Characterization of the hexahydropolyprenols of Aspergillus fumigatus Fresenius.
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ABSTRACT:
SUBMITTER: Stone KJ
PROVIDER: S-EPMC1270265 | biostudies-other | 1967 Feb
REPOSITORIES: biostudies-other
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Project description:The total mycelial lipid of Aspergillus fumigatus was analysed and over half of its hexahydropolyprenol was shown to be esterified with fatty acids. Comparison of the fatty acid content of the prenyl esters with the sterol ester and the total lipid indicated a marked predominance of saturated fatty acids in the polyprenyl esters. The predominant acids esterified to the prenols were palmitic acid, linoleic acid, oleic acid, lignoceric acid, stearic acid and palmitoleic acid. Most of the unesterified polyprenol was found in the mitochondrial fraction, but the esterified prenol was equally distributed throughout the cell fractions. This distribution was unlike that found for ergosteryl ester in the same tissue.
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Project description:Aspergillus fumigatus (Fresenius), IMI 246651, A.T.C.C. 46324, produces two beta-glucosidase enzymes, cotton-solubilizing activity, xylanase and endoglucanase enzymes which can be separated by gel-filtration chromatography. The major endoglucanase does not bind to concanavalin A-Sepharose and does not stain with periodic acid/Schiff reagent. It is homogeneous on polyacrylamide isoelectric focusing (pI = 7.1) and has a mol.wt. of 12500 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The endoglucanase produces glucose and a mixture of oligosaccharides from cellulose; the purified enzyme has a small dextranase activity. It is stable at 50 degrees C and pH 6.
| S-EPMC1152145 | biostudies-other
Project description:A strain of Aspergillus fumigatus Fresenius, isolated from spoiled hay, converts melilotic acid (o-hydroxyphenylpropionic acid) and o-coumaric acid into 4-hydroxycoumarin and dicoumarol. The sequence is shown to be melilotic acid (I) [Formula: see text] coumaric acid (IV) [Formula: see text] beta-hydroxymelilotic acid (II) [Formula: see text] beta-oxomelilotic acid (III) [Formula: see text] 4-hydroxycoumarin (VI), on the basis of (1) studies on the formation of postulated intermediates, (2) experiments with isotopically labelled materials and (3) sequential enzyme induction. In the presence of semicarbazide, o-coumaraldehyde is formed from o-coumaric acid: there is no evidence, however, that this lies on the normal metabolic pathway.
| S-EPMC1178855 | biostudies-other
Project description:1. The mycelium of Aspergillus fumigatus has been shown to incorporate mevalonate into squalene, ubiquinone, ergosterol and hexahydroprenol. 2. The (3)H/(14)C ratio in ubiquinone, biosynthesized from [2-(14)C-(4R)-4-(3)H(1)]mevalonate, is the same as in the squalene; essentially no (3)H was incorporated from [2-(14)C-(4S)-4-(3)H(1)]mevalonate, indicating the biosynthesis of biogenetically trans-isoprene units. 3. The (3)H/(14)C ratio for ergosterol (from ;4R-mevalonate') was 3:5, showing that the proton at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25. 4. As (3)H from both mevalonates was incorporated into the hexahydroprenols the biosynthesis of both cis- and trans-isoprene units must occur. 5. The saturated omega- and psi-isoprene units are shown to be biogenetically trans, as are two of the unsaturated residues. 6. The saturated alpha- and unsaturated beta-isoprene residues are both biogenetically cis. 7. An inexplicable loss of approximately half of the olefinic protons from the cis-portion of hexahydroprenol occurs; possible reasons for this loss are discussed. 8. Increase in chain length of the hexahydroprenols is by a cis addition. 9. A biosynthesis of hexahydroprenols by addition of cis-isoprene units to all-trans-geranylgeranyl pyrophosphate, or a dihydro or tetrahydro derivative thereof, is suggested.
