Role of Ess1 in growth, morphogenetic switching, and RNA polymerase II transcription in Candida albicans.

Candida albicans is a fungal pathogen that causes potentially fatal infections among immune-compromised individuals. The emergence of drug resistant C. albicans strains makes it important to identify new antifungal drug targets. Among potential targets are enzymes known as peptidyl-prolyl cis/trans isomerases (PPIases) that catalyze isomerization of peptide bonds preceding proline. We are investigating a PPIase called Ess1, which is conserved in all major human pathogenic fungi. Previously, we reported that C. albicans Ess1 is essential for growth and morphogenetic switching. In the present study, we re-evaluated these findings using more rigorous genetic analyses, including the use of additional CaESS1 mutant alleles, distinct marker genes, and the engineering of suitably-matched isogenic control strains. The results confirm that CaEss1 is essential for growth in C. albicans, but show that reduction of CaESS1 gene dosage by half (δ/+) does not interfere with morphogenetic switching. However, further reduction of CaEss1 levels using a conditional allele does reduce morphogenetic switching. We also examine the role of the linker α-helix that distinguishes C. albicans Ess1 from the human Pin1 enzyme, and present results of a genome-wide transcriptome analysis. The latter analysis indicates that CaEss1 has a conserved role in regulation of RNA polymerase II function, and is required for efficient termination of small nucleolar RNAs and repression of cryptic transcription in C. albicans

<i>URA3</i> and <i>APE2</i> mRNA expression levels are not significantly altered in heterozygous <i>ESS1</i> mutants and controls.

<p>qRT-PCR showing a quantitative measurement of <i>URA3</i> and <i>APE2</i> mRNA expression in the indicated strains. (<b>A</b>, <b>C</b>) Cells were grown at 30°C in complete synthetic medium (CSM), CSM minus uracil, or CSM minus histidine and leucine as appropriate for each strain. (<b>B</b>, <b>D</b>) Cells were grown at 37°C in serum-containing medium (10% FBS in YPD). Strains are as described in legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g003" target="_blank">Figure 3</a>.</p

Changes in gene expression based on RNA-sequencing results.

<p>The log₂ fold changes are based on results from the Cuffdiff program in the Galaxy server. Cuffdiff was used to select significant gene expression changes depending on whether the <i>p</i> value was greater than the allowed false discovery rate after Benjamini-Hochberg correction for multiple-testing. The highest and lowest log₂ fold changes of the genes considered to be significantly differentially expressed in each group are documented it in the table.</p

Structure-function analysis of CaEss1.

<p>(<b>A</b>) X-ray crystallographic structure of the human homolog, Pin1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Ranganathan1" target="_blank">[40]</a> and CaEss1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Li1" target="_blank">[50]</a>. Substitutions made in the structured linker α-helix of CaEss1 to construct the helix mutant (<i>hm</i>) strains are indicated. (<b>B</b>) Complementation of CaEss1 linker mutants in <i>S. cerevisiae</i>. Plasmids encoding the indicated mutant proteins (independent clone isolates of linker-swapped plasmid, pDS426(sw) and helix substitution plasmid, pDS426(pm)) were separately transformed into a <i>ts</i>-mutant strain of <i>S. cerevisiae</i> (<i>Scess1^H164R</i>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Wu1" target="_blank">[42]</a>. Plasmids were constructed using a pRS426 backbone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a>. Serial dilution assays (1∶5) shows the growth of these independent transformants (a, b). Growth at 37°C, the restrictive temperature for the <i>S. cerevisiae ess1</i> mutant, indicates complementing activity. The CaEss1 helix substitution mutant complements but the linker swap mutant does not. pGDCaEss1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Devasahayam1" target="_blank">[52]</a> (pRS426 backbone) was used as a positive control and pRS426 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a> was used as an empty vector control. (<b>C</b>) Western analysis of whole cell extracts of <i>S. cerevisiae</i> expressing the indicated CaEss1 proteins. A total of 15 µg of protein was used per lane and the blot was probed using anti-CaEss1 polyclonal antibody at a 1∶500 dilution. For the mutant strains, two independent clone isolates of pDS413(sw) (lanes 3 and 4) and pDS413(pm) (lanes 5 and 6) were transformed into <i>S. cerevisae</i> and analyzed. Plasmids were constructed using a pRS413 backbone <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Sikorski1" target="_blank">[88]</a>. The <i>S. cerevisiae</i> strain used (CBW22; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone.0059094-Wilcox1" target="_blank">[44]</a> does not express endogenous Ess1 protein but is viable due to a suppressor mutation (<i>ess1</i>Δ<i>srb10</i>Δ). The linker-swapped protein appears to be absent, or present at a very low level compared to the vector control, while the helix mutant protein is easily detected. pGD-CaESS1 (pRS413 backbone) encoding the wild-type protein (Devasahayam and Hanes, unpublished) was used as a positive control, and pRS413 was used as the empty vector control.</p

<i>CaESS1</i> is expressed at reduced levels in heterozygous mutants as expected.

