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Eukaryotic Cell, December 2003, p. 1266-1273, Vol. 2, No. 6
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.6.1266-1273.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

EAP1, a Candida albicans Gene Involved in Binding Human Epithelial Cells

Fang Li and Sean P. Palecek^*

Department of Chemical and Biological Engineering, University of Wisconsin—Madison, Madison, Wisconsin 53706

Received 10 June 2003/ Accepted 2 October 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
Candida albicans adhesion to host tissues contributes to its virulence and adhesion to medical devices permits biofilm formation, but we know relatively little about the molecular mechanisms governing C. albicans adhesion to materials or mammalian cells. Saccharomyces cerevisiae provides an attractive model system for studying adhesion in yeast because of its well-characterized genetics and gene expression systems and the conservation of signal transduction pathways among the yeasts. In this study, we used a parallel plate flow chamber to screen and characterize attachment of a flo8 S. cerevisiae strain expressing a C. albicans genomic library to a polystyrene surface. The gene EAP1 was isolated as a putative cell wall adhesin. Sequence analysis of EAP1 shows that it contains a signal peptide, a glycosylphosphatidylinositol anchor site, and possesses homology to many other yeast genes encoding cell wall proteins. In addition to increasing adhesion to polystyrene, heterologous expression of EAP1 in S. cerevisiae and autonomous expression of EAP1 in a C. albicans efg1 homozygous null mutant significantly enhanced attachment to HEK293 kidney epithelial cells. EAP1 expression also restored invasive growth to haploid flo8 and flo11 strains as well as filamentous growth to diploid flo8/flo8 and flo11/flo11 strains. Transcription of EAP1 in C. albicans is regulated by the transcription factor Efg1p, suggesting that EAP1 expression is activated by the cyclic AMP-dependent protein kinase pathway.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Candida albicans is the most common fungal pathogen of humans (45). Typically, candidiasis manifests as superficial mucosal diseases, but it also frequently results in systemic infections of immunocompromised patients. Approximately 30% mortality results from systemic candidiasis in susceptible individuals, such as diabetics, surgical patients, and hosts with human immunodeficiency virus infection (65).

Among the factors involved in C. albicans pathogenesis are adhesion of C. albicans to host epithelial and endothelial cells and the dimorphic transition of C. albicans between the ellipsoid yeast form and various filamentous forms: germ tubes, pseudohyphae, and hyphae (36, 41, 45, 53). Signals involved in the yeast-hyphae transition, including temperature, pH, and chemical stimuli, often also lead to increases in adhesin expression (6, 7). Two transcription factors, Cph1p and Efg1p, are required for hyphae formation, adhesion to and penetration of multilayers of human epidermal tissue, and virulence in a mouse model (9, 37).

A number of C. albicans genes encoding adhesins, including ALS1, ALA1, and HWP1, have been identified, but their roles in C. albicans pathogenesis remain unclear. ALA1 and ALS1 were identified based on their abilities to confer upon Saccharomyces cerevisiae the capacity to adhere to extracellular matrix proteins or human umbilical vein endothelial cells (11, 18). HWP1 encodes an adhesion receptor that operates through a transglutaminase-mediated mechanism (59). Ala1p, Als1p, and Hwp1p are predicted to be cross-linked to the ß-1,6-glucans of the cell wall of C. albicans (61). Furthermore, Hwp1p and Als1p function downstream of the transcription factor Efg1p (12, 55). INT1, which was initially characterized as a C. albicans adhesion receptor, has similarity to S. cerevisiae BUD4 and contains a conserved transmembrane region found in the human -integrin gene (14). INT1 expression in S. cerevisiae is sufficient to direct the adhesion of this yeast to HeLa cells (14, 15). Int1p also colocalizes with septins and is involved in axial bud site selection in C. albicans (16). Furthermore, disruption of ALS1, HWP1, or INT1 in C. albicans attenuates virulence in mouse models (12, 15, 59).

