Strains and growth conditions. C. albicans
strains used in this study are listed in Table . The strains were routinely inoculated from overnight YPD cultures (10 g yeast extract, 20 g peptone, and 20 g glucose per liter) into fresh medium and grown at 30°C on a rotary shaker for 6 h. Hyphae were induced in YPD plus 10% heat-inactivated fetal calf serum at 37°C or in α-MEM plus 2% glucose at 37°C. Difco yeast nitrogen base (YNB; without amino acids; Becton Dickinson, Heidelberg, Germany) supplemented with 75 mM ammonium sulfate and 2% glucose was used as synthetic medium. Spider medium contained 1% nutrient broth, 0.2% K2
, and 1% mannitol as a carbon source (18
). For growth on plates, 1.5% agar was added to the medium.
Deletion of SUN41 and reconstitution of mutant strains. SUN41
was deleted in C. albicans
strain SC5314 by FLIP-mediated, site-specific recombination according to the methods of Reuß et al. (29
). Briefly, one pair of sequences flanking the SUN41
coding sequence was amplified by PCR using the following primers: orf6.2071_FLR1a_Apa1 (5′-GCTGGGGCCCAACCCACTGTGGCATATGAA-3′) and orf6.2071_FLR1a_Xho1 (5′-CCGCTCGAGTGCTGGAAGTACTGCACATAATTT-3′) for FLR1; orf6.2071_FLR2_Not1 (5′-ATAAGAATGCGGCCGCACCCCTTTTTCTCTTCTTCCTT-3′) and orf6.2071_FLR2_Sac2 (5′-TCCCCGCGGACCGAAAAATCTTTGGCAGA-3′) for FLR2. Unique cleavage sites were embedded in the primer sequences for directed ligation of the flanking regions into the plasmid pSFS2A (29
). FLR1 was cloned into the vector after ApaI and XhoI digestion, and FLR2 was cloned after NotI and SacII digestion, resulting in the plasmid pSFS2A-sun1a-2. Reconstituted mutants were produced in a similar way. The SUN41
open reading frame, including 1,031 bp upstream, was PCR amplified with the primers orf6.2071_FLR1a_Apa1 and 2071_end_rev_Xho1 (5′-CCGCTCGAGGGAAGAAGAGAAAAAGGGGTATC-3′) and cloned together with FLR2 into pSFS1A (29
) using the restriction sites ApaI/XhoI and NotI/SacII for FLR2. C. albicans
strains were transformed by electroporation as described previously (29
Deletion and reconstitution were confirmed by Southern blotting and real-time PCR.
Isolation of chromosomal DNA and Southern hybridization.
Chromosomal DNA from C. albicans
cells was isolated as described according to the method of Hoffman and Winston (9
). Southern blot analysis was performed according to standard protocols using 25 μg of BclI-digested genomic DNA. To show the gene deletion, the blots were probed with a DNA fragment that was PCR amplified using primers for FLR2 (see above). The reconstitution was detected by a probe amplified with SUN41_Rev_4 (5′-ACCGTTGTTGGAGGTGGTAG-3′) and 2071_end_rev_Xho1. After hybridization, Southern blots were exposed to phosphor screens (Fujifilm, Duesseldorf, Germany) for 24 h. The screens were scanned using a FLA-5100 scanner (Fujifilm).
Growth rate determinations.
The doubling times of the C. albicans strains were determined by growing them in liquid YPD medium on a rotary shaker at 30°C. At 1-hour intervals, an aliquot was removed and sonicated briefly, followed by measurement of the optical density at 600 nm (OD600).
Cell size determination.
Cells were grown under standard conditions in YPD and, after brief sonication, cells were collected by centrifugation. The cell pellet was incubated in Brilliant Blue staining solution to contrast cells followed by two washing steps in phosphate-buffered saline (PBS). Resuspended cells were imaged using light microscopy, and the contrast of cells to background was enhanced using Adobe Photoshop software. Afterward, the area of single cells was determined with the software Scion Image (Scion Corporation). The software Origin (OriginLab Corporation) was used to analyze the data. Two sample independent t tests were performed.
