Terreic Acid Research Articles

Novel components in the nuclear factor-kappa B (NF-κB) signaling pathways of endothelial cells under hyperglycemic-ischemic conditions

Diabetes worsens the outcomes of a number of vascular disorders including peripheral arterial disease (PAD) at least in part through induction of chronic inflammation. However, in experimental PAD, recovery requires the nuclear factor-kappa B (NF-κB) activation. Previously we showed that individually, both ischemia and high glucose activate the canonical and non-canonical arms of the NF-κB pathway, but prolonged high glucose exposure specifically impairs ischemia-induced activation of the canonical NF-κB pathway through activation of protein kinase C beta (PKCβ). Although a cascade of phosphorylation events propels the NF-κB signaling, little is known about the impact of hyperglycemia on the canonical and non-canonical NF-κB pathway signaling. Moreover, signal upstream of PKCβ that lead to its activation in endothelial cells during hyperglycemia exposure have not been well defined. In this study, we used endothelial cells exposed to hyperglycemia and ischemia (HGI) and an array of approximately 250 antibodies to approximately 100 proteins and their phosphorylated forms to identify the NF-κB signaling pathway that is altered in ischemic EC that has been exposed to high glucose condition. Comparison of signals from hyperglycemic and ischemic cell lysates yielded a number of proteins whose phosphorylation was either increased or decreased under HGI conditions. Pathway analyses using bioinformatics tools implicated BLNK/BTK known for B cell antigen receptor (BCR)-coupled signaling. Inhibition of BLNK/BTK in endothelial cells by a specific pharmacological inhibitor terreic acid attenuated PKC activation and restored the IκBα degradation suggesting that these molecules play a critical role in hyperglycemic attenuation of the canonical NF-κB pathway. Thus, we have identified a potentially new component of the NF-κB pathway upstream of PKC in endothelial cells that contributes to the poor post ischemic adaptation during hyperglycemia.

In High Glucose Exposed Endothelial Cells BLNK and BTK are Novel Components of the Nuclear Factor-kappa B Pathway

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFκB signaling in ischemic endothelial cells (EC). We have previously shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFκB pathway through activation of protein kinase C β (PKCβ). However, the signaling pathways upstream of PKCβ involved in this high glucose induced desensitization of NFκB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFκB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates from NG, HG, NGI and HGI groups were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spots by the signal intensity of the corresponding non-phosphorylated spots for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling by associating with the scaffold protein BLNK. However, neither of these proteins has been known to be expressed in ECs or play a role in EC function. In this study, we confirmed BLNK and BTK protein and transcript expression in ECs. Inhibition of BTK by a selective inhibitor terreic acid decreased PKCβ-S661 phosphorylation and restored NFκB activation through IκBα in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as potentially new components of the NFκB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. National Heart, Lung, and Blood Institute (R01 HL130399) to AOD. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

6-MSA, a secondary metabolite distribution hub with multiple fungal destinations.

6-methylsalicylic acid is a small, simple polyketide produced by a broad spectrum of fungal species. Since fungi obtained the ability to synthesize 6-MSA from bacteria through a horizontal gene transfer event, it has developed into a multipurpose metabolic hub from where numerous complex compounds are produced. The most relevant metabolite from a human perspective is the small lactone patulin as it is one of the most potent mycotoxins. Other important end products derived from 6-MSA include the small quinone epoxide terreic acid and the prenylated yanuthones. The most advanced modification of 6-MSA is observed in the aculin biosynthetic pathway, which is mediated by a non-ribosomal peptide synthase and a terpene cyclase. In this short review, we summarize for the first time all the possible pathways that takes their onset from 6-MSA and provide a synopsis of the responsible gene clusters and derive the resulting biosynthetic pathways.