Two different signaling pathways, a mitogen-activated protein kinase (MAPK) cascade and a cyclic AMP (cAMP)-dependent pathway, have been identified to regulate the morphogenetic switch from yeast to pseudohyphae in S. cerevisiae (17, 42). In S. cerevisiae, both pathways converge on Flo11p, a transmembrane protein that mediates cell flocculation and adhesion to plastic and permits filament formation and invasive growth during nitrogen and carbon source starvation (13, 23, 49, 52). Based on the evolutionary conservation of fungal signal transduction pathways, MAPK and cAMP pathways have been identified in C. albicans (3). Functional homologues of the components of the S. cerevisiae MAPK cascade have been identified in C. albicans, such as CST20, HST7, CEK1, and CPH1 (5). The cAMP-dependent pathway in C. albicans includes EFG1, the homologue of S. cerevisiae PHD1, and is believed to be regulated by cAMP-protein kinase A Tpk2p downstream of Ras and adenylyl cyclase (37, 51, 58, 60). However, important differences exist between S. cerevisiae and C. albicans adhesion and dimorphic growth. S. cerevisiae is not able to grow in hyphal forms, whereas C. albicans is more morphologically diverse. The Tup1p transcriptional repressor is required for normal pseudohyphal growth in S. cerevisiae, while Tup1p functions as a repressor of hyphal development in C. albicans (4).

In this study, we isolated EAP1, a novel C. albicans adhesin which can mediate adhesion of S. cerevisiae and C. albicans cells to polystyrene and epithelial cells. Expression of EAP1 can also restore haploid invasive growth and diploid pseudohyphal formation to adhesion-deficient S. cerevisiae. Finally, we demonstrate that EAP1 expression in C. albicans is under the regulation of the transcription factor Efg1p.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Yeast strains, media, and genetic methods. The yeast strains used in this study are listed in Table 1. Strains were derived in the 1278b genetic background (22, 34) using standard genetic methods. Standard yeast culture media and filamentous growth media were prepared as previously described (1, 31). Synthetic low-ammonium (SLAD) medium contained 50 µM ammonium sulfate. Uracil was added to SLAD medium to a concentration of 0.2 mM to make SLAD plus Ura. Galactose was added to medium to replace glucose in order to express genes within plasmids containing the S. cerevisiae GAL1 promoter. Yeast cells were transformed using lithium acetate transformation (19).

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TABLE 1. Yeast strains used in this study

PCR disruption (39) was used to replace FLO8 and FLO11 in both MATa and MAT 1278b cells with the kanMX G418 resistance or HIS3 marker. FLO8 positions +1 to +2400, relative to the start site, were deleted and FLO11 positions -26 to + 4170 were deleted.

To overexpress EAP1 in C. albicans, the C. albicans URA3 gene was obtained as a SacII-XbaI fragment from pDDB57 (66) and inserted into SacII/XbaI-digested pBluescript KS(+) (Stratagene) to generate pAU1. A 1.1-kb region upstream of ACT1 from C. albicans SC5314 genomic DNA was amplified by PCR and cloned into the KpnI and XhoI sites of pAU1 to yield pAU2 (63). The entire open reading frame (ORF) of EAP1 flanked by an upstream ClaI site and a downstream SspI site was amplified by PCR and cloned into pAU2 cut with ClaI and SmaI. The resulting construct (pAU3) was linearized by cutting in the EAP1 ORF DNA with PstI to direct integration to the native EAP1 locus. The linearized pAU3 was used to transform a ura3/ura3 efg1/efg1 C. albicans strain. Ura⁺ clones were selected, and integration of the third copy of EAP1 was verified by PCR.

Parallel plate flow chamber cell adhesion assay. The parallel plate flow chamber (Glycotech, Rockville, Md.) consists of a flow deck that fits inside a 60-mm-diameter petri dish. A silicone rubber gasket is placed between the flow deck and the 60-mm petri dish to form the flow chamber. A peristaltic pump connected to the inlet of the flow chamber provides relatively constant velocity flow through the chamber. The shear stress generated by the flow at the bottom surface of the flow chamber detaches yeast cells from the surface. The shear stress is defined as = 3Qµ/2wh², where Q is the volumetric flow rate, µ is the viscosity, 2 h is the height of the flow field, and w is the width of the flow field. The volumetric flow rate was varied to obtain the desired shear stress. After assembly, the flow chamber was placed on a motorized X-Y stage (Prior) of an Olympus IX70 inverted microscope.

