24 research outputs found
Assays for <i>HAC1</i> mRNA splicing.
<p>(A) RT-PCR analysis. The <i>S. cerevisiae</i> strains containing either empty vector or pRS415-ADH-CgIRE1, in which <i>C. glabrata IRE1</i> was expressed under the control of the <i>S. cerevisiae ADH1</i> promoter, were incubated in SC-leu broth in the presence and absence of 1.5 µg/ml tunicamycin (TM) for 3 h. <i>S. cerevisiae</i> strains: WT, BY42-1; Δ<i>ire1</i>, BY42I-1; and Δ<i>ire1</i>+<i>CgIRE1</i>, BY42I-2. The <i>C. glabrata</i> wild-type strain CBS138 was incubated in SC broth in the presence and absence of TM and dithiothreitol (DTT) at the indicated concentrations for 1 and 3 h. RT-PCR products of the entire <i>HAC1</i> mRNA in <i>S. cerevisiae</i> (left panel) and <i>C. glabrata</i> (right panel) were electrophoresed on a 1% agarose gel. (B) Northern blot analysis. <i>S. cerevisiae</i> WT (BY4742) cells were treated with 1.5 µg/ml TM or 5 mM DTT for 1 h (left panel). <i>C. glabrata</i> cells were treated with 10 µg/ml TM or 10 mM DTT for 3 h (right panel). Both <i>ScHAC1</i> and <i>CgHAC1</i> probes were generated from the 5′ regions of the <i>HAC1</i> ORFs. The same blot was probed for <i>HAC1</i> mRNA, stripped, and then probed for <i>ACT1</i> mRNA. The asterisks indicate potential splicing intermediates (*: 5′ exon plus the intron; **: 5′ exon alone). <i>C. glabrata</i> strains: WT, CBS138; Δ<i>ire1</i>, TG121; and Δ<i>hac1</i>, TG141.</p
qRT-PCR validation of transcriptional profiles in the presence of tunicamycin (TM).
<p>Logarithmic-phase <i>C. glabrata</i> cells were incubated in the presence and absence of 10 µg/ml TM. qRT-PCR was performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003160#s4" target="_blank">Materials and Methods</a>. The means and standard deviations for three independent experiments are shown. <i>C. glabrata</i> strains: WT, 2001T; Δ<i>cnb1</i>, TG161; Δ<i>crz1</i>, TG171; Δ<i>slt2</i>, TG151; Δ<i>ire1</i>, TG121; and Δ<i>hac1</i>, TG141.</p
Dissection of Ire1 functions required for the ER stress response in <i>C. glabrata</i>.
<p>(A) Both the kinase and nuclease activity of Ire1 are required for cell growth in the presence of ER stress. The <i>C. glabrata</i> Δ<i>ire1</i> mutant was transformed with an empty vector or a plasmid containing wild-type <i>IRE1</i>, kinase-dead <i>IRE1</i> (<i>IRE1</i>-KD), or nuclease-dead <i>IRE1</i> (<i>IRE1</i>-ND). Logarithmic-phase cells were adjusted to 2×10<sup>7</sup> cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto synthetic complete medium without tryptophan (SC-trp) plates in the presence and absence of tunicamycin (TM) and dithiothreitol (DTT) at the indicated concentrations. Plates were incubated at 30°C for 48 h. <i>C. glabrata</i> strains: WT+vector, TG11; Δ<i>ire1</i>+vector, TG122; Δ<i>ire1</i>+<i>IRE1</i>, TG123; Δ<i>ire1</i>+<i>IRE1</i>-ND, TG125; and Δ<i>ire1</i>+<i>IRE1</i>-KD, TG124.</p
Functional complementation assays of the <i>IRE1</i> and <i>HAC1</i> orthologs in <i>S. cerevisiae</i> and <i>C. glabrata</i>.
