14 research outputs found
Basic measurements of radiation at station Sapporo (2017-02)
<div><p>During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The <i>hpdA</i> gene of the dimorphic fungus <i>Penicillium marneffei</i>, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in <i>P</i>. <i>marneffei</i> inhibits <i>ex vivo</i> yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including <i>hpdA</i>, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (<i>hmgR</i>) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of <i>hpdA</i> results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells.</p></div
Genes of the tyrosine catabolic cluster are required for hyphal growth on tyrosine and phenylalanine as a nitrogen or carbon source at 25°C.
<p>Growth of the wildtype, Δ<i>wA</i>, Δ<i>hpdA</i>, Δ<i>hmgA</i>, Δ<i>hmgX</i>, Δ<i>maiA</i>, Δ<i>hmgR</i>, and Δ<i>areA</i> strains on carbon and nitrogen free medium (C and N free), on ammonium as the sole nitrogen source (gluc NH<sub>4</sub>), on phenylalanine as the sole nitrogen source (gluc phe), on phenylalanine as the sole carbon source (phe NH<sub>4</sub>), on tyrosine as the sole nitrogen source (gluc tyr) or on tyrosine as the sole carbon source (tyr NH<sub>4</sub>) after 14 days at 25°C.</p
AreA negatively regulates expression of tyrosine catabolism genes.
<p>A. RNA was isolated from wildtype (WT), Δ<i>hmgR</i> and Δ<i>areA</i> strains grown in liquid culture for 2 days at 25°C (A and C) or 6 days at 37°C (B) and transferred into media containing ammonium (NH<sub>4</sub>), alanine (Ala) or tyrosine (Tyr) as the sole nitrogen source at 25°C (A) or at 37°C (B) for 4 hours, or media containing tyrosine (Tyr) or both tyrosine and ammonium (Tyr NH<sub>4</sub>) at 25°C for 4 hours (C). Expression of <i>hpdA</i>, <i>maiA</i>, <i>fahA</i> and a <i>H3</i> loading control was detected by RT PCR.</p
Paralogues of tyrosine catabolic cluster genes.
<p>* The <i>hpdB</i> copy is truncated and lacks the cd07250 and cd08342 domains so is likely to be non-functional.</p><p><sup>#</sup> Copies not present in cluster.</p><p><sup><i>a</i></sup><i>T</i>. <i>stipitatus fahA</i> and <i>maiA</i> are misannotated in the <i>Talaromyces stipitatus</i> ATCC 10500 genomic database as a single fused gene named <i>fahA</i> (TSTA_065590).</p><p>Organisms are as follows: Pm; <i>Penicillium marneffei</i>, Ts; <i>Talaromyces stipitatus</i>, An; <i>Aspergillus nidulans</i>; Af; <i>Aspergillus fumigatus</i>, Hc; <i>Histoplasma capsulatum</i> and Pb; <i>Paracoccidioides brasiliensis</i>.</p><p>Paralogues of tyrosine catabolic cluster genes.</p
The tyrosine induced expression of genes of the catabolism cluster requires the HmgR transcription factor.
<p>RNA was isolated from wildtype (<i>hmgR</i><sup>+</sup>) and Δ<i>hmgR</i> strains grown in liquid culture for 2 days at 25°C or 6 days at 37°C and transferred into media containing ammonium (NH<sub>4</sub>) or tyrosine (Tyr) as the sole nitrogen source at 25°C or 37°C for 4 hours. Expression of <i>hpdA</i> (PMAA_031950), <i>hmgA</i> (PMAA_031960), <i>hmgX</i> (PMAA_031980), <i>fahA</i> (PMAA_031990), <i>maiA</i> (PMAA_032000), <i>mfpA</i> (PMAA_031010) and <i>hmgR</i> (PMAA_032020) was detected by RT PCR.</p
Pyomelanin produced via the tyrosine catabolism pathway is under nitrogen metabolite repression.
<p>A. Wildtype grown for 14 days at 37°C on ANM plus ammonium (NH<sub>4</sub>) or tyrosine (Tyr) as the sole nitrogen source or plus both ammonium and tyrosine (NH<sub>4</sub> Tyr). Pyomelanin production via tyrosine catabolism is under nitrogen metabolite repression. B. Wildtype P. marneffei grown on L-DOPA medium for 14 days at 37°C. C. Pyomelanin formation on ANM plus alanine and tyrosine for 14 days at 37°C in the wildtype, Δ<i>wA</i>, Δ<i>hpdA</i>, Δ<i>hmgA</i>, Δ<i>hmgX</i>, Δ<i>maiA</i>, Δ<i>hmgR and</i> Δ<i>areA</i> strains after 14 days growth at 37°C. D. Wildtype, Δ<i>hmgR</i> and Δ<i>areA</i> grown for 14 days at 37°C on ANM plus ammonium and tyrosine. The Δ<i>hmgR</i> mutant produces increased pyomelanin and the Δ<i>areA</i> mutant produces decreased pyomelanin under this growth condition.</p
Growth of yeast cells on tyrosine and phenylalanine as a nitrogen or carbon source at 37°C requires genes of the tyrosine catabolic cluster.
<p>Growth after 14 days at 37°C of the wildtype, Δ<i>wA</i>, Δ<i>hpdA</i>, Δ<i>hmgA</i>, Δ<i>hmgX</i>, Δ<i>maiA</i>, Δ<i>hmgR</i> and Δ<i>areA</i> strains on carbon and nitrogen free medium (C and N free), on ammonium as the sole nitrogen source (gluc NH<sub>4</sub>), on phenylalanine as the sole nitrogen source (gluc phe), on phenylalanine as the sole carbon source (phe NH<sub>4</sub>), on tyrosine as the sole nitrogen source (gluc tyr) or on tyrosine as the sole carbon source (tyr NH<sub>4</sub>).</p
The catabolism of tyrosine.