Yeast cells were cultured overnight in the appropriate medium, as indicated below in Results. Cells were then pelleted and suspended in 0.1 M sodium phosphate buffer, pH 6.0. After brief sonication to break cell lumps, the cell suspension was pumped into the flow chamber and incubated for 3 h to allow the cells to settle on the surface of the petri dish. The detachment assay was performed by increasing the flow rate of the sodium phosphate buffer (0.1 M; pH 6.0), and thus the shear stress, in a stepwise manner. For each applied shear stress, three fields were selected under the microscope and images were captured using a digital camera (Nikon Spot) and the Metavue software package. The number of cells remaining attached to the surface was automatically identified and counted by Metavue based on contrast of the cells and cell sizes. The adhesion of cells was quantified as the average of the fraction of cells in each of the selected three fields remaining attached after exposure to an applied shear force for 15 min.

Selection for adherent clones of S. cerevisiae. The genomic library of C. albicans SC5314 constructed in pYesR was kindly provided by Yue Fu and Scott Filler (11). Expression of the genes within the library is regulated by the S. cerevisiae GAL1 promoter. This genomic library was transformed into S. cerevisiae SPY308 (MAT ura3-52 his3::hisG leu2::hisG flo8::kan^r). Cells carrying the genomic library were cultured overnight at 30°C in synthetic complete medium lacking uracil (SC-ura) and containing galactose as a carbon source. Cells were pelleted and resuspended in 0.1 M sodium phosphate buffer, pH 6.0. After sonicating for 30 s, the cell suspension was added to the parallel plate flow chamber and incubated at room temperature for 3 h. Nonadherent cells were removed by applying a shear stress of 2.5 dyne/cm², and the fraction of attached cells was measured by image analysis. Adherent cells were recovered by placing solid medium to cover the flow path of the parallel plate flow chamber and incubating the plate overnight at 30°C. Cells were scraped from the solid medium, repooled, and cultured in liquid medium again. The selection for adherent clones was repeated four times to purify the pool. At the end of this selection procedure cells were plated to obtain individual colonies, and plasmids were isolated from those colonies. Approximately 50 individual colonies were sequenced. An oligonucleotide corresponding to a region in the GAL1 promoter was used as the primer to obtain the sequence of the insert DNA adjacent to the GAL1 promoter (48). The obtained sequences were compared to the sequence of the C. albicans genome (http://www-sequence.stanford.edu/group/candida).

Adhesion to human kidney epithelial cells. 293 human kidney epithelial cells were grown to confluent monolayer in six-well tissue culture plates in minimum essential medium (Invitrogen) containing 10% horse serum (Invitrogen). The cells were washed twice in phosphate-buffered saline (PBS) containing Mg²⁺ and Ca²⁺ (PBS⁺⁺) at 37°C. Yeast adhesion to 293 cells was measured essentially as described by Fu et al. (11). Briefly, yeast cells (500 cells/µl) suspended in PBS⁺⁺ were sonicated for 30 s and added to confluent monolayer 293 human kidney epithelial cells incubated for 1 h at 37°C. The initial number of yeast cells in this inoculum was confirmed by colony counting. Nonadherent yeast cells were rinsed away from the 293 cells using PBS⁺⁺. Next, trypsin was added to the wells and the yeast cells were suspended in water and plated on yeast extract-peptone-dextrose (YPD) agar. The number of adherent yeast cells was determined by colony counting, and adherence was expressed as the fraction of cells remaining attached.

Pseudohyphal growth assay. The pseudohyphal growth assay was performed essentially as described by Gimeno et al. (20). Strains to be tested were streaked on SLAD plus Ura plates containing 2% galactose to obtain single cells. Cultures were grown at 30°C for 2 days, and representative colonies were photographed.

Agar invasion assay. Strains to be tested were patched on SC-ura plates containing 2% galactose. Cells were grown at 30°C for 1 day, and the plates were photographed. Next, the plates were rinsed with running water to remove nonadherent cells and the plates were photographed again (50).

Northern blot analysis. C. albicans strains were cultured in liquid YPD medium overnight at 30°C. Cultures were diluted 10-fold into fresh YPD medium supplemented with 10 or 20% serum if indicated and incubated for 2 h at 37°C. Total RNA was extracted from cells by phenol-chloroform followed by ethanol precipitation. For each sample, 40 µg of total RNA was separated by electrophoresis on a formaldehyde gel and transferred by capillary action to a nylon membrane. DNA probes (800-bp region of EAP1 and 1,000-bp region of ACT1 ORFs) were amplified and radiolabeled by PCR. Hybridization and washes were performed according to the methods of Sambrook et al. (54).

