A Low Concentration of Citreoviridin Prevents Both Intracellular Calcium Deposition in Vascular Smooth Muscle Cell and Osteoclast Activation In Vitro.

Vascular calcification (VC) and osteoporosis are age-related diseases and significant risk factors for the mortality of elderly. VC and osteoporosis may share common risk factors such as renin-angiotensin system (RAS)-related hypertension. In fact, inhibitors of RAS pathway, such as angiotensin type 1 receptor blockers (ARBs), improved both vascular calcification and hip fracture in elderly. However, a sex-dependent discrepancy in the responsiveness to ARB treatment in hip fracture was observed, possibly due to the estrogen deficiency in older women, suggesting that blocking the angiotensin signaling pathway may not be effective to suppress bone resorption, especially if an individual has underlying osteoclast activating conditions such as estrogen deficiency. Therefore, it has its own significance to find alternative modality for inhibiting both vascular calcification and osteoporosis by directly targeting osteoclast activation to circumvent the shortcoming of ARBs in preventing bone resorption in estrogen deficient individuals. In the present study, a natural compound library was screened to find chemical agents that are effective in preventing both calcium deposition in vascular smooth muscle cells (vSMCs) and activation of osteoclast using experimental methods such as Alizarin red staining and Tartrate-resistant acid phosphatase staining. According to our data, citreoviridin (CIT) has both an anti-VC effect and anti-osteoclastic effect in vSMCs and in Raw 264.7 cells, respectively, suggesting its potential as an effective therapeutic agent for both VC and osteoporosis.

[Electrophysiological Effect of Citreoviridin on Human InducedPluripotent Stem Cell-derived Cardiomyocytes].

Citreoviridin (CTV) is a mycotoxin produced by various fungi, including Penicillium citreonigrum. One of the toxicities reportedly associated with CTV is neurotoxicity. CTV is also suspected to be associated with acute cardiac beriberi (also known as "Shoshin-kakke") and Keshan disease, which can have adverse effects on the heart, so the in vivo and in vitro toxicity of CTV on the heart or cardiomyocytes in experimental animal models have been reported. However, the toxicity of CTV for the human heart, especially its electrophysiological effect, remains poorly understood. Therefore, to investigate the electrophysiological effect of CTV on the human cardiomyocytes, we conducted a multi-electrode array (MEA) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The MEA revealed that 30 µmol/L of CTV stopped the beating of hiPSC-CMs, and the field potential duration and first peak amplitude were shortened at 10 µmol/L. Before the hiPSC-CMs stopped beating, the length of the inter-spike interval varied two- to four-fold. These results demonstrated that CTV induced an electrophysiological disturbance on human cardiomyocytes. This is first paper to elucidate the electrophysiological effect of CTV on human heart directly and may aid in analyzing the risk associated with CTV to ensure food safety.

Exosomal miR-181a-2-3p derived from citreoviridin-treated hepatocytes activates hepatic stellate cells trough inducing mitochondrial calcium overload.

Increasing evidences indicate the vital role of exosomes-mediated intercellular communication in the pathogenesis of liver fibrosis. However, the underlying mechanisms are still not clearly defined. In this study, we found that citreoviridin (CIT), a mycotoxin and ectopic ATP synthase (e-ATPS) inhibitor, induced liver fibrosis in mice. The exosomes derived from CIT-treated L-02 hepatocytes activated hepatic stellate cells (HSC) LX-2. With exosomal small RNA sequencing, we found 156 differentially expressed miRNAs in the exosomes from CIT-treated L-02 cells, and the predicted target genes of exosomal miRNAs were enriched in calcium signaling pathway. The exosomes from CIT-treated L-02 cells induced mitochondrial calcium accumulation in LX-2 cells. And pharmacological inhibition of mitochondrial calcium uptake relieved exosomes-activated fibrogenic response in LX-2 cells. The miR-181a-2-3p that was predicted to target-regulate mitochondrial calcium uptake 1 (MICU1) was significantly increased in the exosomes from CIT-treated L-02 cells. Exosomes-induced reduction of MICU1, mitochondrial calcium overload and activation of LX-2 cells were reversed by AntagomiR-181a-2-3p. In this study, we pointed out that exosomal miR-181a-2-3p from CIT-treated hepatocytes induced mitochondrial calcium accumulation and activated HSC subsequently through inhibiting the expression of MICU1, shedding new light on the mechanism underlying liver fibrosis and CIT hepatotoxicity.

A target and efficient synthetic strategy for structural and bioactivity optimization of a fungal natural product.

