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Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV, Amsterdam, The Netherlands
Correspondence
Grazyna Sosinska
sosinska{at}science.uva.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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0.02 % (v/v) O2. Using tandem MS and immunoblot analysis, we identified 15 covalently linked glycosylphosphatidylinositol (GPI) proteins in isolated walls (Als1, Als3, Cht2, Crh11, Ecm33, Hwp1, Pga4, Pga10, Phr2, Rbt5, Rhd3, Sod4, Ssr1, Ywp1, Utr2) and 4 covalently linked non-GPI proteins (MP65, Pir1, Sim1/Sun42, Tos1). Five of them (Als3, Hwp1, Sim1, Tos1, Utr2) were absent in cells grown in rich medium. Immunoblot analysis revealed that restricted O2 availability resulted in higher levels of the non-GPI protein Pir1, a putative β-1,3-glucan cross-linking protein, and of the GPI-proteins Hwp1, an adhesion protein, and Pga10 and Rbt5, which are involved in iron acquisition. Addition of the iron chelator ferrozine at saturating levels of O2 resulted in higher cell wall levels of Hwp1 and Rbt5, suggesting that the responses to hypoxic conditions and iron restriction are related.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). It is also well equipped to form biofilms on abiotic surfaces such as dentures and medical devices (Blankenship & Mitchell, 2006; Ramage et al., 2006). Covalently linked cell-wall proteins (CWPs) play an important role in initiating and maintaining mucosal infections and biofilms (De Groot et al., 2007; Li et al., 2007; Naglik et al., 2006; Nobile et al., 2006; Richard & Plaine, 2007; Ruiz-Herrera et al., 2006; Sundstrom, 1999; Zhao et al., 2006; Zupancic & Cormack, 2007). This is consistent with their cellular location because covalently linked CWPs form an external protein layer surrounding the internal skeletal polysaccharide layer of the wall, and thus come directly into contact with biotic and abiotic surfaces. As in Saccharomyces cerevisiae, the cell-wall proteome of C. albicans consists of 15 or more covalently linked CWPs and includes structural proteins, adhesion proteins, carbohydrate-active proteins, proteases, superoxide dismutases and iron-acquisition proteins (Albrecht et al., 2006; De Groot et al., 2004; Garcera et al., 2005; Mao et al., 2003; Weissman & Kornitzer, 2004). In agreement with the outcome of similar studies carried out with S. cerevisiae, genomic transcript profiling studies of C. albicans indicate that the composition of the cell-wall proteome may vary considerably with the growth conditions of the organism. For example, both yeast and hyphal walls seem to possess specific CWPs (Sohn et al., 2003; Staab et al., 1999). The expression of several CWP-encoding genes changes when Candida cells become associated with different human epithelia or with abiotic surfaces (Garcia-Sanchez et al., 2004; Sohn et al., 2006), when transferred to blood (Fradin et al., 2005), or when confronted with environmental stress conditions such as low or high pH (Bensen et al., 2004), the presence of azoles in the medium (De Backer et al., 2001; Liu et al., 2005), which interferes with sterol synthesis, or the presence of caspofungin, which inhibits the synthesis of the skeletal polysaccharide β-1,3-glucan and thereby causes cell-wall stress (Liu et al., 2005). Iron deprivation and hypoxic conditions are also known to affect the expression of CWP-encoding genes (Lan et al., 2004; Setiadi et al., 2006). Collectively, these observations strongly suggest that the composition of the cell-wall proteome may vary considerably, both qualitatively and quantitatively, depending on environmental cues.
Many Candida infections involve colonization of the vaginal mucosal layer (Fidel & Sobel, 2002; Sobel, 2007). About three-quarters of all women suffer from vaginitis at least once during their life time. Vaginal environmental conditions are characterized by a relatively low pH, a high partial pressure of CO2 (approx. 6 %, v/v) and low to vanishing partial pressures of O2 (
10 %, v/v).
