Plasticity of Candida albicans Biofilms

Abstract

Candida albicans, the most pervasive fungal pathogen that colonizes humans, forms biofilms that are architecturally complex. They consist of a basal yeast cell polylayer and an upper region of hyphae encapsulated in extracellular matrix. However, biofilms formed in vitro vary as a result of the different conditions employed in models, the methods used to assess biofilm formation, strain differences, and, in a most dramatic fashion, the configuration of the mating type locus (MTL). Therefore, integrating data from different studies can lead to problems of interpretation if such variability is not taken into account. Here we review the conditions and factors that cause biofilm variation, with the goal of engendering awareness that more attention must be paid to the strains employed, the methods used to assess biofilm development, every aspect of the model employed, and the configuration of the MTL locus. We end by posing a set of questions that may be asked in comparing the results of different studies and developing protocols for new ones. This review should engender the notion that not all biofilms are created equal.

FIG 1

General steps in the formation of a bacterial biofilm. The general developmental program provides us with a contextual framework for defining a C. albicans biofilm, since it includes a number of analogous steps.

FIG 2

The approximate number of papers published per year on Candida albicans biofilms began to increase at a nearly exponential rate after the development of the first in vitro model by Hawser and Douglas, from 1994 to 1998.

FIG 3

The Douglas model, established by Hawser and Douglas (168), has served as the basis for a number of models with variations in one or more conditions. (A) Procedure used by Hawser et al. (179). (B) Representation of the cellular and architectural development of a C. albicans biofilm, using a variation of the Douglas model in which sLee's medium was used to grow cells planktonically, RPMI 1640 (MOPS) medium was used to support biofilm development, and the cultures were rocked to mix the media (38). In panel B, cells in the yeast-phase cell monolayer and polylayer are represented by spheres, germ tubes are represented by tubes, hyphae are represented by vertical tubes, and ECM is represented by gray fibers.

FIG 4

Microscopic images of yeast-phase cells and nuclei in a relatively confluent monolayer, the hyphal upper layer, and both top and side images of confocal projections of biofilms forming in the Douglas model, using planktonic cells grown in sLee's medium, RPMI 1640 (MOPS) medium for biofilm formation, and a silicone elastomer disc as the substrate. (A) Differential interference contrast (DIC) microscopy image of an adhering yeast-phase cell monolayer after 90 min of incubation of planktonic cells in the model well. (B) Nuclear staining with Syto 9, imaged by fluorescence microscopy. (C) Increased magnification of the monolayer formed after 90 min, imaged by DIC microscopy. (D) Side view of a projection image of 500 CLSM scans of a 48-h C. albicans biofilm stained with calcofluor. (E) SEM image of a 48-h C. albicans biofilm prepared using methods that removed the ECM from a collapsed biofilm. (F) SEM image of a 48-h C. albicans biofilm prepared using methods that left the collapsed ECM adhering to the hyphae and substratum. (Republished from reference with permission of the Society for General Microbiology; permission conveyed through Copyright Clearance Center, Inc.) (G) Top and side views of projection images, each composed of 500 CLSM scans, of developing biofilms prepared using a variation of the Douglas model, at 0, 4, 16, and 24 h.

FIG 5

Catheter, denture, and subcutaneous models developed for C. albicans biofilm formation. Panels cite references (A), (B), (C), (D), and (E).

FIG 6

Comparison of budding cell phenotypes of planktonic cultures of C. albicans strain SC5314 grown in culture tubes shaken for 24 and 48 h, using eight different media to obtain planktonic yeast-phase cell cultures for in vitro C. albicans biofilm studies. Budding cells were scored as cells with buds exhibiting a diameter of less than two-thirds that of the mother cell and still attached to the mother cell. The percent unbudded cells in culture is presented in the upper left corner of each panel. Between 500 and 1,100 cells were scored for each preparation. Arrows point to examples of budding cells. Compositions of the media can be found in Table 1.

FIG 7

Example showing why a single time point might be invalid for comparing biofilms. Strain differences, mutations, antifungal drugs, and different model conditions may affect when things happen, not if they happen. A hypothetical example is presented for a difference in timing in the first step of the biofilm development program for two strains, “a” and “b,” which results in an increase in the time it takes for the formation of the yeast-phase cell polylayer for strain “b.” Germination, hypha elongation, and ECM deposition take the normal amounts of time but are shifted in terms of the total amount of time. Data from a single time point at 15 or 33 h would reveal differences that could be interpreted as absolute, when in fact they simply reflect differences in timing.

FIG 8

To understand the profound difference in biofilms formed by MTL-heterozygous and MTL-homozygous cells, white-opaque switching, opaque cell mating, and the role of α pheromone in the formation of MTL-homozygous biofilms must be taken into account. (A) MTL homozygosis and white-opaque (Wh-Op) switching. (B) Shmoo formation induced by pheromones of opposite mating types and pheromone-directed chemotropism in the process of fusion. (C) MTL-homozygous white cell biofilms are induced by the unorthodox release of pheromone of the opposite mating type by rare opaque cells which appear through switching. In this example, white a/a cells produce minority opaque a/a cells. The latter release α pheromone in an unorthodox fashion, which induces white a/a cells to form a biofilm. Op, opaque; hy, hyphae; bp, basal cell polylayer; ECM, extracellular matrix.

FIG 9

Demonstration of chemotropism in an early MTL-homozygous white cell biofilm. The seeded a/a opaque cell was stained green with fluorescein-conjugated ConA, and the seeded α/α opaque cell was stained red with rhodamine-conjugated ConA. The conjugation tubes that formed are stained blue. Note how they extended toward each other by sensing gradients of alternative mating type pheromone released by the opaque cells, in the process of chemotropism. (Reproduced from reference with permission.)