Drugs have been largely inspired from natural products, while enzymes underlying their biosynthesis have enabled complex structures and diverse bioactivities. Nevertheless, the high enzyme specificity and limited in vivo precursor types have restricted the natural product reservoir, but Nature has imprinted natural products with active sites, which can be readily modified by chemosynthesis with various functional groups for more favorable druggability. Here in the less exploited fungal natural products, we introduced CtvA, a polyketide synthase for a mycotoxin citreoviridin biosynthesis in Aspergillus, into an endophytic fungus Calcarisporium arbuscula to expand tetrahydrofuran (THF) into a dioxabicyclo-octane (DBO) ring moiety based on versatility and promiscuity of the aurovertin biosynthetic enzyme. Alternative acylations on the hydroxyl groups essential for cell toxicity by chemosynthesis produced compounds with improved anti-tumor activities and pharmacokinetics. Thus, we showed an effective strategic way to optimize the fungal natural product efficiently for more promising drug development.

An Aspergillus nidulans Platform for the Complete Cluster Refactoring and Total Biosynthesis of Fungal Natural Products.

Fungal natural products (NPs) comprise a vast number of bioactive molecules with diverse activities, and among them are many important drugs. However, the yields of fungal NPs from native producers are usually low, and total synthesis of structurally complex NPs is challenging. As such, downstream derivatization and optimization of lead fungal NPs can be impeded by the high cost of obtaining sufficient starting material. In recent years, reconstitution of NP biosynthetic pathways in heterologous hosts has become an attractive alternative approach to produce complex NPs. Here, we present an efficient, cloning-free strategy for the cluster refactoring and total biosynthesis of fungal NPs in Aspergillus nidulans. Our platform places our genes of interest (GOIs) under the regulation of the robust asperfuranone afo biosynthesis gene machinery, allowing for their concerted activation upon induction. We demonstrated the utility of our system by creating strains that can synthesize high-value NPs, citreoviridin (1), mutilin (2), and pleuromutilin (3), with good to high yield and purity. This platform can be used not only for producing NPs of interests (i.e., total biosynthesis) but also for elucidating cryptic biosynthesis pathways.

Aurovertin B exerts potent antitumor activity against triple-negative breast cancer in vivo and in vitro via regulating ATP synthase activity and DUSP1 expression.

Aurovertin B, a natural compound from Calcarisporium arbuscular, exhibits potent antiproliferative activity particularly against triple-negative breast cancer cells (TNBC), while having less cytotoxicity on normal breast cell MCF10A. However, very little is known about the in vivo antitumor activity of aurovertin B and the possible mechanism of the selective effect on triple-negative breast cancer cells. In this study, flow cytometry and DAPI staining analysis showed that aurovertin B treatment in human triple-negative breast cancer cell MDA-MB-231 could induce more apoptotic cells than taxol treatment group. Furthermore, the present study also revealed that aurovertin B induced apoptosis was due to regulation of ATP synthase activity rather than changes in gene expression. Interestingly, the cancer genome atlas (TCGA) data analysis implied that the expression level of DUSP1, a member of the dual-specificity phosphatases, was highly downregulated in breast tissue of TNBC patients compared with their adjacent normal tissues. Real-time PCR and western blot analyses further demonstrated that aurovertin B could dramatically increase mRNA and protein expression levels of DUSP1 in MDA-MB-231 cells but not in MCF10A cells. The potent anti-tumor activity of aurovertin B was further verified in a human MDA-MB-231 xenograft mouse model.

Whole Genome Analysis Revealed the Genes Responsible for Citreoviridin Biosynthesis in Penicillium citreonigrum.

Citreoviridin (CTV) is a mycotoxin that is produced by Aspergillus terreus, Eupenicillium ochrosalmoneum and Penicillium citreonigrum, and CTV has been detected in a wide range of cereal grains throughout the world. Furthermore, it is especially a serious problem in regions where rice is consumed as a staple food. Moreover, CTV is a well-known yellow rice toxin, and outbreaks of beriberi have occurred due to consumption of rice that is contaminated by CTV even in the recent years. Although CTV biosynthetic genes of A. terreus have been described, those of P. citreonigrum remain unclear, which is concerning since P. citreonigrum is the main cause of CTV contamination in rice. In the present study, we determined the draft genome of the P. citreonigrum strain IMI92228 and revealed the presence of all four genes that form a gene cluster and that are homologous to the CTV biosynthesis genes of A. terreus. The expression of these four homologous genes were highly correlated with CTV production, suggesting that they may play an important role in CTV biosynthesis in P. citreonigrum. We concluded that the gene cluster is a CTV biosynthesis cluster of P. citreonigrum. The findings will contribute to the understanding of the biosynthetic pathway of CTV and will ultimately lead to improvements in the CTV management of agricultural products.

