Unless otherwise stated, C. albicans strain SC5314, kindly provided by Dr. A. Mitchell—Carnegie Mellon University, USA; C. albicans clinical isolate L296 (high biofilm-forming strain), kindly provided by Dr. Arnaldo L. Colombo—UNIFESP, LEMI, Brazil; Saccharomyces cerevisiae strain S288C, kindly provided by Dr. Beatriz A. Castilho—UNIFESP, Brazil and the reference C. albicans strain ATCC90029 (low biofilm-forming strain), were grown in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) overnight with rotary agitation at 30°C. Cells were counted in a hemacytometer (Hirschmann EM Techcolor) and adjusted to the desired concentration immediately before the experiments. To produce hyphae, C. albicans yeasts were diluted to 1.0 × 10⁷ cells/ml in fetal bovine serum containing 5 mg/ml dextrose and incubated at 37°C with agitation for 3 h. Escherichia coli strain DH5-α was used for plasmid transformation. Strain BL21 (DE3) was used for recombinant protein expression. E. coli strains were grown overnight in LB medium supplemented with 100 μg/ml ampicilin or 15 μg/ml kanamicyn at 37°C with constant agitation.

The complete coding region of C. albicans enolase gene was PCR amplified from genomic DNA with the following oligonucleotides: EnoexpresF:(5'-CGGGATCCATGTCTTACGCCACTAAAATC-3') and EnoexpresR: (5'-ATAGTTTAGCGGCCGCTTACAATTGAGAAGCCTTT-3'). The insert was subcloned between Not I and BamHI restriction sites in plasmid pET-28a (+) (Novagen), in fusion with a Histidine tag at the N-terminal portion according to (Sambrook et al., 1989). This construction was transformed into E. coli BL21 (DE3). An overnight culture was inoculated into LB medium containing Kanamycin (15 μg/mL) and C. albicans enolase expression was then induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 2 h. After centrifugation, cells were lysed and the recombinant protein (His₆-enolase) was purified under native conditions in pre-packed Ni-sepharose columns (Clontech) according to the manufacturer's instructions. The eluted protein was dialyzed for 36 h at 15°C against phosphate-buffered-saline (PBS) pH 7.6. Protein concentration was determined with the Bradford assay (Bradford, 1976) and by SDS-PAGE analyses. To assess the purity of the recombinant protein, samples were analyzed by Coomassie blue and silver nitrate staining SDS-polyacrylamide gel electrophoresis.

Purified His₆₋enolase was used to generate specific immune rabbit polyclonal sera. One hundred and fifty microgram of the protein with complete and incomplete Freund's adjuvant (Sigma) for the first and the other two doses respectively, at 20-days intervals, was subcutaneously injected in 2 adult female New Zealand white rabbits. To obtain the pre-immune sera, aliquots of blood were collected before the first immunization. Whole blood was collected 55 days after the first immunization. Sera were obtained by incubating the blood for 30 min at 37°C and overnight at 4°C, followed by centrifugation under 1000 rpm for 15 min at 4°C. All sera were stored at −80°C.

5 × 10⁸ yeasts were resuspended in lysis solution (20 mM Tris pH 8.0, 2 mM MgCl₂, 2 mM EGTA and 150 mM NaCl) with protease inhibitors (1 mM phenylmethylsufonyl fluoride (PMSF), 2 mM benzamidin, 1 μg/ml leupeptin, 10 μg/ml pepstatin A, 4 μg/ml aprotinin, and 1 μg/ml antipain) and phosphatase inhibitors (10 mM Sodium Pyrophosphate and 1 mM Sodium Fluoride). Cell suspensions were vortexed and lysis was done by sequential freezing/thawing. After centrifugation at 13,000 rpm, for 15 min at 4°C, the supernatant (soluble fraction) was separated from the cellular debris and stored at −80°C prior to each use. For spent medium extraction, C. albicans SC5314 cultures in minimum and YPD media were grown at 37°C for 1–3 days. After incubation, 5 ml of media were separated from cells by centrifugation, filtered sterilized, concentrated 500× and frozen at −80°C. E. coli cell lysates of 10⁷ cells were prepared by sequential freezing/boiling procedures and the supernatant was separated from the cellular debris by centrifugation at 13,000 rpm for 5 min at 4°C, and stored at −80°C.

C. albicans SC5314 biofilm was induced according to the protocol described elsewhere (Hawser and Douglas, 1994), with minor modifications. Biofilms were grown on 150 mm² culture bottles, using minimal medium (0.67% YNB, Difco) supplemented with 50 mM glucose. Biofims were allowed to grow during 66 h, with medium replacement every 24 h. The spent medium was removed and biofilms were washed twice with 20 ml of cold PBS to remove non-adherent cells. 10 ml of cold PBS were added into each bottle and using cell scrappers, all biofilms and cells were detached from the bottles. The biofilm suspension was vigorously vortexed to disassemble cells from biofilm matrix, which were separated by centrifugation at 4000 rpm, 8 min, 4°C. Biofilm matrix supernatant was then filtered (0.22 μm filters, TPP), concentrated 500 times using Centricon Plus–20 (5000 mW—Millipore) and stored at −80°C.