Top Abstract Introduction Materials and Methods Results Discussion References

 
FLO8 and FLO11 are required for S. cerevisiae adhesion to polystyrene. A parallel plate flow chamber permits quantitative reproducible adhesion measurements between yeast cells and a surface by applying a known, regulatable shear stress under conditions of laminar flow (40, 64). This shear stress is felt as a shear force, which can detach the cells into the bulk medium or roll them along the surface. A schematic diagram of the flow system is shown in Fig. 1. Shear forces of different magnitudes are obtained by varying flow rate, chamber height, and/or fluid viscosity. Yeast cell adhesion can be quantified as the ratio of the number of attached cells after exposure to an applied shear force to the initial number of attached cells under zero force.

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FIG. 1. Schematic diagram of the flow chamber used for yeast detachment measurements. (A) Side view of the chamber apparatus; (B) diagram of flow system.

We used a parallel plate flow chamber assay to characterize adhesion of wild-type haploid S. cerevisiae strain 1278b cells to the surface of an untreated polystyrene petri dish. As expected, increasing shear stress decreased the fraction of adherent cells (Fig. 2). An extremely low shear stress removed over 40% of the cells, which were most likely just resting on the surface (Fig. 2). As shear stress increased, the fraction of cells remaining attached to the surface decreased in a roughly linear manner up to about 350 dyne/cm². We also measured adhesion as a function of shear stress in flo11 and flo8 strains. FLO11 encodes a cell surface protein involved in adhesion, invasive growth, and filamentous growth (23, 38), and FLO8 encodes a transcription factor required for expression of the flocculins FLO1 and FLO11 (28, 29). Less than 5% of flo8 cells and flo11 cells remained attached at shear stresses as low as 3 dyne/cm² (Fig. 2), demonstrating that FLO8 and FLO11 are required for S. cerevisiae adhesion to polystyrene and suggesting that Flo11p is the protein that mediates this adhesion. S. cerevisiae strains that overexpress FLO11, such as srb8 (46), showed increased adhesion to polystyrene compared to the wild-type strain (data not shown).

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FIG. 2. Effect of applied shear stress on yeast cell detachment from polystyrene. S. cerevisiae strains SKY760 (wild type), SPY308 (flo8), and SPY309 (flo11) were grown in YPD and incubated on the surface of a petri dish in the parallel plate flow chamber for 3 h. Phosphate buffer (0.1 M; pH 6.0) flowed through the chamber for 15 min at a controlled flow rate, and shear stress was calculated. The fraction of cells adhering after flow was determined via image analysis of three fields, containing 600 to 800 cells each, prior to flow. Error bars represent the ranges of three separate experiments.

Identification of EAP1, a C. albicans gene that increases S. cerevisiae adhesion to polystyrene. Based on the observation that an S. cerevisiae flo8 strain is much less adherent to polystyrene than the wild-type strain, we used the parallel plate flow chamber to apply shear stress to select adherent clones of an S. cerevisiae haploid flo8 strain expressing a C. albicans genomic library (11). The S. cerevisiae GAL1 promoter regulates expression of the C. albicans genes within the library. Cells carrying the genomic library were cultured in minimal medium plus galactose overnight at 30°C. The cell suspension was added to the parallel plate flow chamber and incubated at room temperature for 3 h. Nonadherent cells were removed by applying a shear stress of 2.5 dyne/cm², and the fraction of attached cells was measured. This selection procedure was repeated four times, after which the fraction of cells remaining attached at 2.5 dyne/cm² shear stress increased from 1 to 73%. One clone was found to exhibit significantly greater adhesion compared to S. cerevisiae cells harboring empty plasmids. The plasmid contained in this clone was designated as pYE-1. The rescued plasmid was transformed back into an S. cerevisiae flo8 strain and found to increase adhesion to polystyrene (Fig. 3).

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FIG. 3. Eap1p mediates adhesion of S. cerevisiae to polystyrene. S. cerevisiae strains SPY308 (flo8) and SPY309 (flo11) containing either pYesR (empty vector) or pYE-1 (containing EAP1) were grown in minimal medium containing galactose and incubated on the surface of a petri dish in a parallel plate flow chamber for 3 h. Phosphate buffer (0.1 M; pH 6.0) flowed through the chamber for 15 min at a controlled flow rate, and shear stress was calculated. The fraction of cells adhering after flow was determined via image analysis of three fields, containing 600 to 800 cells each, prior to flow. Error bars represent the ranges of three separate experiments.