Diversity, antibacterial and phytotoxic activities of culturable endophytic fungi from Pinellia pedatisecta and Pinellia ternata

BackgroundEndophytic fungi of medicinal plants, as special microorganisms, are important sources of antibacterial compounds. However, the diversity and antibacterial activity of endophytic fungi from Pinellia Tenore have not been systematically studied.ResultsA total of 77 fungi were isolated from roots, stems, leaves, and tubers of Pinellia ternata and P. pedatisecta. All fungi were belonged to five classes and twenty-five different genera. Biological activities tests indicated that 21 extracts of endophytic fungi exhibited antibacterial activities against at least one of the tested bacteria, and 22 fermentation broth of endophytic fungi showed strong phytotoxic activity against Echinochloa crusgalli with the inhibition rate of 100%. Furthermore, four compounds, including alternariol monomethyl ether (1), alternariol (2), dehydroaltenusin (3) and altertoxin II (4), and three compounds, including terreic acid (5), terremutin (6), citrinin (7), were isolated from Alternaria angustiovoidea PT09 of P. ternata and Aspergillus floccosus PP39 of P. pedatisecta, respectively. Compound 5 exhibited strong antibacterial activities against Escherichia coli, Micrococcus tetragenus, Staphylococcus aureus, and Pseudomonas syringae pv. actinidiae with the inhibition zone diameter (IZD) of 36.0, 31.0, 33.7, 40.2 mm and minimum inhibitory concentration (MIC) values of 1.56, 3.13, 1.56, 1.56 μg/mL respectively, which were better than or equal to those of positive gentamicin sulfate. The metabolite 7 also exhibited strong antibacterial activity against P. syringae pv. actinidiae with the IZD of 26.0 mm and MIC value of 6.25 μg/mL. In addition, the compound 7 had potent phytotoxic activity against E. crusgalli with the inhibition rate of 73.4% at the concentration of 100 μg/mL.ConclusionsHence, this study showed that endophytic fungi of P. ternata and P. pedatisecta held promise for the development of new antibiotic and herbicide resources.

RF04 | PSUN163 In High Glucose Exposed Endothelial Cells BTK is a Novel Component of the Nuclear Factor-Kappa B Pathway

Abstract Peripheral artery disease (PAD) is atherosclerotic occlusion of vessels outside the heart and most commonly affects the lower extremities. Diabetes worsens PAD outcomes, but the mechanisms involved are poorly understood. A key component of perfusion recovery in experimental PAD is induction of NFkB signaling in ischemic endothelial cells (EC). We have shown that prolonged exposure of ECs to high glucose before simulated ischemic exposure resulted in impairment of the canonical NFkB pathway through activation of protein kinase C β (PKC β). However, the signaling pathways upstream of PKC β involved in this high glucose induced desensitization of NFkB signaling are not known. We used arrays of antibodies to approximately 100 proteins known to participate in the NFkB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (NG) or high glucose (HG) were subjected to ischemia for 24 hours (NGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spot by the signal intensity of the corresponding non-phosphorylated spot for each protein. Differential expression between samples, were calculated by dividing the phosphorylation ratio of the NGI and HGI with that of the NG and HG controls, respectively. A combination of pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) and analysis of differential phosphorylation between NGI and HGI samples suggested involvement of B cell linker/adapter protein (BLNK)/Bruton's tyrosine kinase (BTK). BTK plays a key role in B cell antigen receptor (BCR)-coupled signaling but not known to be expressed in ECs or play a role in EC function. Here we confirmed BTK expression in ECs by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid or by shRNA mediated knockdown decreased PKCβ-S661 phosphorylation and restored NFkB activation in ECs suggesting a critical role of BLNK/BTK in the EC. Thus, we have identified BLNK/BTK as a potentially new component of the NFkB pathway in ECs that may be a therapeutic target to improve PAD outcomes in diabetes. Presentation: Saturday, June 11, 2022 1:06 p.m. - 1:11 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Production of Fungal Quinones: Problems and Prospects

Fungal quinones can be used for a variety of applications, such as pharmaceuticals, food colorants, textile dyes, and battery electrolytes. However, when producing quinones by fungal cultivation, many considerations arise regarding the feasibility of a production system, such as the quinone yield, purity, ease of extraction, and the co-production of mycotoxins. In this work, we display the initial screening of filamentous fungi for quinone production and evaluate their potential for future optimization. We investigated toluquinone (TQ) potentially produced by Penicillium cf. griseofulvum, terreic acid (TA) produced by Aspergillus parvulus and A. christenseniae, and anthraquinone (AQ) monomers and dimers produced by Talaromyces islandicus. The strains grew on various agar and/or liquid media and were analyzed by ultra-high-performance liquid chromatography–diode array detection–quadrupole time-of-flight mass spectrometry (UHPLC-DAD-QTOF MS). In the case of AQs, feature-based molecular networking (FBMN) was used for the identification of AQ analogs. TQ was not observed in the production strains. TA constituted one of the major chromatogram peaks and was secreted into the growth medium by A. parvulus. The AQs constituted many major chromatogram peaks in the mycelium extracts and endocrocin and citreorosein were observed extracellularly in small amounts.

