Abstract
The ability of Candida albicans to switch from yeast to hyphal growth is essential for its virulence. The walls and especially the covalently attached wall proteins are involved in the primary host–pathogen interactions. Three hyphal induction methods were compared, based on fetal calf serum, the amino sugar N-acetylglucosamine (GlcNAc) and the mammalian cell culture medium Iscove’s modified Dulbecco’s medium (IMDM). GlcNAc and IMDM were preferred, allowing stable hyphal growth over a prolonged period without significant reversion to yeast growth and with high biomass yields. We employed Fourier transform-MS combined with a 15N-metabolically labelled reference culture as internal standard for relative quantification of changes in the wall proteome upon hyphal induction. A total of 21 wall proteins were quantified. Our induction methods triggered a similar response characterized by (i) a category of wall proteins showing strongly increased incorporation levels (Als3, Hwp2, Hyr1, Plb5 and Sod5), (ii) another category with strongly decreased levels (Rhd3, Sod4 and Ywp1) and (iii) a third one enriched for carbohydrate-active enzymes (including Cht2, Crh11, Mp65, Pga4, Phr1, Phr2 and Utr2) and showing only a limited response. This is, to our knowledge, the first systematic, quantitative analysis of the changes in the wall proteome of C. albicans upon hyphal induction. Finally, we propose new wall-protein-derived candidates for vaccine development.
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Eight supplementary tables are available with the online version of this paper.
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Edited by: K. Haynes
Introduction
The opportunistic fungal pathogen Candida albicans is a leading cause of fungal death in a clinical setting (Wisplinghoff et al., 2004). In the immunocompetent population, C. albicans colonizes skin and mucosal surfaces which can lead to acute infections. Over 80 % of all women experience at least one bout of vaginal candidiasis in their lifetime. In immunosuppressed patients, life-threatening bloodstream infections occur regularly and are often fatal if not diagnosed in time. In addition, the emergence of antifungal-resistant strains in hospitals around the world highlights the need for better treatment options and new antifungals and vaccines.
The cell wall of C. albicans is the initial site of host–pathogen interactions and is an obvious target for the development of antifungals and vaccines. It is composed of chitin, a glucan layer and mannoproteins. Most wall proteins are covalently linked to β-1,6-glucans by modified glycosylphosphatidylinositol (GPI) anchors. The genome of C. albicans contains 115 predicted GPI-proteins that can be potentially linked to the wall (Richard & Plaine, 2007). Most of these proteins are only expressed under specific conditions and our previous work suggests that there are no more than approximately 20–30 wall proteins expressed at any given time (Klis et al., 2010). GPI wall proteins generally show a modular structure with an N-terminal conserved domain followed by a highly O-glycosylated serine- and threonine- (S/T)-rich domain that can also include a repeat region (Klis et al., 2009). In particular, mannosylation of wall proteins is important for limiting wall porosity (De Groot et al., 2005) and might play a role in β-glucan masking (Wheeler et al., 2008). Wall proteins show a variety of functions ranging from adhesion (Hoyer et al., 1998; Staab et al., 1999) and iron acquisition (Almeida et al., 2008) to tissue invasion (Schaller et al., 2005) and defence against the immune response (Frohner et al., 2009). Recently, the response of the wall proteome to changes in ambient pH has been described (Sosinska et al., 2011). The adhesins Als1 and Als3 as well as Hwp1 and Hyr1 have been used to vaccinate mice against C. albicans infections, resulting in reduced fungal burden and strongly improved survival (Luo et al., 2010; Spellberg et al., 2006).
The yeast-to-hypha transition is the most prominent morphological change in the C. albicans life cycle. Hyphal cells are important for the formation of biofilms (Chandra et al., 2001; Nobile & Mitchell, 2005; Nobile et al., 2006) as well as the invasion of host tissues, while yeast cells are thought to be important for dispersal in the host. The transition also triggers the secretion of degrading enzymes (Naglik et al., 2004) and the production of proteins that protect against oxidative stress (Martchenko et al., 2004). Mutant strains locked in either the yeast or the hyphal morphotype have been shown to be avirulent (Lo et al., 1997) and efficient transition from one stage to the other is important for full virulence.