There are two possibilities for the increased adherence of the flo8 strain containing pYE-1. First, the C. albicans gene contained in pYE-1 could encode a cell wall protein of C. albicans that can directly mediate S. cerevisiae adhesion to the substrate. Alternatively, the C. albicans gene contained in pYE-1 could encode a protein that enhances activity or expression of an S. cerevisiae adhesin, such as Flo11p. To test whether pYE-1 requires FLO11 to increase adhesion, we transformed pYE-1 into the S. cerevisiae haploid flo11 strain and measured adhesion as a function of shear stress (Fig. 3). S. cerevisiae haploid flo11 cells harboring pYE-1 exhibited enhanced adhesion to polystyrene (Fig. 3), indicating that pYE-1 does not increase adhesion via FLO11 and likely encodes an adhesion molecule. Both flo8 and flo11 strains harboring pYE-1 demonstrated a higher fraction of cells adhering to the surface than wild-type yeast without pYE-1, and those adhering possessed a higher affinity for the surface. Also, the flo8 strain with pYE-1 had a slightly higher adhesion than the flo11 strain with pYE-1.

Sequence analysis of the adherence-promoting gene. An oligonucleotide corresponding to a region in the GAL1 promoter was used as the primer to obtain the sequence of the insert DNA adjacent to the GAL1 promoter (48). Sequence analysis revealed a 1,962-bp ORF capable of encoding a 653-residue polypeptide (ORF number 6.5354). This gene was named EAP1 (enhanced adherence to polystyrene). Sequence analysis showed that Eap1p possesses homology to Hwp1p of C. albicans and certain cell wall proteins of S. cerevisiae, such as Flo11p and Aga1p. Furthermore, the consensus motif YTTWCPL present in Eap1p is conserved in many additional yeast cell wall proteins, including Hwp1p, the C. albicans chitinase Cht2p, the S. cerevisiae flocculation protein Flo1p, the -agglutinin subunit Aga1p, the pheromone-regulated protein Fig2p, and the cell wall protein Sed1p (55) (Fig. 4). All of these proteins are either known or predicted to be glycosylphosphatidylinositol (GPI)-anchored cell surface proteins. Eap1p also contains a serine/threonine-rich sequence that may provide glycosylation sites. Analysis of the deduced amino acid sequence predicts the existence of an N-terminal signal sequence (44). The analysis of the C-terminal sequence suggests a GPI-attached site and a valine at the -5 site that is important for incorporation into the cell wall (24, 25).

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FIG. 4. Alignment of the C. albicans Eap1p sequence with sequences of known adhesin and cell wall proteins containing the conserved YTTWCPL motif. R1, R2, etc. indicate consecutive repeats within the same protein. Residues conserved in at least 50% of the sequences within the region of overlap are boxed.

Expressing EAP1 enhances the adhesion of S. cerevisiae cells to 293 cells. Adhesion to host epithelial and endothelial cells is hypothesized to be a critical step involved in the pathogenesis of C. albicans (45). To test whether EAP1 mediates the adhesion of yeast cells to epithelial cells, yeast cell suspensions were added to confluent monolayers of 293 human embryonic kidney cells grown in six-well tissue culture plates and incubated for 1 h at 37°C. Nonadherent yeast cells were dislodged by gentle agitation followed by a rinse. Adherent yeast cells were collected and quantified by trypsinizing and detaching the monolayer from the plate and then transferring the suspension to a YPD plate to count the number of yeast colonies. Adhesion is expressed as the fraction of yeast cells remaining attached to the 293 cell monolayer after rinsing. Shear flow detachment was unsuitable for this assay, since placing the 293 monolayer in the flow chamber often peeled the cell monolayer from the substratum. Also, the rough surface of the cell monolayer disrupted the laminar flow pattern, complicating the calculation of shear stress. Less than 1% of flo8 S. cerevisiae cells carrying empty vector were able to adhere to 293 cells, whereas over 35% of S. cerevisiae haploid flo8 cells expressing EAP1 adhered to 293 cell monolayers (Fig. 5). Wild-type C. albicans also exhibited similar adhesion to 293 cells, as did S. cerevisiae expressing EAP1 (Fig. 5). Therefore, EAP1 expression is sufficient to mediate yeast attachment to human epithelial cells. The transcription factor Efg1p is an essential regulator of morphogenesis, cell wall remodeling, and virulence of C. albicans (33, 37, 57). Since the Ura status of isogenic mutants affects the adhesion of C. albicans (2), we used an efg1/efg1 Ura⁺ strain to study its adhesion to 293 cells. The adhesion of efg1/efg1 C. albicans cells to 293 cells was decreased compared to that of wild-type C. albicans cells, but complementing EFG1 in the efg1/efg1 null mutant restored adhesion to wild-type levels (Fig. 5). EAP1 under the control of the constitutive ACT1 promoter was integrated into the efg1/efg1 strain (efg1/efg1 EAP1/EAP1::pACT1-EAP1 strain). The resulting strain regained the ability to adhere to 293 cells (Fig. 5).

