Report

Bakers' Yeast, a Model for Fungal Biofilm Formation

See allHide authors and affiliations

Science  02 Feb 2001:
Vol. 291, Issue 5505, pp. 878-881
DOI: 10.1126/science.291.5505.878

Abstract

Biofilms are formed by the aggregation of microorganisms into multicellular structures that adhere to surfaces. Here we show that bakers' yeast Saccharomyces cerevisiae can initiate biofilm formation. When grown in low-glucose medium, the yeast cells adhered avidly to a number of plastic surfaces. On semi-solid (0.3% agar) medium they formed “mats”: complex multicellular structures composed of yeast-form cells. Both attachment to plastic and mat formation require Flo11p, a member of a large family of fungal cell surface glycoproteins involved in adherence. The ability to study biofilm formation in a tractable genetic system may facilitate the identification of new targets for antifungal therapy.

Many microorganisms have the ability to grow in association with a surface in an aggregate of cells called a biofilm. Biofilms have taken center stage with the increasing recognition of their role in human infections. Pathogenic bacteria and fungi can form biofilms on the inert surfaces of implanted prosthetic devices such as catheters and on fragments of dead tissue. In the protected microenvironment of a biofilm, the pathogens are more resistant to antimicrobial therapies (1,2).

Little is known about fungal biofilms because many of the organisms that form these structures are not amenable to genetic approaches (1, 3). In search of a model system for fungal biofilms, we investigated whether the well-characterized bakers' yeastS. cerevisiae can form biofilms. Bacteria are said to form biofilms if they adhere to plastic (1). We found thatS. cerevisiae adhered to polystyrene plates (Fig. 1), and the cells remained adherent even after repeated washes (4). The yeast cells also adhered to polypropylene and, to a lesser degree, to polyvinylchloride (PVC) (5). These results suggested that S. cerevisiae can initiate biofilm formation.

Figure 1

Adherence of Saccharomyces to the surface of polystyrene. Yeast was grown in SC [synthetic complete media (26)] with 2% (w/v) glucose and harvested at an optical density at 600 nm (OD600) of 0.5 to 1.5. Cells were then washed once in sterile H2O, resuspended to 1.0 OD600 in SC with 0, 0.1, or 2% glucose, and transferred by pipette (100 μl) into wells of a 96-well polystyrene plate (Falcon Microtest flat bottom plate, 35-1172; Becton-Dickinson Labware). Cells that adhered to polystyrene were visualized by staining with crystal violet (4). (A) Adherence to plastic at a low glucose concentration. The cells were incubated for 0, 1, 3, or 6 hours at 30°C. The time (hours) after inoculation is indicated above the wells, and glucose concentrations (%) are shown below. (B) Flo11p was required for adherence. Yeast strains were resuspended in SC + 0.1% glucose before transfer to the plate. All of the wild-type and mutant strains used were isogenic (9). The numbers at the top indicate minutes after addition to the plate. (C) Quantitation of the results shown in (B) (27). Each data point is the average of three samples: (▪)MATα FLO11, (•) MAT a FLO11, (▴) MAT aFLO11/FLO11, (□) MATαflo11Δ, (○) MAT a flo11Δ, (▵) MAT aflo11Δ/flo11Δ. (D) The cells in the wells shown in (B) were photographed at 100× magnification with a Zeiss Telaval 31 inverted microscope. Incubation in the well was for 0 min (left) and 180 min (right). Bar, 50 μm.

The adherence of yeast to plastic was enhanced as the glucose concentration was lowered, but it was reduced in the complete absence of glucose, suggesting that there is a requirement for active metabolism (Fig. 1A). Diploid cells did not adhere as well as haploids in this assay (Fig. 1, B and C). Examination of the attached cells by microscopy revealed that they were round yeast-form cells (Fig. 1D).

Because bacterial biofilm formation requires cell surface adhesins (1), we disrupted FLO11, a yeast gene encoding a cell surface glycoprotein that is required for adhesion to agar (6, 7), and FLO8, a yeast gene that encodes a regulatory protein required for FLO11expression (8). Isogenic strains (9) lacking either FLO11 ( flo11Δ; Fig. 1, B to D) orFLO8 (flo8Δ) adhered poorly to polystyrene even in low glucose.

The role of Flo11p in the adherence of Saccharomyces to plastic may be similar to that of the glycopeptidolipids (GPLs) expressed on the cell surface of Mycobacterium smegmatis, a nonflagellated bacterium. Mycobacterium smegmatis mutants defective in GPL synthesis are defective in both biofilm formation and in a distinct colonial behavior called “sliding motility,” suggesting an intimate connection between the two phenotypes (10, 11). Sliding motility is defined as a form of surface motility “ … produced by the expansive forces of the growing bacterial population in combination with cell surface properties that favor reduced friction between the cells and the substrate” (10, p. 4348).

