Dual Lifetimes for Complexes between Glutathione‑<i>S</i>‑transferase (hGSTA1-1) and Product-like Ligands Detected by Single-Molecule Fluorescence Imaging

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione <i>S</i>-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate

Bypass of <i>Candida albicans</i> Filamentation/Biofilm Regulators through Diminished Expression of Protein Kinase Cak1

<div><p>Biofilm formation on implanted medical devices is a major source of lethal invasive infection by <i>Candida albicans</i>. Filamentous growth of this fungus is tied to biofilm formation because many filamentation-associated genes are required for surface adherence. Cell cycle or cell growth defects can induce filamentation, but we have limited information about the coupling between filamentation and filamentation-associated gene expression after cell cycle/cell growth inhibition. Here we identified the CDK activating protein kinase Cak1 as a determinant of filamentation and filamentation-associated gene expression through a screen of mutations that diminish expression of protein kinase-related genes implicated in cell cycle/cell growth control. A <i>cak1</i> <u>d</u>iminished e<u>x</u>pression (DX) strain displays filamentous growth and expresses filamentation-associated genes in the absence of typical inducing signals. In a wild-type background, expression of filamentation-associated genes depends upon the transcription factors Bcr1, Brg1, Efg1, Tec1, and Ume6. In the <i>cak1</i> DX background, the dependence of filamentation-associated gene expression on each transcription factor is substantially relieved. The unexpected bypass of filamentation-associated gene expression activators has the functional consequence of enabling biofilm formation in the absence of Bcr1, Brg1, Tec1, Ume6, or in the absence of both Brg1 and Ume6. It also enables filamentous cell morphogenesis, though not biofilm formation, in the absence of Efg1. Because these transcription factors are known to have shared target genes, we suggest that cell cycle/cell growth limitation leads to activation of several transcription factors, thus relieving dependence on any one.</p></div

DX mutant isolate comparison.

<p>Multiple isolates of each DX strain indicated were obtained from a single transformation of a heterozygous deletion mutant. RNA was extracted from cells grown for 4 hr at 30°C in YPD and used for nanoString expression analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>). Hierarchal clustering of gene expression data was performed using MeV software. Fold change values were obtained by dividing normalized expression values for each mutant strain by the wild-type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up.) Strains are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>.</p

DX mutants in protein kinase-related genes.

<p>DX mutants in protein kinase-related genes.</p

DX mutant complementation.

<p>A. Cell morphology. Strains were stained with Calcofluor White after 4 hr growth at 30°C in YPD medium. Strains: <i>cak1</i> DX (CW1428), <i>cak1</i> DX comp (CW1431), <i>cln3</i> DX (CW1437), <i>cln3</i> DX comp (CW1440), <i>kin28</i> DX (CW1082), <i>kin28</i> DX comp (CW1085), <i>ctk1</i> DX (CW1076), <i>ctk1</i> DX comp (CW1078), <i>snf1</i> DX (CW1138), <i>snf1</i> DX comp (CW1141). B. Cek1 phosphorylation. Immunoblots were performed on yeast extracts from mid-log phase cells grown for 5 hr at 30°C. Strains for lanes 1, 3, 6, 8, 11, 12, 15, and 18 are His^-; strains for lanes 2, 4, 5, 7, 9, 10, 13, 14, 16, 17, and 19 are His⁺. The lanes show experiments with the strain in parentheses: 1 (DAY286), 2 (CW1078), 3 (CW1005), 4 (CW1075), 5 (CW1076), 6 (TA72), 7 (TA86), 8 (CW1041), 9 (CW1082), 10 (CW1085), 11 (DAY286), 12 (CW927), 13 (CW1138), 14 (CW1141), 15 (CW995), 16 (CW1111), 17 (CW1114), 18 (TA72), 19 (TA86). C. Gene expression profiles. RNA was extracted from cells grown for 4 hr at 30°C in YPD and used for nanoString expression analysis (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>). Hierarchal clustering of gene expression data was performed using MeV software. Fold change values were obtained by dividing normalized expression values for each mutant or complemented strain by the wild type strain (DAY185) for each of the probes. The color scale represents Log2 fold change compared to wild type. (Blue limit: 10-fold down; yellow limit: 10-fold up.) Strains are listed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s004" target="_blank">S3 Table</a>.</p

Phenotype and core filamentation gene expression of <i>cak1</i> DX <i>brg1Δ/Δ ume6Δ/Δ</i> mutant strain.