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FIG. 5. Eap1p mediates adhesion of S. cerevisiae to 293 human embryonic kidney cells. (a) S. cerevisiae strain SPY308 (flo8) transformed with vector pYesR (empty vector); (b) S. cerevisiae strain SPY308 (flo8) transformed with pYE-1 (EAP1); (c) C. albicans SC5315; (d) S. cerevisiae strain SKY760 (wild type); (e) C. albicans HLC52 (efg1/efg1::URA3); (f) C. albicans HLC74 (efg1/efg1/EFG1); (g) C. albicans strain SPY312 (efg1/efg1 EAP1/EAP1::pACT1-EAP1). Cells were grown in minimal medium containing galactose and incubated on a confluent 293 cell monolayer for 1 h at 37°C. The cells were rinsed with PBS containing Ca2+ and Mg2+ to remove nonadherent cells. The 293 cell monolayer and associated yeast were detached by trypsinization and added to a YPD plate. Adhesion is quantified as the number of colonies formed on the YPD plate divided by the number of yeast initially added to the 293 monolayer. Error bars represent the standard deviations of three separate experiments. Statistical significance was determined by using Student's t test.

Expression of EAP1 restores haploid invasive growth and diploid pseudohyphal formation to adhesion-deficient S. cerevisiae strains. Flo8p and Flo11p are required for haploid invasive growth and diploid pseudohyphal formation of S. cerevisiae (32, 35, 38). S. cerevisiae haploid flo8 and flo11 strains carrying empty vectors were unable to invade agar on synthetic medium lacking uracil (Fig. 6A), and diploid flo8/flo8 or flo11/flo11 cells failed to form any filaments on SLAD medium (Fig. 6B). However, the ability to penetrate agar and form filaments could be restored by the expression of EAP1 in these strains (Fig. 6). In fact, EAP1 expression induced a stronger filamentous phenotype than that observed in the wild-type 1278b strain on SLAD medium. Thus, EAP1 expression rescued several of the flo11 defects in S. cerevisiae.

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FIG. 6. Expression of EAP1 restores haploid invasive growth and diploid pseudohyphal formation to adhesion-deficient S. cerevisiae. (A) EAP1 mediates S. cerevisiae invasive growth. (a) S. cerevisiae strain SPY308 (flo8) transformed with vector pYesR (empty vector); (b) S. cerevisiae strain SPY308 (flo8) containing vector pYE-1 (EAP1); (c) S. cerevisiae strain SPY309 (flo11) containing pYesR; (d) S. cerevisiae strain SPY309 (flo11) containing vector pYE-1; (e) S. cerevisiae strain SKY760 (wild type). Cells were patched on SC-ura medium containing galactose and allowed to grow overnight at 37°C. The plate was photographed and then held under gently running water for several minutes and photographed again. (B) Expression of EAP1 restores diploid pseudohyphal formation to a flo11/flo11 strain. (a) S. cerevisiae strain SPY311 (flo8/flo8) transformed with vector pYesR;, (b) S. cerevisiae strain SPY311 (flo8/flo8) transformed with pYE-1; (c) S. cerevisiae strain SKY2021 (flo11/flo11) transformed with vector pYesR; (d) S. cerevisiae strain SKY2021 (flo11/flo11) transformed with pYE-1; (e) S. cerevisiae strain SKY756 (wild type). Cells were streaked onto synthetic low-ammonia medium containing galactose. Colonies were allowed to grow at 30°C for 2 days and then were photographed.

