoids (SECs) have been found to be widely distributed in plant pathogenic fungi. However, the natural and ecological functions of these pathways and their metabolites still remain cryptic. In this study, the whole PKS-TPS hybrid pathway in the predominant nematode-trapping fungus Arthrobotrys oligospora was first proposed according to all the intermediates and their derivatives from all the A. oligospora mutants with a deficiency in each gene involved in SEC biosynthesis. Most mutants displayed significantly increased trap formation which was correlated with alteration of the ammonia level. Further analysis revealed that the main metabolites involved in ammonia metabolism were largely increased in most mutants. However, significantly retarded colonization in soil were observed in most mutants compared to the wild-type strain due to significantly decreased antibacterial activities. Our results suggested that A. oligospora used the PKS-TPS hybrid pathway for fungal soil colonization via decreasing fungal nematode-capturing ability. This also provided solid evidence that boosting fungal colonization in soil was the secondary metabolite whose biosynthesis depended on a PKS-TPS hybrid pathway.

Novel Polyketide-Terpenoid Hybrid Metabolites from a Potent Nematicidal Arthrobotrys oligospora Mutant ΔAOL_s00215g278.

Here, we reported that detailed investigation on trace targeted metabolites from nematode-trapping fungus Arthrobotrys oligospora mutant with deletion of P450 gene AOL_s00215g278 led to isolation of 9 new polyketide-terpenoid hybrid derivatives, including four new glycosides of the key precursor farnesyl hydrotoluquinol (1) and, surprisingly, four new sesquiterpenyl epoxy-cyclohexenoids (SECs) analogues. Among them, two major target metabolites 1 and 14 displayed moderate nematode inhibitory ability. Moreover, the mutant lacking AOL_s00215g278 could form far more nematode-capturing traps within 6 h in contact with nematodes and show rapid potent nematicidal activity with killing 93.7% preys, though deletion of the P450 gene resulted in dramatic decrease in fungal colony growth and failure to produce fungal conidia. The results unequivocally revealed that gene AOL_s00215g278 should be involved in not only the SEC biosynthetic pathway in the nematode-trapping fungus A. oligospora but also fungal conidiation and nematicidal activity.

Two CRISPR/Cas9 Systems Developed in Thermomyces dupontii and Characterization of Key Gene Functions in Thermolide Biosynthesis and Fungal Adaptation.

Thermomyces dupontii, a widely distributed thermophilic fungus, is an ideal organism for investigating the mechanism of thermophilic fungal adaptation to diverse environments. However, genetic analysis of this fungus is hindered by a lack of available and efficient gene-manipulating tools. In this study, two different Cas9 proteins from mesophilic and thermophilic bacteria, with in vivo expression of a single guide RNA (sgRNA) under the control of tRNAGly, were successfully adapted for genome editing in T. dupontii We demonstrated the feasibility of applying these two gene editing systems to edit one or two genes in T. dupontii The mesophilic CRISPR/Cas9 system displayed higher editing efficiency (50 to 86%) than the thermophilic CRISPR/Cas9 system (40 to 67%). However, the thermophilic CRISPR/Cas9 system was much less time-consuming than the mesophilic CRISPR/Cas9 system. Combining the CRISPR/Cas9 systems with homologous recombination, a constitutive promoter was precisely knocked in to activate a silent polyketide synthase-nonribosomal peptide synthase (PKS-NRPS) biosynthetic gene, leading to the production of extra metabolites that did not exist in the parental strains. Metabolic analysis of the generated biosynthetic gene mutants suggested that a key biosynthetic pathway existed for the biosynthesis of thermolides in T. dupontii, with the last two steps being different from those in the heterologous host Aspergillus Further analysis suggested that these biosynthetic genes might be involved in fungal mycelial growth, conidiation, and spore germination, as well as in fungal adaptation to osmotic, oxidative, and cell wall-perturbing agents.IMPORTANCEThermomyces represents a unique ecological taxon in fungi, but a lack of flexible genetic tools has greatly hampered the study of gene function in this taxon. The biosynthesis of potent nematicidal thermolides in T. dupontii remains largely unknown. In this study, mesophilic and thermophilic CRISPR/Cas9 gene editing systems were successfully established for both disrupting and activating genes in T. dupontii In this study, a usable thermophilic CRISPR/Cas9 gene editing system derived from bacteria was constructed in thermophilic fungi. Chemical analysis of the mutants generated by these two gene editing systems identified the key biosynthetic genes and pathway for the biosynthesis of nematocidal thermolides in T. dupontii Phenotype analysis and chemical stress experiments revealed potential roles of secondary metabolites or their biosynthetic genes in fungal development and adaption to chemical stress conditions. These two genomic editing systems will not only accelerate investigations into the biosynthetic mechanisms of unique natural products and functions of cryptic genes in T. dupontii but also offer an example for setting up CRISPR/Cas9 systems in other thermophilic fungi.