| S-EPMC1270544 | biostudies-other
Project description:In the last few years, increasing numbers of viruses infecting fungi have been identified. In this study, we used an in silico approach for the analysis of deep RNA sequencing data in order to discover and characterize putative genomic ssRNA or dsRNA mycovirus sequences in Aspergillus fumigatus. RNA sequencing reads of A. fumigatus strains were mapped against the A. fumigatus Af293 reference genome. Unmapped reads were collected for de novo assembly. Contigs were analyzed by Blastx comparison with a mycovirus protein database. Assembled viral genomes were used as template for remapping of RNA sequencing reads. In total, deep RNA sequencing results from 11 A. fumigatus strains were analyzed for the presence of mycoviral genomic RNAs. In 9 out of 11 strains, putative mycoviral RNA genomes were identified. Three strains were infected with two different mycovirus species. Two strains were infected with Aspergillus fumigatus polymycovirus type-1 (AfuPmV-1). Four strains contained fully recovered genomic RNA of unknown narna-like viruses designated as Aspergillus fumigatus narnavirus-1 and Aspergillus fumigatus narnavirus-2 (AfuNV-1 and AfuNV-2). Both viruses showed 38% amino acid sequence identity to Beihai narna-like virus-21. Three strains contained partially recovered genomic RNA of an unknown narna-like virus. Two strains contained fully recovered genomic RNAs of an unknown partitivirus designated as Aspergillus fumigatus partitivirus-2 (AfuPV-2) which showed 50% amino acid sequence identity to Alternaria alternata partitivirus-1. Finally, one strain contained fully recovered genomic RNA of an unknown mitovirus designated as Aspergillus fumigatus mitovirus-1 (AfuMV-1) which showed 34% amino acid sequence identity to Sclerotina sclerotiorum mitovirus. In silico analysis of deep RNA sequencing results showed that a majority of the A. fumigatus strains used here were infected with mycoviruses. Four novel A. fumigatus RNA mycoviruses could be identified: two different Aspergillus fumigatus narna-like viruses, one Aspergillus fumigatus partitivirus, and one Aspergillus fumigatus mitovirus.
| S-EPMC6059430 | biostudies-literature
Project description:Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the ?fkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the ?fkbp12-2 and ?fkbp12-3 strains didn't show any discernable phenotype, the ?fkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.
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Project description:Extracellular vesicles (EVs) are membranous compartments produced by yeast and mycelial forms of several fungal species. One of the difficulties in perceiving the role of EVs during the fungal life, and particularly in cell wall biogenesis, is caused by the presence of a thick cell wall. One alternative to have better access to these vesicles is to use protoplasts. This approach has been investigated here with Aspergillus fumigatus, one of the most common opportunistic fungal pathogens worldwide. Analysis of regenerating protoplasts by scanning electron microscopy and fluorescence microscopy indicated the occurrence of outer membrane projections in association with surface components and the release of particles with properties resembling those of fungal EVs. EVs in culture supernatants were characterized by transmission electron microscopy and nanoparticle tracking analysis. Proteomic and glycome analysis of EVs revealed the presence of a complex array of enzymes related to lipid/sugar metabolism, pathogenic processes, and cell wall biosynthesis. Our data indicate that (i) EV production is a common feature of different morphological stages of this major fungal pathogen and (ii) protoplastic EVs are promising tools for undertaking studies of vesicle functions in fungal cells.IMPORTANCE Fungal cells use extracellular vesicles (EVs) to export biologically active molecules to the extracellular space. In this study, we used protoplasts of Aspergillus fumigatus, a major fungal pathogen, as a model to evaluate the role of EV production in cell wall biogenesis. Our results demonstrated that wall-less A. fumigatus exports plasma membrane-derived EVs containing a complex combination of proteins and glycans. Our report is the first to characterize fungal EVs in the absence of a cell wall. Our results suggest that protoplasts represent a promising model for functional studies of fungal vesicles.