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Citreoviridin induces myocardial apoptosis through PPAR-γ-mTORC2-mediated autophagic pathway and the protective effect of thiamine and selenium.

Citreoviridin (CIT), a mycotoxin and ATP synthase inhibitor, is regarded as one of aetiology factors of cardiac beriberi and Keshan disease. Thiamine (VB1) and selenium (Se) improve the recovery of these two diseases respectively. The underlying mechanisms of cardiotoxic effect of CIT and cardioprotective effect of VB1 and Se have not been fully elucidated. In this study, we found that ectopic ATP synthase was more sensitive to CIT treatment than mitochondrial ATP synthase in H9c2 cardiomyocytes. CIT inhibited the transcriptional activity of peroxisome proliferator activated receptor gamma (PPAR-γ) in mice hearts and H9c2 cells. PPAR-γ agonist attenuated the inhibitory effect of CIT on mechanistic target of rapamycin complex 2 (mTORC2) and stimulatory effect of CIT on autophagy in cardiomyocytes. CIT induced apoptosis through lysosomal-mitochondrial axis in cardiomyocytes. PPAR-γ agonist and autophagy inhibitor alleviated CIT-induced apoptosis and accelerated cardiac biomarker. VB1 and Se accelerated the basal transcriptional activity of PPAR-γ in mice hearts and H9c2 cells. Furthermore, VB1 and Se reversed the effect of CIT on PPAR-γ, autophagy and apoptosis. Our findings defined PPAR-γ-mTORC2-autophagy pathway as the key link between CIT cardiotoxicity and cardioprotective effect of VB1 and Se. The present study would shed new light on the pathogenesis of cardiomyopathy and the cardioprotective mechanism of micronutrients.

The In Vivo and In Vitro Toxicokinetics of Citreoviridin Extracted from Penicillium citreonigrum.

Citreoviridin (CTVD), a mycotoxin called yellow rice toxin, is reported to be related to acute cardiac beriberi; however, its toxicokinetics remain unclear. The present study elucidated the toxicokinetics through in vivo experiments in swine and predicted the human toxicokinetics by comparing the findings to those from in vitro experiments. In vivo experiments revealed the high bioavailability of CTVD (116.4%) in swine. An intestinal permeability study using Caco-2 cells to estimate the toxicokinetics in humans showed that CTVD has a high permeability coefficient. When CTVD was incubated with hepatic S9 fraction from swine and humans, hydroxylation and methylation, desaturation, and dihydroxylation derivatives were produced as the predominant metabolites. The levels of these products produced using human S9 were higher than those obtained swine S9, while CTVD glucuronide was produced slowly in human S9 in comparison to swine S9. Furthermore, the elimination of CTVD by human S9 was significantly more rapid in comparison to that by swine S9. These results suggest that CTVD is easily absorbed in swine and that it remains in the body where it is slowly metabolized. In contrast, the absorption of CTVD in humans would be the same as that in swine, although its elimination would be faster.

Developmental exposure of citreoviridin transiently affects hippocampal neurogenesis targeting multiple regulatory functions in mice.

To investigate the developmental exposure effect of citreoviridin (CIT) on postnatal hippocampal neurogenesis, pregnant ICR mice were dietary exposed to CIT at 0, 1, 3 and 10 ppm from gestation day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without CIT exposure. Male offspring were analyzed. At 10 ppm on PND 21, weak changes suggestive of neural stem cell reduction and progenitor cell proliferation were observed. Number of hilar CALB1+ interneurons reduced, suggesting an influence on neurogenesis. In contrast, number of hilar SST+ interneurons increased and Bdnf and Ntrk2 transcripts upregulated in the dentate gyrus, suggesting a facilitation of BDNF-TRKB signaling for progenitor cell proliferation. Transcript expression changes of an outside regulatory system suggested suppressed function of GABAergic interneurons, especially of PVALB+ interneurons for compensation on neural stem cell reduction. At ≥ 3 ppm, number of ARC+ mature granule cells increased, and at 10 ppm, number of hilar GRIA1+ cells increased and Gria2 and Gria3 upregulated, suggesting an operation of AMPA receptor membrane trafficking on the increase of ARC-mediated synaptic plasticity. On PND 77, all the transcript expression changes of the neurogenesis regulatory system except for Grin2d were inverted, suggesting an operation of a homeostatic mechanism on CIT-induced disruptive neurogenesis. Simultaneous downregulation of Grin2a and Grin2d suggests suppression of GABAergic interneuron function to adjust neurogenesis at the normal level. The no-observed-adverse-effect level of CIT for offspring neurogenesis was determined to be 1 ppm, translating to 0.13-0.51 mg/kg body weight/day of maternal oral exposure.