<p>(<b>A</b>) Quantitative reverse transcription PCR (qRT-PCR) shows <i>CaESS1</i> mRNA expression levels in the indicated strains. (<b>B</b>) Western blot analysis showing the expression of CaEss1 protein in the indicated strains. A total of 3 µg of protein was used per lane. The blot was probed using anti-CaEss1 polyclonal antibody at a 1:500 dilution. CaEss1 is ∼19 kD. Strains correspond to those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g002" target="_blank">2:</a> (<i>ESS1</i>/<i>ESS1</i>) <i>URA3</i> at <i>CaESS1</i> is R6; (<i>ess1</i>Δ/<i>ESS1</i>) <i>URA3</i> at <i>CaESS1</i> is CaGD1; (<i>ESS1</i>/<i>ESS1</i>) <i>HIS1</i>/<i>LEU2</i> is CaDS-B5; (<i>ess1</i>Δ/<i>ESS1</i>) <i>HIS1</i>/<i>LEU2</i> is CaDS-B5.5.</p

Primers used in this study.

<p>Lower case sequences added to obtain appropriate length. Bold indicates a mutation or restriction site.</p

RNA-sequencing indicates transcription readthrough at SnoRNA loci.

<p>Results are visualized using Integrated Genome Viewer. The total number of RNA-sequencing reads (y-axis) and their position along the chromosome (x-axis) is indicated in grey. Forward oriented reads are indicated in red; reverse oriented reads are indicated in blue. Not all reads (blue, red) are visible. Results for the CaEss1 <i>ts</i>-mutant strain are in the upper panels, and results for the isogenic control are in the lower panels (<b>A</b>–<b>C</b>). Solid bars indicate the previously annotated gene positions, while the dotted (grey) boxes indicate the positions implied based on actual transcript data from RNA-sequencing of the the wild-type strain (<i>ESS1</i>/<i>ESS1</i>) (not shown) and the control strain (lower panels). A putative CUT is indicated in panel (B). Approximate positions of primers used for strand-specific cDNA synthesis are shown (black), as are the positions of the primer sets (green) used for qRT-PCR in (D). The positions of likely readthrough transcription are identified by red dashed arrows. (<b>D</b>) Results of qRT-PCR to detect readthrough transcripts for different snoRNA genes (x-axis), expressed as a fold-change (y-axis) of <i>ts</i>-mutant (CaDS-C) over isogenic control (CaDS-FC), normalized to <i>ACT1</i>. For RNA-sequencing, cell growth conditions are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-t002" target="_blank">Table 2</a> (shift to 42°C, then serum induction at 42°C), for qRT-PCR, samples were serum-induced at 37°C.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

<i>CaESS1</i> is essential for the growth of <i>C. albicans</i>.

<p>(<b>A</b>) Western analysis of <i>C. albicans</i> whole cell extracts to detect expression of wild-type CaEss1 (TAA) and a larger protein product encoded by the <i>Caess1^TGC</i> “readthrough” allele (TGC). The level of read-through protein is reduced significantly at 60 min and is nearly absent after 2 hrs at non-permissive temperature (42°C). A total of 7.5 µg of protein was used per lane. The blot was probed using anti-CaEss1 polyclonal antibody at a 1∶500 dilution. (<b>B</b>) Serial dilution (1∶3) of cells of the indicated genotype grown on solid medium (YPD) at different temperatures. The readthrough strain, <i>Caess1</i>Δ/<i>Caess1^TGC</i> shows a clear temperature-sensitive phenotype at 42°C, but no growth defect at 37°C. (<b>C</b>) Filamentation on the indicated solid medium (4 days) (upper two rows), and germ tube formation in liquid Spider medium (2 hrs) (lower row). Upper two rows are reproduced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059094#pone-0059094-g002" target="_blank"><b>Figure 2</b></a> for comparison. In (B) and (C), CaDS-B5 (<i>CaESS1</i>/<i>CaESS1</i>) is used as the wild-type, and is an isogenic control for both the TAA and TGC strains.</p

Function of <i>C. albicans</i> Ess1 linker mutants in <i>S. cerevisiae</i>.

<p>Host strain (MATa ura3-1 leu2-3, 112 trp1-1 can1-100 ade2-1 his3-11, 15 [phi+] ess1Δ<i>TRP1</i>+YEpESS1) is an <i>ess1</i>Δ<i>TRP1</i> mutant of <i>S. cerevisiae</i> (Wu and Hanes, 2000, unpublished) covered by a 2 µM, <i>URA3</i> plasmid expressing <i>ESS1</i> (YEpEss1). Cells were transformed with the indicated plasmids (all 2 µM, <i>HIS3</i>) and plated on complete synthetic media (CSM) minus uracil (ura), histidine (his) and tryptophan (trp). Colonies were picked and passaged (20 ul into 3ml) for three successive overnights in liquid CSM minus his. After 3 days, individual colonies were patched onto CSM minus his trp plates, and replica-plated to CSM minus ura (for Ura+), 1mg/ml 5-FOA (for Ura−), and CSM minus his trp (for total patch number) and scored for uracil prototrophy after 1 day. For the helix subsitution mutants, three independent clone isolates of helix substitution plasmid, pDS413(pm) were tested. For the linker-swapped mutants, two independent clone isolates of plasmid, pDS413(sw) were tested.</p