The yeast-to-hypha transition is tightly regulated by a regulatory network, which includes among others the transcription factors Efg1, Cph1 and Tup1 (reviewed by Biswas et al., 2007), which are activated by morphogenetic stimuli such as the presence of serum, the amino sugar N-acetylglucosamine (GlcNAc) (Simonetti et al., 1974) or specific amino acids (Maidan et al., 2005), as well as upon interaction with innate immune cells like macrophages or neutrophils (Lorenz et al., 2004). Transcriptional studies have indicated that on the transcript level, the yeast-to-hypha transition leads to numerous changes in the expressions of genes encoding proteins involved in both intracellular processes and the composition of the cell surface (Kadosh & Johnson, 2005; Nantel et al., 2002; Sohn et al., 2003). However, transcriptional studies only describe an upregulation or downregulation of gene expression and not the abundance of the final protein. To date the comparison of genome-wide transcription and proteomic studies is limited (Monteoliva et al., 2011) and our understanding of the proteome dynamics is still in its infancy.
In this study we present a comprehensive proteomic analysis based on highly accurate mass determination by Fourier transform (FT)-MS and the use of an internal standard consisting of 15N-metabolically labelled wall proteins for the relative quantification of the changes in the wall proteome of C. albicans upon the yeast-to-hypha transition. We show that hyphal induction triggers a specific response of the wall proteome independent of the induction method. In addition, we identify four categories of wall proteins, depending on morphotype and growth temperature. Many of the quantified proteins promise to be useful for diagnostic and therapeutic purposes.
Methods
Strains, growth conditions and induction of hyphal growth.
All chemicals were obtained from Sigma-Aldrich unless otherwise stated. C. albicans SC5314 (Gillum et al., 1984) was pre-cultured in liquid yeast peptone dextrose (YPD) medium (10 g yeast extract l−1, 20 g peptone l−1 and 20 g glucose l−1) in a rotary shaker at 200 r.p.m. and 30 °C overnight. The next day the overnight culture was used to inoculate flasks containing 50 ml YNB-S [6.7 g yeast nitrogen base (YNB) l−1, 20 g sucrose l−1, 75 mM 3-(N-morpholino)-2-hydroxypropanesulphonic acid (MOPSO), pH 7.4] at OD600 0.5, which corresponds to an initial dry weight of 0.15 mg ml−1 (the dry weight was determined by drying the cell pellet of a 50 ml sample overnight at 65 °C in a pre-weighed centrifuge tube). The cultures were incubated for 18 h at either 30 or 37 °C and 200 r.p.m.
Hyphal growth was induced by supplementing YNB-S with either 5 mM GlcNAc or 10 % fetal calf serum (FCS), or by using 2 % proline as sole carbon source. In addition, C. albicans was incubated in the cell culture media Iscove’s modified Dulbecco’s medium (IMDM-S), α-modified minimum essential medium (α-MEM-S) or Roswell Park Memorial Institute medium 1640 (RPMI 1640-S) at 37 °C overnight [all supplemented with 75 mM MOPSO, pH 7.4, and 20 g sucrose l−1 (sucrose supplement indicated by “-S”)].
Metabolic 15N-labelling of the reference cultures.
To ensure maximal 15N-loading, C. albicans cells from a pre-culture were used to inoculate a second pre-culture to OD600 0.05 in YNB-S with 15N-labelled ammonium sulfate as the sole nitrogen source (Spectra Stable Isotopes; 15N content >99 %) and buffered with 75 mM tartaric acid to pH 4. Tartaric acid was used because it has two pKa values close to pH 4 (4.37 and 3.02 at 37 °C) and is not metabolized by C. albicans (our observations). The culture was incubated overnight at 30 °C and 200 r.p.m. This 15N-labelled pre-culture was used to inoculate two 600 ml cultures of 15N-labelled YNB-S, either buffered with 75 mM tartaric acid at pH 4 or with 75 mM MOPSO at pH 7.4, to OD600 0.1. The cultures were incubated at 37 °C and 200 r.p.m. for 18 h. The 15N-labelled cells of both cultures were harvested and combined 1 : 1; their walls were isolated, divided into aliquots, freeze-dried and stored at −80 °C.
Cell wall preparation.