Novel Polyketide-Terpenoid Hybrid Metabolites and Increased Fungal Nematocidal Ability by Disruption of Genes 277 and 279 in Nematode-Trapping Fungus Arthrobotrys oligospora.

Nematode-trapping fungus Arthrobotrys oligospora can produce a type of sesquiterpenyl epoxy-cyclohexenoid (SEC) metabolites that are regarded as characteristic chemtaxonomic markers. Here, we reported investigation on the functions of a putatively cupin-like family gene 277 and a dehydrogenase gene 279 by gene engineering, chemical metabolite profiling and phenotype analysis. Ten targeted metabolites were isolated from two mutants Δ277 and Δ279 and four novel metabolites including three polyketide-terpenoid (PK-TP) hybrid ones were characterized. Metabolite C277-1 from mutant Δ277 shared the characteristic feature of the first and simplest PK-TP hybrid precursor, prenyl toluquinol, and metabolites C279-1 and C279-2 from mutant Δ279 shared the basic carbon skeleton of the key PK-TP hybrid precursor, farnesyl toluquinol, for biosynthesis of SEC metabolites. These results suggested that gene 277 should be involved in biosynthesis of the second prenyl unit for farnesyl toluquinol precursor, and gene 279 might be responsible for the diagnostic epoxy formation. Further analysis revealed that genes 277 and 279 might play roles in fungal conidiation, predatory trap formation, and nematode-capturing ability.

Integrated Immunomagnetic Bead-Based Microfluidic Chip for Exosomes Isolation.

Exosomes are essential early biomarkers for health monitoring and cancer diagnosis. A prerequisite for further investigation of exosomes is the isolation, which is technically challenging due to the complexity of body fluids. This paper presents the development of an integrated microfluidic chip for exosomes isolation, which combines the traditional immunomagnetic bead-based protocol and the recently emerging microfluidic approach, resulting in benefits from both the high-purity of the former and the automated continuous superiority of the latter. The chip was designed based on an S-shaped micromixer with embedded baffle. The excellent mixing efficiency of this micromixer compared with Y-shaped and S-shaped micromixers was verified by simulation and experiments. The photolithography technique was employed to fabricate the integrated microfluidic chip, and the manufacturing process was elucidated. We finally established an experimental platform for exosomes isolation with the fabricated microfluidic chip built in. Exosomes isolation experiments were conducted using this platform. The distribution and morphology of the isolated exosomes were observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Quantitative size analyses based on transmission electron micrographs indicated that most of the obtained particles were between 30 and 150 nm. Western blot analyses of the isolated exosomes and the serum were conducted to verify the platform's capability of isolating a certain subpopulation of exosomes corresponding to specified protein markers (CD63). The complete time for isolation of 150 µL serum samples was approximately 50 min, which was highly competitive with the reported existing protocols. Experimental results proved the capacity of the established integrated microfluidic chip for exosomes isolation with high purity, high integrity, and excellent efficiency. The platform can be further developed to make it possible for practical use in clinical applications as a universal exosomes isolation and characterization tool.

Metabolites from Two Dominant Thermophilic Fungal Species Thermomyces lanuginosus and Scytalidium thermophilum.

Thermomyces lanuginosus and Scytalidium thermophilum are among the most ubiquitous thermophilic fungi in compost and soil. Chemical study on these two prevalent strains collected from Yunnan led to isolation of 23 metabolites, including one new metabolite, therlanubutanolide, and 15 known compounds, isolated from the YGP culture broth of Thermomyces lanuginosus and 7 known compounds isolated from Scytalidium thermophilum, respectively. Therlanubutanolide shared the quite similar features of the same carbon skeleton and saturation as natural hexadecanoic acids. This was the first reported discovery of such a lactone as natural occurring metabolite. All the compounds were reported for the first time from thermophilic fungi. Among them, N-[(2S,3R,4E,8E)-1,3-dihydroxy-9-methyloctadeca-4,8-dien-2-yl]acetamide was for the first time reported to be a naturally occurring metabolite and its NMR data was first provided in this study. A type of PKS-derived metabolites, three 3,4-dihydronaphthalen-1(2H)-ones, which were widely found in plant pathogenic fungi as phytotoxins and reported to have antimicrobial activity, were obtained from both dominant thermophilic fungi. The frequent occurrence of such PKS phytotoxins in these two thermophilic fungi might suggest particular ecological interest.

