Telomeric <i>ORFs</i> (<i>TLO</i>s) in <i>Candida</i> spp. Encode Mediator Subunits That Regulate Distinct Virulence Traits

<div><p>The <i>TLO</i> genes are a family of telomere-associated ORFs in the fungal pathogens <i>Candida albicans</i> and <i>C. dubliniensis</i> that encode a subunit of the Mediator complex with homology to Med2. The more virulent pathogen <i>C. albicans</i> has 15 copies of the gene whereas the less pathogenic species <i>C. dubliniensis</i> has only two (<i>CdTLO1</i> and <i>CdTLO2</i>). In this study we used <i>C. dubliniensis</i> as a model to investigate the role of <i>TLO</i> genes in regulating virulence and also to determine whether <i>TLO</i> paralogs have evolved to regulate distinct functions. A <i>C. dubliniensis tlo1</i>Δ/<i>tlo2</i>Δ mutant is unable to form true hyphae, has longer doubling times in galactose broth, is more susceptible to oxidative stress and forms increased levels of biofilm. Transcript profiling of the <i>tlo1</i>Δ/<i>tlo2</i>Δ mutant revealed increased expression of starvation responses in rich medium and retarded expression of hypha-induced transcripts in serum. ChIP studies indicated that Tlo1 binds to many ORFs including genes that exhibit high and low expression levels under the conditions analyzed. The altered expression of these genes in the <i>tlo1</i>Δ/<i>tlo2</i>Δ null mutant indicates roles for Tlo proteins in transcriptional activation and repression. Complementation of the <i>tlo1</i>Δ/<i>tlo2</i>Δ mutant with <i>TLO1</i>, but not <i>TLO2</i>, restored wild-type filamentous growth, whereas only <i>TLO2</i> fully suppressed biofilm growth. Complementation with <i>TLO1</i> also had a greater effect on doubling times in galactose broth. The different abilities of <i>TLO1</i> and <i>TLO2</i> to restore wild-type functions was supported by transcript profiling studies that showed that only <i>TLO1</i> restored expression of hypha-specific genes (<i>UME6, SOD5</i>) and galactose utilisation genes (<i>GAL1</i> and <i>GAL10</i>), whereas <i>TLO2</i> restored repression of starvation-induced gene transcription. Thus, Tlo/Med2 paralogs encoding Mediator subunits regulate different virulence properties in <i>Candida</i> spp. and their expansion may account for the increased adaptability of <i>C. albicans</i> relative to other <i>Candida</i> species.</p></div

The relationship between Tlo1 binding and gene expression.

<p>(A) A plot of Tlo1 enrichment scores for the 367 most highly enriched genes versus the expression levels of those genes in YEPD broth (extracted from microarray data). The categories of highly expressed and repressed genes are color-coded; TCA = Tricarboxylic acid cycle; Sulfur AA = sulphur amino acid biosynthesis; N scavenging = Nitrogen scavenging; NAG = GlcNac metabolism; GPI = glycophosphatidylinositol anchor). (B) Analysis of the expression of Tlo1-enriched groups of genes in the wild type in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant indicated that the highly expressed, highly enriched genes exhibited reduced expression whereas the poorly expressed and repressed genes exhibited increased expression in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant. The change in expression in these groups, with the exception of the genes involved on translation, was significant (ANOVA, P<0.05).</p

Localization of Tlo1-DNA interaction by ChIp-Chip analysis.