Expression of EAP1 in C. albicans is under the regulation of EFG1. The morphological transformation of C. albicans from yeast form to hyphal and pseudohyphal growth can be induced by serum at 37°C. Northern blot analysis was used to determine whether the transcription of EAP1 was morphogenically regulated in C. albicans. EAP1 was transcribed both in YPD medium at 30°C and YPD medium supplemented with 10% fetal bovine serum at 37°C after 120 min of growth (Fig. 7A).

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FIG. 7. Northern blot analysis of the expression of EAP1. (A) RNA was prepared from C. albicans SC5314 (wild type) and HLC52 (efg1/efg1) grown in YPD at 30°C (N) or in YPD supplemented with 10% fetal bovine serum at 37°C (I). A 40-µg aliquot of total RNA was applied, and transcripts were detected using probes specific for EAP1 and ACT1 (loading control). (B) RNA was prepared from C. albicans SC5314 (wild type), HLC52 (efg1/efg1), and HLC74 (efg1/efg1/EFG1) grown in YPD supplemented with 20% fetal bovine serum at 37°C. A 60-µg aliquot of total RNA was applied, and transcripts were detected using probes specific for EAP1 and ACT1 (loading control).

No EAP1 expression was detected by Northern blot analysis in an efg1/efg1 mutant in YPD medium supplemented with 10% fetal bovine serum at 37°C or in YPD in the absence of serum at 30°C (Fig. 7A). The transcription of EAP1 was restored in an efg1/efg1 mutant complemented with EFG1 in YPD medium supplemented with 20% fetal bovine serum, although at a reduced level (Fig. 7B). Therefore, Efg1p is required for EAP1 expression even though EAP1 is not induced during the dimorphic switch in response to serum and 37°C.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we identified EAP1, a C. albicans gene that enhances cell adhesion to human epithelial cells as well as to polystyrene when expressed in S. cerevisiae. EAP1 expression also complements flo11 mutations in S. cerevisiae, restoring invasive growth to haploid flo8 and flo11 strains as well as filamentous growth to diploid flo8/flo8 and flo11/flo11 strains. Transcription of EAP1 in C. albicans requires the transcription factor Efg1p, a key regulator of hyphal growth in the cAMP-protein kinase A pathway.

To identify potential C. albicans adhesion receptors, we used a parallel plate shear flow assay to screen a genomic library expressed in S. cerevisiae. Results obtained from this assay were quantitatively accurate and consistent, in contrast with results from many qualitative adhesion assays that rely on operator technique (e.g., shaking or pipetting). Desired forces can be applied to yeast cells by varying the volumetric flow rates, solution viscosity, or the dimensions of the flow path. The high resolution of this system enables us to isolate and quantify subtle difference in adhesion between different strains. We are also capable of identifying trends of cell adhesion with increasing shear forces. We found that FLO8 and FLO11 are required for S. cerevisiae adhesion when polystyrene is used as the substratum. This finding agrees with a previous report (49). Haploid flo11 cells exhibited slightly greater adherence to polystyrene than flo8 cells (Fig. 2), probably because Flo8p can activate the transcription of other genes encoding adhesins, such as FLO1, which can also contribute to the adhesion to polystyrene in a less efficient manner than FLO11 (29).

Although EAP1 was initially found by selecting transformants adhering to polystyrene, this gene concomitantly confers upon S. cerevisiae the ability to adhere to human kidney epithelial cells. It is feasible to modify the parallel plate flow chamber assay to select adherent clones to more biologically realistic surfaces by functionalizing the polystyrene and reacting with or adsorbing extracellular matrix proteins to the surface. This assay may also prove promising in testing roles of putative adhesins in systemic candidiasis by mimicking the physical forces and the surface characteristics in the host. Glee et al. investigated adhesion interactions of C. albicans with endothelial monolayers under simulated physiologic shear stress using capillary tubes and demonstrated that hydrophobic C. albicans cells have a higher binding affinity than hydrophilic C. albicans cells for endothelial cells and other C. albicans cells (21). In our assay we were not able to identify known C. albicans adhesins, such as ALS1, ALA1, and HWP1, perhaps because the adhesins encoded by these genes do not bind to polystyrene as strongly as Eap1 and these clones were lost in the multiple rounds of selection.