Sesquiterpenyl Epoxy-Cyclohexenoids and their Signaling Functions in Nematode-Trapping Fungus Arthrobotrys oligospora.

In this study, we purified three new sesquiterpenyl epoxy-cyclohexenoid (SEC) analogues, arthrobotrisin D (11) and its two derivatives, from nematode-trapping fungus Arthrobotrys oligospora. Our results revealed that arthrobotrisin type SEC metabolites could be detected in all the test fungal strains from geographically distinct regions grown on different nutrient media, indicative of unique diagnostic character as chemical indicators for A. oligospora. The time course designs over short-term intervals of the fungus under direct contact and indirect contact with living or dead nematodes revealed that arthrobotrisin B and D (6 and 11) displayed significant relationships (positive or negative correlation) with fungal saprophytic and pathogenic stages during a nematode predation event. Interestingly, fungus on nutrient-limiting medium conducive to fungal trap formation could rapidly drop the concentration levels of arthrobotrisins B and D within 6 h when dead nematodes were around, in great contrast to that for living nematodes. Moreover, only in the fungal strain under direct contact with living dominant soil bacteria, arthrobotrisins B and D exhibited significant increase in amounts. Among them, the new SEC, arthrobotrisin D (11) was found to be a key unique metabolic signal for fungal colony growth and fungal interaction with prey and bacteria. Our study suggested that chemical analysis of SEC metabolites in A. oligospora provides a window into the fungal growth status and much valuable information about ecological environments associated with the nematode infections.

The Velvet Proteins VosA and VelB Play Different Roles in Conidiation, Trap Formation, and Pathogenicity in the Nematode-Trapping Fungus Arthrobotrys oligospora.

The velvet family proteins VosA and VelB are involved in growth regulation and differentiation in the model fungus Aspergillus nidulans and other filamentous fungi. In this study, the orthologs of VosA and VelB, AoVosA, and AoVelB, respectively, were characterized in the nematode-trapping fungus Arthrobotrys oligospora, which captures nematodes by producing trapping devices (traps). Deletion of the AovelB gene resulted in growth defects in different media, and the aerial hyphae from the ΔAovelB mutant lines were fewer in number and their colonies were less dense than those from the wild-type (WT) strain. The ΔAovelB mutants each displayed serious sporulation defects, and the transcripts of several sporulation-related genes (e.g., abaA, flbC, rodA, and vosA) were significantly down-regulated compared to those from the WT strain. Furthermore, the ΔAovelB mutant strains became more sensitive to chemical reagents, including sodium dodecyl sulfate and H2O2. Importantly, the ΔAovelB mutants were unable to produce nematode-capturing traps. Similarly, extracellular proteolytic activity was also lower in the ΔAovelB mutants than in the WT strain. In contrast, the ΔAovosA mutants displayed no obvious differences from the WT strain in these phenotypic traits, whereas conidial germination was lower in the ΔAovosA mutants, which became more sensitive to heat shock stress. Our results demonstrate that the velvet protein AoVelB is essential for conidiation, trap formation, and pathogenicity in A. oligospora, while AoVosA plays a role in the regulation of conidial germination and heat shock stress.

AoStuA, an APSES transcription factor, regulates the conidiation, trap formation, stress resistance and pathogenicity of the nematode-trapping fungus Arthrobotrys oligospora.

The APSES protein family comprises a conserved class of fungus-specific transcriptional regulators. Some members have been identified in partial ascomycetes. In this study, the APSES protein StuA (AoStuA) of the nematode-trapping fungus Arthrobotrys oligospora was characterized. Compared with the wild-type (WT) strain, three ΔAoStuA mutants grew relatively slowly, displayed a 96% reduction in sporulation capacity and a delay in conidial germination. The reduced sporulation capacity correlated with transcriptional repression of several sporulation-related genes. The mutants were also more sensitive to chemical stressors than the WT strain. Importantly, the mutants were unable to produce mycelial traps for nematode predation. Moreover, peroxisomes and Woronin bodies were abundant in the WT cells but hardly found in the cells of those mutants. The lack of such organelles correlated with transcriptional repression of some genes involved in the biogenesis of peroxisomes and Woronin bodies. The transcript levels of several genes involved in the cAMP/PKA signalling pathway were also significantly reduced in the mutants versus the WT strain, implicating a regulatory role of AoStuA in the transcription of genes involved in the cAMP/PKA signalling pathway that regulates an array of cellular processes and events. In particular, AoStuA is indispensable for A. oligospora trap formation and virulence.