RESUMEN
Using CRISPR-Cas9 technology and a microhomology-mediated end-joining repair system, we substituted genes of the gliotoxin pathway in Aspergillus fumigatus with genes responsible for chetomin biosynthesis from Chaetomium cochliodes, leading to the production of three new epipolythiodioxopiperazines (ETPs). This work represents the first successful endeavor to produce ETPs in a non-native host. Additionally, the simultaneous disruption of five genes in a single transformation marks the most extensive gene knockout event in filamentous fungi to date.
Asunto(s)
Aspergillus fumigatus , Gliotoxina , Piperazinas , Aspergillus fumigatus/metabolismo , Aspergillus fumigatus/genética , Piperazinas/química , Piperazinas/metabolismo , Gliotoxina/biosíntesis , Gliotoxina/química , Estructura Molecular , Chaetomium/metabolismo , Chaetomium/química , Sistemas CRISPR-CasRESUMEN
The number of patients suffering from fungal diseases has constantly increased during the last decade. Among the fungal pathogens, the airborne filamentous fungus Aspergillus fumigatus can cause chronic and fatal invasive mold infections. So far, only three major classes of drugs (polyenes, azoles, and echinocandins) are available for the treatment of life-threatening fungal infections, and all present pharmacological drawbacks (e.g., low solubility or toxicity). Meanwhile, clinical antifungal-resistant isolates are continuously emerging. Therefore, there is a high demand for novel antifungal drugs, preferentially those that act on new targets. We studied urease and the accessory proteins in A. fumigatus to determine their biochemical roles and their influence on virulence. Urease is crucial for the growth on urea as the sole nitrogen source, and the transcript and protein levels are elevated on urea media. The urease deficient mutant displays attenuated virulence, and its spores are more susceptible to macrophage-mediated killing. We demonstrated that this observation is associated with an inability to prevent the acidification of the phagosome. Furthermore, we could show that a nickel-chelator inhibits growth on urea. The nickel chelator is also able to reverse the effects of urease on macrophage killing and phagosome acidification, thereby reducing virulence in systemic and trachea infection models. IMPORTANCE The development of antifungal drugs is an urgent task, but it has proven to be difficult due to many similarities between fungal and animal cells. Here, we characterized the urease system in A. fumigatus, which depends on nickel for activity. Notably, nickel is not a crucial element for humans. Therefore, we went further to explore the role of nickel-dependent urease in host-pathogen interactions. We were able to show that urease is important in preventing the acidification of the phagosome and therefore reduces the killing of conidia by macrophages. Furthermore, the deletion of urease shows reduced virulence in murine infection models. Taken together, we identified urease as an essential virulence factor of A. fumigatus. We were able to show that the application of the nickel-chelator dimethylglyoxime is effective in both in vitro and in vivo infection models. This suggests that nickel chelators or urease inhibitors are potential candidates for the development of novel antifungal drugs.
RESUMEN
A chemical investigation of the filamentous fungus Aspergillus californicus led to the isolation of a polyketide-nonribosomal peptide hybrid, calipyridone A (1). A putative biosynthetic gene cluster cpd for production of 1 was next identified by genome mining. The role of the cpd cluster in the production of 1 was confirmed by multiple gene deletion experiments in the host strain as well as by heterologous expression of the hybrid gene cpdA inAspergillus oryzae. Moreover, chemical analyses of the mutant strains allowed the biosynthesis of 1 to be elucidated. The results indicate that the generation of the 2-pyridone moiety of 1 via nucleophilic attack of the iminol nitrogen to the carbonyl carbon is different from the biosynthesis of other fungal 2-pyridone products through P450-catalyzed tetramic acid ring expansions. In addition, two biogenetic intermediates, calipyridones B and C, showed modest inhibition effects on the plaque-forming ability of SARS-CoV-2.
