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Fungal biofilms are heterogeneous, surface-associated colonies comprised of filamentous hyphae (chains of elongated cells), pseudohyphal cells, yeast-form cells, and various forms of extracellular matrix. When grown on a substratum under liquid culture medium, the microbial fungus Candida albicans forms dense biofilms that range in thickness from 100 to 600μm. Apical hyphae in the medium and invasive hyphae in the substratum may add greatly to the thickness and complexity of the biofilm. Because of the heterogeneity of the structure, and the large refractive index differences between cell walls, cytoplasm, and medium, fungal biofilms appear optically opaque. For fixed specimens that can be transferred out of an aqueous medium, refractive index matching methods provide a high degree of clarification. Confocal scanning, 2-photon scanning, or selective-plane illumination microscopy then can be used to obtain high-quality image data spanning the full thickness of the biofilm. Using refractive index matching and confocal microscopy, we have imaged many interesting features within wild-type, mutant, and engineered biofilms, including cellular phenotypes that vary with position, the effect of growth conditions, and gene expression through reporter constructs. This approach greatly expands the range of microscopical studies, allowing researchers to observe and quantify specific phenomena within medically or industrially relevant forms of microbial growth., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The fungus Candida albicans is a major source of device-associated infection because of its capacity for biofilm formation. It is part of the natural mucosal flora and thus has access to available niches that can lead to infection. In this chapter we discuss the major properties of C. albicans biofilms and the insight that has been gleaned from their genetic determinants. Our specific areas of focus include biofilm structure and development, cell morphology and biofilm formation, biofilm-associated gene expression, the cell surface and adherence, the extracellular matrix, biofilm metabolism, and biofilm drug resistance.

A biofilm is a surface-associated microbial community. Diverse fungi are capable of biofilm growth. The significance of this growth form for infection biology is that biofilm formation on implanted devices is a major cause of recurrent infection. Biofilms also have limited drug susceptibility, making device-associated infection extremely difficult to treat. Biofilm-like growth can occur during many kinds of infection, even when an implanted device is not present. Here we summarize the current understanding of fungal biofilm formation, its genetic control, and the basis for biofilm drug resistance., (Copyright © 2014 Cold Spring Harbor Laboratory Press; all rights reserved.)

Unlabelled: 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.