The cell pellet was collected by centrifugation, frozen in liquid nitrogen and ground into a fine powder with a pestle and mortar. This step ensured highly efficient breakage, especially in the case of hyphal growth, and reduced the amount of cytosolic contaminants in the final sample. Cell walls were prepared as described elsewhere (de Groot et al., 2004). Briefly, the finely ground cell pellet was washed several times with PBS (8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4 per l; pH 7.4 with HCl) and then subjected to breakage in a FastPrep bead beater (Savant Intruments) with glass beads (0.25–0.50 mm, 12–16 runs for 45 s at speed 6.0) in the presence of a protease inhibitor cocktail. Full breakage was controlled by light-microscopic inspection. The pellet was washed several times with 1 M NaCl and stored overnight at 4 °C. The following day, the pellet was washed several times with MilliQ water and then boiled four times for 10 min in SDS extraction buffer [150 mM NaCl, 2 % (w/v) SDS, 100 mM Na-EDTA, 100 mM β-mercaptoethanol, 50 mM Tris/HCl, pH 7.8], washed with MilliQ water and lyophilized overnight. The resulting purified wall pellets were either stored at −80 °C until needed or directly reduced and S-alkylated (Yin et al., 2005). The wall pellets were treated with reducing solution (10 mM dithiothreitol in 100 mM NH4HCO3) and incubated for 1 h at 55 °C. After centrifugation the supernatant was replaced by alkylating solution (65 mM iodoacetamide in 100 mM NH4HCO3) for 45 min at room temperature in the dark. Alkylation was stopped by incubating the samples in 55 mM dithiothreitol/100 mM NH4HCO3 for 5 min. Subsequently, samples were washed six times with 50 mM NH4HCO3 and either frozen in liquid nitrogen and stored at −80 °C or directly used for experiments.
14N/15N mixing and sample preparation for MS analysis.
Our quantification workflow is summarized in Fig. 1(a). The freeze-dried, reduced and alkylated walls of both the 14N-query and the 15N-reference walls were weighed and resuspended in 50 mM ammonium bicarbonate buffer and mixed to obtain a 1 : 1 mixture based on dry weight (2 mg : 2 mg). The mixed walls were digested for 18 h using 2 µg Trypsin Gold (Promega) and subsequently desalted using a C18 tip column (Varian). The amount of peptides was determined using a NanoDrop ND-1000 (Isogen Life Science) at 205 nm as described before (Desjardins et al., 2009; Sorgo et al., 2010). For each run a sample containing 0.8 µg peptides was used.
(a) Workflow to obtain both peptide passports and 14N : 15N-peptide pair ratios of the 14N-query culture with respect to the 15N-labelled reference culture. The 15N internal standard is cancelled out when two 14N-query cultures are compared with each other. (b) Representative comparison of a hyphal induction condition with a non-induced control. Positive values for a protein indicate a strong increase in abundance upon hyphal induction and vice versa.
MS analysis and data processing.
Accurate mass data were acquired using an ApexQ FT ion cyclotron resonance mass spectrometer (Bruker Daltonik) equipped with a 7T magnet and a CombiSource coupled to an Ultimate 3000 (Dionex) HPLC system with a PepMap100 C18 (5 µm particle size, 10 nm pore size, 300 µm inner diameter × 5 mm length) precolumn and a PepMap100 C18 (5 µm particle size, 10 nm pore size, 300 µm inner diameter × 250 mm length) analytical column (Dionex).
Samples (3 µl) containing 0.8 µg of tryptic peptides in a 0.1 % trifluoroacetic acid aqueous solution were loaded onto the precolumn. Following injection, a linear gradient (from 0.1 % formic acid/100 % H2O to 0.1 % formic acid/40 % CH3CN/60 % H2O) was applied over a period of 120 min at a flow rate of 3 µl min−1. During elution a chromatogram of up to 1850 high-resolution electrospray ionization-FT-MS spectra was recorded using an MS duty cycle of about 3 s.
The data were processed using the Data Analysis 3.4 software program (Bruker Daltonik). The 1800 mass spectra were batch-wise extracted from the chromatogram, and the monoisotopic masses of the peptides were determined using Bruker’s peak recognition technology SNAP II. Mass calibration was achieved by selective extraction and subsequent summation of 12 mass spectra from the chromatogram corresponding to MSMS/MASCOT identified tryptic peptides originating from wall proteins (see Supplementary Tables S1–S2, available with the online version of this paper). With the calculated masses of these calibrant peptides the summed spectrum was mass calibrated and the resulting calibration parameters were applied to all spectra in the chromatogram. This resulted in a mass calibration of better than 1.5 p.p.m. over the entire chromatogram for all analyses.