BLNK/BTK: Novel Components in the NF-kappa B Pathways of Endothelial Cells in Diabetic Condition

Peripheral artery disease (PAD) is atherosclerotic occlusion of vessel outside the heart that most commonly affects the lower extremities. The effects of PAD-related ischemia are exacerbated under diabetic hyperglycemic conditions. Under ischemic conditions, an adaptive induction of the NFkB pathways is in vascular endothelial cells is required for recovery. We have recently shown that prolonged exposure of cells to high glucose before ischemia resulted in impairment of the canonical NFkB pathway through decrease in IkBa degradation. However, the signaling pathways involved in hyperglycemia and ischemia mediated effects on the NFkB pathways are not well understood. Since the NFkB signaling pathways propagate through a cascade of phosphorylation events, we used arrays of antibodies to approximately 100 proteins known to participate in the NFkB pathway to identify the changes in their phosphorylation states in human umbilical vein endothelial cells (HUVEC). Cells grown for three days either in culture medium with normal glucose (LG) or high glucose (HG) were subjected to ischemia for 24 hours (LGI and HGI, respectively). Cell lysates were then incubated with the array of antibodies printed on glass slides (Full Moon Biosystems, Sunnyvale, CA) and fluorescent signals were digitally recorded and normalized. The change in protein phosphorylation was calculated by dividing the intensity of the phosphorylated spot by the signal intensity of the corresponding non-phosphorylated spot for each protein. Differential expression between LG and LGI samples, and HG and HGI were calculated by dividing the phosphorylation ratio of the LGI and HGI with that of the LG and HG controls, respectively. A threshold of 1.5-fold increase or decrease was used to determine changes. Compared to the LG, LGI samples had 26 protein sites with increased phosphorylation whereas 36 sites had decreased phosphorylation. Similarly, compared to HG, HGI samples increased phosphorylation of 25 protein sites and decreased phosphorylation of 40 sites. A Venn-diagram analysis of LGI and HGI sites revealed 8 sites with an increase and 12 sites with a decrease in phosphorylation were specific to HGI. Pathway analyses using bioinformatics tools on 65 modulated phosphorylation sites in HGI (represented by 35 genes) suggested involvement of B cell linker/adapter protein (BLNK)/Bruton’s tyrosine kinase (BTK) that are critical for B cell antigen receptor (BCR)-coupled signaling. BTK expression in EC was confirmed by immunoblotting. Inhibition of BTK by a specific inhibitor terreic acid restored IkBa degradation in EC grown in high glucose suggesting a critical role of BLNK/BTK in diabetic ischemia. Thus, we have identified BLNK/BTK as potentially new components of the NFkB pathway in endothelial cells that contributes to the poor recovery during hyperglycemic ischemia.

Heterologous pathway assembly reveals molecular steps of fungal terreic acid biosynthesis

Terreic acid is a potential anticancer drug as it inhibits Bruton’s tyrosine kinase; however, its biosynthetic molecular steps remain unclear. In this work, the individual reactions of terreic acid biosynthesis were determined by stepwise pathway assembly in a heterologous host, Pichia pastoris, on the basis of previous knockout studies in a native host, Aspergillus terreus. Polyketide synthase AtX was found to catalyze the formation of partially reduced polyketide 6-methylsalicylic acid, followed by 3-methylcatechol synthesis by salicylate 1-monooxygenase AtA-mediated decarboxylative hydroxylation of 6-methylsalicylic acid. Our results show that cytochrome P450 monooxygenase AtE hydroxylates 3-methylcatechol, thus producing the next product, 3-methyl-1,2,4-benzenetriol. A smaller putative cytochrome P450 monooxygenase, AtG, assists with this step. Then, AtD causes epoxidation and hydroxyl oxidation of 3-methyl-1,2,4-benzenetriol and produces a compound terremutin, via which the previously unknown function of AtD was identified as cyclooxygenation. The final step involves an oxidation reaction of a hydroxyl group by a glucose-methanol-choline oxidoreductase, AtC, which leads to the final product: terreic acid. Functions of AtD and AtG were determined for the first time. All the genes were reanalyzed and all intermediates and final products were isolated and identified. Our model fully defines the molecular steps and corrects previous results from the literature.

