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.

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.

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.

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.

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.

Characterization and functional analysis of calcium/calmodulin-dependent protein kinases (CaMKs) in the nematode-trapping fungus Arthrobotrys oligospora.

Ca2+/calmodulin-dependent protein kinases (CaMKs) are unique second-messenger molecules that impact almost all cellular processes in eukaryotes. In this study, five genes encoding different CaMKs were characterized in the nematode-trapping fungus Arthrobotrys oligospora. These CaMKs, which were retrieved from the A. oligospora genome according to their orthologs in fungi such as Aspergillus nidulans and Neurospora crassa, were expressed at a low level in vitro during mycelial growth stages. Five deletion mutants corresponding to these CaMKs led to growth defects in different media and increased sensitivity to several environmental stresses, including H2O2, menadione, SDS, and Congo red; they also reduced the ability to produce conidia and traps, thus causing a deficiency in nematicidal ability as well. In addition, the transcriptional levels of several typical sporulation-related genes, such as MedA, VelB, and VeA, were down-regulated in all ΔCaMK mutants compared with the wild-type (WT) strain. Moreover, these mutants exhibited hypersensitivity to heat shock and ultraviolet-radiation stresses compared with the WT strain. These results suggest that the five CaMKs in A. oligospora are involved in regulating multiple cellular processes, such as growth, environmental stress tolerance, conidiation, trap formation, and virulence.

Integrated Metabolomics and Morphogenesis Reveal Volatile Signaling of the Nematode-Trapping Fungus Arthrobotrys oligospora.

The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species Arthrobotrys oligospora Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms.

Long Non-Coding RNA Reprogramming (ROR) Promotes Cell Proliferation in Colorectal Cancer via Affecting P53.

BACKGROUND Colorectal cancer (CRC) remains one of the most common lethal malignant tumors worldwide. The correlation between lncRNAs expression and CRC development has not been well identified in the recent literature. This study focused on the role of lncRNA-ROR on CRC progression and development. MATERIAL AND METHODS Quantitative real-time PCR (qRT-PCR) assay was conducted to identify the expression level of lncRNA-ROR. Cell proliferation and viability were examined by MTT assay and colony formation assay. Cell cycle distribution and apoptosis were detected by flow cytometry. Expressions of p53, p21, and FAS protein levels were assessed by Western blotting. CRC cells transfected with lncRNA-shRNA were injection into nude mice to identify the function of lncRNA-ROR on tumorigenesis in vivo. RESULTS The expression level of lncRNA-ROR was elevated in CRC tissues when compared to adjacent tissues (n=78). lncRNA-ROR knockdown significantly suppressed cell proliferation and viability, while lncRNA-ROR overexpression had the opposite effect. Decreased lncRNA-ROR expression enhanced cell apoptosis and triggered cell cycle arrest in G0/G1 phase, while elevated lncRNA-ROR expression presented the opposite effect. Protein levels of p53 and p53 target genes were affected by lncRNA-ROR in vitro, and downregulation of lncRNA-ROR impeded tumorigenesis in vivo. CONCLUSIONS Our study demonstrates that lncRNA-ROR participates in controlling CRC proliferation, viability, and apoptosis, partially by modulating p53, which provides potential and prospective therapeutic targets for CRC.

New Bioactive Macrocyclic Diterpenoids from Euphorbia helioscopia.

Three new macrocyclic diterpenoids, euphoscopoids A - C (1 - 3), including two new jatrophanes and a new lathyrane, were isolated from the whole plant of Euphorbia helioscopia. Their structures were elucidated by spectroscopic methods. Antifeedant and cytotoxic activities of these isolates were evaluated. All compounds showed significant antifeedant activity against a generalist plant-feeding insect, Helicoverpa armigera, with EC50 values ranging from 2.05 to 4.34 µg/cm2 . In addition, compound 2 showed moderate cytotoxicity against tumor cell lines NCI-H1975, HepG2, and MCF-2, while compounds 1 and 3 were not active at 80 µm. The results suggested not only the defensive function of macrocyclic diterpenoids in E. helioscopia against insect herbivores, but also their potential applications as new natural insect antifeedants.

Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate.

Pneumocandins produced by the fungus Glarea lozoyensis are acylated cyclic hexapeptides of the echinocandin family. Pneumocandin B0 is the starting molecule for the first semisynthetic echinocandin antifungal drug, caspofungin acetate. In the wild-type strain, pneumocandin B0 is a minor fermentation product, and its industrial production was achieved by a combination of extensive mutation and medium optimization. The pneumocandin biosynthetic gene cluster was previously elucidated by a whole-genome sequencing approach. Knowledge of the biosynthetic cluster suggested an alternative way to produce exclusively pneumocandin B0. Disruption of GLOXY4, encoding a nonheme, α-ketoglutarate-dependent oxygenase, confirmed its involvement in l-leucine cyclization to form 4S-methyl-l-proline. The absence of 4S-methyl-l-proline abolishes pneumocandin A0 production, and 3S-hydroxyl-l-proline occupies the hexapeptide core's position 6, resulting in exclusive production of pneumocandin B0. Retrospective analysis of the GLOXY4 gene in a previously isolated pneumocandin B0-exclusive mutant (ATCC 74030) indicated that chemical mutagenesis disrupted the GLOXY4 gene function by introducing two amino acid mutations in GLOXY4. This one-step genetic manipulation can rationally engineer a high-yield production strain.

New insights into the echinocandins and other fungal non-ribosomal peptides and peptaibiotics.