The primary amino acid sequence of EAP1 shares features of highly glycosylated yeast cell wall proteins with N-terminal signal peptides and C-terminal sites mediating GPI anchor addition (24). Hwp1p, Ala1p, and Als1p from C. albicans and Epa1p from Candida glabrata are members of this class of proteins (62). Eap1p also contains a valine five residues upstream from the site that determines linkage to ß (1,6)-glucan of the cell wall (25). According to the above features, EAP1 has been predicted to be a new C. albicans adhesin by searching the C. albicans genome for proteins in the GPI cell wall protein class (62).

When expressed in S. cerevisiae, EAP1 exhibits functional similarity to S. cerevisiae FLO11. FLO11 encodes a cell wall protein required for both invasion and pseudohyphae formation by wild-type S. cerevisiae, presumably by enabling cell-cell and cell-substrate adhesion (38, 47). Cells of the S. cerevisiae strain 1278b with deletions of FLO11 do not form pseudohyphae as diploids nor invade agar as haploids (38). C. albicans EAP1 complements the deletion of FLO11 in S. cerevisiae to restore both haploid invasive growth and diploid pseudohyphae formation. Increasing adhesion alone appears to be sufficient to restore invasion and filament formation. The hyperfilamentation observed in S. cerevisiae cells expressing EAP1 (Fig. 6) might be due to greater adhesion exhibited by this strain than by the wild-type strain. Expression of S. cerevisiae FIG2 or FLO10 encoding GPI-anchored proteins mediating adhesion can bypass the requirement for FLO11 for both filamentation and invasion (23). Likewise, inhibiting mother-daughter separation following cytokinesis by mutating CTS1, ACE2, or EGT2 restores invasive growth in flo8 and flo11 strains (10, 27, 30). Since morphogenesis in S. cerevisiae and C. albicans is governed in part by the same signal transduction pathways, MAPK cascade and cAMP-dependent pathway, and EAP1 is under the regulation of EFG1, it is possible that EAP1 is also involved in the morphogenesis in C. albicans.

EAP1 is transcribed both in cells grown in YPD at 30°C and in YPD supplemented with 10% fetal bovine serum at 37°C. Nantel et al. reported that the transcription of EAP1 is induced by twofold when yeast cells are cultured in YPD supplemented with 10% serum at 37°C for 6 h by using a DNA microarray of the C. albicans genome (43). We found that there was no significant EAP1 induction after 6 h of culture using Northern blot analysis (data not shown). The presence of EAP1 under both noninducing conditions and hyphae-inducing conditions suggests that EAP1 might be involved in adhesion of yeast cells to mammalian cells before hyphal formation and in the process of forming hyphae.

The cell surface component that Eap1p binds remains unclear. An increase in C. albicans hydrophobicity increases binding to fibronectin (56); adhesion to both cell surfaces and polystyrene may be explained by such a mechanism. The sequence of Eap1p shows slight homology to yeast lectins, and certain lectins, such as C. glabrata Epa1p, can mediate attachment to mammalian cells, while others, including S. cerevisiae flocculins, cannot (8). C. glabrata Epa1p binds N-acetyl lactosamine glycoconjugates (8), but ligands for other Candida spp. adhesins have not been conclusively identified.

In summary, these results demonstrate that C. albicans EAP1 encodes a protein that permits S. cerevisiae cell adhesion to epithelial cells as well as invasive and filamentous growth. In C. albicans, EAP1 functions downstream of Efg1p in the cAMP-PKA pathway.

 
We thank Scott Filler and Yue Fu for providing the C. albicans genomic library, Aaron Mitchell for providing pDDB57, and Gerry Fink for providing the C. albicans strains used in this study.

This work was supported by a Whitaker Foundation grant (RG-01-0421 to S.P.P.).

 
* Corresponding author. Mailing address: Department of Chemical and Biological Engineering, 1415 Engineering Dr., Madison, WI 53706. Phone: (608) 262-8931. Fax: (608) 262-5434. E-mail: palecek{at}engr.wisc.edu.

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Eukaryotic Cell, December 2003, p. 1266-1273, Vol. 2, No. 6
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