Cell lysates and proteins were resuspended in 4 × Laemmli buffer, boiled for 5 min, separated by SDS-PAGE (Laemmli, 1970) and transferred to poly (vinylidene difluoride) membranes (GE Healthcare) (Towbin et al., 1979). Membranes were blocked overnight at 4°C with 5% non-fat dry milk in PBS-0.1% tween 20 and probed with: Pre-immune rabbit serum (1:500 in PBS); anti-His₆-enolase (1:2000 in PBS); anti-His₆-tag antibody—Santa Cruz Biotechnology (1:1000 in PBS). Membranes were then incubated with anti-rabbit IgG or anti-mouse IgG secondary antibodies (Sigma) at 1:1000 for 1 h in an orbital shaker. Detection was performed with the ECL-PLUS (GE Healthcare) according to the manufacturer's suggestions.

C. albicans SC5314 was grown in minimum medium at 37°C until the stationary phase. The culture was separated into two batches. The first batch was then separated again. One flask was maintained at room temperature and the other at 37°C without shaking for 5 h. The second batch was rapidly centrifuged to separate the cells from conditioned medium, filtered and then separated again. One flask was maintained at room temperature and the other at 37°C without shaking for 5 h. Aliquots of 5 ml were removed in different time points: 0 min, 30 min, 1, 2, 3, 4, and 5 h, concentrated 500 times (to 10 μ l) and stored at −80°C.

C. albicans SC5314 was grown in YPD medium, overnight, at 37°C and 200 rpm. Two aliquots of 10⁷ yeasts were separated. The first aliquot was fixed in 3.5% formaldehyde for 1 h, washed in PBS and incubated overnight with anti His₆-enolase (diluted 1:80 in PGN + 1% saponin) (permeabilized cells). The second aliquot was first incubated with anti His₆-enolase without saponin and then was fixed (non-permeabilized). After consecutive washes in PBS, cells were incubated with anti-rabbit IgG marked with fluorescein (FITC) (diluted 1:100 in PBS) for one 1 h, at room temperature. After two washes, the number of yeasts was estimated with a fluorescent cytometer Becton-Dickinson, counting 10⁴ cells. As a control, permeabilized and non-permeabilized cells were incubated with pre-immune rabbit serum. For the enolase uptake assay, a culture of C. albicans SC5314 was processed as described above. Five aliquots of 2 × 10⁴ cells were separated. The first aliquot was incubated overnight at 4°C with the elution buffer of the recombinant protein purification and the other four aliquots were incubated with different concentrations of His₆-enolase: 1.25, 2.5, 5, and 10 μg. After washes with PBS, cells were fixed with 3.5% paraformaldehyde for 1 h. Then, cells were incubated with anti-His₆ (diluted 1:20 in PGN) for 1 h, washed and incubated with anti-rabbit-IgG labeled with FITC (diluted 1:50 in PBS). As control, the recombinant enolase was coupled to latex beads (100 μ g of His₆-enolase to 10⁸ beads) and incubated in coupling buffer (200 mM sodium carbonate, 500 mM sodium chloride, pH 8.5) overnight, at 37°C, with agitation. Beads were subsequently washed and blocked with BSA solution in PBS (20 mg/ml) and then incubated with anti-His₆ (diluted 1:20 in PGN) for 1 h. After this period, they were washed and incubated with anti-mouse IgG labeled with FITC (diluted 1:50 in PGN). After two washes cells were analyzed in a Becton-Dickinson flow cytometer (10,000 events).

C. albicans were centrifuged, washed with PBS and fixed with 3.5% formaldehyde in PBS for 1 h. After incubation, cells were washed at least five times with PBS and then 10 μ l were dripped on to glass slides to dry at room temperature. For biofilm formation, glass slides were coated with poly-lysine (10 mg/ml) (Sigma), dried and the biofilm was formed according to the protocol previously described. Slides containing yeasts, hyphae and biofilm were permeabilized with 1% saponin (BDH, Amersham, UK) in PGN (0.2% gelatin, 0.1% NaN₃ in PBS) or incubated only with PGN for blocking (non-permeabilized cells) for 15 min. The coverslips were incubated with anti-His₆-enolase (diluted 1:80 in PGN or PGN + saponin) overnight at 4°C and washed with PBS. They were subsequently incubated with anti-rabbit-IgG conjugated with Cy3 (Sigma) diluted 1:100 in calcofluor white (10 mg/ml) for 1 h, in the dark. After washing with PBS, coverslips were mounted in glycerol buffered with 100 mM Tris pH 8.6 and 0.1% p-phenylenediamine. Slides were examined under a confocal microscope (BioRad 1024-UV) coupled to a Zeiss Axiovert 100 microscope. Alternatively, images were acquired in an epifluorescence microscope.