Non-ribosomal peptide synthetases (NRPSs) are a primary modality for fungal peptidic natural product assembly and are responsible for some of the best known, most useful, and most destructive fungal metabolites. Through genome sequencing and computer-assisted recognition of modular motifs of catalytic domains, one can now confidently identify most NRPS biosynthetic genes of a fungal strain. The biosynthetic gene clusters responsible for two of the most important classes of NRP fungal derived drugs, cyclosporine and the echinocandins, have been recently characterized by genomic sequencing and annotation. Complete biosynthetic gene clusters for the pneumocandins and echinocandins have been mapped at the genetic level and functionally characterized to some extent. Genomic sequencing of representative strains of most of the variants in the echinocandin family, including the wild-type of the three fungal strains employed for industrial-scale production of caspofungin, micafungin and anidulofungin, has enabled characterization of the basic architecture of the echinocandin NRPS pathways. A comparative analysis of how pathway genes cause variations in lipoinitiation, biosynthesis of the non-proteinogenic amino acids, amino acid substitutions, and hydroxylations and sulfonations of the core peptide and contribute to the molecular diversity of the family is presented. We also review new information on the natural functions of NRPs, the differences between fungal and bacterial NRPSs, and functional characterization of selected NRPS gene clusters. Continuing discovery of the new fungal nonribosomal peptides has contributed new structural diversity and potential insights into their biological functions among other natural peptides and peptaibiotics. We therefore provide an update on new peptides, depsipeptides and peptaibols discovered in the Fungi since 2009.

Biologically active dichapetalins from Dichapetalum gelonioides.

A phytochemical investigation of the toxic tropical plant Dichapetalum gelonioides led to the isolation and identification of 14 new dichapetalins (1-14) and the known dichapetalins A (15) and K (16). The structures of the new compounds were determined by analyses of their NMR, MS, electronic circular dichroism, and X-ray diffraction data. The esterification at C-25 by 4-hydroxyphenylpropanoic acid and the hydroxylation at C-2' are unique in this unusual class of natural products. In addition to the known cytotoxicity, an array of biological activities, including antifeedant, nematicidal, antifungal, and NO and AChE inhibitory activities, were observed for this class of compounds. These findings suggested that dichapetalin hybrid triterpenoids as a class have broad biologically active cellular functions including defense against insect herbivores and pathogens.

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.

Genomic and proteomic analyses of the fungus Arthrobotrys oligospora provide insights into nematode-trap formation.

Nematode-trapping fungi are "carnivorous" and attack their hosts using specialized trapping devices. The morphological development of these traps is the key indicator of their switch from saprophytic to predacious lifestyles. Here, the genome of the nematode-trapping fungus Arthrobotrys oligospora Fres. (ATCC24927) was reported. The genome contains 40.07 Mb assembled sequence with 11,479 predicted genes. Comparative analysis showed that A. oligospora shared many more genes with pathogenic fungi than with non-pathogenic fungi. Specifically, compared to several sequenced ascomycete fungi, the A. oligospora genome has a larger number of pathogenicity-related genes in the subtilisin, cellulase, cellobiohydrolase, and pectinesterase gene families. Searching against the pathogen-host interaction gene database identified 398 homologous genes involved in pathogenicity in other fungi. The analysis of repetitive sequences provided evidence for repeat-induced point mutations in A. oligospora. Proteomic and quantitative PCR (qPCR) analyses revealed that 90 genes were significantly up-regulated at the early stage of trap-formation by nematode extracts and most of these genes were involved in translation, amino acid metabolism, carbohydrate metabolism, cell wall and membrane biogenesis. Based on the combined genomic, proteomic and qPCR data, a model for the formation of nematode trapping device in this fungus was proposed. In this model, multiple fungal signal transduction pathways are activated by its nematode prey to further regulate downstream genes associated with diverse cellular processes such as energy metabolism, biosynthesis of the cell wall and adhesive proteins, cell division, glycerol accumulation and peroxisome biogenesis. This study will facilitate the identification of pathogenicity-related genes and provide a broad foundation for understanding the molecular and evolutionary mechanisms underlying fungi-nematodes interactions.

A Trojan horse mechanism of bacterial pathogenesis against nematodes.

Understanding the mechanisms of host-pathogen interaction can provide crucial information for successfully manipulating their relationships. Because of its genetic background and practical advantages over vertebrate model systems, the nematode Caenorhabditis elegans model has become an attractive host for studying microbial pathogenesis. Here we report a "Trojan horse" mechanism of bacterial pathogenesis against nematodes. We show that the bacterium Bacillus nematocida B16 lures nematodes by emitting potent volatile organic compounds that are much more attractive to worms than those from ordinary dietary bacteria. Seventeen B. nematocida-attractant volatile organic compounds are identified, and seven are individually confirmed to lure nematodes. Once the bacteria enter the intestine of nematodes, they secrete two proteases with broad substrate ranges but preferentially target essential intestinal proteins, leading to nematode death. This Trojan horse pattern of bacterium-nematode interaction enriches our understanding of microbial pathogenesis.

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.

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.

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.

Thermolides, potent nematocidal PKS-NRPS hybrid metabolites from thermophilic fungus Talaromyces thermophilus.

Macrocyclic PKS-NRPS hybrid metabolites represent a unique family of natural products mainly from bacteria with broad and outstanding biological activities. However, their distribution in fungi has rarely been reported, and little has been reported regarding their nematocidal activity. Here we describe an unprecedented class of PKS-NRPS hybrid metabolites possessing a 13-membered lactam-bearing macrolactone, thermolides A-F (1-6) from a thermophilic fungus Talaromyces thermophilus. We showed that 1 and 2 displayed potent inhibitory activity against three notorious nematodes with LC(50) values of 0.5-1 µg/mL, as active as commercial avermectins. This work provided a new class of promising lead compounds for nematocide discovery.