Genome-Wide Expression Profiles Drive Discovery of Novel Compounds that Reduce Binge Drinking in Mice.

Transcriptome-based drug discovery has identified new treatments for some complex diseases, but has not been applied to alcohol use disorder (AUD) or other psychiatric diseases, where there is a critical need for improved pharmacotherapies. High Drinking in the Dark (HDID-1) mice are a genetic model of AUD risk that have been selectively bred (from the HS/Npt line) to achieve intoxicating blood alcohol levels (BALs) after binge-like drinking. We compared brain gene expression of HDID-1 and HS/Npt mice, to determine a molecular signature for genetic risk for high intensity, binge-like drinking. Using multiple computational methods, we queried LINCS-L1000 (Library of Integrated Network-Based Cellular Signatures), a database containing gene expression signatures of thousands of compounds, to predict candidate drugs with the greatest potential to decrease alcohol consumption. Our analyses predicted novel compounds for testing, many with anti-inflammatory properties, providing further support for a neuroimmune mechanism of excessive alcohol drinking. We validated the top 2 candidates in vivo as a proof-of-concept. Terreic acid (a Bruton's tyrosine kinase inhibitor) and pergolide (a dopamine and serotonin receptor agonist) robustly reduced alcohol intake and BALs in HDID-1 mice, providing the first evidence for transcriptome-based drug discovery to target an addiction trait. Effective drug treatments for many psychiatric diseases are lacking, and the emerging tools and approaches outlined here offer researchers studying complex diseases renewed opportunities to discover new or repurpose existing compounds and expedite treatment options.

A High-Throughput Screen for Yeast Replicative Lifespan Identifies Lifespan-Extending Compounds

Progress in aging research is constrained by the timerequirement of measuring lifespans. Even the most rapid model for eukaryotic aging, the replicative lifespan of Saccharomyces cerevisiae, is technically limited to only several lifespan measurements each day. Here we report a 384-well plate-based technique to measure replicative lifespan, termed High-Life. Using the High-Life technique, a single researcher can compare lifespan for more than 1,000 conditions per day. We validated the technique with long-lived mutant strains and the lifespan-extending compound ibuprofen. We also applied this technique to screen a small compound library for lifespan extension. Two hits, terreic acid and mycophenolic acid, were validated on our single-cell replicator device and found to extend mean replicative lifespan by 15% and 20%, respectively. Together, we report a technique for high-throughput lifespan measurement, and we identify two lifespan-extending compounds. Our technique could be used to efficiently drive early-stage discovery of pro-longevity therapeutics.

Antioxidant compounds produced by Pseudocercospora sp. ESL 02, an endophytic fungus isolated from Elaeocarpus sylvestris

ObjectiveTo isolate endophytic fungi from Elaeocarpus sylvestris (E. sylvestris) and to isolate antioxidant compounds from a potential source fungus. MethodsEndophytic fungi were isolated from fresh leaves and stems of E. sylvestris and identified based on DNA analysis. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was used to evaluate the antioxidant activity of the fungi. The potential antioxidant fungus was further studied to isolate antioxidant compounds. The isolated compounds were identified by melting point analysis, optical rotation, spectral analysis using a UV spectrophotometer, high resolution fast atom bombardment mass spectrometry, X-ray crystallography analysis, 1H nuclear magnetic resonance analysis and 13C nuclear magnetic resonance analysis. The isolated compounds were evaluated with DPPH radical scavenging, reducing power, and β-carotene bleaching assays. ResultsSeven endophytic fungi were successfully isolated from E. sylvestris and identified as Pestalotiopsis sp. EST 01, Pestalotiopsis sp. EST 02, Diaporthales sp. EST 03, Meyerozyma sp. EST 04, Diaporthales sp. EST 05, Pestalotiopsis sp. ESL 01, and Pseudocercospora sp. ESL 02. Of the seven fungi, Pseudocercospora sp. ESL 02 had the highest antioxidant activity [IC50 = (30.54 ± 0.88) μg/mL]. From that fungus, two compounds identified as terreic acid (1) and 6-methylsalicylic acid (2) were isolated with an IC50 of DPPH radical scavenging activity of (0.22 ± 0.02) mmol/L and (3.87 ± 0.27) mmol/L, respectively. The compounds also had good activities from the reducing power and β-carotene bleaching assays. ConclusionsThe Pseudocercospora sp. ESL 02 fungus isolated from E. sylvestris looks promising as a novel source of terreic acid.

