36 research outputs found

    HapX of <i>A</i>. <i>benhamiae</i> is important for transcription of iron regulatory genes during iron limitation.

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    <p>(A) Transcript levels of transcription factors HapX and SreA during iron starvation (-Fe) and iron-replete conditions (+Fe). (B) Transcript levels of the genes <i>cccA</i>, <i>hemA</i>, <i>lysF</i> and <i>cycA</i> during iron starvation (-Fe) and iron-replete conditions (+Fe). Transcript levels measured by quantitative RT-PCR analysis are presented relative to those of <i>A</i>. <i>benhamiae</i> wild type during iron-replete conditions. Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> mutant were statistically significant during iron starvation (2way ANOVA; ** significant at P < 0.01, *** significant at P < 0.001).</p

    HapX-dependent siderophore biosynthesis of <i>A</i>. <i>benhamiae</i> during iron starvation.

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    <p>(A) Quantification of extracellular siderophores produced by <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain during iron starvation (-Fe) and iron sufficiency (+Fe). Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> were statistically significant during iron starvation (2way ANOVA; *** significant at P < 0.001). (B) Quantification of the extracellular siderophores ferrichrome C and ferricrocin in supernatant extracts of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain after cultivation for 5 d at 30°C during iron starvation by HPLC analysis. Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> were statistically significant for ferrichrome C (2way ANOVA; * significant at P < 0.05) (C) Postulated biosynthesis pathway of the siderophores ferricrocin and ferrichrome C (based on the information from <i>A</i>. <i>fumigatus</i>) and genomic organization of the genes <i>sidC</i> (ARB_07686) and <i>sidA</i> (ARB_07687) of <i>A</i>. <i>benhamiae</i>. (D) Quantitative RT-PCR analysis of the transcript level of the genes <i>sidA</i> (ornithine monooxygenase), <i>sidC</i> (NRPS) and <i>hmg1</i> (HMG-CoA reductase) of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain during iron starvation (-Fe) and iron-replete conditions (+Fe). Transcript levels are presented relative to those of <i>A</i>. <i>benhamiae</i> wild type during iron-replete conditions. Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> were statistically significant during iron starvation (2way ANOVA; *** significant at P < 0.001).</p

    Generation of <i>A</i>. <i>benhamiae</i> Δ<i>hapX</i> mutants and reconstituted strains.

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    <p>(A) For deletion of the <i>hapX</i> locus (white arrow) in the wild-type strain <i>A</i>. <i>benhamiae</i> LAU2354-2 (bottom) a DNA cassette, containing the hygromycin resistance gene <i>hph</i> (black arrow) under control of the <i>gpd</i> promoter (P<sub><i>gpd</i></sub>, bent arrow) together with the termination sequence fragment T<sub><i>trpC</i></sub> (filled circle) flanked by <i>hapX</i> upstream and downstream regions (<i>hapX</i><sub>up</sub> and <i>hapX</i><sub>down</sub>, solid lines), was used (top). (B) For reinsertion of the <i>hapX</i> gene into its original locus in the Δ<i>hapX</i> mutants a DNA cassette, containing the coding region of <i>hapX</i> and the neomycin resistance gene <i>neo</i> (lined arrow) under control of the <i>A</i>. <i>benhamiae</i> actin promoter (P<sub><i>ACT1</i></sub>, bent arrow) together with the <i>Candida albicans</i> actin termination sequence fragment T<sub><i>ACT1</i></sub> (blank circle) flanked by <i>hapX</i> upstream and downstream regions (<i>hapX</i><sub>up</sub> and <i>hapX</i><sub>down</sub>, solid lines), was used. (C) Southern blot of <i>Nde</i>I-digested genomic DNA of the wild-type strain <i>A</i>. <i>benhamiae</i> LAU2354-2, Δ<i>hapX</i> mutants and <i>hapX</i><sup><i>C</i></sup> reconstituted strains with <i>hapX</i>-specific probe 1. The probes which were used for Southern analysis of the transformants are indicated by the black bars. Only the following relevant restriction sites are given in panels a and b: A, <i>Apa</i>I; B, <i>Bam</i>HI; H, <i>Hin</i>dIII; Nd, <i>Nde</i>I; <i>Not</i>I. The sizes of the hybridizing fragments are given on the left and their identities on the right.</p

    Identification of siderophores produced by <i>A</i>. <i>benhamiae</i>.