In this study, we have analysed the cell-wall proteome of C. albicans cells grown in vagina-simulative medium (VSM) at 6 % CO2 (v/v) and at O2 levels ranging from 7 to 0.01 % (v/v). We also studied the effect of iron availability by growing the cells at 7 % O2 with an iron chelator. Our results show that under these conditions a new set of CWPs is incorporated into the walls compared to cells grown in rich medium at pH 5–5.6 (De Groot et al., 2004). At restrictive O2 concentrations the levels of at least four CWPs increase, i.e. the structural protein Pir1, the adhesion protein Hwp1 and, in particular, Pga10 and Rbt5, two iron-acquisition proteins. These observations are consistent with the notion that hypoxic conditions may lead to a cellular response that involves an increased scavenging capacity for iron.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. VSM was modified from Moosa et al. (2004) and Owen & Katz (1999) and consisted of 58 mM NaCl, 18 mM KOH, 2 mM Ca(OH)2, 1.75 mM glycerol, 6.7 mM urea, 33 mM glucose, and 6.7 g yeast nitrogen base (YNB) l–1 (Difco). In addition, lactic acid (22 mM; pKa=3.85) and acetic acid (17 mM; pKa=4.76), which are natural compounds in the vaginal fluid, were added to maintain the pH at 4.2. Strains were pre-cultured to saturation in YPD medium [1 % (w/v) Bacto-yeast extract; 2 % (w/v) Bacto-peptone; 2 % glucose (w/v)] at 30 °C and 200 r.p.m. The cells were then inoculated in VSM in batch fermenters (working volume 0.5 l) at 37 °C with an aeration rate of 0.5 l min–1 and stirred at 200 r.p.m. The cells were cultured overnight under ambient air to an OD600 of 0.1; aeration was subsequently switched to gas mixtures, consisting of N2, 0.01–7 % (v/v) O2 and 6 % (v/v) CO2, which leads to dissolved O2 concentrations that correspond to values measured in the human vagina (Wagner & Ottesen, 1982). The cells were harvested at an OD600 of 1. To induce iron restriction, 1 mM ferrozine [5,6-diphenyl-3-(2-pyridyl)-1,2,4-triazine-4',4''-disulfonic acid; Sigma-Aldrich] was added to overnight cultures at OD600 0.1. Culturing was continued until an OD600 1 was reached and the cells were collected for cell-wall analysis. The OD600 of cultures was measured using a Shimadzu model UV mini 1240 spectrophotometer (OD600=1 corresponds to 1.5x107 cells ml–1). To determine the percentage of budded cells (the budding index), 200 cells were taken from two independent cultures for each condition and counted. Relative growth rates were determined by following the OD600 of three separate cultures in the range OD600 0.1 to 1. A reference cell culture grown in rich medium (pH 5.5) (Table 2) was obtained as follows. The medium consisted of 20 g glucose l–1, 10 g Casamino acids l–1 (Becton Dickinson), 6.7 g YNB l–1 (Difco), 110 µg leucine ml–1, 55 µg tyrosine ml–1, 55 µg tryptophan ml–1 and 55 µg adenine sulfate ml–1, pH 5.5, as described by De Groot et al. (2004). The cells were cultured overnight in a batch fermenter aerated with atmospheric air with stirring at 200 r.p.m. at 30 °C and were harvested at mid-exponential phase.
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). Cultures were harvested in the exponential phase of growth, washed with cold demineralized water and with 10 mM Tris/HCl buffer, pH 7.5, and disintegrated in a Bio-Savant Fast Prep 120 machine (Qbiogene), using 0.25–0.50 mm diameter glass beads (Emergo BV). A protease inhibitor mixture (Sigma-Aldrich) was added to protect CWPs from degradation by intracellular proteases. To remove non-covalently linked proteins associated with the wall preparation, crude cell walls were washed with 1 M NaCl and boiled twice for 5 min in 2 % (w/v) SDS, 150 mM NaCl, 100 mM Na-EDTA, 100 mM β-mercaptoethanol, 50 mM Tris/HCl, at pH 7.8. SDS-extracted walls were washed three times with demineralized water and freeze-dried.
Sample preparation for MS analysis.
For MS analysis, CWPs were modified by reduction and S-alkylation as follows. Cell walls were treated with the reducing reagent 10 mM dithiothreitol in 100 mM NH4HCO3 for 1 h at 55 °C. The samples were then cooled and the walls were alkylated by incubating them for 45 min at room temperature in the dark in 65 mM iodoacetamide in 100 mM NH4HCO3. The reaction was quenched by incubating the walls in 55 mM dithiothreitol in 100 mM NH4HCO3 for 5 min. Prior to digestion with trypsin, the walls were washed three times with 50 mM NH4HCO3. Trypsin digestion of S-alkylated CWPs was carried out as described by Yin et al. (2005). Cell walls were incubated overnight at 37 °C in the presence of sequencing-grade trypsin (Roche Applied Science) using a CWP/enzyme ratio of 50 : 1.
MS identification of covalently linked CWPs.