Polyketides in Aspergillus terreus: biosynthesis pathway discovery and application.

The knowledge of biosynthesis gene clusters, production improving methods, and bioactivity mechanisms is very important for the development of filamentous fungi metabolites. Metabolic engineering and heterologous expression methods can be applied to improve desired metabolite production, when their biosynthesis pathways have been revealed. And, stable supplement is a necessary basis of bioactivity mechanism discovery and following clinical trial. Aspergillus terreus is an outstanding producer of many bioactive agents, and a large part of them are polyketides. In this review, we took polyketides from A. terreus as examples, focusing on 13 polyketide synthase (PKS) genes in A. terreus NIH 2624 genome. The biosynthesis pathways of nine PKS genes have been reported, and their downstream metabolites are lovastatin, terreic acid, terrein, geodin, terretonin, citreoviridin, and asperfuranone, respectively. Among them, lovastatin is a well-known hypolipidemic agent. Terreic acid, terrein, citreoviridin, and asperfuranone show good bioactivities, especially anticancer activities. On the other hand, geodin and terretonin are mycotoxins. So, biosynthesis gene cluster information is important for the production or elimination of them. We also predicted three possible gene clusters that contain four PKS genes by homologous gene alignment with other Aspergillus strains. We think that this is an effective way to mine secondary metabolic gene clusters.

Escherichia coli N-Acetylglucosamine-1-Phosphate-Uridyltransferase/Glucosamine-1-Phosphate-Acetyltransferase (GlmU) Inhibitory Activity of Terreic Acid Isolated from Aspergillus terreus

Secondary metabolite of Aspergillus terreus, terreic acid, is a reported potent antibacterial that was identified more than 60 years ago, but its cellular target(s) are still unknown. Here we screen its activity against the acetyltransferase domain of a bifunctional enzyme, Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU). An absorbance-based assay was used to screen terreic acid against the acetyltransferase activity of E. coli GlmU. Terreic acid was found to inhibit the acetyltransferase domain of E. coli GlmU with an IC50 of 44.24 ± 1.85 µM. Mode of inhibition studies revealed that terreic acid was competitive with AcCoA and uncompetitive with GlcN-1-P. It also exhibited concentration-dependent killing of E. coli ATCC 25922 up to 4× minimum inhibitory concentration and inhibited the growth of biofilms generated by E. coli. Characterization of resistant mutants established mutation in the acetyltransferase domain of GlmU. Terreic acid was also found to be metabolically stable in the in vitro incubations with rat liver microsome in the presence of a NADPH regenerating system. The studies reported here suggest that terreic acid is a potent antimicrobial agent and support that E. coli GlmU acetyltransferase is a molecular target of terreic acid, resulting in its antibacterial activity.

Combining UHPLC-High Resolution MS and Feeding of Stable Isotope Labeled Polyketide Intermediates for Linking Precursors to End Products.

We present the results from stable isotope labeled precursor feeding studies combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry for the identification of labeled polyketide (PK) end-products. Feeding experiments were performed with (13)C8-6-methylsalicylic acid (6-MSA) and (13)C14-YWA1, both produced in-house, as well as commercial (13)C7-benzoic acid and (2)H7-cinnamic acid, in species of Fusarium, Byssochlamys, Aspergillus, and Penicillium. Incorporation of 6-MSA into terreic acid or patulin was not observed in any of six evaluated species covering three genera, because the 6-MSA was shunted into (2Z,4E)-2-methyl-2,4-hexadienedioic acid. This indicates that patulin and terreic acid may be produced in a closed compartment of the cell and that (2Z,4E)-2-methyl-2,4-hexadienedioic acid is a detoxification product toward terreic acid and patulin. In Fusarium spp., YWA1 was shown to be incorporated into aurofusarin, rubrofusarin, and antibiotic Y. In A. niger, benzoic acid was shown to be incorporated into asperrubrol. Incorporation levels of 0.7-20% into the end-products were detected in wild-type strains. Thus, stable isotope labeling is a promising technique for investigation of polyketide biosynthesis and possible compartmentalization of toxic metabolites.

