Tools to make Stachybotrys chartarum genetically amendable: Key to unlocking cryptic biosynthetic gene clusters.

The soil and indoor fungus Stachybotrys chartarum can induce respiratory disorders, collectively referred to as stachybotryotoxicosis, owing to its prolific production of diverse bioactive secondary metabolites (SMs) or mycotoxins. Although many of these toxins responsible for the harmful effects on animals and humans have been identified in the genus Stachybotrys, however a number of SMs remain elusive. Through in silico analyses, we have identified 37 polyketide synthase (PKS) genes, highlighting that the chemical profile potential of Stachybotrys is far from being fully explored. Additionally, by leveraging phylogenetic analysis of known SMs produced by non-reducing polyketide synthases (NR-PKS) in other filamentous fungi, we showed that Stachybotrys possesses a rich reservoir of untapped SMs. To unravel natural product biosynthesis in S. chartarum, genetic engineering methods are crucial. For this purpose, we have developed a reliable protocol for the genetic transformation of S. chartarum and applied it to the ScPKS14 biosynthetic gene cluster. This cluster is homologous to the already known Claviceps purpurea CpPKS8 BGC, responsible for the production of ergochromes. While no novel SMs were detected, we successfully applied genetic tools, such as the generation of deletionand overexpression strains of single cluster genes. This toolbox can now be readily employed to unravel not only this particular BGC but also other candidate BGCs present in S. chartarum, making this fungus accessible for genetic engineering.

Insights into Ergochromes of the Plant Pathogen Claviceps purpurea.

Claviceps purpurea is an ergot fungus known for its neurotropic alkaloids, which have been identified as the main cause of ergotism, a livestock and human disease triggered by ergot consumption. Tetrahydroxanthone dimers, the so-called ergopigments, presumably also contribute to this toxic effect. Overexpression of the cluster-specific transcription factor responsible for the formation of these pigments in C. purpurea led to the isolation of three new metabolites (8-10). The new pigments were characterized utilizing HRMS, NMR techniques, and CD spectroscopy and shown to be xanthone dimers. Secalonic acid A and its 2,4'- and 4,4'-linked isomers were also isolated, and their absolute configuration was investigated. The contribution of secalonic acid A, its isomers, and new metabolites to the toxicity of C. purpurea was investigated in HepG2 and CCF-STTG1 cells. Along with cytotoxic properties, secalonic acid A was found to inhibit topoisomerase I and II activity.

Identification of the polyketide synthase PKS7 responsible for the production of lecanoric acid and ethyl lecanorate in Claviceps purpurea.

Claviceps purpurea is a plant pathogenic fungus which is still highly relevant in modern agriculture as it infects grasses such as rye and wheat. The disease caused by the consumption of contaminated grain or flour has been known since the Middle Ages and is termed ergotism. The main cause for the toxicity of this fungus is attributed to the ergot alkaloids. Apart from these alkaloids and the ergochromes known as ergot pigments, the secondary metabolism of C. purpurea is not well investigated. This study demonstrated the function of the polyketide synthase PKS7 in C. purpurea by determining the effect of its overexpression on metabolite profiles. For the first time, the depsides lecanoric acid, ethyl lecanorate, gerfelin, and C10-deoxy gerfelin were discovered as secondary metabolites of C. purpurea. Additionally, to estimate the contribution of isolated secondary metabolites to the toxic effects of C. purpurea, lecanoric acid, ethyl lecanorate, and orsellinic acid were tested on HepG2 and CCF-STTG1 cell lines. This study provides the first report on the function of C. purpurea PKS7 responsible for the production of depsides, among which lecanoric acid and ethyl lecanorate were identified as main secondary metabolites.

Comparative genomics of geographically distant Fusarium fujikuroi isolates revealed two distinct pathotypes correlating with secondary metabolite profiles.

Fusarium fujikuroi causes bakanae ("foolish seedling") disease of rice which is characterized by hyper-elongation of seedlings resulting from production of gibberellic acids (GAs) by the fungus. This plant pathogen is also known for production of harmful mycotoxins, such as fusarins, fusaric acid, apicidin F and beauvericin. Recently, we generated the first de novo genome sequence of F. fujikuroi strain IMI 58289 combined with extensive transcriptional, epigenetic, proteomic and chemical product analyses. GA production was shown to provide a selective advantage during infection of the preferred host plant rice. Here, we provide genome sequences of eight additional F. fujikuroi isolates from distant geographic regions. The isolates differ in the size of chromosomes, most likely due to variability of subtelomeric regions, the type of asexual spores (microconidia and/or macroconidia), and the number and expression of secondary metabolite gene clusters. Whilst most of the isolates caused the typical bakanae symptoms, one isolate, B14, caused stunting and early withering of infected seedlings. In contrast to the other isolates, B14 produced no GAs but high amounts of fumonisins during infection on rice. Furthermore, it differed from the other isolates by the presence of three additional polyketide synthase (PKS) genes (PKS40, PKS43, PKS51) and the absence of the F. fujikuroi-specific apicidin F (NRPS31) gene cluster. Analysis of additional field isolates confirmed the strong correlation between the pathotype (bakanae or stunting/withering), and the ability to produce either GAs or fumonisins. Deletion of the fumonisin and fusaric acid-specific PKS genes in B14 reduced the stunting/withering symptoms, whereas deletion of the PKS51 gene resulted in elevated symptom development. Phylogenetic analyses revealed two subclades of F. fujikuroi strains according to their pathotype and secondary metabolite profiles.