Instrument and analysis settings were as described by Yin et al. (2005). Tryptic peptides (5–10 µg) were desalted and concentrated on a C18-ZipTip pre-column (Millipore) and fractionated using a nano-LC system (PepMap C18; LC Packings, Dionex). Eluted peptides were directly ionized by electrospray in a Micromass quadrupole time-of-flight mass spectrometer (Waters). The ions from the survey spectrum were automatically selected for fragmentation in a collision chamber using Masslynx software. Tandem MS spectra of ionized peptide fragments were analysed with Biolynx and Masslynx Pepseq software. Proteins were identified by comparison of the identified amino acid peptide sequences with in silico digests of the proteins translated from Assembly 19 of the C. albicans SC5314 genome sequence (www.candidagenome.org/) using Mascot software. Several precautions were taken to optimize the reproducibility of the results over time and to monitor drift of the equipment. (1) The nano-LC column was cleaned daily by thoroughly washing it with 100 % solvent B (acetonitrile+0.1 % formic acid). (2) To prevent carry over from previous samples, at least one empty injection was run between each sample. (3) The amount of sample loaded in the LC step of each run was measured using an A214 chromatogram. (4) To control the efficiency of ionization and to verify that the run-to-run variation of the ion intensity was <20 %, the total ion count (TIC) profile of each LC/MS/MS run was determined. (5) To verify the accuracy of MS selection, the instrument was calibrated daily with 1 pmol cytochrome c (Dionex). (6) Each sample (containing 1 pmol protein) was run at least three times, and in two of them the exclusion list was used to ensure complete coverage. (7) The general performance of the LC/MS/MS system was checked monthly with a cytochrome c digest to verify that a similar number of peptides of similar Mascot score were identified.
Isolation of CWPs for immunoblot analysis.
Glycosylphosphatidylinositol (GPI)-modified CWPs were released by treating cell-wall material with recombinant Trichoderma harzianum endo-β-1,6-glucanase (Bom et al., 1998) as described by Kapteyn et al. (2001). Freeze-dried cell walls (4 mg) were incubated overnight with 2.5 µl [0.16 U (mg cell walls)–1] enzyme and 2 µl of a protease inhibitor mixture (Sigma-Aldrich) in 200 µl 50 mM sodium phosphate buffer, pH 5.5, at 37 °C overnight. To release mild alkali-extractable CWPs, cell walls were incubated with 30 mM NaOH at 4 °C for 17 h with gentle shaking; the reaction was stopped by neutralization with 30 mM acetic acid (Mr
a et al., 1997). In each lane, the equivalent of 0.15 mg dried walls (corresponding to 1 mg dried biomass) was applied.
Immunoblot analysis of CWPs.
CWPs were separated by electrophoresis using linear 3–8 % polyacrylamide gradient gels in Tris/acetate (Invitrogen). The separated proteins were transferred onto an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore). To reduce non-specific staining by the antisera, the membrane-blotted proteins were first incubated with 50 mM periodic acid in 100 mM sodium acetate (pH 4.5) prior to the blocking step. Immunoblot analysis was performed with polyclonal Hwp1 (Staab et al., 1996) or Pga10 antiserum (Weissman & Kornitzer, 2004), diluted 1 : 10 000 in 5 % (w/v) milk powder in PBS buffer, pH 7.4, for 2 h. S. cerevisiae Pir2 antiserum (Russo et al., 1992) was diluted 1 : 50 000 and incubated with the blots overnight to enhance interaction with the probed proteins. After washing with PBS, the membranes were incubated with goat anti-rabbit antiserum, conjugated with peroxidase (GARPO) at a dilution of 1 : 10 000 in 5 % (w/v) milk powder in PBS buffer, pH 7.4. Proteins were visualized using Enhanced Chemiluminescence (ECL) (Amersham Biosciences).
Quantazyme sensitivity.
Quantazyme sensitivity of intact cells was measured as described by Kapteyn et al. (2001). Exponentially growing cells were centrifuged and washed twice with 50 mM Tris/HCl, pH 7.5. The OD600 of the cell suspensions was adjusted to 1 and the cells were pre-incubated for 1 h with 40 mM β-mercaptoethanol in 50 mM Tris/HCl buffer, pH 7.5. Quantazyme (from Oerskovia xanthineolytica; Quantum Biotechnologies) was added at 20 U (ml cell suspension)–1. Incubation was carried out at 30 °C, and the decrease in OD600 was measured at 5 min intervals.
Determination of the polypeptide and chitin content of isolated cell walls.