C. albicans strains were grown under standard conditions to exponential phase and imaged using an Olympus BX 60 microscope (Olympus, Hamburg, Germany).
Scanning electron microscopy.
C. albicans strains for scanning electron microscopy were grown under standard conditions to exponential phase. Cells were washed two times with H2O and dropped onto glass slides. After freezing, cells were dried by lyophilization and were sputtered according to standard protocols. Scanning electron microscopy was performed using the 1530 VP electron microscope (LEO).
Transmission electron microscopy.
Samples for transmission electron microscopy were prepared according to the protocol described by Reinhard et al. (28
). Briefly, 3 × 107
blastospores of each strain were collected by centrifugation at 1,600 × g
after 6 h of growth in liquid YPD medium and fixed for 1 h by addition of glutaraldehyde at a final concentration of 2.5%. The samples were then washed with YPD and postfixed for 1 h with 1% osmium tetroxide at room temperature. The fixed cells were dehydrated through a graded series of acetone and embedded in Spurr's resin (Sigma-Aldrich). Thin sections were stained with uranyl acetate and lead citrate and then imaged with a Zeiss EM 10 electron microscope.
Cultures of C. albicans strains were inoculated from overnight cultures, grown to an optical density at 600 nm of 1, and gently sonicated. Series of 10-fold dilutions were prepared, and approximately 105, 104, 103, 102, and 101 cells were spotted onto YPD plates supplemented with 10% serum, YNB plates with Congo red (300 μg/ml), or α-MEM plates. As a control, cells were applied to YPD plates without any supplement. The plates were documented after incubation at 30°C or 37°C for 5 days using a digital camera (Easyshare Z7590; Kodak GmbH, Stuttgart, Germany) or a flatbed digital scanner (Epson Expression 1680 Pro).
C. albicans strains were inoculated from overnight cultures and grown to exponential phase in liquid YPD medium which was supplemented with Congo red (300 μg/ml).
C. albicans strains were inoculated from overnight YPD plates to fresh YPD plates (with or without 10% fetal calf serum). After growth for 48 h (30°C or 37°C for hyphae), cell films were washed for 2 min with double-distilled H2O on a circular shaker at 75 rpm. The same procedure was used for plates containing additional Congo red (300 μg/ml).
Cells were treated with the recombinant β-1,3-glucanase Quantazyme ylg (MP Biomedicals, Illkirch, France) according to the protocol of the manufacturer. The OD600 was measured before and every minute after addition of 500 U/ml enzyme. Data were plotted against time, and the time required for a 50% decrease in the OD was determined as the half-life for spheroplast lysis.
The human colorectal adenocarcinoma cell line Caco-2 (ATCC HTB-37) was grown in 75 cm2 tissue culture flasks (Greiner Bio-One, Frickenhausen, Germany). Dulbecco's modified Eagle's medium (Gibco) supplemented with 10% heat-inactivated fetal calf serum, 1 mM sodium pyruvate, and 1% gentamicin was used as the medium. Cells were cultivated at 37°C under 5% CO2. Just before reaching confluence, Caco-2 cell cultures were split 1:5 by standard methods.
Twenty-four-well polystyrene plates were employed to study the adhesion behavior of C. albicans
on different surfaces as described previously (5
). The quantitative data were based on four experiments.
Biofilms from different C. albicans
strains were produced on sterilized, polystyrene, flat-bottom 96-well microtiter plates (Greiner Bio-One) as described previously (12
) with some modifications. Briefly, 100 μl of a standardized cell suspension (3 × 107
cells/ml) was transferred into each well (eight replicas) of a microtiter plate and incubated for 1.5 h at 37°C to allow the yeast to adhere to the surfaces of the wells (15
). As controls, eight wells of the microtiter plate were handled in an identical fashion, except that no Candida
suspensions were added. Following the adhesion phase, the cell suspensions were aspirated and each well was washed twice with 150 μl of PBS to remove loosely adherent cells. A total of 100 μl of yeast nitrogen base medium was then transferred into each of the wells, and the plates were incubated at 37°C. The biofilms were allowed to develop for 48 h and then the yeasts were quantified by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay (see below).
XTT reduction assay.