<p>(A) A map of the right arm of <i>C. dubliniensis</i> chromosome 5 showing Tlo1-HA enriched areas, including the subtelomeric and Major Repeat Sequence (MRS) regions. Arrows indicate the position of individual ORFs. All chromosomes can be visualised here: <a href="http://bioinf.gen.tcd.ie/jbrowse/?data=cdub" target="_blank">http://bioinf.gen.tcd.ie/jbrowse/?data=cdub</a>. (B) ChIP-chip Enrichment profiles for <i>PUT1</i>, <i>ACT1</i>, <i>CLN1</i> and CD36_51290. Double headed arrows show approximate positions of amplimers used in QRTPCR confirmation experiments. (C) QRT-PCR confirmation of the enrichment levels shown in panel B using gene-specific primers and an intergenic region (IGR)-specific primer set (Chr5 521680–521729 bp)(<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004658#pgen.1004658.s014" target="_blank">Table S5</a>). Amplification was carried out on immunoprecipitated chromatin in anti-HA antibody samples (HA-IP) and no antibody (-AB IP) negative control samples. Relative enrichment values refer to the quantity of DNA amplified from anti-HA immunoprecipitated samples (‘enriched’) relative to total input chromatin (‘non-enriched’).</p

Microarray gene expression profiling of the <i>med3</i>Δ mutant in YEPD and 10% (v/v) serum.

<p>(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) and <i>med3</i>Δ mutants relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% serum (1 h). The fold change (Log₂) relative to wild-type is color coded as indicated in the lower panel. (B) Venn diagrams illustrating the numbers of genes commonly regulated greater than 1.5-fold in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) and <i>med3</i>Δ mutants following growth in YEPD broth or 10% serum. The box below indicates major functional classes of commonly regulated genes identified by GO term analysis using the GO Term finder at CGD (<a href="http://www.candidagenome.org/" target="_blank">http://www.candidagenome.org/</a>). (C) Graph plotting the expression levels (Log₂ fold change versus wild-type) of genes associated with selected GO terms in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) and <i>med3</i>Δ mutants during growth in 10% (v/v) serum.</p

Biochemical analysis of Mediator in <i>C. dubliniensis</i> wild-type and <i>tlo1</i>Δ/<i>tlo2</i>Δ.

<p>Lysates from untagged WT <i>C. dubliniensis</i> (Lane 1), a Med8-6His-Flag-tagged WT strain (Lane 2), a Med8-6His-Flag-tagged <i>tlo1</i>Δ/<i>tlo2</i>Δ strain (Lane 3), a Med8-6His-Flag-tagged <i>med3</i>Δ strain (Lane 4) and a Tlo1-6His-Flag-tagged WT strain (Lane 5) were subjected, in parallel, to multiple chromatographic separations. The elutions from the IMAC (Talon) step were analyzed by 8% SDS-PAGE. Proteins were revealed by staining with silver. Bands that are present in WT but clearly absent in the <i>tlo1</i>Δ/<i>tlo2</i>Δ and <i>med3</i>Δ Mediator are indicated in parenthesis on the right. The absence of these proteins in the <i>tlo1</i>Δ/<i>tlo2</i>Δ Mediator was confirmed by mass spectroscopy.</p

Microarray gene expression profiling of the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant in YEPD and 10% serum.

<p>(A) A heat map generated in GeneSpring GX12 showing all 1.5 fold regulated genes in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant relative to wild type strain Wü284 during exponential growth in YEPD broth and following inoculation in 10% (v/v) serum (1 h and 3 h). The fold change (Log₂) relative to wild-type is color coded as indicated in the lower panel. GO terms associated with up and down regulated clusters are indicated on the left. (B) A cartoon metabolic map of the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant showing the changes in expression of genes involved in energy metabolism during growth in YEPD. Genes in green exhibit a 1.5-fold or greater reduction in expression relative to wild-type whereas those in red exhibited a 1.5-fold increase in expression. Genes in black were not significantly changed.</p

Reintroduction of <i>TLO1</i> or <i>TLO2</i> in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant restores wild-type levels of expression of overlapping and distinct sets of genes.

