13 research outputs found
Elevated catalase expression in a fungal pathogen is a double-edged sword of iron
We thank our colleagues in the Aberdeen Fungal Group, Lloyd Peck (British Antarctic Survey) and John Helmann (Cornell University) for insightful discussions. We thank Christophe dâEnfert and Melanie Legrand (Institut Pasteur) for help with the design of barcodes and provision of the CIp10-PTET-GTw overexpression vector and CEC2908 strain. We are grateful to the following Core Facilities at the University of Aberdeen for their excellent technical assistance, advice and support: the Medical Research Facility; the Centre for Genome Enabled Biology and Medicine; the Iain Fraser Cytometry Centre; the Microscopy and Histology Facility; Aberdeen Proteomics; and the qPCR Facility.Peer reviewedPublisher PD
Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins
Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins
Impact of catalase on resistance to neutrophil killing.
<p><i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, black, Ca2084), <i>cat1</i>Î (red, Ca Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) pre-grown with 0 or 20 with ÎŒM doxycycline (pale blue and blue, respectively) were exposed to human neutrophils for 2 h, and then fungal survival assayed. Each data point represents the mean for three replicates from one healthy donor. The data were analysed using one-way ANOVA with Tukeyâs post-hoc test: *, <i>p</i> †0.05; **, <i>p</i> †0.01.</p
Manipulation of catalase levels in <i>C</i>. <i>albicans</i>.
<p>Catalase activities were measured in protein extracts from mid-exponential <i>C</i>. <i>albicans</i> cultures containing 0 or 20 ÎŒM doxycycline (- or + Dox, respectively): <i>cat1</i>Î, Ca2089; wild-type, WT, Ca2084; red, <i>tetON-CAT1</i> isolates, Ca2040, Ca2043, Ca2046 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). Immediately before harvesting, wild-type and <i>cat1</i>Î cultures were exposed to 0 or 5 mM H<sub>2</sub>O<sub>2</sub> for one hour. Means and standard deviations from three independent replicate experiments are shown, and the data were analysed using one-way ANOVA with Tukeyâs post-hoc test: *, <i>p</i> †0.05; **, <i>p</i> †0.01; ***, <i>p</i> †0.001; ****, <i>p</i> †0.0001.</p
In the absence of stress, elevated catalase levels impose a fitness defect on <i>C</i>. <i>albicans</i> that is suppressed by iron supplementation.
<p><b>(A)</b> The growth of <i>C</i>. <i>albicans tetON-CAT1</i> isolates (1, 4, 10) was monitored (OD<sub>600</sub>) in YPD containing 0 or 20 ÎŒM doxycycline (Ca2040, Ca2043, Ca2046; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). The impact of <i>tetON-CAT1</i> induction upon fitness was assayed by subtracting the OD after growth in the absence of doxycycline (OD<sub>-Dox</sub>) from the OD in the presence of doxycycline (OD<sub>+Dox</sub>). Data represent the means and standard deviations from three independent experiments. <b>(B)</b> The impact of iron on the fitness of <i>C</i>. <i>albicans tetON-CAT1</i> strain 1 (Ca2040) was measured in YPD cultures containing 0 or 20 ÎŒM doxycycline plus different concentrations of FeCl<sub>3</sub>. Data represent the means and standard deviations from three independent experiments. <b>(C)</b> The effect of <i>tetON-CAT1</i> expression on genes involved in iron assimilation and homeostasis was assessed by qRT-PCR of specific transcripts (relative to the <i>ACT1</i> mRNA internal control) during growth of <i>C</i>. <i>albicans tetON-CAT1</i> isolate 1 (Ca2040) in YPD containing 0 or 20 ÎŒM doxycycline. The data, which represent the means and standard deviations from three replicate measurements, are normalised relative to the corresponding transcript level in wild type cells grown with doxycycline.</p
Impact of catalase levels on host colonisation during systemic infection.