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Project description:UnlabelledThe mating efficiency of 50 Aspergillus fumigatus isolates from both clinical and environmental sources was analyzed. Forty isolates completed the sexual cycle in 4 weeks with variable levels of fertility designated high, medium, or low. Two opposite-mating-type strains exhibiting the highest fertility, AFB62 (MAT1-1), isolated from a case of invasive aspergillosis, and AFIR928 (MAT1-2), isolated from the environment, were chosen as the supermater pair. Single cleistothecia obtained from a cross of the two strains harbored a minimum of 1 × 10(4) ascospores. The viability of ascospores increased with the age of the fruiting body, 17% at 4 weeks and reaching 95% at 20 weeks. AFB62 and AFIR928 were equally virulent in two different murine models, despite differences in their sources. High recombination frequencies were observed when the closely linked genes alb1 (AFUA_2G17600) and abr2 (AFUA_2G17530) were used as genetic markers. Comparative genome hybridization analyses revealed that only 86 genes (ca. 0.86% of the genome) are significantly diverged between AFB62 and AFIR928. The high fertility in a relatively short period, combined with a high degree of virulence and a high recombination frequency, demonstrates that the mating pair AFB62 and AFIR928 provides an excellent tool for genetic studies of A. fumigatus.ImportanceAspergillus fumigatus is a heterothallic fungal pathogen that causes life-threatening infections in immunocompromised hosts. Although heterothallism facilitates genetic study via recombinational analysis, previous work showed that a 6-month incubation period is required for the completion of sexual reproduction in this species. Such a long incubation period impedes progress in genetic research. To discover a highly fertile (supermater) pair that can complete the sexual cycle in a considerably shorter period, we screened 50 strains collected from various geographic regions for mating efficiency. We identified a highly virulent pair of supermaters that can be an invaluable tool for genetic study.
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Project description:5'-Phosphodiesterase (5'-PDE) catalyzes the hydrolysis of ribonucleic acid to obtain a mixture of ribonucleotides, such as 5'-guanosine monophosphate and 5'-adenosine monophosphate. In this study, a 5'-PDE was newly isolated and purified from Aspergillus fumigatus. Following purification, this enzyme exhibited a specific activity of 1036.76 U/mg protein, a molecular weight of 9.5 kDa, and an optimal temperature and pH for enzyme activity of 60°C and 5.0, respectively. However, its activity was partially inhibited by Fe3+, Cu2+, and Zn2+, but slightly improved by the presence of K+ and Na+. Additionally, chemical-modification experiments were also applied to investigate the structural information of 5'-PDE, in which the residues containing carboxyl and imidazole groups were essential for enzyme activity based on their localization in the 5'-PDE active site. Furthermore, purified 5'-PDE could specifically catalyze the synthesis of ribonucleotides with a Vmax 0.71 mmol/mg·min and a KM of 13.60 mg/mL.
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Project description:Trimeric G proteins play a central role in the G protein signaling in filamentous fungi and Gα subunits are the major component of trimeric G proteins. In this study, we characterize three Gα subunits in the human pathogen Aspergillus fumigatus. While the deletion of gpaB and ganA led to reduced colony growth, the growth of the ΔgpaA strain was increased in minimal media. The germination rate, conidiation, and mRNA expression of key asexual development regulators were significantly decreased by the loss of gpaB. In contrast, the deletion of gpaA resulted in increased conidiation and mRNA expression levels of key asexual regulators. The deletion of gpaB caused a reduction in conidial tolerance against H2O2, but not in paraquat (PQ). Moreover, the ΔgpaB mutant showed enhanced susceptibility against membrane targeting azole antifungal drugs and reduced production of gliotoxin (GT). The protein kinase A (PKA) activity of the ΔganA strain was severely decreased and protein kinase C (PKC) activity was detected all strains at similar levels, indicating that all G protein α subunits of A. fumigatus may be a component of the cAMP/PKA signaling pathway and appear to possess the PKC signaling pathway as an alternative backup pathway to compensate for PKA depletion. Collectively, the three Gα subunits regulate growth, germination, asexual development, resistance to oxidative stress, and GT production differently via the PKA or PKC signaling pathway. The function of GanA of A. fumigatus was elucidated for the first time.
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