The Functions of Mediator in Candida albicans Support a Role in Shaping Species-Specific Gene Expression

The Mediator complex is an essential co-regulator of RNA polymerase II that is conserved throughout eukaryotes. Here we present the first study of Mediator in the pathogenic fungus Candida albicans. We focused on the Middle domain subunit Med31, the Head domain subunit Med20, and Srb9/Med13 from the Kinase domain. The C. albicans Mediator shares some roles with model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, such as functions in the response to certain stresses and the role of Med31 in the expression of genes regulated by the activator Ace2. The C. albicans Mediator also has additional roles in the transcription of genes associated with virulence, for example genes related to morphogenesis and gene families enriched in pathogens, such as the ALS adhesins. Consistently, Med31, Med20, and Srb9/Med13 contribute to key virulence attributes of C. albicans, filamentation, and biofilm formation; and ALS1 is a biologically relevant target of Med31 for development of biofilms. Furthermore, Med31 affects virulence of C. albicans in the worm infection model. We present evidence that the roles of Med31 and Srb9/Med13 in the expression of the genes encoding cell wall adhesins are different between S. cerevisiae and C. albicans: they are repressors of the FLO genes in S. cerevisiae and are activators of the ALS genes in C. albicans. This suggests that Mediator subunits regulate adhesion in a distinct manner between these two distantly related fungal species

Berberine and plant stanols synergistically inhibit cholesterol absorption in hamsters

The present study was conducted to determine the efficacy and underlying mechanism of berberine (BBR), plant stanols (PS) and their combination on plasma lipids. Male Golden Syrian hamsters were randomly divided into 4 groups (n = 15/group) and fed a cornstarch\u2013casein\u2013sucrose-based diet containing 0.15 percent cholesterol and 5 percent fat. Three treatment groups were supplemented with 0.17 percent (equivalent to 100 mg kg 121 d 121) BBR, 1 percent PS, or a combination of both (BBRPS) for 4 wk. At the end of the study, plasma lipids were analyzed with enzymatic methods, cholesterol absorption and synthesis using stable isotope tracer methodology, and gene and protein expressions in the liver and small intestine using real-time PCR and Western blot, respectively. BBR and PS significantly lowered plasma total- and nonHDL-cholesterol levels, and BBRPS markedly improved cholesterol-lowering efficacy compared to BBR or PS alone. Further examinations revealed that BBR and PS both inhibited cholesterol absorption and by contrast, increased cholesterol synthesis, and exerted a synergistic action when they were combined. Plasma total or nonHDL-cholesterol levels were significantly correlated with cholesterol absorption rates. BBR upregulated sterol 27-hydroxlase gene expression and BBRPS increased both cholesterol-7\u3b1-hydroxylase and sterol 27-hydroxlase gene expressions. BBR and PS also synergistically decreased plasma triacylglycerides. These findings suggest that the cholesterol-lowering action of BBR might involve a combination of inhibition of cholesterol absorption and stimulation of bile acid synthesis. The combination of BBR and PS improves cholesterol-lowering efficacy through a synergistic action on cholesterol absorption, in addition to synergistically reducing plasma triacylglycerols in hamsters.Peer reviewed: YesNRC publication: Ye

Cell wall integrity is linked to mitochondria and phospholipid homeostasis in Candida albicans through the activity of the post-transcriptional regulator Ccr4-Pop2

Peer reviewed: YesNRC publication: Ye

The roles of Med31 in adhesion are different between <i>S. cerevisiae</i> and <i>C. albicans</i>.

<p>A) Colonies of wild type <i>S. cerevisiae</i> Σ1278b and the <i>med31Δ</i> mutant were grown on YPD plates at 30°C and photographed. The <i>flo11Δ</i> strain was used as the negative control, to show smooth colony morphology in the absence of <i>FLO11</i>. B) Expression of <i>FLO11</i> was tested by qPCR after 90 min in 0.2% glucose synthetic complete media, the condition used for biofilm formation in C). Levels of <i>FLO11</i> were normalized to <i>ACT1</i>, and expressed related to the wild type, which was set to 1. Shown are averages from at least three independent biological repeats and the standard error. C) The ability of the <i>S. cerevisiae med31Δ</i> mutant to adhere to polystyrene was assessed in 0.2% glucose synthetic complete media as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#s4" target="_blank">Materials and Methods</a>. Quantification was performed by crystal violet staining. At least three independent cultures were used, assayed in quadruplicates. For the mutant, two independently constructed deletion strains were used and gave equivalent results. The <i>flo11Δ</i> mutant was assayed in parallel as a negative control and showed no adherence at any of the time points (not shown). p&lt;0.001.</p

Regulation of adhesins and Ace2-dependent genes by <i>MED20</i> and <i>SRB9/MED13</i> in <i>C. albicans</i>.