For each FT-MS analysis the resulting array of up to 1800 monoisotopic mass lists was exported as a MASCOT generic file (.mgf). Ion abundances in the exported array of monoisotopic mass lists were the spectral intensities of the most abundant isotope summed over all charge states for each peptide. The exported .mgf file was imported into the in-house-developed CoolToolBox software program (de Koning et al., 2006; Heijnis et al., 2011; Kasper et al., 2007; Müller et al., 2009; Popolo et al., 2008; Sosinska et al., 2011). From the imported array of up to 1800 monoisotopic mass spectra the CoolToolBox program constructed up to 1000 peptide ion chromatograms. For each peptide ion chromatogram the mass and retention time were determined at the apex of the chromatogram profile and the abundance was summed over the ion chromatogram profile.
The Ultimate liquid chromatography (LC)-MS data processing resulted in a peptide monoisotopic mass list with corresponding abundances and LC retention times. Peptide assignments of the ion masses were obtained by matching the processed LC-FT-MS data from the tryptic peptides within a mass window of 1.5 p.p.m. with the masses of tryptic peptides from corresponding 14N- and 15N- C. albicans wall proteins, obtained from an in silico digestion of the C. albicans database of 153 potential processed wall proteins (Sorgo et al., 2010). The peptide elution profiles were shorter than 25 s. As expected the retention behaviour of the 14N- and 15N-peptides was similar, with the 15N-peptides consistently eluting only a few seconds before the corresponding 14N-peptides. Using this criterion combined with the accurate masses of both 14N- and 15N-peptides, the CoolToolBox program automatically picked out all 14N/15N-peptide pairs using a peptide pair retention time window of only 10 s over the total gradient of 120 min. This resulted in unique series of assigned tryptic peptide pairs with the corresponding 14N/15N isotopic ratios for all identified wall proteins (see Supplementary Tables S3–S7, available with the online version of this paper). Further validation of the 14N/15N peptide pair assignments was obtained by matching their retention times with the retention times of the corresponding peptides identified with a LC-FT-MS-MS/MASCOT analysis of tryptic digests of a C. albicans 14N-proteome obtained under identical experimental LC conditions.
For each identified wall peptide pair the CoolToolBox program searched the in silico digest tryptic peptide database generated from the 14N- and 15N-wall proteome database for possible alternative assignments within a mass window of 1.5 p.p.m. For (i) identical peptide sequences in other proteins, for (ii) alternative peptide sequences with the same number of nitrogen atoms and for (iii) alternative peptide sequences with a different number of nitrogen atoms. The numbers of each of the three alternative assignments are listed in the comprehensive Supplementary Tables S3–S7. These numbers indicate the uniqueness of the peptide pairs used for protein quantification. At least three biological replicates of all conditions were measured (Supplementary Tables S3–S7). After analysing the runs as described above, 21 proteins were quantified for all samples (Tables 1 and 3). To evaluate the accuracy of our measurements, the biological relative standard error (RSE) was calculated by averaging the RSE for each protein over all biological replicates. The biological variation was in the range of 18–45 % (Table 3) with a median value of 27 %. We quantified between one and 13 peptide pairs per protein (Table 3). For a complete list of all identified peptide pairs see Supplementary Table S8 (available with the online version of this paper).
Undef., Undefined.
Results
Relative quantification method for wall proteins
A metabolically labelled 15N-reference culture enables relative quantification of unlabelled 14N-query cultures. These can originate from highly different growth conditions and sources such as planktonic cultures, biofilms, and infected tissues and organs, irrespective of nitrogen sources. To obtain a wide representation of wall proteins, and since the culturing pH is known to have a strong effect on the mode of growth and the composition of the wall proteome (Sosinska et al., 2011), we used an acidic pH (pH 4) and a pH close to neutral (pH 7.4) for metabolic 15N-labelling of C. albicans. This allows for systematically collecting query : reference ratios of individual wall proteins belonging to wall proteomes obtained in this and later studies and for a future meta-analysis of the data. To ensure maximal 15N-loading of the cells, we grew them in two consecutive overnight cultures with the stable nitrogen (15N) isotope present in the form of ammonium sulfate (Oda et al., 1999) as sole nitrogen source. We used tryptic shaving of the cell wall (Yin et al., 2005) for optimal release of covalently anchored wall proteins. The high FT-MS mass accuracy of better than 3 p.p.m. together with a narrow elution time window allowed us to set strict rules for the identification of proteins. Our mass spectrometric analysis was dominated by a very abundant peptide (IYDQLPECAK) that was derived from three proteins (Csa1, Pga10 and Rbt5), making it unsuitable for quantification (see discussion below).