Cell walls (4 mg) were suspended in 100 µl 1 M NaOH. The suspension was incubated at 100 °C for 10 min, cooled and neutralized with 100 µl 1 M HCl. Insoluble material was pelleted by centrifugation and the supernatant was used for protein determination using the bicinchoninic acid protein assay (Pierce). A calibration curve was prepared by using BSA treated in the same way as the cell wall samples.
Chitin content was measured using the method described by Kapteyn et al. (2001). NaOH-extracted (4 mg; see protein determination above) cell walls were hydrolysed for 17 h in 1 ml 6 M HCl at 100 °C. Samples were evaporated under a stream of air and resuspended in 1 ml de-mineralized water. To 0.1 ml sample, 0.1 ml 1.5 M Na2CO3 in 4 % acetylacetone was added and the mixture was boiled for 20 min. After cooling, 0.7 ml 96 % ethanol and 0.1 ml 1.6 g p-dimethylaminobenzaldehyde in 30 ml concentrated HCl and 30 ml 96 % (v/v) ethanol were added before incubation for 1 h at room temperature. A calibration curve was prepared by measuring A520 in a concentration range of 0–40 µg glucosamine ml–1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) (Methods). The medium further contained urea and glycerol and was buffered at pH 4.2 by making use of the physiological buffers found in the vaginal fluid, i.e. 22 mM lactic acid (pKa 3.85) and 17 mM acetic acid (pKa 4.76) (Owen & Katz, 1999). Glucose, which is present in the vaginal fluid at a concentration of 33 mM, served as the main carbon source. The cells were grown at 37 °C. The microenvironment of the human vagina is characterized by lower O2 and higher CO2 levels compared to atmospheric values; (Wagner & Ottesen, 1982). The O2 concentrations measured in the vaginal fluid vary from almost 0 to maximally 0.11 mM [equivalent to 10 % (v/v) partial O2 pressure], depending on the day of the menstrual cycle and the method used for measuring gas tensions. Vaginal CO2 levels, however, vary only slightly during the menstrual cycle (6–8 %, v/v) (Wagner & Ottesen, 1982). Therefore, in this study we supplied O2 to cells in concentrations equivalent to a partial O2 pressure between 0.01 and 7 % (v/v), in combination with 6 % (v/v) CO2, using batch fermenters with controlled aeration.
The growth rate of C. albicans on the medium selected and under an atmosphere of 7 % O2/6 % CO2 equals 0.60 h–1 (corresponding to a generation time of 70 min; see Fig. 1), which is considerably faster than under atmospheric conditions in the same medium (0.45 h–1, corresponding to a generation time of 90 min). As S. cerevisiae can fix CO2 via pyruvate carboxylase to form the citric acid cycle intermediate oxaloacetate, and as higher CO2 concentrations up to 6 % result in increased CO2 fixation (Cazzulo et al., 1968; Liener & Buchanan, 1951), the increased growth rate might be caused by the elevated CO2 concentration. Fig. 1 further shows that at 7 % (v/v), O2 is present at a saturating concentration with respect to the growth rate. The maximal growth rate of C. albicans in batch culture decreases significantly, however, when the O2 level is lowered to less than 0.5 % (v/v) O2 and is halved at 0.02 % (v/v), which corresponds to a dissolved O2 concentration of approximately 0.23 µM. Further experiments were carried out using an O2 level of 0.02 % (v/v).
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). This was not reflected in significant changes in the total polypeptide content of the walls, which was 2.4 % at both 7 % O2 (v/v) and 0.02 % O2 (v/v). The respective chitin contents of these wall preparations were 3.5 and 2.9 %. The small differences in these cell-wall components may imply that the increased Quantazyme resistance of C. albicans is due to an altered protein composition of its wall (see next section).