<p>A) Cells from the indicated strains were grown in yeast form (YPD) and relative mRNA levels for the indicated genes determined by qPCR. <i>ACT1</i> was used for normalization. Similar results were obtained when <i>THD3</i> was used as the normalization control (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s005" target="_blank">Figure S3</a>). Shown are averages for three independent biological repeats and the standard error. B) Yeast cell morphology of the wild type and Mediator mutants was assessed in YPD at 30°C. The scale bar is 20 µm.</p

Cell wall–related genes differentially expressed in the <i>C. albicans med31ΔΔ</i> mutant.

<p>Cell wall–related genes differentially expressed in the <i>C. albicans med31ΔΔ</i> mutant.</p

The <i>med31ΔΔ</i> mutant is defective for filamentation and virulence in an animal host.

<p>A) The worm <i>C. elegans</i> was infected by wild type <i>C. albicans</i>, the <i>med31ΔΔ</i> mutant and the <i>med31ΔΔ</i>+<i>MED31</i> complemented strain and the appearance of penetrative filamentation was monitored daily over a period of seven days. The worms were photographed with a 40× magnification objective. B) The percentage of worm filamentation was determined after three days of infection. Shown are averages of 4 experiments and the standard deviation. The p value was &lt;0.002 for both the comparison of the mutant with the wild type, and the mutant with the complemented strain. C) The ability of the <i>med31ΔΔ</i> mutant to kill worms was determined in the first 80 h post infection, when most of the worm death due to penetrative filamentation occurs. Three independent experiments were performed and equivalent results obtained. A representative experiment is shown. The <i>med31ΔΔ</i> mutant killed the <i>C. elegans</i> host with delayed kinetics compared to the wild type and the reconstituted strains (blue line- <i>med31ΔΔ</i>, grey line- WT, red line- <i>med31ΔΔ</i>+<i>MED31</i>). The log-rank test used for statistical analysis returned a p value of 0.0032 for the wild type versus <i>med31ΔΔ</i> mutant comparison, while there was no significant difference between the wild type and the <i>med31ΔΔ</i>+<i>MED31</i> complemented strain p = 0.5572).</p

Med31 is required for cytokinesis and filamentous growth of <i>C. albicans</i>.

<p>A) Cultures of wild type C. <i>albicans</i>, the <i>med31ΔΔ</i> mutant and the complemented <i>med31ΔΔ</i>+<i>MED31</i> strain were grown to log phase and cells were observed by microscopy using DIC for bright field (left panel) or through the DAPI filter for calcofluor white staining (right panel). Strains lacking Med31 display a cell separation defect, forming cell chains with the cells attached at the mother-daughter junction, as judged by staining with calcofluor white. B) To determine the proportion of cell chains at least 200 cells were counted for each of the strains. Cell counts were performed after a brief 1 s sonication to disperse cell aggregates. Shown are averages of three independent experiments and the standard error. C) Wild type or mutant strains were streaked on plates containing filamentation-inducing media and incubated at 37°C for 4–5 days. The colonies were photographed using a stereo dissecting microscope. The <i>med31ΔΔ</i> mutant was unable to undergo filamentous differentiation on plates in all media tested. The mutant was also compromised for filamentation in liquid media (data shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s007" target="_blank">Figure S5</a>).</p

Med31 is required for the expression of Ace2-dependent genes and adhesins in yeast and hyphal growth.

<p>A) Cells from <i>med31ΔΔ</i> or <i>ace2ΔΔ</i> mutants were grown in YPD at 30°C (yeast morphology) or in Spider media at 37°C to induce filamentous growth, and levels of the indicated genes determined by quantitative PCR. The expression of the indicated genes was normalised to <i>ACT1</i> and expressed relative to wild type levels, which were set to 1. Equivalent results were obtained when the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding gene <i>TDH3</i> was used for normalization (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613.s005" target="_blank">Figure S3</a>). Shown are the averages of at least three independent experiments and the standard error. B) The promoter regions (2 kb) of <i>ALS1</i>, <i>ALS3</i> and <i>EAP1</i> were searched for putative Ace2 binding motifs from <i>S. cerevisiae</i> (RRCCAGC) or <i>C. albicans</i> (MMCCASC) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613-Mulhern1" target="_blank">[57]</a>. The three motifs conforming to <i>C. albicans</i> consensus sequences were found for the <i>ALS3</i> promoter, whereas none were found for the other two genes. The activating regions A1 and A2 in the <i>ALS3</i> promoter were mapped by <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002613#pgen.1002613-Argimon1" target="_blank">[59]</a>: A1: from −321 bp to −471 bp (upstream of the START codon); A2: from −1049 to −1438 bp.</p