The adhesion assays were based on a modification of the protocol previously described by (Segal and Sandovskylosica, 1995). Five week-old male Balb/c mice were purchased from University of São Paulo - USP, Brazil. Experiments were performed in accordance with guidelines for animal use and care of the Federal University of São Paulo—UNIFESP and approved by the Ethics Committee in Research. Mice were maintained in pathogen free conditions and aged 8–12 weeks for the experiments. Mouse integral intestine was aseptically removed from sacrificed animals and a cut of approximately 0.4 inches was done after the end of the stomach and before the caecum. The small intestine (duodenum, jejunum, and ileum) was cut longitudinally, rinsed once in sterile PBS to remove aggregates and opened on a flat surface. Mice intestinal tissue disks (6 mm in diameter) were obtained with a sterile metal punch. Inhibition of C. albicans adhesion to mouse intestinal disks coated with recombinant enolase was performed as follows: disks were added to eppendorf tubes containing increasing concentrations of His₆-enolase to a final volume of 1 ml in PBS. These tubes were then incubated for 2 h at 37°C in an orbital shaker. Disks were rinsed twice in PBS, added to eppendorf tubes containing 2.5 × 10⁶ C. albicans in PBS and incubated for 2 h at 37°C. Disks were rinsed 5 times to remove non adherent cells, homogenized and eluted in 1 ml of PBS. Pre-coated C. albicans adhesion assays were performed by preincubating 5.2 × 10⁶ C. albicans yeasts with different concentrations of purified anti His₆-enolase for 2 h at 37°C. Cells were centrifuged at 3000 g for 3 min at 4°C, washed 3 times and eluted. Disks were added to cell suspensions and processed as previously described. All elutions were serially diluted and plated on YPD medium containing 34 μg of chloramphenicol/ml. Plates were incubated for 48 h at 30°C, and the number of attached microorganisms per tissue disk was estimated by counting C. albicans colony forming units (cfu).

The intracellular role of enolase as a glycolytic pathway enzyme is widely known. To investigate its potential extracellular role, suggested by its presence in the biofilm matrix, we produced a recombinant C. albicans enolase (Figure 1) and an anti-His₆-enolase polyclonal rabbit serum. We found that native enolase can be detected in the biofilm matrix of C. albicans SC5314 and in cell lysates of three different C. albicans strains including the SC5314, one clinical isolate identified as a high biofilm producer (L296) and a reference strain that is a low biofilm producer (ATCC90029), used as control (Padovan et al., 2009) (Figure 1C). Only one band was observed in the biofilm matrix and also the homologous enolase in the cell lysate of S. cerevisiae S288C. A weaker signal was observed after 3 days in YPD medium (Figure 1C).

Since it has been reported that enolase is present on the cell surface of yeasts (Pancholi, 2001), we tested the ability of the anti His₆-enolase polyclonal rabbit serum to detect cell surface enolase in biofilm-forming cells. Flow cytometry data (Figure 2A) showed that enolase could be detected on the surface of 14% non-permeabilized yeasts cells, while in permeabilized cells; enolase was detected in 40% (Figure 2B). The growth conditions of cells used in this experiment (see Flow cytometry) might have induced the formation of germ-tubes and pseudo-hyphae, possibly explaining the flatline shift in fluorescence observed in Figures 2A,B. To verify whether cells in the biofilm also had enolase on their surface, immunofluorescence microscopy images of yeasts and hyphae were compared with biofilms (Figure 3). Figures 3A–D show that enolase was not recognized by the pre-immune serum. Figures 3E–H show enolase covering the cell surface of non-permeabilized yeasts. Diffused and stronger labeling was observed in permeabilized cells (Figure 3K) although several cells were not labeled. Figures 3M,N showed strong enolase signal in permeabilized biofilm-forming cells, both in yeasts attached to the slide, at the basal layer, as in hyphae and buds at the upper part of the biofilm. Non-permeabilized biofilm-forming cells exhibited less enolase signal on their surface (Figures 3O,P). Strikingly, in non-permeabilized biofilm-forming cells, the co-localization of enolase with calcofluor occurred in a few cells. These data support previous findings that enolase is present on the cell surface of C. albicans yeasts and indicate for the first time that enolase is also present on the surface of hyphae and biofilm-forming cells (yeasts and hyphae).

