S accharomyces cerevisiae [PSI ϩ ], the infectious misfolded amyloid form of the protein Sup35, is one of the better-characterized yeast prions (1)(2)(3)(4)(5). Sup35 is an essential protein with three domains, an N-terminal Asn/Gln-rich aggregation-prone domain, a middle domain, and a C-terminal domain that mediates translation termination. In [psi Ϫ ] yeast cells, Sup35 is properly folded, whereas in [PSI ϩ ] yeast cells, most of the Sup35 is misfolded into amyloid seeds, resulting in significant readthrough at stop codons because of the absence of functional Sup35. The conversion of properly folded Sup35 into its misfolded amyloid conformation can occur either spontaneously or by seeding with misfolded Sup35. Once conversion occurs, the [PSI ϩ ] prion conformation continues to pass from generation to generation, with new seeds constantly being generated to maintain a steady-state level of seeds in the yeast population.New seeds are produced when preexisting seeds are severed by Hsp104, a member of the triple-A ATPase family (6). Hsp104 is composed of six identical subunits that form a hexameric ring. Threading of Sup35 molecules through the pore of the Hsp104 hexamer severs the prion seeds in an ATP-dependent reaction (7). Inactivation of the Hsp104 ATPase activity inhibits its severing activity, which in turn cures [PSI ϩ ] and other prions by causing the seeds to dilute out as the cells divide (3,(8)(9)(10)(11)(12)(13)(14).Surprisingly, unlike other yeast prions, the [PSI ϩ ] prion is also cured by overexpression of Hsp104 (3), and even though this was shown more than a decade ago, the mechanism of this curing is still not understood. However, it does appear that the mechanism of [PSI ϩ ] curing by Hsp104 overexpression is very different from the mechanism of curing by Hsp104 inactivation. First, the kinetics of [PSI ϩ ] curing when Hsp104 is overexpressed are very different from the kinetics of curing when Hsp104 is inactivated (8,15,16 One model that has been proposed for the curing of [PSI ϩ ] by the overexpression of Hsp104 is that it involves disaggregation or dissolution of the prion seeds (3,4,8). The role of Hsp104 in this dissolution process could be due to its severing activity, which reduces the size of the prion seeds and concomitantly increases the number of seeds. . This observation was supported by measuring the mobility of GFP-labeled Sup35 in the cytosol and also by measuring the soluble fraction of GFP-labeled Sup35 in cell lysates; both measurements showed an increase in the level of free Sup35. Therefore, in contrast to severing activity, trimming activity reduces the size of the prion seeds without increasing their number.Recently, two other models have been proposed for the curing of [PSI ϩ ] by Hsp104 overexpression. One model proposes that the overexpression of Hsp104 blocks fragmentation of the amyloid fiber (21). This model was based on the observation that when Hsp104 was overexpressed, rather than being recruited to the NMG fibrils by Ssa1, it bound directly to the amyloid ...

Candida albicans, like other pathogens, can form complex biofilms on a variety of substrates. However, as the number of studies of gene regulation, architecture, and pathogenic traits of C. albicans biofilms has increased, so have differences in results. This suggests that depending upon the conditions employed, biofilms may vary widely, thus hampering attempts at a uniform description. Gene expression studies suggest that this may be the case. To explore this hypothesis further, we compared the architectures and traits of biofilms formed in RPMI 1640 and Spider media at 37°C in air. Biofilms formed by a/␣ cells in the two media differed to various degrees in cellular architecture, matrix deposition, penetrability by leukocytes, fluconazole susceptibility, and the facilitation of mating. Similar comparisons of a/a cells in the two media, however, were made difficult given that in air, although a/a cells form traditional biofilms in RPMI medium, they form polylayers composed primarily of yeast cells in Spider medium. These polylayers lack an upper hyphal/matrix region, are readily penetrated by leukocytes, are highly fluconazole susceptible, and do not facilitate mating. If, however, air is replaced with 20% CO 2 , a/a cells make a biofilm in Spider medium similar architecturally to that of a/␣ cells, which facilitates mating. A second, more cursory comparison is made between the disparate cellular architectures of a/a biofilms formed in air in RPMI and Lee's media. The results demonstrate that C. albicans forms very different types of biofilms depending upon the composition of the medium, level of CO 2 in the atmosphere, and configuration of the MTL locus.T he opportunistic fungal pathogen Candida albicans, like opportunistic bacterial pathogens (1, 2), forms biofilms on the surfaces of a variety of tissues and synthetic devices introduced into hosts (3-5). Traditionally, biofilms serve the purpose of anchoring populations of pathogens to a surface and providing a conditioned microenvironment that facilitates growth, survival, and intercellular signaling (6, 7). Biofilms also serve to exclude host and therapeutic challenges, such as antibodies, white blood cells, and antibiotics (8, 9). Bacterial biofilms, which were initially the most intensely studied, have been shown to be architecturally complex, adhering at their base to a targeted surface and composed of multiple cell types embedded in a self-generated complex extracellular matrix (10, 11). Many of the characteristics of bacterial biofilms have been shown to be exhibited by biofilms formed by C. albicans, including drug resistance (12-15), impermeability to low-and high-molecular-weight molecules (15), and resistance to penetration by human polymorphonuclear leukocytes (PMNs) (15, 16). However, studies both to characterize the traits of C. albicans biofilms and to elucidate the molecular mechanisms regulating formation do not always provide the same results, most notably transcription profiles, mutational analyses of the signal transduction pathways, and t...

Candida albicans remains the most pervasive fungal pathogen colonizing humans. The majority of isolates from hosts are heterozygous at the mating type locus (MTLa/α), and a third of these have recently been shown to be capable of switching to the opaque phenotype. Here we have investigated the roles of two transcription factors (TFs) Sfl2 and Efg1, in repressing switching in a/α strains. Deleting either gene results in the capacity of a/α cells to switch to opaque en masse under facilitating environmental conditions, which include N-acetylglucosamine (GlcNAc) as the carbon source, physiological temperature (37°C), and high CO2 (5%). These conditions are similar to those in the host. Our results further reveal that while glucose is a repressor of sfl2Δ and efg1Δ switching, GlcNAc is an inducer. Finally, we show that when GlcNAc is the carbon source, and the temperature is low (25°C), the efg1Δ mutants, but not the sfl2Δ mutants, form a tiny, elongate cell, which differentiates into an opaque cell when transferred to conditions optimal for a/α switching. These results demonstrate that at least two TFs, Sfl2 and Efg1, repress switching in a/α cells and that a/α strains with either an sfl2Δ or efg1Δ mutation can switch en masse but only under physiological conditions. The role of opaque a/α cells in commensalism and pathogenesis must, therefore, be investigated. IMPORTANCE More than 95% of Candida albicans strains isolated from humans are MTLa/α, and approximately a third of these can undergo the white-to-opaque transition. Therefore, besides being a requirement for MTL-homozygous strains to mate, the opaque phenotype very likely plays a role in the commensalism and pathogenesis of nonmating, a/α populations colonizing humans.