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Aspergillus/metabolismo , Piridonas/metabolismo , Aspergillus oryzae/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Eliminación de Gen , Humanos , Familia de Multigenes/genética , Policétidos/metabolismo , Policétidos/farmacología , Piridonas/farmacología , Pirrolidinonas/metabolismo , Pirrolidinonas/farmacología , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19RESUMEN
A chemical investigation of Aspergillus californicus IBT 16748 led to the isolation of two new oxepine-pyrimidinone-ketopiperazine type nonribosomal peptides oxepinamides L (1) and M (2). Their structures were characterised by spectroscopic analysis including HRESIMS, 1D and 2D NMR. The absolute structure of 1 was assigned by ECD calculation. The antibacterial and cytotoxic properties of 1 were evaluated.
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Oxepinas , Pirimidinonas , Aspergillus , Estructura Molecular , Péptidos , Pirimidinonas/farmacologíaRESUMEN
New infectious diseases and increase in drug-resistant microbial pathogens emphasize the need for antibiotics with novel mode-of-action. Tetramates represented by fungi-derived tenuazonic acid and bacterial polycyclic tetramate macrolactams (PTMs) are an important family of natural products with a broad spectrum of antimicrobial activities. Despite their potential application as new antibiotics, it remains unknown how PTMs function. In this study, genomic mining revealed that PTM biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria, and we investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A that previously has only been isolated from a Gram-negative bacterium was obtained after a scale-up fermentation, isolation, and structure elucidation through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis. In addition, these three tetramates were for the first time to be confirmed as metallophores and the stoichiometry of the complexes were shown to be Fe(III)(equisetin)3/Fe(III)(equisetin)2 and Fe(III)(ikarugamycin)2, respectively. Meanwhile, we found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction. Their antibacterial activity was reduced by adding the radical scavenger, vitamin C. Altogether, our work demonstrates that equisetin and PTMs can act as metallophores and their antimicrobial mechanism is possibly mediated through Fenton chemistry.
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In recent years, there has been an increasing demand for the replacement of synthetic food colorants with naturally derived alternatives. Filamentous fungi are prolific producers of secondary metabolites including polyketide-derived pigments, many of which have not been fully characterized yet. During our ongoing investigations of black aspergilli, we noticed that Aspergillus homomorphus turned yellow when cultivated on malt extract agar plates. Chemical discovery guided by UV and MS led to the isolation of two novel yellow natural products, and their structures were elucidated as aromatic α-pyrones homopyrones A (1) and B (2) by HRMS and NMR. Combined investigations including retro-biosynthesis, genome mining, and gene deletions successfully linked both compounds to their related biosynthetic gene clusters. This demonstrated that homopyrones are biosynthesized by using cinnamoyl-CoA as the starter unit, followed by extension with three malonyl-CoA units, and lactonization to give the core hybrid backbone structure. The polyketide synthase AhpA includes a C-methylation domain, which however seems to be promiscuous since only 2 is C-methylated. Altogether, the homopyrones represent a rare case of hybrid phenylpropanoid- and polyketide-derived natural products in filamentous fungi. KEY POINTS: ⢠Homopyrones represent a rare type of fungal polyketides synthesized from cinnamic-CoA. ⢠CRISPR/Cas9 technology has been firstly applied in Aspergillus homomorphus.
Asunto(s)
Policétidos , Aspergillus , Hongos , Sintasas PoliquetidasRESUMEN
Five new polyketides were isolated from the rare filamentous fungus Aspergillus californicus IBT 16748 including calidiol A (1); three phthalide derivatives califuranones A1, A2, and B (2-4); and a pair of enantiomers (-)-calitetralintriol A (-5) and (+)-calitetralintriol A (+5) together with four known metabolites (6-9). The structures of the new products were established by extensive spectroscopic analyses including HRMS and 1D and 2D NMR. The absolute configurations of two diastereomers 2 and 3 and the enantiomers (-5) and (+5) were assigned by comparing their experimental and calculated ECD data, whereas the absolute configuration of 4 was proposed by analogy. Compound 1 showed moderate activity against methicillin-resistant Staphylococcus aureus.