<p>A. In vitro biofilms. Strains were grown under in vitro biofilm conditions for 48 hr, then visualized by confocal microscopy. Cross-sectional views are shown. Strains: <i>CAK1</i> (DAY185), <i>cak1</i> DX <i>brg1Δ/Δ ume6Δ/Δ</i> (CW1715), <i>cak1</i> DX <i>CAK1 brg1Δ/Δ ume6Δ/Δ</i> (CW1720). B. Cell morphology. Cell cultures were grown for 4 hr at 37°C in YPD, then fixed and stained with Calcofluor White prior to visualization. C. Cell morphology. Cell cultures were grown for 4 hr at 30°C in YPD, then fixed and stained with Calcofluor White prior to visualization. D. RNA levels for environmental response genes were determined by nanoString for strains grown for 4 hr at 37°C in YPD (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s006" target="_blank">S5 Table</a>). Normalized expression levels are shown for core filamentation genes in the strains indicated. Symbols: * = p < 0.05; ** = p < 0.01 for comparison of <i>cak1</i> DX <i>brg1Δ/Δ ume6Δ/Δ</i> to <i>cak1</i> DX <i>CAK1 brg1Δ/Δ ume6Δ/Δ</i> strains.</p

Apical projections of biofilm and measurements of biofilm matrix components.

<p>A. Strains were grown under in vitro biofilm conditions for 48 hr, then visualized by confocal microscopy. Side projections of a biofilm of each strain is shown. B. Apical view projections of the same biofilms shown in panel A were obtained using maximum intensity Z-projection of 100 planes at 0.9 μm step-size at the distance indicated from the basal layer. C. Biofilms were grown for matrix isolation. Ten ml of matrix was collected for each strain, biomass collected and dried, and quantitation of matrix total protein, carbohydrate, and normalized values of β-1,3 glucan were determined. Strains: WT (DAY185), <i>cak1</i> DX (CW1428), <i>cak1</i> DX comp (CW1431). Triplicate samples for all but <i>cak1</i> DX carbohydrate (duplicate) determinations. ** = p<0.01 for comparison of either <i>cak1</i> DX or <i>cak1</i> DX comp to WT.</p

DX mutant phenotypes.

<p>Strains were stained with Calcofluor White after 4 hr growth at 30°C in YPD medium. Expression levels of core filamentation genes in mutants grown for 4 hr at 30° in YPD are expressed as fold-change to the wild type; complete data are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006487#pgen.1006487.s003" target="_blank">S2 Table</a>. Strains: WT (DAY286), <i>cln3</i> DX (CW994), <i>cak1</i> DX (CW1003), <i>kin28</i> DX (CW1041), <i>gin4</i> DX (CW900), <i>dbf2</i> DX (CW914), <i>ctk1</i> DX (CW1005), <i>ipl1</i> DX (CW1038), <i>snf1</i> DX (CW927), <i>sak1</i> DX (CW995), <i>ire1</i> DX (CW906), <i>mck1</i> DX (CW1006), <i>cdc28</i> DX (CW991), <i>cmk2</i> DX (CW999), <i>cdc7</i> DX (CW993), <i>sha3</i> DX (CW1010).</p

Expression of <i>ZAP1</i> and novel Zap1 dependent genes.

<p>Strains were grown in Spider medium for 8 hr at 37°C and QRTPCR assays were used to determine RNA levels for of <i>ZAP1, ORF19.4652, PGA39</i> and <i>QDR1.</i> RNA levels were normalized to control <i>TDH3</i> RNA and then expressed as relative units compared to each RNA in the wild-type strain. Strains included wild type (DAY185), <i>zap1Δ/Δ</i> (CJN1201), <i>zcf28Δ/Δ</i> (JF144), <i>zcf28Δ/Δ+ZAP1-OE</i> (JFY261), <i>try2−/−</i> (EHY97), <i>try2Δ/Δ+ZAP1-OE</i> (JFY337), <i>try3−/−</i> (EHY30), and <i>try3−/−+ZAP1-OE</i> (JFY251).</p

Summary of adherence mutant properties.

<p>Footnotes:</p>a<p>These columns list each mutant according to the mutated gene (orf19 numbers and gene names).</p>b<p><i>S. cerevisiae</i> orthologs or best hits, or transcription factor classes, are indicated as listed in the Candida Genome Database.</p>c<p>Column that indicates whether a deletion transcription factor mutant was available for adherence testing. All deletion mutants were created in the SN152 parent strain as described in Homann et al. 2009.</p>d<p>Column that indicates whether a deletion transcription factor mutant was created and test for adherence. Strains were created in the BWP17 background and genotypes are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s006" target="_blank">Table S4</a>.</p>e<p>These columns list the relative adherence for each mutant strain, and for each mutant strain derivative that carries the <i>ZAP1-OE</i> allele. The complete dataset for adherence measurements is in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s003" target="_blank">Table S1</a>. All of the mutants and <i>ZAP1-OE</i> strains were insertion homozygotes except for <i>ace2, arg81, bcr1, crz2, snf5,</i> and <i>zfu2</i>, which were deletion homozygotes.</p>f<p>This column lists the number of genes that were differentially expressed in each mutant compared to the wild-type control strain DAY185, as indicated by nanoString profiling. A cutoff p value of 0.05 was applied. Complete data and p values are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002525#ppat.1002525.s004" target="_blank">Table S2</a>.</p