To determine whether Saccharomyces displays aFLO11-dependent phenotype similar to sliding motility, we inoculated strains onto YPD plates containing 0.3% rather than 2% agar. On this low agar concentration, S. cerevisiaeexhibited an elaborate pattern of multicellular growth resulting in a confluent mat (Fig. 2). The low concentration of agar required for formation of this structure is similar to that which triggers the sliding motility of M. smegmatis (10,11).

Figure 2

Mat formation bySaccharomyces. Isogenic yeast strains (9) were inoculated onto YPD agar plates (0.3 or 2%) with a toothpick 1 to 2 days after the plates were poured. The plates were then wrapped with parafilm and incubated at 25°C. (A) to (G) Formation of a single mat by a MATα strain (9) over time on a 0.3% agar plate. The same plate was photographed after (A) 2, (B) 4, (C) 5, (D) 6, (E) 7, (F) 9, and (G) 13 days at 25°C. (H) The same MATα strain on a 2% agar YPD plate after 13 days at 25°C. (I) to (K) Mating type affected the morphology of mats. Compare the MAT a strain (I) with theMATα strain (G), both grown for 13 days on YPD-0.3% agar. (J and K) AMAT a/α diploid is delayed in spoke appearance [compare (E, MATα) and (J, MATa/α), both at 7 days of growth]. By 13 days the diploid resembles the haploids [compare (G, MATα), (I, MATa), and (K, MATa/α)]. (L) FLO11 function is required for mat formation. A flo11Δ strain after 13 days of growth on a YPD–0.3% agar plate. Bar, 1 cm.

When inoculated in the center of 0.3% agar plates, S. cerevisiae produced a flat mat covering a larger surface than that of the same strain inoculated on 2% agar (Fig. 2, G and H). This structure grew in a radial form both on circular and square petri dishes and ultimately covered most of the agar, achieving a mean diameter of 7.8 ± 0.57 cm after 13 days (12). The mature structure had a central hub made of a network of cables (Fig. 3A) and radial spokes emanating from the hub. Spokes formed reproducibly within a defined range with a mean of 14.4 spokes ± 4.5 (12). The spokes and hub were more distinct at 25°C than at 30°C. The number of cells produced by mat formation on 0.3% agar was 7.6 times greater (day 12) than that in a colony produced on 2% agar by the same strain (13). The formation of mats and spokes, like adherence to plastic, was sensitive to glucose concentration; reduction in the glucose concentration resulted in a more rapid formation of spokes and hubs (14).

Figure 3

Structure of the Saccharomyces mats. (A) The parallel cables in this figure formed the white spokes seen in Fig. 2G (MATα). The spokes seemed to emanate from the network of cables originating in the hub. The lighter color of the spoke contrasted with the smoother edge of the mat. (B) The flo11Δ mat (Fig. 2L) was smooth with no substructure. (C) The MAT a strain (Fig. 2I) had a network of cables that extended to the edge of the mat and formed narrower and more frequent spokes. The photographs in (A), (B), and (C) were made at 2.5× magnification through a Technival 2 dissecting microscope. Bar, 2 mm. (D) A scanning electron micrograph of the yeast-form cells that comprise the MATα mat was made at 5500× magnification. Bar, 2 μm.

The ability of S. cerevisiae to form the mat structure was FLO11-dependent. Growth of a flo11Δ strain on a 0.3% agar plate produced a mass of cells with a smaller diameter and without the characteristic morphology of a FLO11 mat (Figs. 2L and 3B). The FLO11 mat after 12 days on 0.3% agar contained ∼1.6 times the number of cells as a flo11Δ strain, whereas on 2% agar a flo11Δ strain produced a colony with ∼1.5 times as many cells as an isogenic FLO11strain (13). FLO8, a regulator ofFLO11 expression, was also required for mat formation.

The FLO11 gene is also required for filamentous growth, a morphological switch from the yeast form to multicellular pseudohyphae (invasive chains of elongated cells), that is induced by conditions of nitrogen starvation. Filamentous growth requires components of a signaling cascade of the mitogen-activated protein kinase (MAP kinase) family for maximal transcription ofFLO11 (15–17). Strains carrying mutations (9) in genes encoding components of this MAP kinase pathway (e.g., the MAP kinase kinase kinase ste11and the transcription factor ste12) that reduce filamentation also formed mats more slowly than the wild type. AlthoughFLO11 expression is required for both filamentous growth and mat formation, the cells in both FLO11 and flo11Δstrains were yeast form and not pseudohyphal (Fig. 3D), as determined by light microscopy and scanning electron microscopy.