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7 % (v/v) and at 6 % CO2 (v/v) in VSM, we applied LC followed by tandem MS (LC/MS/MS) of CWP-derived peptides. In combination with immunoblot analysis we detected 19 proteins, including 15 known or predicted GPI proteins, and four non-GPI proteins (Table 2
, Fig. 3). For comparison, cell walls obtained from a fermenter-grown reference culture grown in rich medium were also analysed. Importantly, the GPI-proteins Als3, Hwp1 and Utr2, and the non-GPI proteins Sim1 and Tos1, were only found in VSM cultures. Additionally, in VSM cultures a specific peptide confirming the presence of Rbt5 was detected, which was not identified previously (De Groot et al., 2004). The transglucosylase Phr2 was identified in the walls at all tested O2 concentrations, consistent with its function at acidic pHs (De Bernardis et al., 1998; Fonzi, 1999; Mühlschlegel & Fonzi, 1997). Phr2 peptides were also identified in the reference culture, probably due to the fact that during culture the pH of the reference culture dropped from 5.5 to 4.9. This is consistent with the known pH-dependency of PHR2 expression (Mühlschlegel & Fonzi, 1997). Als3, an adhesion protein that mimics mammalian cadherins (Phan et al., 2007), was also identified in all VSM samples (20 identifications of Als3 peptides; data not shown), whereas no Als3 peptides were found in the reference culture. This is consistent with the frequent occurrence of ALS3 transcripts in vaginal samples during C. albicans infection (Cheng et al., 2005). Als3 has been described as a hypha-specific protein (Argimon et al., 2007; Hoyer et al., 1998); however, as our samples did not contain hyphae, our observations demonstrate that the synthesis of Als3 may also occur in non-hyphal cells under selective environmental conditions. Table 2 also shows that the peptides released by trypsin from GPI-modified CWPs and identified by tandem MS were predominantly found in the N-terminal region of these proteins, consistent with the observation that this region generally is less serine- and threonine-rich and therefore less glycosylated (Chen et al., 1995). No such tendency was observed for the non-GPI proteins.
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), we found increased levels of Pir1 protein in cell-wall extracts obtained with mild alkali, when the O2 level decreased below 0.02 % (v/v) (Fig. 3a). As Pir1 protein is a potential β-1,3-glucan cross-linking protein and essential for cell-wall integrity (Klis et al., 2007; Martinez et al., 2004), this might explain the increased Quantazyme resistance of intact cells grown at low O2 levels.
As the expression of HWP1 is increased in non-hyphal cells grown under hypoxic conditions (Setiadi et al., 2006), we also quantitatively analysed Hwp1 levels. Fig. 3(b) shows that Hwp1 was indeed present in the walls isolated from cells grown under vagina-simulative conditions at all O2 concentrations tested and that its level increased at hypoxic O2 levels. Nevertheless, the highest levels observed under these conditions were still significantly lower than the levels found in hyphal walls; Hwp1 was not found in walls of cells grown in rich medium even after overexposure of the immunoblots (data not shown).
The transcript levels of RBT5, which encodes a predicted GPI protein involved in iron acquisition (De Groot et al., 2003; Weissman & Kornitzer, 2004), also showed a considerable increase under hypoxic conditions (Setiadi et al., 2006). Fig. 3(c) shows that an antiserum raised against Pga10, a homologue of Rbt5 and also involved in iron acquisition (Weissman & Kornitzer, 2004), identified two bands with molecular masses of about 100 and 130 kDa at an O2 concentration of 0.5 % (v/v). To investigate if these bands represented Rbt5 and Pga10 or other members of the family (Weissman & Kornitzer, 2004), we analysed the homozygous single-deletion mutants and the double deletant. Fig. 4 shows that the two bands incorporated in the cell wall of the control strain (CAF2-1) were not present in the double-deletion strain and that only a single band is detected in each of the single mutant strains grown under low-O2 conditions. This is consistent with the notion that the 100 kDa band corresponds to Rbt5 and the 130 kDa band to Pga10. Interestingly, PGA10 and RBT5 belong to the same transcriptional module as HWP1 (level 16, module 16; see also the Candida Genome Database – www.candidagenome.org; Ihmels et al., 2005), showing that these three genes are co-regulated under several other test conditions as well.