<p>(A) A graph plotting the change in expression of genes up regulated (red lines) or down regulated (blue lines) in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant following complementation with either <i>TLO1</i> (<i>+TLO1</i>) or <i>TLO2</i> (<i>+TLO2</i>) in YEPD or (B) 10% (v/v) serum. (C) Heat map of selected genes that were differentially expressed in the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant during growth in YEPD broth (C) or 10% (v/v) serum (D) that exhibited a <i>TLO1</i> or <i>TLO2</i> specific restoration to wild-type in expression. Colors in the first column (<i>tlo1</i>Δ/<i>tlo2</i>Δ) represent the fold change in expression of the indicated genes in <i>tlo</i>ΔΔ vs wild-type; colors in columns 2 and 3 (+<i>TLO1</i> and +<i>TLO2</i>) represent the fold change in expression of the indicated genes following reintegration of <i>TLO1</i> or <i>TLO2</i> versus <i>tlo</i>ΔΔ. (E to H) QRT-PCR of the indicated genes was carried out following 30, 60 and 180 min incubation in 10% (v/v) serum at 37°C. Gene expression was determined using Sybr green technology and gene-specific levels are expressed relative to <i>ACT1</i>. Primer sequences are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004658#pgen.1004658.s014" target="_blank">Table S5</a>.</p

Structure and expression of <i>C. dubliniensis TLO</i> genes.

<p>(A) <i>TLO1</i> and <i>TLO2</i> encode proteins of 320 and 355 amino acids respectively that exhibit 81% identity in the N-terminal Med2-like domain, and 50% identity in the C-terminal portion of the protein. In addition, Tlo2 has an internal triplet repeat, indicated. (B) Expression of <i>TLO1</i> and <i>TLO2</i> was measured by QRT-PCR using gene-specific primers during growth in YEPD broth at 37°C in early exponential (1.5 h-post inoculation), mid-exponential (8 h) and early stationary phase (18 h) in strain Wü284 (WT). The effect of deletion of either <i>TLO1</i> or <i>TLO2</i> on the expression of the other paralog was also analysed. (C) Expression of reintegrated copies of <i>TLO1</i> and <i>TLO2</i> in plasmid pCDRI in the <i>tlo1</i>Δ/<i>tlo2</i>Δ strain.</p

Deletion of <i>TLOs</i> has different effects on biofilm formation compared to a <i>med31</i>Δ mutant.

<p>(A) Gene Set Enrichment Analysis (GSEA) plot showing differential expression of genes down-regulated in a <i>C. albicans med31</i>Δ mutant in the <i>tlo1</i>Δ/<i>tlo2</i>Δ mutant (i.e. they are upregulated in the <i>tlo1</i>Δ/<i>tlo2</i>Δ mutant) (B) Biofilm formation was assessed in the indicated strains on plastic surfaces at 37°C in the presence or absence of 5% CO₂ as indicated. Adherent biomass determined by an XTT reduction assay. The experiment was carried out in triplicate on three separate occasions (n = 9) and error bars represent the standard deviation from the mean. <i>P</i> values above figure are the result of one-way ANOVA with Tukey's post hoc test.</p

Phenotypic analysis of <i>C. dubliniensis tloΔ</i> and <i>med3</i>Δ mutants.

<p>The rate of formation of true hyphae in water supplemented with 10% serum was measured in the <i>tlo1</i>Δ, <i>tlo2</i>Δ and <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutants relative to Wü284 (A), in the <i>TLO1</i> and <i>TLO2</i> reintegrants (<i>tlo</i>ΔΔ+<i>TLO1/2</i>)(B) and in the <i>med3</i>Δ mutant (C). Measurements were taken over a period of 5 h by counting the number of cells producing unconstricted germ-tubes and expressing this value as a percentage of the whole population. (D) Cellular morphology of Wü284, the <i>tlo1</i>Δ/<i>tlo2</i>Δ (<i>tlo</i>ΔΔ) mutant and <i>TLO1</i> and <i>TLO2</i> reintegrated strains in 10% serum following staining with calcofluor white. (E) Oxidative stress susceptibility in <i>tlo</i>Δ mutants. Susceptibility to oxidative stress was assessed by spotting 20 µl aliquots of 10-fold serially diluted suspensions (10⁶ to 10³ cells/ml) of the indicated strains on YEPD agar plates containing 7 mM H₂O₂ or 100 mM menadione. Plates were incubated for 48 h at 37°C.</p