The XTT reduction assay used was a modification of the methods described previously (15
). Briefly, XTT (Sigma-Aldrich) solution (1 mg/ml in PBS) and menadione (Sigma) solution (1 M in acetone) were prepared immediately before each assay. XTT solution was mixed with the menadione solution at a ratio of 1,000:1 by volume. The biofilms were first washed two times with 200 μl of PBS, and then 100 μl of the XTT-menadione solution was added to each well. The plate was then incubated in the dark for 2 h at 37°C. Following incubation, 80-μl aliquots of solution were transferred to new wells and the color change in the solution was measured at 490 nm with a microtiter plate reader (SpectraMax Plus microplate spectrophotometer; Molecular Devices, Ltd., Sunnyvale, CA). The absorbance values of the controls were then subtracted from the values of the test wells to eliminate spurious results due to background interference.
C. albicans cells were grown in suspension cultures as described before. Cells were collected by centrifugation at 1,600 × g and dropped into liquid nitrogen for generating cell pearls. These cell pearls were stored at −80°C. In order to isolate total RNA, the cell pearls were broken mechanically by grinding in a precooled Retsch mill (Retsch, Haan, Germany), and RNA was further purified using the RNeasy mini kit (Qiagen, Hilden, Germany), following the instructions of the manufacturer. The quality of the isolated RNA was checked by agarose electrophoresis prior to analysis.
A selection of all enzymes annotated as modifying the major backbone of the cell wall was tested. Amounts of cDNA of selected genes in the sun41Δ mutant strain were compared to the wild type by using TDH3 (glyceraldehyde-3-phosphate dehydrogenase) as a reference and assuming an equal amount of transcript of this gene in the samples. Additionally, the transcript amounts of the genes of interest were compared to the amount of TDH3 transcript to get an idea about the abundance of the respective genes. One μg total RNA was transcribed to cDNA using the QuantiTec reverse transcription kit (Qiagen). Two ng of this cDNA was used to perform real-time PCR in a LightCycler 480 (Roche, Mannheim, Germany) with the LightCycler 480 probes master kit according to the manufacturer's instructions on 96-multiwell plates. Probes used (as indicated) originated from the human Universal ProbeLibrary (Roche), and gene-specific primers (TIB MOLBIO, Berlin, Germany) were as follows: for BGL2 (orf19.4565), 5′-TGAAGCTGAAAAGGAAGCTTTG-3′, 5′-GCTTCAGAACCAACCAAGAAA-3′, and probe 22; for PHR1 (orf19.3829), 5′-TCTGGTGGAAGCTCCAAATC-3′, 5′-GGTGCTGCTGCTTGATGAT-3′, and probe 6; for ACF2 (orf19.3417), 5′-TCAAACATCAGCAACAACCAA-3′, 5′-TGTATGGATTGGGGCATCTT-3′, and probe 89; for ENG1 (orf19.3066), 5′-CACCAACAGTTTTCGCAAGA-3′, 5′-TTGGTTTACCGTTGTTGTCG-3′, and probe 18; for SCW11 (orf19.3893), 5′-ACCACACAATCACCTTCCACT-3′, 5′-TTGGACTAGAGGTTGAGGTTGAG-3′, and probe 11; for XOG1 (orf19.2990), 5′-TGCTAAATGGTTGAATGGTGTC-3′, 5′-GCATTATCGTAAGCACCCTCA-3′, and probe 82; for ACE2 (orf19.6124), 5′-TCATCGCCAGAACCACATT-3′, 5′-GGATATCTGTTGCGGTGGTT-3′, and probe 60; for SIM1 (orf19.5032), 5′-CCAGTTGTTTTGGGATCTGG-3′, 5′-TGGGTTTGGAATCAATGACA-3′, and probe 131; for CBK1 (orf19.4909), 5′-AACCACAACAGCAGCAACAA-3′, 5′-GCTGCTGCTGGAATATTTGC-3′, and probe 5; for CHT3 (orf19.7586), 5′-GGTGCTGCTGGATCTTATGG-3′, 5′-CCCAAAGAGTATGAGCAAATTGT-3′, and probe 59; for SUN41 (orf19.3642), 5′-CAGGTACTGAAAATATGGTTATTCCA-3′, 5′-TGATCAACAACGGTAATGACAGA-3′, and probe 10; for PHR2 (orf19.6081), 5′-GTTTCATTAGCCGACTACTTTGC-3′, 5′-TGTTTATACCGAAAAAGTCAGCAG-3′, and probe 9; for TDH3 (orf19.6814), 5′-GCCGTCAACGATCCATTC-3′, 5′-AGAATCGTATTTGAACATGTAAGCA-3′, and probe 50. The LC480 software (Roche) was used to analyze the data.