<p>A. Tyrosine is catabolised to the toxic compound 4-hydroxyphenylpyruvate by an unknown mechanism. Phenylalanine can be degraded by the prephenate pathway to 4-hydroxyphenylpyruvate. 4-hydroxyphenylpyruvate dioxygenase (HpdA) catalyses the conversion of 4-hydroxyphenylpyruvate to homogentisate. Homogentisate can either be oxidized and polymerized to form the brown pigment pyomelanin or metabolised to 4-maleylacetoacetate by homogentisate 1,2-dioxygenase (HmgA). 4-maleylacetoacetate is metabolised to 4-fumarylacetoacetate by maleylacetoacetate isomerase (MaiA). Fumarlacetoacetate hydrolase (FahA) catalyses the conversion of 4-fumarylacetoacetate to acetoacetate and fumarate, which can be utilized as carbon sources via the TCA cycle. B. Genes required for the catabolism of tyrosine are located in a gene cluster which is conserved in filamentous (<i>Talaromyces stipitatus</i>; Ts, <i>Aspergillus nidulans</i>; An and <i>Aspergillus fumigatus</i>; Af, light grey box) and dimorphic (<i>P</i>. <i>marneffei</i>; Pm, <i>Paracoccidioides brasiliensis</i>; Pb, <i>Coccidioides immitis</i>; Ci, <i>Histoplasma capsulatum</i>; Hc and <i>Blastomyces dermatitidis</i>; Bd, dark grey boxes) fungi. Tyrosine catabolism genes are coloured as follows: <i>hpdA</i>; red, <i>hmgA</i>; blue, <i>hypW</i>; aqua, <i>hmgX</i>; yellow, <i>fahA</i>; green, <i>maiA</i>; pink and <i>hmgR</i>; orange. Flanking genes (PMAA_031940, PMAA_032030, TSTA_065640, TSTA_065560, AN1900, AN1892, AFUA_2G04190, AFUA_2G04270, PADG_08469, PADG_08463, CIMG_01309, CIMG_01315, HCEG_08529, HCEG_08533, HCEG_03254, HCEG_03257, BDDG_05746, BDDG_05738, BDDG_08624 and BDDG_12986) with no characterized role in tyrosine catabolism are shown in grey (same shade if orthologous). <i>hypW</i> is present only in <i>P</i>. <i>marneffei</i> (PMAA_032010) and <i>T</i>. <i>stipitatus</i> (TSTA_065580). <i>A</i>. <i>nidulans</i> contains an internal gene (AN1894) with no characterized role in tyrosine catabolism (light green). <i>T</i>. <i>stipitatus fahA</i> and <i>maiA</i> are misannotated in the <i>Talaromyces stipitatus</i> ATCC 10500 genomic database as a single fused gene named <i>fahA</i> (TSTA_065590). The cluster has been divided into two in <i>H</i>. <i>capsulatum</i> and <i>B</i>. <i>dermatitidis</i>. <i>P</i>. <i>brasiliensis</i>, <i>C</i>. <i>immitis</i>, <i>H</i>. <i>capsulatum</i> and <i>B</i>. <i>dermatitidis</i> lack a <i>hmgR</i> orthologue. Gene orthologues used to generate this Figure are as follows: <i>hpdA</i> (PMAA_031950, TSTA_065630, AN1899, AFUA_2G04200, PADG_08468, CIMG_01310, HCEG_08530 and BDDG_05744), <i>hmgA</i> (PMAA_031960, TSTA_065620, AN1897, AFUA_2G04220, PADG_08466, CIMG_01312, HCEG_08532 and BDDG_05741), <i>hypW</i> (PMAA_031970 and TSTA_065610), <i>hmgX</i> (PMAA_031980, TSTA_065600, AN1898, AFUA_2G04210, PADG_08467, CIMG_01311, HCEG_08531 and BDDG_05742), <i>fahA</i> (PMAA_031990, TSTA_065590, AN1896, AFUA_2G04230, PADG_08465, CIMG_01313, HCEG_03255 and BDDG_08623), <i>maiA</i> (PMAA_032000, AN1895, AFUA_2G04240, PADG_08464, CIMG_01314, HCEG_03256 and BDDG_08618), <i>mfpA</i> (PMAA_031010, TSTS_065580), and <i>hmgR</i> (PMAA_032020, TSTA_065570, AN1893 and AFUA_2G04262).</p
Analysis of induction in cadmium chloride-treated cells transfected with TFBS-UR plasmids.
<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with cadmium. (A) Microarray-based detection of TF derived activation of UR expression. (B) qPCR-based detection of TF-derived activation of UR expression. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The grey bar represents treatment-independent changes in the system. TFBS marked with * represent treatment-dependent effects on the TF library. Numerical data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s004" target="_blank">Table S3</a>. A statistical analysis of the qPCR assay data is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone-0050521-g003" target="_blank">Figure 3</a>.</p
Induction of selected TFBS-directed UR expression in HEK293 cells after treatment with cadmium, dexamethasone, TPA and forskolin.
<p>HEK293 cells transfected with a plasmid pool, that included the plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050521#pone.0050521.s003" target="_blank">Table S2</a> and pRL-SV40 and were subsequently treated with drugs of interest. (A) MRE-directed UR expression after treatment with cadmium. (B) GRE-directed UR expression after treatment with dexamethasone. (C) NF-κB-directed UR expression after treatment with TPA. (D) CREB-directed UR expression after treatment with forskolin. Values are presented as log2 treatments of the fold induction of the TFBS-directed UR expression after treatment with the inducer of interest. The error bars are calculated as 1 standard error of the mean each way.</p