Candida albicans forms two types of biofilm in RPMI 1640 medium, depending upon the configuration of the mating type locus. In the prevalent a/␣ configuration, cells form a biofilm that is impermeable, impenetrable by leukocytes, and fluconazole resistant. It is regulated by the Ras1/cyclic AMP (cAMP) pathway. In the a/a or ␣/␣ configuration, white cells form a biofilm that is architecturally similar to an a/␣ biofilm but, in contrast, is permeable, penetrable, and fluconazole susceptible. It is regulated by the mitogen-activated protein (MAP) kinase pathway. The MTL-homozygous biofilm has been shown to facilitate chemotropism, a step in the mating process. This has led to the hypothesis that specialized MTL-homozygous biofilms facilitate mating. If true, then MTL-homozygous biofilms should have an advantage over MTL-heterozygous biofilms in supporting mating. We have tested this prediction using a complementation strategy and show that minority opaque a/a and ␣/␣ cells seeded in MTLhomozygous biofilms mate at frequencies 1 to 2 orders of magnitude higher than in MTL-heterozygous biofilms. No difference in mating frequencies was observed between seeded patches of MTL-heterozygous and MTL-homozygous cells grown on agar at 28°C in air or 20% CO 2 and at 37°C. Mating frequencies are negligible in seeded patches of both a/␣ and a/a cells, in contrast to seeded biofilms. Together, these results support the hypothesis that MTL-homozygous (a/a or ␣/␣) white cells form a specialized "sexual biofilm."

MTL-homozygous (a/a or ␣/␣) white cells form a complex sexual biofilm that exhibits the same architecture as that of MTLheterozygous (a/␣) pathogenic biofilms. However, the former is regulated by the mitogen-activated protein (MAP) kinase pathway, while the latter is regulated by the Ras1/cyclic AMP (cAMP) pathway. We previously demonstrated that in the formation of an MTL-homozygous, mature (48 h) sexual biofilm in RPMI 1640 medium, the MAP kinase pathway targets Tec1 rather than Cph1, the latter of which is the target of the same pathway, but for the opaque cell mating response. Here we continued our analysis of the role of Tec1 by comparing the effects of deleting TEC1 on initial adhesion to silicone elastomer, high-resolution confocal microscopy assessments of the stages and cellular phenotypes during the 48 h of biofilm development, human white cell penetration, and biofilm fragility. We show that although Tec1 plays only a minor role in initial adhesion to the silicone elastomer, it does play a major role in the growth of the basal yeast cell polylayer, vertical extension of hyphae and matrix deposition in the upper portion of the biofilm, final biofilm thickness, penetrability of human white blood cells, and final biofilm integrity (i.e., resistance to fluid flow). These results provide a more detailed description of normal biofilm development and architecture and confirm the central role played by the transcription factor Tec1 in the biofilm model employed here.C andida albicans forms biofilms with different functional characteristics, depending upon the configuration of the MTL locus (1-4). After 48 h in pH-stabilized RPMI 1640 medium at 37°C in air, a/␣ cells form a biofilm on silicone elastomer that is approximately 100 m thick and composed of a basal yeast cell polylayer (ϳ20 m thick) and an extensive upper layer of vertically oriented hyphae (ϳ80 m thick) that are uniformly distributed and embedded in a dense extracellular matrix. These a/␣ biofilms are firmly attached to the silicone elastomer substratum, highly resistant to penetration by phagocytic human white blood cells, resistant to drugs, such as fluconazole, and impermeable to low-and high-molecular-weight molecules (5). Under exactly the same conditions, a/a and ␣/␣ cells in the white phase of the whiteopaque transition (6) form biofilms that have the same architecture and integrity as those of a/␣ biofilms but differ in that they are readily penetrated by human phagocytic white blood cells, susceptible to fluconazole, and permeable to low-and high-molecularweight molecules (1, 5). MTL-heterozygous and MTL-homozygous biofilms also differ in the capacity to support mating (2, 7). MTL-homozygous biofilms support mating of seeded minority opaque cells at 10 to over 100 times the frequency of that of MTLheterozygous biofilms (7). Because of the differences in the latter pathogenic and mating characteristics, biofilms formed by a/␣ cells, which represent the predominant MTL genotype colonizing hosts (8-12), were deemed "pathogenic," while those ...

In Candida albicans the transcription factor Efg1, which is differentially expressed in the white phase of the white-opaque transition, is essential for expression of the white phenotype. It is one of six transcription factors included in a proposed interactive transcription network regulating white-opaque switching and maintenance of the alternative phenotypes. Ten sites were identified in the EFG1 promoter that differentially bind one or more of the network transcription factors in the white and/or opaque phase. To explore the functionality of these binding sites in the differential expression of EFG1, we generated targeted deletions of each of the 10 binding sites, combinatorial deletions, and regional deletions using a Renilla reniformis luciferase reporter system. Individually targeted deletion of only four of the 10 sites had minor effects consistent with differential expression of EFG1, and only in the opaque phase. Alternative explanations are considered.