Molecular genetic characterization of terreic acid pathway in Aspergillus terreus.

Terreic acid is a natural product derived from 6-methylsalicylic acid (6-MSA). A compact gene cluster for its biosynthesis was characterized. Isolation of the intermediates and shunt products from the mutant strains, combined with bioinformatic analyses, allowed for the proposition of a biosynthetic pathway for terreic acid.

Culture-based and sequence-based insights into biosynthesis of secondary metabolites by Aspergillus terreus ATCC 20542

Aspergillus terreus ATCC 20542 was cultivated in various culture media in order to activate its genome-encoded biosynthetic pathways and explore the secondary metabolic repertoire. In addition to mevinolinic acid (lovastatin) and its precursor monacolin L, a number of other secondary metabolites were found in the analyzed cultures, namely terreic acid, citrinin, (+)-geodin, terrein, and dehydrocurvularin. In contrast to previously described gene clusters responsible for production of lovastatin, monacolin L, (+)-geodin and dehydrocurvularin, the gene clusters of A. terreus associated with the formation of terreic acid, citrinin and terrein still await identification. Putative gene clusters potentially related to citrinin and terreic acid biosynthesis were suggested in the publicly available genome of A. terreus NIH 2624. The functions of putative genes in the previously identified cluster of (+)-geodin biosynthesis were predicted by confronting the annotation results with the proposed biosynthetic pathway and published biochemical studies on the underlying enzymes. Since there were no available data regarding genetic aspects of terrein biosynthesis, the candidate gene cluster potentially responsible for the production of terrein was not suggested.

Differential antibacterial properties of the MurA inhibitors terreic acid and fosfomycin

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus, but its cellular target remains unknown. We recently reported that terreic acid inactivates the bacterial cell wall biosynthetic enzyme MurA in vitro by covalent reaction with residue Cys115 in a similar manner as the MurA-specific antibiotic fosfomycin. To address if terreic acid also targets MurA in vivo, we conducted antibacterial studies using selected E. coli strains in parallel with fosfomycin. While overexpression of MurA conferred resistance to fosfomycin, it did not protect cells treated with terreic acid. Furthermore, flow cytometry revealed that the antibiotic action of terreic acid appears to be primarily bacteriostatic, as opposed to the bactericidal action observed for fosfomycin. Combined, the data suggest that MurA is not the molecular target of terreic acid and that the antibiotic activity of terreic acid proceeds through a different mechanism of action. The methodology applied here provides a reliable and convenient tool to rapidly assess the potential of newly discovered in vitro inhibitors to target residue Cys115 of MurA in the cell.

Isolation of Antioxidant Compounds from Aspergillus Terreus LS01

The antioxidative activity of an ethyl acetate extract from Aspergillus terreus LS01was evaluated using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen-peroxide-scavenging free radical scavenging, and β-caroten-linoleat model assay and the antioxidants in the extract were isolated and identified. Silica gel column chromatography was used to separate the extract into five fractions (F1-F5). Fraction 3 possessed significant antioxidative activity with an IC 50 of 19.91μg/ml. Fraction 3 was further separated by column chromatography and yielded the crystalline compounds 1 and 2, which were identified as terreic acid and terremutin, respectively, on the basis of UV-vis spectra, MS and NMR analyses. Terreic acid and terremutin exhibited the highest level of DPPH free radical scavenging activity with IC 50 values of 0.115±4.02 and 0.114±2.19 mM, respectively. These compounds also showed scavenging activity of hydrogen peroxide radicals which terreic acid exhibited higher than terremutin with 74.07±1.48% and 33.74±2.81% respectively. In the β-caroten-linoleat model assay, the inhibition of terreic acid retained 26.01±1.14% and terremutin retained 32.29±2.23%. This is the first report on antioxidative activity of terreic acid and terremutin from A.terreus . The findings indicated that A. terreus could be considered as potential source of natural antioxidant products.