<p>(A) <i>C. glabrata IRE1</i> was expressed under the control of the <i>S. cerevisiae ADH1</i> promoter in the <i>S. cerevisiae</i> Δ<i>ire1</i> mutant. Logarithmic-phase cells were adjusted to 2×10<sup>7</sup> cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto agar plates in the presence and absence of 0.5 µg/ml tunicamycin (TM). Plates were incubated at 30°C for 48 h. <i>S. cerevisiae</i> strains: WT+vector, BY42-1; Δ<i>scire1</i>+vector, BY42I-1; and Δ<i>scire1</i>+<i>CgIRE1</i>, BY42I-2. (B) <i>C. glabrata HAC1</i> was expressed under the control of the <i>S. cerevisiae ADH1</i> promoter in the <i>S. cerevisiae</i> Δ<i>hac1</i> mutant. Expression levels of representative UPR target genes in <i>S. cerevisiae</i> were analyzed by qRT-PCR as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003160#s4" target="_blank">Materials and Methods</a>. Results are presented as fold expression relative to the levels in the wild-type control. The means and standard deviations for three independent experiments are shown. <i>S. cerevisiae</i> strains: WT, BY42-2; schac1, BY42H-1; and schac1+CgHAC1, BY42H-2. (C) <i>C. glabrata HAC1</i> was expressed under the control of the <i>S. cerevisiae ADH1</i> promoter in the <i>S. cerevisiae</i> Δ<i>hac1</i> and Δ<i>ire1</i> mutants. The assay was performed as in part A. <i>S. cerevisiae</i> strains: WT+vector, BY42-2; Δ<i>schac1</i>+vector, BY42H-1; and Δ<i>schac1</i>+<i>CgHAC1</i>, BY42H-2; Δ<i>scire1</i>+vector, BY42I-3; and Δ<i>scire1</i>+<i>CgHAC1</i>, BY42I-4. (D) The induced form of <i>S. cerevisiae HAC1</i>, denoted as <i>ScHAC1<sup>i</sup></i>, was expressed under the control of the <i>S. cerevisiae PGK1</i> promoter in the <i>S. cerevisiae</i> Δ<i>hac1</i> and Δ<i>ire1</i> mutants. The assay was performed as in part A. <i>S. cerevisiae</i> strains: WT+vector, BY42-2; Δ<i>schac1</i>+vector, BY42H-1; and Δ<i>schac1</i>+<i>ScHAC1<sup>i</sup></i>, BY42H-3; Δ<i>scire1</i>+vector, BY42I-3; and Δ<i>scire1</i>+<i>ScHAC1<sup>i</sup></i>, BY42I-5. (E) <i>ScHAC1<sup>i</sup></i> was expressed under the control of the <i>S. cerevisiae PGK1</i> promoter in the <i>C. glabrata</i> wild-type and Δ<i>ire1</i> mutant strains. The assay was performed as in part A except TM concentration (1.5 µg/ml). <i>C. glabrata</i> strains: WT+vector, TG11; WT+<i>ScHAC1<sup>i</sup></i>, TG13; Δ<i>cgire1</i>+vector, TG122; and Δ<i>cgire1</i>+<i>ScHAC1<sup>i</sup></i>, TG126.</p
Genome-wide gene expression profiles in response to tunicamycin (TM) exposure for 3 h.
<p>Hierarchical clustering of genes whose expression levels were changed more than 2-fold after treatment with 1.5 µg/ml TM for 3 h. Genes were clustered with centroid linkage. <i>C. glabrata</i> strains: WT, 2001T; Δ<i>cnb1</i>, TG161; Δ<i>crz1</i>, TG171; Δ<i>slt2</i>, TG151; and Δ<i>ire1</i>, TG121.</p
Effects of <i>IRE1</i> deletion on stress response in wild-type and Δ<i>cnb1</i> backgrounds in <i>C. glabrata</i>.
<p>(A) Ire1 plays a role in azole tolerance in <i>C. glabrata</i> when calcineurin is absent. Logarithmic-phase cells of each <i>C. glabrata</i> strain were adjusted to 2×10<sup>7</sup> cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto synthetic complete (SC) plates containing either fluconazole (FLC) or voriconazole (VRC) at the indicated concentrations. Plates were incubated at 30°C for 48 h. MICs were determined by a broth microdilution method. (B) Ire1 and calcineurin serve redundant roles in cell growth under certain stress conditions in <i>C. glabrata</i>. The assay was performed as in part A. <i>C. glabrata</i> strains: Wild-type, CBS138; Δ<i>ire1</i>, TG121; Δ<i>cnb1</i>, TG161; and Δ<i>cnb1</i> Δ<i>ire1</i>, TG1612.</p
Sequence analysis of <i>C. glabrata</i> Hac1.
<p>(A) Sequence alignment of bZIP domains in Hac1 homologs from fungi and humans. The DNA binding domain is underlined. GenBank accession number: <i>Candida glabrata</i> Hac1, XP_448761; <i>Saccharomyces cerevisiae</i> Hac1, NP_116622; <i>Candida albicans</i> Hac1, XP_718538; <i>Aspergillus fumigatus</i> HacA, XP_748727; <i>Cryptococcus neoformans</i> Hxl1, XP_568439; and <i>Homo sapiens</i> Xbp1, NP_005071. (B) A schematic representation of the putative splice sites and ORF lengths in <i>C. glabrata</i> and <i>S. cerevisiae HAC1</i>. The <i>HAC1</i> ORFs and bZIP domains were shown in grey and blue boxes, respectively. In <i>S. cerevisiae</i>, the intron (purple box) in the uninduced form of <i>HAC1</i> (<i>HAC1<sup>u</sup></i>) is excised in the induced form of <i>HAC1</i> (<i>HAC1<sup>i</sup></i>). Red arrowheads and asterisks indicate putative splice sites and stop codons, respectively. Blue arrows indicate primers that were used for RT-PCR assays to examine <i>HAC1</i> splicing. (C) Alignment of the RNA sequence surrounding the predicted intron in <i>C. glabrata</i> and <i>S. cerevisiae HAC1</i>. In <i>S. cerevisiae</i>, <i>HAC1</i> mRNA is known to form stem-loop structures that enclose the intron between the two loops of seven residues (shown in red) held in place by short stems (yellow and green boxes). The predicted 5′ and 3′ splice sites and intron lengths in the <i>S. cerevisiae</i> and <i>C. glabrata HAC1</i> mRNAs are indicated.</p
<i>C. glabrata</i> Ire1 is required for virulence in a murine model of disseminated candidiasis.
