Biofilms are a principal form of microbial growth and are critical to development of clinical infection. They are responsible for a broad spectrum of microbial infections in the human host. Many medically important fungi produce biofilms, including Candida [1], Aspergillus [2], Cryptococcus [3], Trichosporon [4], Coccidioides [5], and Pneumocystis [6]. In this review we emphasize common features among fungal biofilms, and point toward genes and pathways that may have conserved roles.

Biofilms are complex surface-associated cell populations embedded in an ECM that possess distinct phenotypes compared to their planktonic cell counterparts. Nutrients, quorum-sensing molecules, and surface contact are contributory factors. C. albicans biofilms are comprised primarily of yeast-form and hyphal cells, both of which are required for biofilm formation [1]. Formation is a sequential process involving adherence to a substrate (either abiotic or mucosal surface), proliferation of yeast cells over the surface, and induction of hyphal formation [1]. ECM accumulates as the biofilm matures, and seems to contribute to cohesion [8]. C. albicans biofilms form on numerous abiotic [9] and biotic surfaces [10]–[12]. In denture stomatitis, a combination of biotic mucosal (the host) and abiotic surface (the denture) biofilm formation exists [13]. Other Candida spp. including C. tropicalis, C. parapsilosis, and C. glabrata form ECM-containing biofilms but do not produce true hyphae [14].

Transcription factors play fundamental roles in both positive and negative regulation of biofilm formation through regulation of hyphal formation and cell surface proteins responsible for adherence [1]. Bcr1, a C₂H₂ zinc finger transcription factor, is a critical determinant of C. albicans biofilm formation in all environments studied to date [10], [12], [13], [19]. Bcr1 seems to be a conserved regulator of biofilm formation, because the Bcr1 ortholog of C. parapsilosis is required for biofilm formation as well [20]. Ace2, another C₂H₂ zinc finger transcription factor, also contributes to C. albicans biofilm formation, probably through its role in adherence as well as hypha formation [21]. The C. albicans transcription factor Efg1, a global regulator of cell surface protein genes and hyphal formation [1], is required for biofilm formation as well. The orthologs or best hits of Bcr1, Ace2, and Efg1, including C. glabrata CAGL0E06116g, CAGL0M04323g, and CAGL0L01771g, and C. parapsilosis CPAG00564, CPAG00148, and CPAG00178, are good candidates for biofilm regulators in those species. Transcription factors with analogous roles to Bcr1, Ace2, and Efg1 of C. albicans in A. fumigatus may be identified amongst the 124 uniquely expressed or upregulated transcription factors identified in biofilm culture by Gibbons et al., 2011 [22]. The A. fumigatus transcription factor LAEA, a regulator of cell type and secondary metabolism gene clusters in A. fumigatus, is highly upregulated in biofilms [22], and it remains to be seen whether it may influence biofilm phenotypes.

Biofilm cells have phenotypes distinct from planktonic cells, and this difference is reflected in greatest detail at the gene expression level. Detailed gene expression profiling comparisons, conducted in both C. albicans and A. fumigatus, have revealed substantial changes in gene expression between biofilm and planktonic cells [22], [26]. Changes in transcription factor expression is characteristic of C. albicans biofilm formation in vitro and in vivo [24]–[26], suggesting biofilm formation to be a highly regulated process. Similarly, almost 50% of the predicted transcription factors of A. fumigatus, including many with roles in asexual and sexual development, are upregulated in biofilms compared to planktonic cells.

Genetic exchange is a feature of bacterial biofilms, mediated in part by extracellular DNA. Although extracellular DNA has been detected in C. albicans biofilms [28], the main mechanism of biofilm-associated genetic exchange involves mating and cell fusion. Most biofilm studies have been conducted with nonmating a/α cells, but biofilm formation of the mating-capable cell types, a/a and α/α, has revealed a unique regulatory pathway intimately tied to pheromone signalling. In order to mate, C. albicans must go through a switch from the white to opaque cell type. Upon switching, α/α opaque cells release a mating pheromone that induces a mating response in a/a opaque cells and vice versa. Pheromone release also induces an adhesive phenotype among the mating-incompetent a/a white cells [29], leading to mixed biofilm formation and ultimately mating [30].

Fungal biofilms reflect a range of architectures. Regulators of biofilm formation may be conserved even among disparate biofilm architectures. From detailed analysis in C. albicans and A. fumigatus, there are numerous candidate genes that could be investigated in other biofilm-forming fungi. In addition to hyphal gene expression, characteristic biofilm gene expression patterns include increased expression of transcription factors and protein synthesis genes. Differential adhesin expression, upregulation of cell wall genes, and increased primary metabolism are features of the biofilm environment. Studies of mating pheromone effects on adherence highlight how a small portion of biofilm constituents can have a significant impact on biofilm formation. The presence of highly drug tolerant persister cells in biofilms (discussed above) is another illustration of the contribution of cell heterogeneity to overall biofilm properties. How other heterogeneous properties among biofilm cells may contribute to the overall development and integrity of pathogenic fungal biofilms will be an interesting question for future research.