Wall protein characteristics and coverage
Table 1 shows that we identified 25 wall-associated proteins with an N-terminal signal peptide in total, 22 of which are predicted to be GPI-anchored. Under our culture conditions we did not consistently detect peptide pairs for the GPI proteins Als1, Als2 and Rbt5 and the non-GPI-protein Fwp1 (Orf19.7104) (Supplementary Tables S3–S7). For example, we found that the level of Als1 was increased approximately fivefold in the walls of cultures grown in the presence of the hyphal inducers fetal calf serum or GlcNAc compared with the reference walls, consistent with its presence in the hyphal base (Coleman et al., 2010) (Supplementary Tables S5 and S6). However, it could not be identified with certainty in the other cultures. The remaining subset of 21 secretory proteins, 19 of which were GPI proteins, was used for further quantitative, comparative analysis. Most of the GPI-proteins have a conserved domain in the N-terminal region, which is also the origin of almost all of the tryptic peptides we detected. Standard mass spectrometric techniques cannot detect all known covalently linked wall proteins. The tryptic peptides can be either too small or too large for the m/z window used as is the case for the tryptic peptides in the N-terminal domain of Hwp1 (Candida Genome Database) (Skrzypek et al., 2010; Sosinska et al., 2011). Glycosylation can interfere with their identification as frequently happens in case of the C-terminal part of GPI-proteins, which is S/T-rich and predicted to be highly glycosylated. Chemical deglycosylation can partially solve this problem as shown for Pga59 and Pga62 (Castillo et al., 2008), but the currently available deglycosylation procedures have ill-defined side-effects. This results in less efficient and reproducible identification of wall proteins and prevents their quantification (our unpublished data). Furthermore, as mentioned in the previous section, identification is sometimes partial, when only a single tryptic peptide is identified that belongs to several members of the same wall protein family.
Under all growth conditions we identified two predicted cytosolic proteins in our wall preparations even after extensive extraction with hot SDS, namely Ssa2 and Tdh3 (Supplementary Tables S3–S7). Ssa2 is a chaperone protein belonging to the Hsp70 family, and evidence has been presented previously that it is present at the hyphal surface (Urban et al., 2003). We found it also in the cell walls of cultures grown at 30 °C and thus completely in the yeast form. Tdh3 is a glycolytic enzyme and has been detected at the cell surface of both yeast cells and hyphae (Gil-Navarro et al., 1997). Since they lack a classical signal peptide, it is not known how these two proteins are retained by the walls and how they reach this location.
Selection of three representative methods for hyphal induction
A variety of conditions induce the yeast-to-hypha transition in C. albicans. We previously showed that culturing cells in the presence of sucrose instead of glucose simulates a low-glucose extracellular environment while allowing high biomass yields (Sorgo et al., 2010). While C. albicans remained in the yeast form at 30 °C even at a hyphal-permissive pH of 7.4, a shift to 37 °C induced some hyphal formation. All growth conditions were tested for biomass formation after 18 h of incubation with an initial dry weight of 0.15 mg ml−1 (Table 2). At 30 °C 7.6 mg biomass ml−1 was obtained after 18 h, whereas at 37 °C the biomass yield was reduced to 5.0 mg ml−1. FCS has been the induction method of choice for decades but due to its animal origin it is not completely defined and varies from batch to batch. Over the 18 h incubation period used in this study in YNB-S+10 % FCS (6.9 mg ml−1), reversion to the yeast form was common. Adding 5 mM GlcNAc (6.4 mg ml−1) to a culture led to strong hyphal induction with more than 90 % hyphae after 18 h. We also tested YNB with 2 % proline as sole carbon source but discarded this induction method because of its low biomass yield (2.5 mg ml−1). To mimic physiological conditions more closely, we tested three FCS-free media regularly used for mammalian cell culture: IMDM-S (5.1 mg ml−1), α-MEM-S (4.9 mg ml−1) and RPMI 1640-S (4.1 mg ml−1). These media maintain hyphal growth even when entering stationary phase with IMDM-S showing the strongest hyphal induction. Based on these results we selected the following hyphal induction media for further study: YNB-S+10 % FCS and two fully synthetic media, namely, YNB-S+5 mM GlcNAc and IMDM-S.