Auranthine, a Benzodiazepinone from Penicillium aurantiogriseum: Refined Structure, Absolute Configuration, and Cytotoxicity.

The structure of the known Penicillium aurantiogriseum-derived secondary metabolite auranthine was refined using a combination of synthetic, spectroscopic, and X-ray diffractometric approaches. Thus, auranthine was shown to be a fused quinazolino benzodiazepinedione (2) bearing an acyclic aliphatic nitrile moiety, thereby significantly differing from the originally proposed structure 1 published in 1986. Its absolute configuration was confirmed by CD spectroscopy and DFT calculations. The cultivation of P. aurantiogriseum was optimized, allowing high production of auranthine. The cytotoxicity profile of auranthine and its semisynthetic analogues is reported. The refined structure of auranthine provides a valid target for the total synthesis of this underexplored natural product and its derivatives.

Diuretic activity and toxicity of some Verbascum nigrum extracts and fractions.

CONTEXT: Verbascum nigrum L. (Scrophulariaceae) is a perennial plant used in folk medicine for the treatment of kidney diseases due to its presumable diuretic properties. OBJECTIVE: We investigated the diuretic activity and toxicity of extracts from different parts of V. nigrum and identified a group of compounds responsible for the biological effect. MATERIALS AND METHODS: Five ethanol extracts from herb, roots, flowers, leaves and stems as well as five fractions of polar compounds isolated from herb of V. nigrum were orally administrated as a single dose of 50 mg/kg to rats. Urinary excretion and electrolyte content were measured at 3 and 6 h after the treatment. The acute toxicity of the V. nigrum extracts and fractions was evaluated in mice. RESULTS: All extracts, except the one prepared from the roots, showed a significant increase of the urine output within first 3 h after their administration. The extract from stems was the most active, inducing urine output of 14.6 ± 0.8 ml/kg BW versus 5.2 ± 1.4 ml/kg BW of the control. It also demonstrated saluretic activity with a natriuretic index 4.1 and a kaliuretic index 3.8. The diuretic activity was correlated with the flavonoid content in the plant organs. Flavonoid fractions demonstrated significant activity; the higher content of flavonoids (expressed as hesperidin) translated into more pronounced diuretic (35.9 ± 2.1 ml/kg BW) and saluretic effects (natriuretic index 4.5 and kaliuretic index 5.4). CONCLUSION: The diuretic activity of traditionally used V. nigrum was validated experimentally. The pharmacological effect was attributed to flavonoids, which accumulated in aerial parts of the plant, mainly in stems.

Total synthesis of (±)-auranthine confirmed its refined structure.

Auranthine, isolated in 1986 from Penicillium aurantiogriseum, is a fungal benzodiazepine. Through the successful total synthesis of (±)-auranthine, we confirmed the refined structure of natural (-)-auranthine. We established that natural (-)-auranthine is a fused quinazolino benzodiazepine dione 1 featuring an acyclic aliphatic nitrile moiety, thereby disproving the proposed structure 2.

Micro-scale screening of genetically modified Fusarium fujikuroi strain extends the apicidin family.

Apicidins are a class of naturally occurring cyclic tetrapeptides produced by few strains within the Fusarium genus. These secondary metabolites have gained significant attention due to their antiprotozoal activity through HDAC inhibition, thereby highlighting their potential for the treatment of malaria. Predominantly, apicidins have been isolated from Fusarium semitectum, offering a deep insight into the biosynthetic pathway responsible for their formation. A similar biosynthetic gene cluster has also been identified in the rice pathogenic fungus F. fujikuroi, leading the discovery of three additional apicidins through genetic manipulation. Routine mass spectrometric screening of these compound-producing strains revealed another metabolite structurally related to previously studied apicidins. By optimizing culture conditions and developing an effective isolation method, we obtained a highly pure substance, whose chemical structure was fully elucidated using NMR and HRMS fragmentation. Further studies were conducted to determine cytotoxicity, antimalarial activity, and HDAC inhibitory activity of this new secondary metabolite alongside the previously known apicidins. This work not only expands the apicidin class with a new member but also provides extensive insights and comparative analysis of apicidin-like substances produced by F. fujikuroi.

Amide-functionalized 1,2,4-Triazol-5-amines as Covalent Inhibitors of Blood Coagulation Factor XIIa and Thrombin.

