Regulatory role of glycerol in Candida albicans biofilm formation

Abstract

Biofilm formation by Candida albicans on medically implanted devices poses a significant clinical challenge. Here, we compared biofilm-associated gene expression in two clinical C. albicans isolates, SC5314 and WO-1, to identify shared gene regulatory responses that may be functionally relevant. Among the 62 genes most highly expressed in biofilms relative to planktonic (suspension-grown) cells, we were able to recover insertion mutations in 25 genes. Twenty mutants had altered biofilm-related properties, including cell substrate adherence, cell-cell signaling, and azole susceptibility. We focused on one of the most highly upregulated genes in our biofilm proles, RHR2, which specifies the glycerol biosynthetic enzyme glycerol-3-phosphatase. Glycerol is 5-fold-more abundant in biofilm cells than in planktonic cells, and an rhr2Δ/Δ strain accumulates 2-fold-less biofilm glycerol than does the wild type. Under in vitro conditions, the rhr2Δ/Δ mutant has reduced biofilm biomass and reduced adherence to silicone. The rhr2Δ/Δ mutant is also severely defective in biofilm formation in vivo in a rat catheter infection model. Expression profiling indicates that the rhr2Δ/Δ mutant has reduced expression of cell surface adhesin genes ALS1, ALS3, and HWP1, as well as many other biofilm-upregulated genes. Reduced adhesin expression may be the cause of the rhr2Δ/Δ mutant biofilm defect, because overexpression of ALS1, ALS3, or HWP1 restores biofilm formation ability to the mutant in vitro and in vivo. Our findings indicate that internal glycerol has a regulatory role in biofilm gene expression and that adhesin genes are among the main functional Rhr2-regulated genes.

Importance: Candida albicans is a major fungal pathogen, and infection can arise from the therapeutically intractable biofilms that it forms on medically implanted devices. It stands to reason that genes whose expression is induced during biofilm growth will function in the process, and our analysis of 25 such genes confirms that expectation. One gene is involved in synthesis of glycerol, a small metabolite that we find is abundant in biofilm cells. The impact of glycerol on biofilm formation is regulatory, not solely metabolic, because it is required for expression of numerous biofilm-associated genes. Restoration of expression of three of these genes that specify cell surface adhesins enables the glycerol-synthetic mutant to create a biofilm. Our findings emphasize the significance of metabolic pathways as therapeutic targets, because their disruption can have both physiological and regulatory consequences.

FIG 1

Biofilm gene expression and RHR2 function. (A) Transcription profiling comparison. RNA-Seq-based expression ratios for biofilm versus planktonic growth conditions (see Table S1 in the supplemental material) were used to define biofilm-upregulated genes (indicated by +) and biofilm-downregulated genes (indicated by −). Common responses among three inocula used in this study, strain SC5314, strain WO-1 white, and strain WO-1 opaque, are indicated by the Venn diagram. An additional 49 genes had divergent responses among the inocula. (B) RHR2 impact on biofilm biomass. Biofilms were grown in Spider or Spider-glycerol medium for 48 h, and the average dry weight was measured (n = 5). Results are expressed relative to the wild type. The strains used were DAY185 (wild type), JVD005 (rhr2Δ/Δ), and JVD006 (rhr2Δ/Δ+pRHR2). (C and D) Confocal imaging of biofilms. Twenty-four-hour biofilms of wild-type, rhr2Δ/Δ, and rhr2Δ/Δ+pRHR2 strains were grown in the media indicated, stained, and imaged. Side-view projections were computed by reslicing the intensity-corrected serial image stack from bottom to top. The resliced stack was then used for maximum-intensity projection. The displayed apical-view projections were pseudocolored to indicate biofilm depth, using the color calibration and scale bar displayed at the top right. The color scale bars correspond to 180 µm (B) or 101 µm (C). Biomass and glycerol levels were quantified from 48-h biofilms as described in Materials and Methods. Glycerol levels were normalized to total cell weight.

FIG 2

RHR2-dependent gene expression and function. (A) Genome-wide analysis. RNA-Seq expression data values for rhr2Δ/Δ+pRHR2 complement were divided by expression data values of the rhr2Δ/Δ mutant to calculate fold change values. Genes with fold changes of ≥1.5 or ≤0.67 are shown. For these differentially regulated genes, their fold change values in biofilm versus those under planktonic conditions were obtained. MultiExperimentViewer (MeV v4.6.2) was then used for hierarchical clustering by average linkage clustering based on Manhattan distance and optimized for gene leaf order. Yellow indicates upregulated genes; blue indicates downregulated genes. (B) Glycerol response of gene expression in the rhr2Δ/Δ mutant. Overnight cultures grown in yeast extract-peptone-dextrose and yeast extract-peptone-glycerol were used to inoculate Spider and Spider-glycerol media, respectively, and cells were grown for an additional 8 h for RNA extraction and qRT-PCR determinations. The table shows gene expression in the rhr2Δ/Δ mutant relative to the wild type. (C) Adherence assays. Cell wall adhesin genes were overexpressed using a constitutive TDH3 promoter in the rhr2Δ/Δ background. Substrate adherence was quantified as described in Materials and Methods and the legend to Fig. 1B. An asterisk above a bar indicates a P value of <0.05 with respect to rhr2Δ/Δ. (D) Biofilm formation assays. Adhesin overexpression strains were used to analyze biofilm formation. The biofilm biomass assay and confocal imaging of biofilms were performed as outlined in Materials and Methods and for panel B. The pseudocolor scale bar corresponds to 161 µm.

FIG 3

RHR2 requirement for biofilm formation in vivo. Strains indicated were inoculated in the rat venous catheter biofilm model, incubated for 24 h, and imaged using scanning electron microscopy. The images are ×100- and ×1,000-magnification views of the catheter lumens, with scale bars corresponding to 200 µm and 20 µm, respectively. The strains used were JVD006 (rhr2Δ/Δ+pRHR2), JVD005 (rhr2Δ/Δ), JVD018 (rhr2Δ/Δ+ALS1-OE), JVD020 (rhr2Δ/Δ+HWP1-OE), and JVD025 (rhr2Δ/Δ+ALS3-OE), from top to bottom, respectively.