15N denotes that peptides that were suitable for quantification were only present in the reference culture, whereas 14N denotes that such peptides were only present in the query culture.
Of the three induction methods used for quantification, FCS showed the weakest hyphal induction after 18 h judging by both visual inspection (Table 2) and the abundance of hyphal indicator proteins (Table 3). FCS is usually not used for long-term induction, because of increasing reversion to the yeast form in time. GlcNAc at millimolar concentrations was shown to be a strong inducer of hyphal growth even for a prolonged period of time. IMDM-S was the strongest hyphal inducer in our study. The abundance of proteins related to hyphal growth (Als3, Hwp2, Plb5, Sod5 and especially Hyr1) was strongly increased in IMDM-S-induced walls while levels of yeast-associated protein (Sod4, Rhd3 and Ywp1) were strongly decreased (Table 3). IMDM-S-grown cells showed, in addition to these proteins, an increase of Cht2, Phr1 and Sap9. A third category of proteins showed relatively limited variation in abundance under all induction conditions. This category mainly consists of proteins involved in cell wall maintenance and remodelling (e.g. Crh11, Ecm33, Mp65, Pga4, Phr2, Ssr1 and Utr2). Conceivably, these proteins serve a core function required for both yeast and hyphal growth and therefore are required to be maintained at a certain level. Fig. 1(b) shows a relative comparison of protein abundances in IMDM-S at 37 °C with YNB-S at 37 °C. The separation into hypha-associated, yeast-associated and morphotype-independent proteins is representative of all inducers (see also Table 3).
The wall proteome response of IMDM- and GlcNAc-grown cells is highly similar
Ranked correlation analysis (Lehmann & D’Abrera, 1975) of their quantified wall proteomes revealed a strong positive correlation [Spearman’s rank correlation coefficient (RS) = 0.88; P = 1×10−7]. These data suggest that the wall proteome response is mainly dependent on the presence, but not the character, of a hyphal inducer. This is supported by the observation that also the combination of FCS/IMDM and FCS/GlcNAc showed a significant positive correlation (RS = 0.67, P = 0.001 and RS = 0.57, P = 0.008, respectively). Consistent with the considerable reversion to the yeast form and the low relative abundance of hyphal indicator proteins, FCS is the only inducer showing a significant correlation with YNB-S at 37 °C (RS = 0.56, P = 0.01), while both GlcNAc and IMDM-S are not significantly correlated with YNB-S at 37 °C.
Identification of three yeast-indicating, five hypha-indicating and two low-temperature-associated proteins
Our data show that Als3, Hwp2, Hyr1, Pbl5 and Sod5 are good indicators of hyphal growth (Table 3) as also suggested by earlier transcriptional studies (Argimón et al., 2007; Hayek et al., 2010; Kadosh & Johnson, 2005; Theiss et al., 2006). These proteins increased upon the temperature change from 30 to 37 °C and increased even more when an inducer was added, showing morphotype association. Rhd3, Sod4 and Ywp1 are indicative of yeast cells because they showed the inverse relationship as indicated by earlier studies concerning Rhd3 and Ywp1(de Boer et al., 2010; Granger et al., 2005). On the other hand, Als4 and Pir1 levels were sharply decreased upon increasing the temperature from 30 to 37 °C and did not change significantly when inducers were added, indicating association with low temperature (Table 3). These results agree qualitatively with transcriptional data, which show that the expression of ALS4 (Zhao et al., 2005) and PIR1 (Sohn et al., 2003) decreases strongly upon switching the temperature from 30 to 37 °C. Pir1 is essential in C. albicans and is involved in wall regeneration and wall organization (Martínez et al., 2004) and probably also in β-1,3-glucan cross-linking (Ecker et al., 2006), while Als4 is important for the infection of buccal epithelial cells (Green et al., 2004). Interestingly, these proteins have also been shown to be contact-dependent, suggesting that they are involved in infections of the skin, where the body temperature is generally lower (Sorgo et al., 2011).
