Mar Drugs. Published online Jan 7. B ; moc. Received Aug 26; Accepted Dec
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All relevant data are within the paper and its Supporting Information files. Abstract Aspergillus flavipes has received considerable interest due to its potential to produce therapeutic enzymes involved in sulfur amino acid metabolism. In natural habitats, A. Sulfur limitation affects virulence and pathogenicity, and modulates proteome of sulfur assimilating enzymes of several fungi. However, there are no previous reports aimed at exploring effects of sulfur limitation on the regulation of A.
In this report, we show that sulfur limitation affects morphological and physiological responses of A. Introduction Aspergillus flavipes is a nutritionally facultative fungus, widely distributed in the rhizosphere [ 1 ] and as an endophyte in various plants [ 2 ].
These enzymes exhibit a remarkable pharmaceutical potential for use against cardiovascular diseases and cancer. Endophytic isolates of A. Gene expression and metabolomic activities of A. Metabolic adaptability based on nutrient availability is increasingly reported as a major modulator of physiological behavior of this fungus. However, detailed metabolic status of A. In this study, we addressed enzymatic, proteomic and transcriptomic responses of this fungus under sulfur limitation conditions.
In filamentous fungi, sulfur containing amino acids, methionine and cysteine, as well as inorganic sulfur are the most metabolized sulfur sources via the methionine-cysteine cycle [ 14 ]. Sulfur uptake, a key step in sulfur assimilation, is mediated by plasma membrane sulfate permease [ 15 ].
This enzyme is highly regulated by sulfur repression metabolite repression. In Aspergillus and Penicillium, this enzyme is encoded by two genes sutA and sutB sulfate transporter of SulP family [ 15 — 17 ]. In Neurospora crassa, sulfate permease I and II are encoded by cys and cys, respectively [ 18 , 19 ]. Transcription of the sulfate permease gene of fungi is strongly regulated by sulfur levels and is repressed by methionine supplementation in the medium [ 20 ].
Transcriptional regulation of sulfur metabolism is dependent on the Sulfur Metabolite Repression SMR system that consists of the metR gene encoding a bZIP transcriptional factor, which controls the expression of all sulfur metabolizing enzymes [ 14 , 21 , 22 ].
The sulfite molecule is further reduced to sulfide by sulfite reductase [ 21 ]. Subsequently, sulfide is incorporated with O-acetylserine or O-acetyl-homoserine to form cysteine and homocysteine by cysteine synthase and homocysteine synthase, respectively [ 19 ].
Simultaneously, cysteine and homocysteine undergo transsulfuration and reverse transsulfuration forming L-methionine, glutathione and polyamines [ 4 ]. Sulfur assimilation and metabolism have been extensively studied in filamentous fungi such as A. It plays an important role in the pathogenicity and virulence of A. From a pharmaceutical perspective, A.
However, there are no reports describing the kinetics of sulfate assimilation and metabolism of sulfur amino acids in A. The objective of this work was to study the molecular expression and proteomic profiling of A. The activity and expression of A. Relationships between gene expression and enzymatic activities of several key enzymes that were investigated in this report are discussed in the context of S availability and limitation.
Amino acids were filter-sterilized 0. Cultures were incubated at the same conditions as above up to 6 days. Fungal tissues were collected by filtration, washed with sterile potassium phosphate buffer pH 7.
Measurement of fungal morphology, growth kinetics, sulfur uptake and glutathione pool The effect of sulfur starvation on morphological growth of A.
After culturing of A. Total glutathione concentration of A. Briefly, the collected mycelial pellets 1. The mixture was centrifuged at xg for 15 min at RT and the supernatant was transferred to new tubes and neutralized with triethanolamine.
Assessment of sulfur metabolizing enzymes activity and intracellular protein For assaying total intracellular protein and activity of sulfur-metabolizing enzymes, mycelial pellets were collected and washed with mM potassium phosphate PP buffer, pH 7.
The homogenate was centrifuged at xg for 15 min and the supernatant was used as a source of enzymes as describe below [ 7 ]. The activity of ATP-sulfurylase was assessed using sodium molybdate assay [ 33 ]. Assay contained 0. The activity of sulfite reductase was measured by sulfide assay [ 34 ]. Blank reactions contained water instead of sodium bisulfite.
Immediately after cooling, 2 mM N,N-dimethyl-p-phenylenediamine in 6. After 20 min, the developed methylene blue color was measured at A nm a microtiter plate reader. The activity of cysteine synthase was determined [ 36 ]. Each reaction contained mM potassium phosphate pH 7. The mixture was boiled for 5 min and the developed color was measured at nm. Glutathione reductase was assayed based on NADH oxidation [ 31 ]. The concentration of total intracellular protein was assessed by Bradford assay Bio-Rad Assay Kit, cat — using bovine serum albumin as standard.
Differential expression of these genes as well as those of the sulfur transcriptional activator metR and sulfur controller scon genes [ 14 , 22 ] in response to sulfur starvation was determined using qPCR. The primer sequences for qPCR analysis of these genes are listed in Table 1.
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Hosts[ edit ] Aspergillus flavus is found globally as a saprophyte in soils and causes disease on many important agriculture crops. Common hosts of the pathogen are cereal grains, legumes, and tree nuts. Specifically, A. It is common for the pathogen to originate while host crops are still in the field; however, symptoms and signs of the pathogen are often unseen. In grains, the pathogen can invade seed embryos and cause infection, which decreases germination and can lead to infected seeds planted in the field. The pathogen can also discolor embryos, damage seedlings, and kill seedlings, which reduces grade and price of the grains.
Рост и распространение[ править править код ] Аспергиллы относят к грибам дейтеромицетам , не имеющим половой стадии развития. С появлением данных анализа ДНК стало более вероятно, что все члены рода аспергилл близкородственны аскомицетам , и их следует считать представителями аскомицетов. Как правило, грибы растут на богатых углеродом субстратах, таких как моносахариды к примеру, глюкоза и полисахариды например, амилоза. Виды аспергилл являются распространённым заражающим фактором крахмалистых продуктов таких, как хлеб и картофель и растут внутри либо на поверхности многих растений и деревьев.