Comparison of Switching and Biofilm Formation between MTL -Homozygous Strains of Candida albicans and Candida dubliniensis

Abstract

Candida albicans and Candida dubliniensis are highly related species that share the same main developmental programs. In C. albicans, it has been demonstrated that the biofilms formed by strains heterozygous and homozygous at the mating type locus (MTL) differ functionally, but studies rarely identify the MTL configuration. This becomes a particular problem in studies of C. dubliniensis, given that one-third of natural strains are MTL homozygous. For that reason, we have analyzed MTL-homozygous strains of C. dubliniensis for their capacity to switch from white to opaque, the stability of the opaque phenotype, CO 2 induction of switching, pheromone induction of adhesion, the effects of minority opaque cells on biofilm thickness and dry weight, and biofilm architecture in comparison with C. albicans. Our results reveal that C. dubliniensis strains switch to opaque at lower average frequencies, exhibit a far lower level of opaque phase stability, are not stimulated to switch by high CO 2 , exhibit more variability in biofilm architecture, and most notably, form mature biofilms composed predominately of pseudohyphae rather than true hyphae. Therefore, while several traits of MTL-homozygous strains of C. dubliniensis appear to be degenerating or have been lost, others, most notably several related to biofilm formation, have been conserved. Within this context, the possibility is considered that C. dubliniensis is transitioning from a hypha-dominated to a pseudohypha-dominated biofilm and that aspects of C. dubliniensis colonization may provide insights into the selective pressures that are involved. In the evolution of species, developmental programs rapidly evolve in response to the selective pressures of environmental change and decay when those selective pressures weaken or disappear (1-3). Decay is most obvious among strains within species with predominately clonal population structures (i.e., species that rarely undergo recombination), since they result in increased strain variability (4-7). A remarkable example of the apparent decay of developmental programs can be found in Candida dubliniensis, a close relative of the more common opportunistic yeast pathogen Candida albicans (8)(9)(10)(11). Candida dubliniensis and Candida albicans diverged approximately 20 million years ago (12), soon after the Eocene/Oligocene period, at approximately the same time primates evolved. The two species share approximately 96% of their genes (13) and undergo similar developmental programs, such as filamentation (14, 15), white-opaque switching (16), and mating (16). However, while these developmental programs appear to have been highly conserved among strains of C. albicans, they are highly variable or diminished among strains of Candida dubliniensis. The two species also exhibit differences in a number of other traits, including the utilization of carbon sources (17-19), adherence to buccal epithelium in glucose-based medium (20), the induction of chlamydospores (21), the adhesive characteristics of mating cells in suspension (1...

Plasticity of Candida albicans Biofilms

“…Nobile et al (190) subsequently demonstrated, in a variation of the Douglas model using Spider medium, that the rate of accumulation of budding yeast cells in the medium decreased between 24 and 60 h. It was not clear whether the yeast-phase cells accumulating in the medium originated in the adhesive yeast cell basal layer, were released from the hyphal upper layer, or were released from yeast cell pockets within the hyphal upper region of the developing biofilm. The lowest rate of release was observed in RPMI 1640 (MOPS) medium, which is consistent with the observation that biofilms formed in this medium contain vertically oriented hyphae with few lateral yeast cells (45,183,184). This is therefore consistent with the suggestion that dispersed cells may be formed by hyphae in media that facilitate lateral yeast cell formation.…”

“…In Fig. 4E, a scanning electron micrograph (SEM) is presented from the top of a collapsed 48-h biofilm prepared such that the extracellular matrix was removed (note the homogeneity of hyphae in the top-view image) (45). In Fig.…”

“…3B. This variation is employed because the architecture of the biofilm formed has been assessed over time in detail (38,45,183,184). In this version of the Douglas model, planktonic cells are grown to stationary phase in sLee's medium to obtain a majority of unbudded yeast-phase cells in stationary phase.…”

“…4D, a side view is presented of a projection image of 500 scans obtained by confocal laser scanning microscopy (CLSM) of a 48-h biofilm of strain SC5314. The image was processed with Imaris 3D image processing and analysis software (Bitplane, Zurich, Switzerland) (45) to accentuate the vertical orientation of the hyphae in the mature biofilm in this model. In Fig.…”

“…For C. albicans, the latter omission has not had a profound effect on most studies, since 90% to 95% of strains are MTL heterozygous (a/␣) (40)(41)(42). However, for the closely related species C. dubliniensis, which shares most of the developmental processes exhibited by C. albicans (43)(44)(45)(46), the omission is indeed worrisome given that one-third of all natural strains are MTL homozygous (44).…”