Isolation of secreted proteins. C. albicans
was inoculated after two washing steps in H2
O in fresh synthetic medium YNB or α-MEM and grown for 7 h. Cells were removed from 10 ml of medium by three rounds of centrifugation (3,500 × g
, 3,500 × g
, and 12,000 × g
). Proteins were precipitated out of the medium by methanol-chloroform (36
). Protein pellets were dissolved in 100 μl 100 mM ammonium bicarbonate. Reduction and alkylation of disulfide chains were achieved by addition of 10 mM dithiothreitol and incubation at 65°C for 15 min, followed by adding iodoacetamide to a final concentration of 20 mM and incubation at room temperature in the dark for 15 min. Thereupon, 1 μg trypsin (sequencing grade, modified; Promega, Mannheim, Germany) was added, and the digest mixture was incubated for 5 h at 37°C before it was stopped by adding trifluoroacetic acid to a final concentration of 1%. Analysis of peptides was performed by tandem mass spectrometry (MS/MS) using matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) or liquid chromatography-coupled electrospray ionization (ESI).
C. albicans was grown in the synthetic medium YNB or α-MEM for 7 h before collecting the supernatant. After precipitation of the proteins, they were resuspended and digested using trypsin. Peptides in a volume of 10 μl were purified by ZipTipC18 (Millipore, Billerica, MA) using a standard protocol and eluted in 1 μl directly on a prespotted AnchorChip target (Bruker Daltonik GmbH, Bremen, Germany). The monoisotopic molecular mass of the peptides was analyzed by MALDI-TOF MS using an Ultraflex II TOF/TOF 200 apparatus (Bruker). The mass spectrometer was set to scan over 700 to 4,000 Da, and the mass spectra were acquired in reflector mode and processed using FlexControl and FlexAnalysis software (Bruker). The most dominant peaks were further microsequenced using post-source decay analysis.
Reverse-phase liquid chromatography (LC)-MS/MS was performed using a Surveyor LC system (Thermo Fisher Scientific, Ulm, Germany) which was coupled to a Finnigan LCQDECA mass spectrometer (Thermo) equipped with an electrospray ion source.
The peptide mixtures were autosampled in 0.1% aqueous trifluoroacetic acid and separated on the analytical column (Jupiter; C18, 1 mm [inner diameter] by 10 cm; Phenomenex) using a linear gradient of 7 to 100% acetonitrile in 0.1% (vol/vol) formic acid for 44 min at a flow rate of 50 μl/min and ionized by an applied voltage of 5 kV to the emitter. The mass spectrometer was operated in data-dependent acquisition mode to automatically switch between MS and MS/MS. Survey MS spectra were acquired for 1 s, and the most intense ions were isolated and sequentially fragmented for 1.5 s by low-energy collision-induced dissociation. The mass spectrometer was set to scan over 300 to 2,000 Da, and the mass spectra were acquired and processed using Xcalibur software (Thermo).
Analysis of mass spectrometric data.
The tandem MS spectra were submitted to the database search program MASCOT (Matrix Science, Great Britain) in order to identify the proteins. Data files were searched against a C. albicans database. This database is based on assembly 19 of the translated open reading frames of the nucleotide sequence of the Stanford Genome Technology Center. The MASCOT search parameters were the following: allowing up to one missed cleavage, a tolerance of 1.5 Da for peptides and 1 Da for MS/MS (MALDI-TOF/TOF, 100 ppm for peptides and 0.5 Da for MS/MS). Probability-based MASCOT scores were used to evaluate protein identifications. Only peptides with P values of <0.05 for random occurrence were considered to be significant.