<p>(A) Groups of 8 immunocompetent mice were intravenously inoculated with 8×10<sup>7</sup> cells for each <i>C. glabrata</i> strain. Bilateral kidneys and spleen were excised 7 days after injection. Appropriate dilutions of organ homogenates were plated, and the numbers of CFU were counted after 2 days of incubation at 30°C. Numbers of recovered CFU from each organ are indicated for individual mice in the scatter plots. The geometric mean is shown as a bar. Statistical analyses were performed using the Kruskal-Wallis test with Dunn's multiple comparison post-test. Asterisks indicate statistically significant differences (*: <i>P</i><0.05; **: <i>P</i><0.01; ***: <i>P</i><0.001). NS indicates no significance (<i>P</i>>0.05). Representative data of two independent experiments are shown. <i>C. glabrata</i> strains: Wild-type, TG11; Δ<i>ire1</i>, TG122; and Δ<i>ire1</i>+<i>IRE1</i>, TG123. (B) Groups of 7 mice were immunosuppressed by intraperitoneal administration of cyclophosphamide (200 mg/kg/day) 72, 48, and 24 h before challenge with <i>C. glabrata</i> cells. The mice were infected intravenously with <i>C. glabrata</i> cells (2×10<sup>7</sup> and 2×10<sup>6</sup> cells/mouse) on Day 0 of the experiment, and survival was monitored for 12 days post-infection. Kaplan-Meier curves were created and compared with the log rank (Mantel-Cox) test. Upper panel (2×10<sup>7</sup> cells/mouse): <i>P</i> = 0.0078 for wild-type vs. Δ<i>ire1</i>, <i>P</i> = 0.0284 for Δ<i>ire1</i> vs. Δ<i>ire1</i>+<i>IRE1</i>, and <i>P</i> = 0.6768 for wild-type vs. Δ<i>ire1</i>+<i>IRE1</i>; lower panel (2×10<sup>6</sup> cells/mouse): <i>P</i> = 0.0043 for wild-type vs. Δ<i>ire1</i>, <i>P</i> = 0.0222 for Δ<i>ire1</i> vs. Δ<i>ire1</i>+<i>IRE1</i>, and <i>P</i> = 0.7440 for wild-type vs. Δ<i>ire1</i>+<i>IRE1</i>.</p
Multiple signaling pathways are coordinately involved in the ER stress response in <i>C. glabrata</i>.
<p>(A) The <i>C. glabrata</i> Δ<i>ire1</i>, Δ<i>cnb1</i>, Δ<i>crz1</i>, and Δ<i>slt2</i> deletion mutants were transformed with either an empty vector or a plasmid containing the corresponding wild-type gene. Logarithmic-phase cells were adjusted to 2×10<sup>7</sup> cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto synthetic complete medium without tryptophan (SC-trp) plates in the presence and absence of tunicamycin (TM) and dithiothreitol (DTT) at the indicated concentrations. Plates were incubated at 30°C for 48 h. (B) Time-kill analysis of the <i>C. glabrata</i> deletion mutants in the presence of TM. Logarithmic-phase cells (5×10<sup>5</sup> CFU/ml) were incubated in SC medium containing 1.5 µg/ml TM. The number of viable cells was determined by plating the appropriate dilutions on YPD plates at the indicated time points. Data are expressed as the percentages of viability relative to the untreated (time point 0) control in each strain. The means and standard deviations for three independent experiments are shown. <i>C. glabrata</i> strains: Wild-type, CBS138; Vector control, TG11; Δ<i>ire1</i>, TG122; Δ<i>ire1</i>+<i>IRE1</i>, TG123; Δ<i>cnb1</i>, TG162; Δ<i>cnb1</i>+<i>CNB1</i>, TG163; Δ<i>crz1</i>, TG172; Δ<i>crz1</i>+<i>CRZ1</i>, TG173; Δ<i>slt2</i>, TG152; Δ<i>slt2</i>+<i>SLT2</i>, TG153; Δ<i>cnb1</i> Δ<i>ire1</i>, TG1612; Δ<i>crz1</i> Δ<i>ire1</i>, TG1712; and Δ<i>slt2</i> Δ<i>ire1</i>, TG1512.</p
Growth assay in the presence of ER stress-inducing agents.
<p>Logarithmic-phase cells of <i>S. cerevisiae</i> and <i>C. glabrata</i> strains were adjusted to 2×10<sup>7</sup> cells/ml, and then 5 µl of serial 10-fold dilutions were spotted onto synthetic complete (SC) plates containing either tunicamycin (TM) or dithiothreitol (DTT) at the indicated concentrations. Plates containing TM and DTT were incubated at 30°C for 24 and 48 h, respectively. <i>S. cerevisiae</i> strains: WT, BY4742; Δ<i>ire1</i>, BY4742Δire1; and Δ<i>hac1</i>, BY4742Δhac1. <i>C. glabrata</i> strains: WT, CBS138; Δ<i>ire1</i>, TG121; and Δ<i>hac1</i>, TG141.</p