To counteract thrombosis, new safe and efficient antithrombotics are required. We herein report the design, synthesis, and biological activity of a series of amide-functionalized acylated 1,2,4-triazol-5-amines as selective inhibitors of blood coagulation factor XIIa and thrombin. The introduction of an amide moiety into the main scaffold of 3-aryl aminotriazoles added certain three-dimensional properties to synthesized compounds and allowed them to reach binding sites in FXIIa and thrombin previously unaddressed by non-functionalized 1,2,4-triazol-5-amines. Among synthesized compounds, one quinoxaline-derived aminotriazole bearing N-butylamide moiety inhibited FXIIa with the IC50 value of 28 nM, whereas the N-phenylamide-derived aminotriazole inhibited thrombin with the IC50 value of 41 nM. Performed mass-shift experiments and molecular modeling studies proved the covalent mechanism of FXIIa and thrombin inhibition by synthesized compounds. In plasma coagulation tests, developed aminotriazoles showed anticoagulant properties mainly affecting the intrinsic blood coagulation pathway, activation of which is associated with thrombosis but is negligible for hemostasis.

Ion identity molecular networking for mass spectrometry-based metabolomics in the GNPS environment.

Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.

Acylated 1H-1,2,4-Triazol-5-amines Targeting Human Coagulation Factor XIIa and Thrombin: Conventional and Microscale Synthesis, Anticoagulant Properties, and Mechanism of Action.

We herein report the conventional and microscale parallel synthesis of selective inhibitors of human blood coagulation factor XIIa and thrombin exhibiting a 1,2,4-triazol-5-amine scaffold. Structural variations of this scaffold allowed identifying derivative 21i, a potent 29 nM inhibitor of FXIIa, with improved selectivity over other tested serine proteases and also finding compound 21m with 27 nM inhibitory activity toward thrombin. For the first time, acylated 1,2,4-triazol-5-amines were proved to have anticoagulant properties and the ability to affect thrombin- and cancer-cell-induced platelet aggregation. Performed mass spectrometric analysis and molecular modeling allowed us to discover previously unknown interactions between the synthesized inhibitors and the active site of FXIIa, which uncovered the mechanistic details of FXIIa inhibition. Synthesized compounds represent a promising starting point for the development of novel antithrombotic drugs or chemical tools for studying the role of FXIIa and thrombin in physiological and pathological processes.

Detection of the Cytotoxic Penitrems A-F in Cheese from the European Single Market by HPLC-MS/MS.

Penitrems are fungal indole diterpene-derived tremorgenic secondary metabolites, which are mainly produced by Penicillium spp. Several cases of intoxications with penitrems and subsequent occurrences of penitrem A in foodstuff underline the need for reliable quantitation methods for the detection of these mycotoxins in food. In this study, a simple and fast high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the quantitative analysis of penitrems A-F in cheese was developed. Therefore, penitrems A-F were isolated from Penicillium crustosum as analytical reference standards. The analysis of 60 cheese samples from the European single market (EU) revealed the occurrence of penitrem A in 10% of the analyzed samples with an average concentration of 28.4 µg/kg and a maximum concentration of 429 µg/kg. In addition to penitrem A, other members of the group of penitrems, namely, penitrems B, C, D, E, and F, were for the first time quantitatively detected in food samples, although in lower concentrations and with lower incidence in comparison to penitrem A. Moreover, we report cytotoxic effects of all penitrems on two cell lines (HepG2 and CCF-STTG1). This clearly underlines their relevance and the importance to analyze food samples in order to get insights into the human exposure toward these mycotoxins.

Influence of Environmental Factors on the Production of Penitrems A-F by Penicillium crustosum.

Filamentous fungi produce a multitude of secondary metabolites, some of them known as mycotoxins, which are toxic to vertebrates and other animal groups in low concentrations. Among them, penitrems, which belong to the group of indole-diterpene mycotoxins, are synthesized by Penicillium and Aspergillus genera and exhibit potent tremorgenic effects. This is the first complex study of the penitrems A-F production under the influence of different abiotic factors, e.g., media, incubation time, temperature, pH, light, water activity, and carbon and nitrogen source as well as oxidative and salt stress. For this purpose, penitrems A-F were isolated from Penicillium crustosum cultures and used as analytical standards. Among the carbon sources, glucose supplemented to the media at the concentration of 50 g/L, showed the strongest inducing effect on the biosynthesis of penitrems. Among nitrogen sources, glutamate was found to be the most favorable supplement, significantly increasing production of these secondary metabolites. CuSO4-promoted oxidative stress was also shown to remarkably stimulate biosynthesis of all penitrems. In contrast, the salt stress, caused by the elevated concentrations of NaCl, showed an inhibitory effect on the penitrem biosynthesis. Finally, cheese model medium elicited exceptionally high production of all members of the penitrems family. Obtained results give insides into the biosynthesis of toxicologically relevant penitrems A-F under different environmental factors and can be utilized to prevent food contamination.

Synthesis, local anaesthetic and antiarrhythmic activities of N-alkyl derivatives of proline anilides.

We describe here the design, synthesis and evaluation of in vivo local anaesthetic and antiarrhythmic activities of a series of N-alkylproline anilides. Most of the compounds demonstrated surface anaesthetic activity higher than that of lidocaine, ropivacaine and bupivacaine. We established that the local anaesthetic activity was sensitive to structural variations in the substitution pattern at the aromatic ring and the type of alkyl group at the proline nitrogen atom. Some of the prepared N-alkylproline anilides possessed significant antiarrhythmic activity with higher therapeutic indexes than the reference drugs.