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), hypoxic conditions are expected to lead to iron deprivation, raising the question whether iron deprivation alone might induce similar changes in the cell-wall proteome as O2 limitation. Fig. 5 shows that the changes in the cell-wall proteome of cells treated with the ferrous iron chelator ferrozine (1 mM, resulting in a limited decrease in relative growth rate from 0.60 to 0.55 h–1) partially overlapped with the changes observed in the cell-wall proteome of cells grown under hypoxic conditions. The levels of Rbt5 and Hwp1 in cell walls increased in both ferrozine-treated cells and in cells grown under hypoxic conditions, but in ferrozine-treated cells the levels of Pir1 and Pga10 were not increased.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Using a combination of tandem MS and immunoblot analysis, we identified 19 CWPs, five of which (Als3, Hwp1, Sim1, Tos1 and Utr2) were not found in a reference culture grown in rich medium. This is in agreement with earlier results (De Groot et al., 2004). This supports the notion that the composition of the cell-wall proteome of C. albicans is tightly controlled. The presence of Phr2 in the cell walls is in agreement with earlier observations showing that PHR2 is specifically expressed at pHs below pH 5.5 and that the resulting protein is directly involved in cell wall assembly and is essential for colonization of the vagina (De Bernardis et al., 1998; Fonzi, 1999; Mühlschlegel & Fonzi, 1997). Immunoblot analysis identified the presence of an additional GPI protein (Hwp1), which was missed by our MS analysis due to the lack of suitable tryptic peptides (i.e. not heavily glycosylated and with a mass that does not exceed the detection limit of the mass spectrometer). Both Als3 and Hwp1 have been described as hypha-specific adhesion proteins (Argimon et al., 2007; Hoyer et al., 1998; Staab et al., 1999). However, as our cultures did only contain yeast and pseudohyphal cells, our results show that both proteins can also be expressed in non-hyphal cells, depending on the specific growth conditions. This is consistent with the observation that HWP1 is moderately expressed in pseudohyphal cells (Snide & Sundstrom, 2006). Interestingly, ALS3 transcripts have been frequently detected in clinical vaginal fluid specimens, suggesting that the adhesion protein Als3 is important for colonization of this environment (Cheng et al., 2005).
O2 limitation affects multiple processes in eukaryotic cells, such as iron uptake, mitochondrial respiration, sterol synthesis and the synthesis of unsaturated fatty acids, all of which at some stage require molecular O2 (Berg et al., 2007; Kosman, 2003; Schweizer, 2004). There are several indications that the hypoxic conditions used in our study result in a molecular response that resembles the response observed when cells grow under iron-restricted conditions. First, when the O2 concentrations were lowered, the levels of the iron-acquisition proteins Pga10 and Rbt5 in the cell wall increased. Second, intact cells became more resistant to the endo-β-1,3-glucanase Quantazyme (Fig. 2) and to the cell-wall-degrading enzyme preparation Zymolyase (data not shown). This is in agreement with the observation that iron limitation in C. albicans results in increased resistance of intact cells to Zymolyase (Sweet & Douglas, 1991). Third, genomic transcript analyses have revealed that iron-acquisition and iron-uptake genes, including RBT5, are not only upregulated in cells grown at low-iron conditions (Lan et al., 2004; Weissman & Kornitzer, 2004), but also under hypoxic conditions (Setiadi et al., 2006). Finally, addition of the iron-chelating compound ferrozine to the medium, at saturating O2 levels, resulted in an increased level of Rbt5 in the wall (Fig. 5). This is in agreement with the observation that the antifungal agent ciclopirox, which is believed to possess iron-chelating properties as well, strongly increases the transcript level of RBT5 (Lee et al., 2005; Sigle et al., 2005). Interestingly, the presence of ferrozine in the medium also caused an increase in the cell wall of the adhesion protein Hwp1, suggesting an additional control mechanism for HWP1 expression. We propose that hypoxic conditions result in reduced iron uptake and competition for iron by iron-containing enzymes and that this might lead in various ways to increased expression of specific CWP-encoding genes. Another explanation for the response to hypoxic conditions may reside in the fact that numerous O2-dependent reactions in the cell are carried out by iron-containing enzymes [e.g. haems in respiration and Fe-dependent sterol synthesis (Kaplan et al., 2006)]. Maintaining a sufficiently high rate of O2-dependent enzymic reactions could therefore be achieved by an increased synthesis of iron-containing enzymes. To be able to do so, an increase in iron-scavenging proteins like PGA10 and RBT5 would indeed be expected. Our results do not exclude that processes such as sterol synthesis and the synthesis of unsaturated fatty acids, which are affected by the low O2 concentrations used in our study, might also cause major changes in (plasma) membrane properties indirectly affecting the cell wall as well. Indeed, several CWPs (Ecm33, Phr2, Pga10, Pir1 and Rbt5; see Table 2) are induced when the cells are treated with azoles, which are known to affect ergosterol synthesis. We conclude that the cell-wall proteome of C. albicans sensitively reflects the environmental conditions and helps the cell to adjust to stress conditions encountered during the infection process. Our results also show that the composition of the cell-wall proteome is tightly controlled, and that multiple signalling pathways are involved in the regulation of its composition.
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ACKNOWLEDGEMENTS |
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Edited by: J. G. Berman
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Received 21 August 2007;
revised 12 October 2007;
accepted 15 October 2007.
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) or at 0.02 % O2/6 % CO2 (
). The values shown are the means of two replicates.
and pga10