15 research outputs found
Table_1_Genomic Organization and Expression of Iron Metabolism Genes in the Emerging Pathogenic Mold Scedosporium apiospermum.DOCX
<p>The ubiquitous mold Scedosporium apiospermum is increasingly recognized as an emerging pathogen, especially among patients with underlying disorders such as immunodeficiency or cystic fibrosis (CF). Indeed, it ranks the second among the filamentous fungi colonizing the respiratory tract of CF patients. However, our knowledge about virulence factors of this fungus is still limited. The role of iron-uptake systems may be critical for establishment of Scedosporium infections, notably in the iron-rich environment of the CF lung. Two main strategies are employed by fungi to efficiently acquire iron from their host or from their ecological niche: siderophore production and reductive iron assimilation (RIA) systems. The aim of this study was to assess the existence of orthologous genes involved in iron metabolism in the recently sequenced genome of S. apiospermum. At first, a tBLASTn analysis using A. fumigatus iron-related proteins as query revealed orthologs of almost all relevant loci in the S. apiospermum genome. Whereas the genes putatively involved in RIA were randomly distributed, siderophore biosynthesis and transport genes were organized in two clusters, each containing a non-ribosomal peptide synthetase (NRPS) whose orthologs in A. fumigatus have been described to catalyze hydroxamate siderophore synthesis. Nevertheless, comparative genomic analysis of siderophore-related clusters showed greater similarity between S. apiospermum and phylogenetically close molds than with Aspergillus species. The expression level of these genes was then evaluated by exposing conidia to iron starvation and iron excess. The expression of several orthologs of A. fumigatus genes involved in siderophore-based iron uptake or RIA was significantly induced during iron starvation, and conversely repressed in iron excess conditions. Altogether, these results indicate that S. apiospermum possesses the genetic information required for efficient and competitive iron uptake. They also suggest an important role of the siderophore production system in iron uptake by S. apiospermum.</p
Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and <i>HSP90</i> gene deleted to evaluate BER MIC<sub>50</sub>, (b) end point comparative RTPCR of genes involved in CW integrity in WT <i>C. albicans</i> cells in presence and absence of BER, (c) and in <i>HSF1</i> conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.
<p>Effect of BER on CW integrity mutants (a) serial dilution assay of calcineurin and MAP kinase pathway and <i>HSP90</i> gene deleted to evaluate BER MIC<sub>50</sub>, (b) end point comparative RTPCR of genes involved in CW integrity in WT <i>C. albicans</i> cells in presence and absence of BER, (c) and in <i>HSF1</i> conditional mutant lane indicates 1: WT, 2: HSF1 TET/hsf1, 3: HSF1/hsf1, 4,5,6,: +Doxy, 7,8,9 :+BER, 10, 11, 12: +Doxy+Ber.</p
Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT <i>C. albicans</i> cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.
<p>Determination of endogenous ROS generation by BER and induction of apoptosis (a) (upper panel) bar graph representing relative fluorescent units when cells were treated with DCFDA in presence and absence of BER, AA is added to revert the ROS production, (lower panel) fluorescent microscopy images of WT <i>C. albicans</i> cells labeled with DCFDA, (b) Cytometric determination FITC Annexin V labeling in WT cells treated with BER.</p
<i>HSF1</i> conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous in presence of BER.
<p><i>HSF1</i> conditional mutant is susceptible to various antifungal drugs (a) susceptibility WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous for BER (b) different classes of antifungal drugs; FLC, CAS, TRB, AMB, and their combination with BER, (c) CW perturbing agents; CFW, CR, SDS (d) TEM images of WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous in presence of BER.</p
TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of <i>HSF1</i> (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.
<p>TF mutant library screening (a) Serial dilution assays of TF mutant strains in the presence of BER, (b) end point comparative RTPCR of <i>HSF1</i> (gene deleted in JMR044) in WT strain (DAY286) in presence and absence of BER.</p
Model depicting pathways affected by BER treatment in <i>C. albicans</i>.
<p>Model depicting pathways affected by BER treatment in <i>C. albicans</i>.</p
Antifungal potential of BER (a) Growth curve of WT <i>C. albicans</i> cells at 100, 150 and 200 µg/ml, (b) serial dilution assays in solid (left panel) and liquid medium for testing BER susceptibility of <i>C. albicans</i> and non albicans species.
<p>(c) Serial dilution assays of <i>CDR1</i> (Gu5) and <i>MDR1</i> (F5) overexpressing and (d) their deletions strains in presence of BER.</p
BER treatment results in dysfunctional mitochondria (a) growth of <i>C. albicans</i> cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in <i>C. albicans</i> WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous strains in presence and absence of BER.
<p>BER treatment results in dysfunctional mitochondria (a) growth of <i>C. albicans</i> cells in non-fermentable carbon source (glycerol) in presence of BER (b) MTR labeling of the active mitochondria by FACS in <i>C. albicans</i> WT cells in presence and absence of BER, bar graph representing number of events gated (c) MTR labeling were also done in WT, <i>HSF1</i> conditional mutant and <i>HSF1</i> heterozygous strains in presence and absence of BER.</p
Gold labeling of cell wall mannan groups in <i>S</i>. <i>boydii</i> germ tubes.
<p>Germ tubes labeled with gold-conjugated concanavalin A (Con A; 5-nm gold particles) showing higher affinity of gold particles to the hyphal part (H) of germ tubes compared to the mother cell (MC) under transmission electron microscopy. Arrow indicates the limit of the outer cell wall layer of the mother cell. Bar: 0.5 ÎĽm.</p
Fluorescence labeling of <i>S</i>. <i>boydii</i> surface carbohydrates with FITC-conjugated lectins.
<p>Germ tubes after labeling with concanavalin A (<b>A</b> and <b>C</b>) or wheat germ agglutinin (<b>B</b> and <b>D</b>) lectins. The same fields are presented under fluorescence (<b>A</b> and <b>B</b>) and phase contrast microscopy (<b>C</b> and <b>D</b>) respectively. Arrows indicate mother cells.</p