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    <p>(A) HPLC chromatogram of the lyophilized culture supernatants of <i>A</i>. <i>benhamiae</i>. Ferrichrome C and ferricrocin were identified as extracellular siderophores. (B) Chemical structures and molecular masses of the siderophores ferrichrome C and ferricrocin produced by <i>A</i>. <i>benhamiae</i>.</p

    HapX of <i>A</i>. <i>benhamiae</i> is important for growth, conidiation and hyphal pigmentation during iron starvation.

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    <p>(A) Cultivation of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain in AMM during iron-replete conditions (+Fe), iron limitation (-Fe), harsh iron starvation (-Fe +BPS) and in the presence of the xenosiderophore deferoxamine (-Fe + DFOM). Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> mutant were statistically significant during iron starvation and in the presence of BPS and DFOM (2way ANOVA; * significant at P < 0.05, ** significant at P < 0.01, *** significant at P < 0.001). (B) Growth of the fungal strains in AMM during iron-replete conditions (+Fe) and iron starvation (-Fe) for 5 d at 30°C led to the formation of a reddish pigmented mycelium of Δ<i>hapX</i> mutant, particularly during iron deficiency.</p

    HapX of <i>A</i>. <i>benhamiae</i> is dispensable for growth on keratin substrates.

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    <p>Cultivation of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain on human hair, finger nails and keratin powder derived from hooves and horns. Scale bar represents 5 mm.</p

    HapX of <i>A</i>. <i>benhamiae</i> under high iron concentrations.

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    <p>(A) Cultivation of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain during iron starvation (-Fe), high iron concentrations (1–10 mM FeSO<sub>4</sub>) and in the presence of the iron chelator BPS on solid medium for 7 d at 30°C. Scale bar represents 5 mm. (B) Cultivation of <i>A</i>. <i>benhamiae</i> wild type, Δ<i>hapX</i> mutant and <i>hapX</i><sup><i>C</i></sup> reconstituted strain in AMM with high iron concentrations (1–7 mM FeSO<sub>4</sub>). Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> mutant were statistically significant between 5 mM and 7 mM Fe (2way ANOVA; ** significant at P < 0.01, *** significant at P < 0.001). (C) Quantitative RT-PCR analysis of the <i>cccA</i> gene under different iron concentrations. The mycelium was cultivated under high iron concentrations (hFe) or shifted for 1 h from -Fe to +Fe (sFe). Data represent the means and standard deviations of three biological replicates. The differences between wild type and Δ<i>hapX</i> mutant were statistically significant during sFe (2way ANOVA; *** significant at P < 0.001).</p

    Additional file 5 of Additional oxidative stress reroutes the global response of Aspergillus fumigatus to iron depletion

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    Results of RT-qPCR measurements. Relative transcription levels were quantified with ΔΔCP = ΔCPtreated – ΔCPcontrol. ΔCPtreated = CPreference gene - CPtested gene measured from treated cultures. ΔCPcontrol = CPreference gene - CPtested gene measured from control cultures or from the iron depleted cultures. CP values stand for the qRT-PCR cycle numbers of crossing points. The fks1 gene was used as reference gene. qRT-PCR data are presented as the mean and S.D. data calculated from three measurements. Significantly higher or lower than 0 ΔΔCP values (up- or down-regulated gene) are marked with red and blue colors, respectively (Student’s t-test, p < 0.05, n = 3). (XLS 42 kb

    Network Modeling Reveals Cross Talk of MAP Kinases during Adaptation to Caspofungin Stress in <i>Aspergillus fumigatus</i>

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    <div><p>Mitogen activated protein kinases (MAPKs) are highly conserved in eukaryotic organisms. In pathogenic fungi, their activities were assigned to different physiological functions including drug adaptation and resistance. <i>Aspergillus fumigatus</i> is a human pathogenic fungus, which causes life-threatening invasive infections. Therapeutic options against invasive mycoses are still limited. One of the clinically used drugs is caspofungin, which specifically targets the fungal cell wall biosynthesis. A systems biology approach, based on comprehensive transcriptome data sets and mathematical modeling, was employed to infer a regulatory network and identify key interactions during adaptation to caspofungin stress in <i>A</i>. <i>fumigatus</i>. Mathematical modeling and experimental validations confirmed an intimate cross talk occurring between the cell wall-integrity and the high osmolarity-glycerol signaling pathways. Specifically, increased concentrations of caspofungin promoted activation of these signalings. Moreover, caspofungin affected the intracellular transport, which caused an additional osmotic stress that is independent of glucan inhibition. High concentrations of caspofungin reduced this osmotic stress, and thus decreased its toxic activity. Our results demonstrated that MAPK signaling pathways play a key role during caspofungin adaptation and are contributing to the paradoxical effect exerted by this drug.</p></div
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