Discussion
Although transcript levels are expected to correlate with protein synthesis rates, it seems rather unlikely that they show a linear correlation with actual protein levels, especially, when the environmental conditions are unstable. Transcript levels are thus expected to provide information about the direction in which the incorporation levels of wall proteins will move but to be much less valuable in predicting the actual changes in protein levels. A variety of studies have shown morphotype-specific changes in mRNA levels in response to hyphal inducers (Braun et al., 2000; Braun & Johnson, 2000; Kadosh & Johnson, 2005; Sohn et al., 2003). However, there are only a few quantitative proteomic studies available to complement these data (Monteoliva et al., 2011). We set out to perform relative quantification of the covalently anchored cell wall proteins of C. albicans with and without induction of hyphal growth. We used a gel-free approach combining a 15N-metabolically labelled standard culture and FT-MS with a mass accuracy of better than 3 p.p.m. over a large dynamic range. In this way, we were able to perform relative quantification of 21 C. albicans wall proteins.
We used three representative methods of hyphal induction. Muramyl dipeptides, the main inducing component of FCS (Xu et al., 2008) and GlcNAc are both derivatives of peptidoglycan and strong inducers of hyphal growth (Alvarez & Konopka, 2007; Simonetti et al., 1974). Hyphal induction by GlcNAc, acting as a signalling molecule, is mainly mediated by the activation of adenylyl cyclase (Castilla et al., 1998), similar to muramyl dipeptides (Xu et al., 2008). IMDM-S, α-MEM-S and RPMI 1640-S contain all amino acids and some vitamins and are all good inducers, with methionine and proline being the most probable cause of induction (Maidan et al., 2005). IMDM-S was preferred because it was a strong inducer and because the cultures remained in the hyphal growth mode until stationary phase, producing large amounts of biomass. Intriguingly, the three selected inducers, although very different, led to a highly similar response of the wall proteome to GlcNAc and IMDM and to a lesser extent also to FCS, which was due to its partial reversion to yeast growth.
Hypha-associated proteins are important for establishing and maintaining an infection
The adhesin Als3 (Argimón et al., 2007), Hwp2 (Sohn et al., 2003), Hyr1 (Bailey et al., 1996), a potential exo-α-sialidase, the fungal-specific phospholipase Plb5 (Theiss et al., 2006) and the superoxide dismutase Sod5 (Martchenko et al., 2004) were identified as indicators of hyphal growth. Strikingly, all these proteins have infection-related roles ranging from adhesion and aggregation [Als3 (Hoyer et al., 1998), Hwp2 (Alsteens et al., 2010; Hayek et al., 2010; Otoo et al., 2008; Ramsook et al., 2010)], homo- and heterologous biofilm formation [Als3 (Chandra et al., 2001; Nobile & Mitchell, 2005; Nobile et al., 2006; Silverman et al., 2010), Hyr1 (Nobile et al., 2006)], facilitated endocytosis via cadherins [Als3 (Phan et al., 2007)], iron acquisition from the host protein ferritin [Als3 (Almeida et al., 2008)], tissue destruction and in vivo organ colonization [Plb5 (Ibrahim et al., 1995; Theiss et al., 2006)] as well as resistance to host defences. Hyr1 confers resistance to neutrophil killing (Luo et al., 2010), while Sod5 is important for coping with the extracellular reactive oxygen species produced by bone-marrow-derived macrophages, myeloid dendritic cells (Frohner et al., 2009), neutrophils and other granulocytes (Fradin et al., 2005). All these features are important for mounting an effective infection and their association with hyphal growth hints at their importance during localized, invasive infections.
Yeast-associated proteins play a role in dissemination and systemic infections
Rhd3, Sod4 and Ywp1 were indicative of yeast growth. For Rhd3 and Ywp1 no functions have yet been described while Sod4 is a superoxide dismutase. Rhd3 is involved in establishing a systemic infection (de Boer et al., 2010). RHD3 knockouts showed a significant decrease in cell wall mannans and resulted in reduced cytokine production in reconstituted human epithelium infections (de Boer et al., 2010), which implies a role in immune evasion. This might be directly related to the morphotype-specific unmasking of β-glucans (Wheeler & Fink, 2006; Wheeler et al., 2008). Ywp1 abundance is strongly decreased in hyphal walls (our data) as is also indicated by transcriptional studies (Sohn et al., 2003) (Table 3). It seems to play a role in dispersal in the host, since knockouts adhere and form biofilms more readily (Granger et al., 2005). It also possesses a propeptide that might be useful for diagnostic purposes (Granger et al., 2005). The abundance of Sod4, a functional homologue of Sod5, is inversely correlated with the degree of hyphal formation (Fradin et al., 2005; Frohner et al., 2009).
