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. 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.