The mats formed by isogenic MAT a,MATα, and MAT a/α diploid strains (9) had distinguishable morphologies. The mats of theMAT a strain were typically smaller in diameter and formed more spokes than the MATα strain (12). In addition, mats formed by theMAT a strain were rougher in texture with more cables, rougher edges, and fewer lobes than mats formed by theMATα strain (compare Fig. 2, I and G, and Fig. 3, A and C).

At early time points, the mats formed by theMAT a/α diploid were markedly different in morphology from that of either haploid (compare Fig. 2, J and E), although the differences lessened with time (compare Fig. 2, G, I, and K). This difference between haploids and diploids is likely to reflect the reduced expression of FLO11 inMAT a/α diploids versus MAT a orMATα haploids. Previous work has shown that FLO11transcription decreases with increasing ploidy (18). Moreover, the adherence of strains to agar also decreased with ploidy, but could be restored by overexpression of FLO11(18). We found that the diameter of the mat and the other detailed features of mat architecture decreased as ploidy increased. The alteration in phenotype was pronounced in tetraploid strains, which have four copies of FLO11 but resembled the attenuated, amorphous structure of flo11Δ strains (Fig. 4).

Figure 4

Increased ploidy reduces mat formation bySaccharomyces. A series of isogenic strains from haploid to tetraploid (18) on YPD-0.3% agar plates after 13 days at 25°C. Previous work (18) had shown that the level ofFLO11 as well as the FLO11-dependent agar invasion phenotype of Σ1278b decreased as the ploidy increased. (A) MATα, (B) MATαα, (C) MATααα, and (D)MATαααα. Bar, 1 cm.

The reproducible structure of the yeast mats raises important questions about its origins. The radial symmetry and the reproducibility of the number of spokes appear to be the hallmarks of a programmed developmental event, but are strongly influenced by the environment. Our data show that the viscosity of the medium, the Flo11p protein on the surface of the yeast cells, and the nutrients in the medium all contribute to the development of this unusual structure.

Although Flo11p is required for both adherence to plastic and mat formation, the molecular basis for this connection remains unclear. One possible explanation for the role of FLO11 in the adherence of yeast cells to plastic, the multicellular morphological behaviors on 0.3% agar plates, and invasive growth is that Flo11p promotes both cell-cell adhesion and cell-surface adhesion. Previous work has shown that Flo11p is required for cells to adhere to each other in filamentous growth and for cells to adhere to the agar (6, 7). Flo11p may have properties distinct from those of other yeast cell surface proteins that enable it to initiate biofilm formation. For example, Flo1p, a related cell surface protein, promotes avid cell-cell adhesion but not cellular adhesion to an inert surface (7).

As noted above, Flo11p may play a role similar to that of theM. smegmatis GPLs, which are thought to be required for biofilm formation and sliding function because they increase surface hydrophobicity (10, 11). Indeed, when we measured the hydrophobicity of the FLO11 and flo11Δ strains by their ability to partition between water and octane (19), we found that only 12% of the FLO11 cells partitioned to the aqueous phase as compared with 91% of theflo11Δ cells, indicating that FLO11 cells were more hydrophobic. Flo11p might therefore increase the adherence ofSaccharomyces to the wells of plastic 96-well plates. The same hydrophobic property may facilitate mat formation on 0.3% agar plates by reducing the interaction of yeast cells with the aqueous surface. Decreasing the adhesion of the cells to the plate's surface would promote the movement of the cells across the plate. Presumably, the patterns arise from a combination of the frictional forces and the cell-cell interactions. The effect of glucose concentration on the development of these various phenotypes is also likely to be related to the repression of FLO11 transcription by glucose (20).

Adherence to plastic is only the initial stage of biofilm formation. In some organisms such as Pseudomonas aeruginosa the biofilm matures to form microcolonies of bacteria that are surrounded by an extracellular matrix (1). Whether the yeast biofilms described here are a prelude to further developmental events is not yet clear. However, our preliminary analyses of Saccharomyces in another assay of biofilm formation (3) suggest that the yeast biofilms may undergo some maturation (21).

Pathogenic fungi such as Candida albicans andCandida glabrata have orthologs of Flo11p and form mats (22), and C. albicans forms biofilms (1, 3). The Flo11p orthologs have been postulated to be virulence genes because when expressed inSaccharomyces they confer the ability to adhere to mammalian cells (23, 24). However, because these pathogenic fungi have many redundant copies of the genes encoding these cell surface glycoproteins, it is difficult to investigate their virulence function by mutational analysis (24,25). The discovery that Saccharomyces can undergo the initial steps of biofilm formation suggests that it may be a useful model for the genetic dissection of the role of these cell surface proteins in pathogenesis. In addition, Saccharomyces may be a valuable tool for screening compounds that block fungal adhesion, a possible new avenue to antifungal therapy.

  • * To whom correspondence should be addressed. E-mail: gfink{at}wi.mit.edu

REFERENCES AND NOTES

View Abstract

Navigate This Article