Morphotype-independent proteins serve housekeeping functions of the wall
Under all induction conditions analysed Cht2, Crh11, Ecm33, Mp65, Pga4, Phr1, Phr2, Ssr1 and Utr2 remained relatively stable in abundance. Intriguingly, they are all involved in cell wall maintenance and remodelling (de Groot et al., 2004). This result is also interesting because it mirrors our observations of the secretome (Sorgo et al., 2010). It should be noted that this set of proteins is stable in abundance with respect to the yeast-to-hypha transition but can be significantly affected by other stresses like azole treatment (Sorgo et al., 2011). Remarkably, the transglycosylase Mp65 is present in both the core set defined for the secretome (Sorgo et al., 2010) and every wall preparation and is largely morphotype-independent. This is in agreement with the observation that transcript levels of Mp65 are stable as well (Sohn et al., 2003). Interestingly, it is not covalently anchored to the wall by any known linkages, raising the question of whether it becomes trapped in the walls during homogenization. Furthermore, Mp65 is already established as a diagnostic biomarker and being immunodominant (Bromuro et al., 1994; Gomez et al., 1996). Its strong immunogenicity and high abundance make it a promising target for vaccine development. Finally, PHR1 and PHR2 are a classical example of pH-controlled wall-protein-encoding genes. They belong to the same family and share a similar function, with PHR1 being strongly expressed at neutral pH and PHR2 strongly expressed at pH <5.5 (De Bernardis et al., 1998). The presence of Phr2 in the walls of cultures grown at neutral pH has been described previously (Sosinska et al., 2011). However, at neutral pH its incorporation level was 36-fold lower than at pH 4, suggesting low wall levels at neutral pH. The consistently lower coverage (4–13 %) of the conserved domain of Phr2 compared with Phr1 (29–41 %) in our experiments, which have all been carried out at pH 7.4 (Table 1), supports this.
New targets for antifungal strategies, vaccine development and early diagnostics
The diverse functions of Als3 make it a promising target for the development of new antifungals and vaccines. Monoclonal antibodies bind to the N-terminal region of Als3 (Brena et al., 2007). Vaccination of immunocompetent mice with the recombinant N terminus of Als3 significantly improved survival (Spellberg et al., 2006). Similarly, vaccination with the N terminus of Hyr1 is immunoprotective in mice (Luo et al., 2010). Our data suggest that the N terminus of Hwp2, Plb5 and Sod5, which are all GPI proteins and are therefore predicted to have their N-terminal region extended into the medium (Klis et al., 2009), might be used in a similar fashion. All the peptides we identified also originate from the N-terminal region, which extends away from the cell wall, and might therefore be used in a peptide-based vaccine. The highly abundant peptide IYDQLPECAK is the beginning of the CFEM domain (Kulkarni et al., 2003), a conserved domain of eight cysteine residues spaced over about 70 amino acids that has been implicated in biofilm formation (Pérez et al., 2006). The cysteine in the peptide is the first of eight cysteines in the CFEM domain, which is present in five surface proteins in C. albicans (Csa1, Csa2, Pga7, Pga10 and Rbt5) and in a divergent form in Ssr1. Rbt5, Pga10 and Csa1 have been shown to be involved in iron acquisition (Weissman & Kornitzer, 2004; Weissman et al., 2008). The CFEM domain is located in their N-terminal region and is not glycosylated, suggesting that it might be accessible on the surface. While all CFEM proteins except for Csa1 have only one copy of this domain, Csa1 has four, indicating a domain expansion. CFEM proteins are also present in other Candida species. The abundance of this particular peptide and the CFEM domain in general make them promising targets for marker and vaccine development.
In conclusion, our data give a first quantitative proteomic snapshot of the changes in the wall proteome during the yeast-to-hypha transition of C. albicans. Our method can be easily expanded to other fungi, plants and micro-organisms. The use of a metabolically labelled reference culture as an internal standard allows (i) the comparison of diverse conditions without having to label them individually and (ii) the generation of a library of wall protein ratios under these conditions. We could further show that very different inducers result in a similar response of the wall proteome. In addition, we identified potential targets, especially in the N-termini of wall proteins, for the development of novel diagnostics as well as for a peptide-based vaccine.
Acknowledgements
We thank two anonymous reviewers for their valuable comments. We also thank Winfried Roseboom for technical assistance. F. M. K. is supported by the EU Programme FP7-214004-2 FINSysB. A. G. S. and C. J. H. are grateful for the support by all members of the FINSysB consortium.