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Antibacterianos/farmacología , Aspergillus/química , Policétidos/farmacología , Antibacterianos/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Policétidos/aislamiento & purificaciónRESUMEN
Two new naphthyl-products calinaphthyltriol A (1) and calinaphthalenone A (2) were isolated from Aspergillus californicus IBT 16748 together with one known compound ophiobolin X (3). Their structures were elucidated by extensive spectroscopic analyses. The absolute configuration of 2 was solved by comparing its optical rotation with data for the known compounds 4, 5, and 6 as well as theoretical calculations. The antibacterial and cytotoxic activities of 1 and 3 were evaluated. Both compounds did not show antibacterial activity (MIC > 96 µg·ml-1) against a few selected clinically relevant Gram positive and Gram negative bacterial strains. However, they showed moderate cytotoxicity against HL-60 cell line with IC50 values of 18 and 24 µg·ml-1, respectively.
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Aspergillus/química , Antibacterianos/farmacología , Antibióticos Antineoplásicos/farmacología , Fermentación , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Células HL-60 , Humanos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Estructura MolecularRESUMEN
Recently, a rare class of nonribosomal peptides (NRPs) bearing a unique Oxepine-Pyrimidinone-Ketopiperazine (OPK) scaffold has been exclusively isolated from fungal sources. Based on the number of rings and conjugation systems on the backbone, it can be further categorized into three types A, B, and C. These compounds have been applied to various bioassays, and some have exhibited promising bioactivities like antifungal activity against phytopathogenic fungi and transcriptional activation on liver X receptor α. This review summarizes all the research related to natural OPK NRPs, including their biological sources, chemical structures, bioassays, as well as proposed biosynthetic mechanisms from 1988 to March 2020. The taxonomy of the fungal sources and chirality-related issues of these products are also discussed.
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Objective: To investigate the chemical constituents from stems of Kadsura longipedunculata. Methods: The constituents were isolated and purified by various chromatographic methods,and their structures were elucidated by spectroscopic analysis and comparison with literatures. Results: Seven compounds were isolated from the stems of Kadsura longipedunculata and were identified as longipedunin D( 1),renchangianin A( 2),renchangianin B( 3),meso-dihydroguaiaretic acid( 4),isolariciresinol-9-O-ß-D-xyloside( 5),(-)-gallocatechin( 6) and( +)-catechin( 7). Conclusion: Compound 1 is a novel lignan,and compounds 2,3 are isolated from this plant for the first time.
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Kadsura , Cromatografía , Guayacol/análogos & derivados , Lignanos , Estructura Molecular , Extractos Vegetales , Tallos de la PlantaRESUMEN
The stamen of lotus, known as Nelumbinis stamen, has been used as the folk medicine and functional food for a long time, which showed good activities of anti-ulcer, anti-thrombosis, analgesic, anti-diarrhea, strengthen uterine contraction. The bioactivities of Nelumbinis stamen were attributed to the existence of flavonoids, its characteristic chemical constituents. A reliable method for comprehensive chemical analysis of flavonoids in Nelumbinis stamen by HPLC-DAD-MS was developed for the first time. The extraction protocol of flavonoids from Nelumbinis stamen was optimized by an orthogonal design. The chromatographic conditions were optimized, which exhibited similar level than that of the UHPLC platform allowing target compound identification in a shorter time with little solvent consumption. Moreover, similarity analysis, hierarchical clustering analysis and principal components analysis were successfully applied to demonstrate the variability of these Nelumbinis stamen samples.
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Flavonoides/química , Flores/química , Lotus/química , Cromatografía Líquida de Alta Presión , Espectrometría de MasasRESUMEN
Systematical and comprehensive statistics, induction and analysis on literature and data of acupuncture and moxibustion treatment of tuberculosis in incunabula are carried out by computer. The results indicate that in treatment of tuberculosis, most select acupoints of the upper back, chest-epigastrium and The Yangming Channel on the leg to promote dispersing function of the lung, strength the spleen and anti-tuberculosis; select acupoints of The Conception Vessel on the lower abdomen and Shenshu (BL 23) and others on the lower back to tonify the kidney and anti-tuberculosis. Clinically, most select moxibustion and acupuncture with reinforcing-reducing manipulation and pricking blood therapy. And referential recipes for clinical treatment are raised.