<p>The nine <i>C</i>. <i>albicans</i> strains were grown separately in medium containing 0 or 20 ΌM doxycycline, mixed in approximately equal proportions, and then this pool of nine barcoded strains used to initiate systemic infection in mice via the tail vein (n = 6 mice per group): wild-type (<i>CAT1</i>) strains 23, 21, 26 (Ca2084, Ca2085, Ca2087); <i>cat1</i>Πstrains 28, 38, 54 (Ca2089, Ca2092, Ca2030); <i>tetON-CAT1</i> strains 1, 4, 10, (Ca2038, Ca2041, Ca2044; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). Mice that received the pool of nine <i>C</i>. <i>albicans</i> strains pre-grown with doxycycline were treated with doxycycline, whereas mice that were infected with the pool of <i>C</i>. <i>albicans</i> strains pre-grown without doxycycline did not (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#sec011" target="_blank">Materials and Methods</a>). After four days, mice were killed by cervical dislocation and the kidneys, brains, livers and spleens harvested. The fungal cells infecting these organs were grown on YPD plates, genomic DNA isolated from each fungal population, and the abundance of each barcode in each fungal population was quantified by barseq. The Relative Abundance of each barcode in each tissue from each mouse was calculated relative to the total number of barcode reads for that specific sample, and then normalised against the proportion for that barcode in the pool of nine <i>C</i>. <i>albicans</i> strains used to infect that mouse. Each symbol represents data for a single mouse. The pairwise comparisons indicated were analysed using the Students t-test: *, <i>p</i> †0.05; **, <i>p</i> †0.01. Data are presented for three of the nine barcoded <i>C</i>. <i>albicans</i> strains: <i>CAT1</i> strain 23, grey and black; <i>cat1</i>Πstrain 28, pink and red; <i>tetON-CAT1</i> strain 1, pale blue and blue; no doxycycline, grey, pink, pale blue; 20 ΌM doxycycline, black, red, blue. These wild type and <i>cat1</i>Πstrains were representative of the other isolates. However, <i>tetON-CAT1-</i>1 behaved differently from the other <i>tetON-CAT1</i> isolates, which were shown subsequently to have lost their phenotype (below).</p
Stochastic differences in catalase expression within a population of <i>C</i>. <i>albicans</i> cells affect resistance to peroxide stress.
<p><b>(A)</b> Western blot of GFP in <i>C</i>. <i>albicans</i> cells grown in YPD at 30°C: GFP, cells expressing GFP from pACT1-GFP (Ca230); <i>CAT1</i>, control cells with no GFP (Ca674); <i>CAT1-GFP</i>, cells expressing Cat1-GFP (Ca2213) (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006405#ppat.1006405.s006" target="_blank">S1 Table</a>). <b>(B)</b> DIC and fluorescence microscopy of log-phase <i>CAT1-GFP</i> cells (Ca2213) exposed to 0 or 5 mM H<sub>2</sub>O<sub>2</sub> for 1 h: scale bar = 5 ÎŒm. <b>(C)</b> The resistance of <i>C</i>. <i>albicans</i> strains to peroxide was assessed by plating onto YPD plates containing 0, 5 or 10 mM H<sub>2</sub>O<sub>2</sub>: <i>CAT1/CAT1</i>, Ca674; <i>CAT1/cat1</i>Î, Ca1862; <i>cat1</i>Î/<i>cat1</i>Î, Ca1864; <i>CAT1-GFP</i>, Ca2213. For each stress condition, the images for all strains were taken from the same plate. <b>(D)</b> Sorting of <i>C</i>. <i>albicans</i> cells of similar size expressing relatively low or high levels of Cat1-GFP within the same population growing on YPD (no stress) by FACS: Cat1-GFP cells, Ca2213; No GFP control, Ca674. <b>(E)</b> The survival of FACS sorted cells with low or high Cat1-GFP levels on YPD plates containing different concentrations of H<sub>2</sub>O<sub>2</sub> (CFU) expressed as a percentage of the survival for the no stress control. Means and standard deviations from three replicates are presented: *, <i>p</i> †0.05; **, <i>p</i> †0.01; ***, <i>p</i> †0.001; ****, <i>p</i> †0.0001.</p
Impact of catalase on resistance to neutrophil killing.
<p><i>C</i>. <i>albicans</i> wild-type (<i>CAT1</i>, black, Ca2084), <i>cat1</i>Î (red, Ca Ca2089) and <i>tetON-CAT1</i> cells (Ca2038) pre-grown with 0 or 20 with ÎŒM doxycycline (pale blue and blue, respectively) were exposed to human neutrophils for 2 h, and then fungal survival assayed. Each data point represents the mean for three replicates from one healthy donor. The data were analysed using one-way ANOVA with Tukeyâs post-hoc test: *, <i>p</i> †0.05; **, <i>p</i> †0.01.</p