New species in Aspergillus section Terrei

Section Terrei of Aspergillus was studied using a polyphasic approach including sequence analysis of parts of the β-tubulin and calmodulin genes and the ITS region, macro- and micromorphological analyses and examination of extrolite profiles to describe three new species in this section. Based on phylogenetic analysis of calmodulin and β-tubulin sequences seven lineages were observed among isolates that have previously been treated as A. terreus and its subspecies by Raper & Fennell (1965) and others. Aspergillus alabamensis, A. terreus var. floccosus, A. terreus var. africanus, A. terreus var. aureus, A. hortai and A. terreus NRRL 4017 all represent distinct lineages from the A. terreus clade. Among them, A. terreus var. floccosus, A. terreus NRRL 4017 and A. terreus var. aureus could also be distinguished from A. terreus by using ITS sequence data. New names are proposed for A. terreus var. floccosus, A. terreus var. africanus, A. terreus var. aureus, while Aspergillus hortai is recognised at species level. Aspergillus terreus NRRL 4017 is described as the new species A. pseudoterreus. Also included in section Terrei are some species formerly placed in sections Flavipedes and Versicolores. A. clade including the type isolate of A. niveus (CBS 115.27) constitutes a lineage closely related to A. carneus. Fennellia nivea, the hypothesized teleomorph is not related to this clade. Aspergillus allahabadii, A. niveus var. indicus, and two species originally placed in section Versicolores, A. ambiguus and A. microcysticus, also form well-defined lineages on all trees. Species in Aspergillus section Terrei are producers of a diverse array of secondary metabolites. However, many of the species in the section produce different combinations of the following metabolites: acetylaranotin, asperphenamate, aspochalamins, aspulvinones, asteltoxin, asterric acid, asterriquinones, aszonalenins, atrovenetins, butyrolactones, citreoisocoumarins, citreoviridins, citrinins, decaturins, fulvic acid, geodins, gregatins, mevinolins, serantrypinone, terreic acid (only the precursor 3,6-dihydroxytoluquinone found), terreins, terrequinones, terretonins and territrems. The cholesterol-lowering agent mevinolin was found in A. terreus and A. neoafricanus only. The hepatotoxic extrolite citrinin was found in eight species: A. alabamensis, A. allahabadii, A. carneus, A. floccosus, A. hortai, A. neoindicus, A. niveus and A. pseudoterreus. The neurotoxic extrolite citreoviridin was found in five species: A. neoafricanus, A. aureoterreus, A. pseudoterreus, A. terreus and A. neoniveus. Territrems, tremorgenic extrolites, were found in some strains of A. alabamensis and A. terreus.

The Fungal Product Terreic Acid Is a Covalent Inhibitor of the Bacterial Cell Wall Biosynthetic Enzyme UDP-N-Acetylglucosamine 1-Carboxyvinyltransferase (MurA),

Terreic acid is a metabolite with antibiotic properties produced by the fungus Aspergillus terreus. We found that terreic acid is a covalent inhibitor of the bacterial cell wall biosynthetic enzyme MurA from Enterobacter cloacae and Escherichia coli in vitro. The crystal structure of the MurA dead-end complex with terreic acid revealed that the quinine ring is covalently attached to the thiol group of Cys115, the molecular target of the antibiotic fosfomycin. Kinetic characterization established that the inactivation requires the presence of substrate UNAG (UDP-N-acetylglucosamine), proceeding with an inactivation rate constant k(inact) of 130 M(-1) s(-1). Although the mechanisms of inactivation are similar, fosfomycin is approximately 50 times more potent than terreic acid, and the structural consequences of covalent modification by these two inhibitors are fundamentally different. The MurA-fosfomycin complex exists in the closed enzyme conformation, with the Cys115-fosfomycin adduct buried in the active site. In contrast, the dead-end complex with terreic acid is open, is free of UNAG, and has the Cys115-terreic acid adduct solvent-exposed. It appears that terreic acid reacts with Cys115 in the closed, binary state of the enzyme, but that the resulting Cys115-terreic acid adduct imposes steric clashes in the active site. As a consequence, the loop containing Cys115 rearranges, the enzyme opens, and UNAG is released. The differential kinetic and structural characteristics of MurA inactivation by terreic acid and fosfomycin reflect the importance of noncovalent binding potential, even for covalent inhibitors, in ensuring inactivation efficiency and specificity.