A <em>Candida</em> Biofilm-Induced Pathway for Matrix Glucan Delivery: Implications for Drug Resistance

<div><p>Extracellular polysaccharides are key constituents of the biofilm matrix of many microorganisms. One critical carbohydrate component of <em>Candida albicans</em> biofilms, β-1,3 glucan, has been linked to biofilm protection from antifungal agents. In this study, we identify three glucan modification enzymes that function to deliver glucan from the cell to the extracellular matrix. These enzymes include two predicted glucan transferases and an exo-glucanase, encoded by <em>BGL2</em>, <em>PHR1</em>, and <em>XOG1</em>, respectively. We show that the enzymes are crucial for both delivery of β-1,3 glucan to the biofilm matrix and for accumulation of mature matrix biomass. The enzymes do not appear to impact cell wall glucan content of biofilm cells, nor are they necessary for filamentation or biofilm formation. We demonstrate that mutants lacking these genes exhibit enhanced susceptibility to the commonly used antifungal, fluconazole, during biofilm growth only. Transcriptional analysis and biofilm phenotypes of strains with multiple mutations suggest that these enzymes act in a complementary fashion to distribute matrix downstream of the primary β-1,3 glucan synthase encoded by <em>FKS1</em>. Furthermore, our observations suggest that this matrix delivery pathway works independently from the <em>C. albicans ZAP1</em> matrix formation regulatory pathway. These glucan modification enzymes appear to play a biofilm-specific role in mediating the delivery and organization of mature biofilm matrix. We propose that the discovery of inhibitors for these enzymes would provide promising anti-biofilm therapeutics.</p> </div

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

Biofilm and planktonic phenotypes of select glucan modification mutant strains.

*<p>Percent biofilm formation compared to reference strain.</p>**<p>Percent of biofilm remaining after exposure to fluconazole at 1000 µg/ml,</p>***<p>Matrix β-1,3 glucan concentration normalized to biofilm burden,</p>****<p>Based upon Candida or Saccharomyces Genome Database.</p>+<p>Strains compared to reference strain DAY185.</p>++<p>Strains compared to reference strain SN250.</p

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

Analysis of glucan modifier double knockout strains.

<p>(A) The glucan modifier double knockout strains and the reference strain were assayed for filamentation in YPD. Representative images from light microscopy are shown (top row). The strains were also assayed for adherence to coverslips following 2 h incubation. Representative light micrographs are shown (bottom row). (B) The double knockouts were examined for overall biofilm growth in both YPD and RPMI by comparing the ODs of the untreated control with those of the reference strain in an XTT assay. Strains were also examined for relative planktonic growth in YPD using a turbidity endpoint. (C) The glucan modifier double knockout strains (<i>bgl2</i>−/− <i>phr1</i>−/− and <i>bgl2</i>−/− <i>xog1</i>−/−), the modifier single knockouts, and the reference strain were assayed for biofilm susceptibility to fluconazole (125 µg/ml shown) using the XTT assay as described above. Data for all XTT assays above are expressed as percent reduction compared to untreated controls. Standard errors are shown.</p

Biofilm drug susceptibility and matrix drug sequestration.

<p>(A) Mature in vitro biofilms from the reference strain and <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− null mutants and complemented strains were assayed for fluconazole susceptibility using the 96-well XTT assay. The figure represents data from three assay replicates of a representative example of 3 biological replicates of the 250 µg/ml fluconazole exposure. (B) Mature in vivo biofilms from the reference strain and <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− null mutants were grown on the rat central venous catheter model and then exposed to a dwell of 250 µg/ml of fluconazole or 0.15 M NaCl for 24 h. Following sonication, the burden of remaining biofilm cells was assayed using viable plate counts. The figure represents the mean and standard deviation from three replicates. The * symbol indicates CFUs were significantly different from the reference strain (p value<0.01) based upon ANOVA. (C) Intact biofilms grown from the reference and glucan modifier mutant strains were exposed to [H3]fluconazole, washed, and harvested. Scintillation counting was performed in triplicate to determine the fluconazole content in the intact biofilms and the isolated matrix. Standard deviations are shown. The * symbol indicates glucan measurements were significantly different (p value<0.03) based upon ANOVA.</p

Impact of glucan modification enzyme mutants on cell wall composition and function of biofilm cells.

<p>(A) Cell walls from reference strain, <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutant biofilms were isolated and fractionated by alkali treatment and enzymatic digestion. ANOVA with pairwise comparisons using the Holm-Sidak method was used to compare the β-1,3 glucan and β-1,6 glucan fractions among the strains <b>*</b>, p<0.05. Assays were performed in triplicate on two occasions. Standard deviations are shown. (B) Reference strain (i) and <i>bgl2</i>−/− (ii), <i>xog1</i>−/− (iii), and <i>phr1</i>−/− (iv) mutant biofilms were collected from 6-well polystyrene plates and imaged using TEM. Scale bars represent 0.25 um. (C) Reference strain and <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutant biofilms were treated with serial dilutions of β-1,3 glucanase, H₂O₂, calcofluor white, or SDS. Compound impact was determined using an XTT reduction assay. Data are expressed as percent remaining biofilm compared to untreated controls. Standard errors are shown. ANOVA with pairwise comparisons using the Holm-Sidak method was used to compare the mutant strains at each drug concentration <b>*</b>, p<0.05.</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

Relationship between β-1,3 glucan synthase and modification enzymes during biofilm growth.

<p>(A) RNA was isolated from reference and <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutant biofilms. Real-time RT-PCR assays were used to measure transcript levels in triplicate. Data are shown as a normalized ratio of transcript in the mutant strain to that in the reference strain. (B) The glucan modifier genes, <i>BGL2</i>, <i>XOG1</i>, and <i>PHR1</i> were placed under the control of an inserted <i>TDH3</i> promoter for overexpression of these genes in the homozygous <i>FKS1</i>−/+ mutant. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown), and drug impact was determined using an XTT reduction assay. The * symbol indicates biofilm susceptibilities were significantly different (p value<0.008) based upon ANOVA with pairwise comparison. (C) The <i>FKS1</i> gene was placed under the control of an inserted <i>TDH3</i> promoter for overexpression of this genes in the <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutants. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown) and drug impact was determined using an XTT reduction assay. (D) The glucan modifier genes, <i>BGL2</i>, <i>XOG1</i>, and <i>PHR1</i> were placed under the control of an inserted <i>TDH3</i> promoter for overexpression of these genes in each of the homozygous <i>bgl2</i>−/−, <i>xog1</i>−/−, and <i>phr1</i>−/− mutants. Biofilms were treated with serial dilutions of fluconazole for 48 h (250 µg/ml shown) and drug impact was determined using an XTT reduction assay. Data for all XTT assays above are expressed as percent reduction compared to untreated controls. Standard errors are shown. Student's <i>t</i> test was used for (C) and (D) to compare the mutant strains at each drug concentration <b>*</b>, p<0.05.</p