Aurovertin B sensitizes colorectal cancer cells to NK cell recognition and lysis.

The natural killer group 2D (NKG2D) receptor on natural killer (NK) cells play an important role in immunosurveillance to cancer cells, which could mediate the eradication of tumor cells through specific interactions with NKG2D ligands on tumor cells. Here we report one natural compound aurovertin B from basidiomycete Albatrellus confluens significantly stimulates the expression of NKG2D ligands on tumor cells, which greatly sensitizes its recognition and lysis by NK cell. It is completely a novel role for aurovertin B to target tumor cells to death mediated by NK cells and our findings indicate aurovertin B may deserve further development as sensitizing agent in NK cell mediated cancer immunotherapy.

Synthesis and production of the antitumor polyketide aurovertins and structurally related compounds.

Aurovertins belong to a family of highly reducing polyketides sharing a polyene α-pyrone-type structure. These compounds comprise aurovertin, asteltoxin, avertoxin, citreoviridin, verrucosidin, and their derivatives, which exihibit potent antitumor, antiviral, and antibacterial activities. Until now, over 40 aurovertins and structurally related compounds have been found in the fungal kingdom. Due to the unique structural feature and interesting bioactivities, significant progresses have been achieved for the structural identification, chemical synthesis, and biosynthesis of the mentioned compounds. Understanding of aurovertin biosynthetic mechanism provides a solid basis for engineering the metabolic pathway of those compounds by rational design and realizing their production in the model fungal host.

Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane.

Rational design for heterologous production of aurovertin-type compounds in Aspergillus nidulans.

Aurovertins are the structurally diverse polyketides that distribute widely in different fungal species. They feature a 2,6-dioxabicyclo[3.2.1]-octane ring in structure and exhibit the potential antitumor activity against breast cancer as F1-ATPase ß subunit inhibitor. In this study, we constructed the biosynthetic pathway of aurovertin in an Aspergillus nidulans host and obtained seven aurovertin-type compounds. Surprisingly, three new aurovertin geometric isomers were characterized. By introducing an inducible promoter xylP(p) in the pathway gene acyltransferase aurG, we can control the product ratios among different aurovertin compounds by adding glucose and/or inducer xylose. The yields of aurovertins could be increased up to about 20 times by adding a constitutive promoter gpdA(p) to transcription factor AurF, which indicates AurF's positive role in the biosynthesis of aurovertin. Taken together, our results provided not only an efficient way to generate bioactive fungal natural products but also realized the rational controlling their yields with designed promoters.

Occurrence of Penicillium brocae and Penicillium citreonigrum, which Produce a Mutagenic Metabolite and a Mycotoxin Citreoviridin, Respectively, in Selected Commercially Available Rice Grains in Thailand.

Commercially available rice grains in Thailand were examined to isolate the monoverticillate Penicillium species responsible for toxic yellowed rice. Penicillium species were obtained from seven out of 10 rice samples tested. Among them, one Penicillium citreonigrum isolate and six Penicillium brocae isolates were morphologically identified. The P. citreonigrum isolate produced the mycotoxin citreoviridin on a yeast extract sucrose broth medium. Mycotoxin surveys showed that citreoviridin was not detected in any samples, but one out of 10 rice samples tested was positive for aflatoxin B1 at a level of 5.9 µg/kg. An Ames test revealed that methanol extracts from rice grains inoculated with selected P. brocae isolates were positive for strains TA100 and YG7108 of Salmonella typhimurium, suggesting the presence of base-pair substitution and DNA alkylation mutagens. Our data obtained here demonstrated that aflatoxin B1 and toxic P. citreonigrum were present on domestic rice grains in Thailand, although limited samples were tested. Penicillium brocae, which may produce mutagenic metabolites, was isolated for the first time from the surface of Thai rice grains.

Citreoviridin induces triglyceride accumulation in hepatocytes through inhibiting PPAR-α in vivo and in vitro.