It could be argued that the presence of enolase in the biofilm matrix is a consequence of cytoplasm leakage of dead cells at the bottom layers of the biofilm, and therefore there would be no specific function for enolase in the biofilm matrix. To test whether native enolase could be detected in stationary cultures, C. albicans SC5314 was grown in minimal medium until reaching the stationary phase and aliquots of the culture medium with and without cells were taken from 0.5, 1, 2, 3, 4, and 5 h to check for the presence of native enolase. A decay in native enolase levels was observed at room temperature, with or without cells (Figure 4A), while a much faster decay was observed at 37°C at 30 min, especially in the cell-free conditioned medium (Figure 4B). To test whether cytosolic proteins could be detected in the biofilm due to cell lysis, we checked for the presence of Sui2p (eIF2α—The alpha subunit of the eukaryotic translation initiation factor 2), a cytoplasmic protein of S. cerevisiae. The S. cerevisiae Sui2p has 84% of similarity and 70% identity with its C. albicans homolog, CaSui2p (orf19.6213). We detected Sui2p and CaSui2p in cell extracts of S. cerevisiae and C. albicans, respectively, while in conditioned media it was weakly detected and not detected at all in the biofilm matrix (Data not shown).

Because enolase was detected on the cell surface of planktonic yeasts and hyphae and on biofilm forming cells, the enolase decay could also be explained by the enolase uptake from the external medium to the cell wall. To test this hypothesis, cultures of C. albicans SC5314 were incubated with 4 different concentrations of His₆-enolase and its presence on the cell surface of yeasts was assessed by flow cytometry. Figure 5 shows that there was no recognition of the recombinant enolase on the surface of C. albicans cells in all the concentrations tested (1.25–10 μ g), as opposed to His₆-enolase coated beads. These data suggest that enolase present on the cell surface is not a product of protein uptake from cell debris lysis.

Taken together the results above suggest that enolase in the biofilm matrix is not a product of cell lysis because it would be rapidly degraded at 37°C as occurred in yeast cell cultures (Figure 4B). Therefore enolase might be functionally secreted to the extracellular environment such as biofilms and to the cell surface where it is stable. These results also indicate that the presence of enolase on the cell surface is not a result of protein uptake from cell debris lysis.

To investigate the role of cell surface enolase in adhesion, we first checked whether C. albicans yeasts preferentially associates with specific parts of mice small intestine in vitro. There was no difference in the number of yeasts adhering to disks obtained from three equal portions (proximal, medial and distal third parts) of small intestine tissues (Figure S1 supplemental). Therefore, we employed disks prepared from the whole small intestine in all our assays. C. albicans yeasts were pre-incubated with different concentrations of affinity-purified rabbit anti-serum and then incubated with mice GI tissue disks. There was no difference in the number of yeasts per tissue disks when C. albicans was blocked with the pre-immune serum or with the anti His₆-CaYlr39cp (an antibody against the intracellular protein CaYlr339cp), used as a negative control. On the other hand, blocking C. albicans with 10 and 20 μg of anti His₆-enolase decreased its adhesion to mice GI tissue disks to approximately 42 and 47 % respectively, in relation to anti His₆-CaYlr339cp (Figure 6A). The inhibition of adhesion seemed to saturate when 10 μg of antibodies were used since twice of its concentration did not decrease proportionately the adhesion of yeasts to GI disks. In an attempt to verify if the decrease in adhesion observed was not due to the potential recognition and blockage of surface proteins rich in histidine regions in C. albicans, yeasts were also blocked with a monoclonal anti-His₆-Tag antibody. There was no difference in adhesion in relation to the negative control (Data not shown). Also, viability assays indicated that this inhibition was not dependent on potential candidacidal activities promoted by the polyclonal antiserum raised against recombinant enolase (Figure S2 supplemental). These results indicate that the blockage of enolase present on the cell surface of C. albicans with specific antibodies against enolase impairs its adhesion to mice GI disks in vitro.

Since the coating of cell surface enolase with antibodies was able to inhibit C. albicans yeasts adhesion to GI disks in vitro, we hypothesized that blocking potential enolase receptors on disks might also have an effect on C. albicans adhesion. To test this hypothesis, mice GI tissue disks were pre-incubated with different concentrations of recombinant His₆-enolase. After incubation, disks were washed and then incubated with C. albicans yeasts. Strikingly, the incubation of disks with 0.5 mg of soluble purified His₆-enolase inhibited C. albicans adhesion to GI disks at 70% in comparison to the purified protein His₆-CaYlr339cp, used as a negative control. The decreased adhesion was dose dependent because incubation of disks with 0.1 and 0.2 mg of enolase inhibited adhesion in 39 and 52 % respectively (Figure 6B). There was no difference in the number of attached C. albicans cells per disk in relation to disks incubated with His₆-CaYlr339cp in PBS or PBS alone. Together, these results indicate that recombinant enolase may be blocking specific host enolase receptor(s) on mice GI disks, thus inhibiting the adhesion of C. albicans.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.