Citreoviridin (CIT) is a mycotoxin produced by Penicillum citreonigrum, Aspergillus terreus and Eupenicillium ochrosalmoneum. CIT occurs naturally in moldy rice and corn. CIT is associated with the development of atherosclerosis in the general population. Alteration in hepatic lipid metabolism is a pathogenic factor in atherosclerosis. However the effect and the underlying mechanism of CIT on hepatic lipid metabolism are largely unknown. In this study, we reported that CIT induced triglyceride accumulation in mice liver and human liver HepG2 cells as shown in oil red O staining. CIT (0.1 mg/kg-0.3 mg/kg) for 6 weeks elevated liver triglyceride contents in mice. CIT inhibited the transactivation activity of peroxisome proliferator-activated receptor-α (PPAR-α) in hepatocyte in vivo and in vitro, as shown by the reduced mRNA levels of PPAR-α target genes which play key roles in lipid metabolism in various aspects. PPAR-α agonist fenofibrate attenuated CIT-induced triglyceride accumulation in HepG2 cells. Furthermore, CIT increased serum total cholesterol/high-density lipoprotein cholesterol ratio, a strong risk factor for cardiovascular disease. In summary, we reported that CIT induced PPAR-α-dependent hepatic triglyceride accumulation and dyslipidemia. Our data will provide new mechanistic insights into CIT-induced lipid alterations.

Role of α/ß interface in F1 ATPase rotational catalysis probed by inhibitors and mutations.

The F1 sector of ATP synthase (FOF1) synthesizes or hydrolyses ATP via a rotational catalysis mechanism that couples chemical reaction with subunit rotation. Phytopolyphenols such as curcumin can inhibit bulk phase F1 ATPase activity by extending the catalytic dwell time during subunit rotation (Sekiya, M., Hisasaka, R., Iwamoto-Kihara, A., Futai, M., Nakanishi-Matsui, M., Biochem. Biophys. Res. Commun. 452 (2014) 940-944). Citreoviridin, a polyene α-pyrone mycotoxin isolated from Penicillium sp, also inhibits ATPase activity. Molecular docking and mutational analysis indicated that these compounds interact with a region near the ß-subunit Arg398 residue that lies at the interface with the α-subunit. Binding of these inhibitors lowered the rotation rate and increased the duration of the catalytic dwell synergistically with substitution of ß-subunit Ser174 to Phe (ßS174F), which rendered the enzyme defective for conformational transmission between ß-subunits of different catalytic stages. Furthermore, substitution of α-subunit Glu402 to Ala (αE402A) in the α/ß-interface also decreased the rotation rate by increasing the duration of the catalytic dwell. Interestingly, this mutation restored the catalytic dwell of the ßS174F variant to that of the wild-type enzyme. These results suggest that the α/ß-interface is involved in conformational changes of the ß-subunit during rotational catalysis.

Screening and Molecular Evolution of a Single Chain Variable Fragment Antibody (scFv) against Citreoviridin Toxin.

Citreoviridin (CIT), a small food-borne mycotoxin produced by Penicillium citreonigrum, is generally distributed in various cereal grains and farm crop products around the world and has caused cytotoxicity as an uncompetitive inhibitor of ATP hydrolysis. A high affinity single chain variable fragment (scFv) antibody that can detect the citreoviridin in samples is still not available; therefore, it is very urgent to prepare an antibody for CIT detection and therapy. In this study, an amplified and assembled scFv from hybridoma was used to construct the mutant phage library by error-prone PCR, generating a 2 × 108 capacity mutated phage display library. After six rounds of biopanning, the selected scFv-5A10 displayed higher affinity and specificity to CIT antigen, with an increased affinity of 13.25-fold (Kaff = 5.7 × 109 L/mol) compared to that of the original wild-type scFv. Two critical amino acids (P100 and T151) distributed in H-CDR3 and L-FR regions that were responsible for scFv-5A10 to CIT were found and verified by oligonucleotide-directed mutagenesis, and the resulting three mutants except for the mutant (P100K) lost binding activity significantly against CIT, as predicated. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect CIT was 25-562 ng/mL with IC50 at 120 ng/mL. The limit of detection was 14.7 ng/mL, and the recovery average was (90.612 ± 3.889)%. Hence, the expressed and purified anti-CIT MBP-linker-scFv can be used to detect CIT in corn and related samples.

Biosynthetic Pathway of the Reduced Polyketide Product Citreoviridin in Aspergillus terreus var. aureus Revealed by Heterologous Expression in Aspergillus nidulans.

Citreoviridin (1) belongs to a class of F1-ATPase ß-subunit inhibitors that are synthesized by highly reducing polyketide synthases. These potent mycotoxins share an α-pyrone polyene structure, and they include aurovertin, verrucosidin, and asteltoxin. The identification of the citreoviridin biosynthetic gene cluster in Aspergillus terreus var. aureus and its reconstitution using heterologous expression in Aspergillus nidulans are reported. Two intermediates were isolated that allowed the proposal of the biosynthetic pathway of citreoviridin.