TY - JOUR
AU - Domínguez, Ángel
AB - By two-dimensional gel electrophoresis (2-DE) and mass spectrometry, we have characterized the polypeptide species present in extracts obtained by 60% ethanol treatment of whole mature (48 h) biofilms formed by a reference strain (CAI4-URA3) and four Candida albicans null mutants for cell-wall-related genes (ALG5, CSA1, MNN9 and PGA10). Null mutants form fragile biofilms that appeared partially split and weakly attached to the substratum contrary to those produced by the reference strain. An almost identical, electrophoretic profile consisting of about 276 spots was visualized in all extracts examined. Proteomic analysis led to the identification of 131 polypeptides, corresponding to 86 different protein species, being the rest isoforms—83 displayed negative hydropathic indexes and 82 lack signal peptide. The majority of proteins appeared at pI between 4 and 6, and molecular mass between 10 and 94 kDa. The proteins identified belonged to the following Gene Ontology categories: 21.9% unknown molecular function, 16.2% oxidoreductase activity, 13.3% hydrolase activity and 41.8% distributed between other different GO categories. Strong defects in biofilm formation appreciated in the cell-wall mutant strains could be attributed to defects in aggregation due to abnormal cell wall formation rather than to differences in the biofilm extracellular matrix composition. Candida albicans, cell wall-related genes; PGA10, CSA1, MNN9, ALG5, null mutants, biofilm, extracellular matrix, proteomic analysis INTRODUCTION Although Candida albicans is a typical human commensal microorganism resident of the mucosal surfaces, skin and both gastrointestinal tract and female genitourinary tract (Underhill and Iliev 2014), it is also regarded as one of the most common human pathogens in the clinical setting, being recognized as major agents of hospital-acquired infection (Pfaller et al. 2012; Guinea 2014). A major factor contributing to the virulence of C. albicans is its ability to form biofilms on implanted medical devices, such as catheters, cardiac valvules and other types of prostheses (Kojic and Darouiche 2004; Ramage, Martínez and López-Ribot 2006; Uppuluri, Pierce and López-Ribot 2009; Kniemeyer et al. 2011). Candida albicans cells in biofilms (sessile cells) display more resistance to antifungal agents than their free-living (planktonic) counterparts and are protected from the host defense mechanisms (Ramage, Wickes and López-Ribot 2001; Jabra-Rizk, Falkler and Meiller 2004; Seneviratne et al. 2008; Ramage et al. 2009). This increased resistance of C. albicans cells is not entirely understood and is likely to be a multifactorial phenomenon (Ramage et al. 2002, 2012; Al-Fattani and Douglas 2006; LaFleur, Kumamoto and Lewis 2006; Perumal, Mekala and Chaffin 2007; Bonhomme and d'Enfert 2013; Desai, Mitchell and Andes 2014; Van Acker, Van Dijck and Coenye 2014). Because biofilms are difficult to eradicate with conventional antifungal therapy and represent a permanent focus of infection, in many cases, removal of the implanted device is absolutely needed which poses a problematic issue due to the patient's conditions, anatomic location or underlying disease (Pappas 2011). Consequently, it is essential to develop new approaches to manage C. albicans (as well as other Candida genus species) biofilm-associated infections to preserve the implanted device. Candida albicans biofilms are composed of different cell types (sessile forms)—yeast cells, pseudohyphal cells and hyphal cells—encased in an extracellular matrix (EM) which is the most distinguishing feature of C. albicans biofilms (Chandra et al. 2001; Nett et al. 2010; Gulati and Nobile 2016). In addition to embed fungal cells, the EM plays important structural (preserving the architectural integrity of biofilm; Ramage et al. 2010; Taff, Nett and Zarnowski 2012; Bonhomme and d'Enfert 2013) and physiological functions, including antifungal drug resistance that poses a critical clinical problem as stated above. This EM is composed of carbohydrates, proteins, lipids and extracellular non-coding DNA (eDNA). Major EM components in C. albicans are proteins (55% [wt/wt]) and carbohydrates (25% [wt/wt]), whereas lipids and eDNA accounted for up to ∼15% (wt/wt) and ∼5% (wt/wt), respectively (Thomas et al. 2006; Nett et al. 2007; Martins et al. 2010; López-Ribot 2014; Zarnowski et al. 2014; Nobile and Johnson 2015; Gulati and Nobile 2016). Identification of EM components depends on the method used for efficient EM isolation that is a challenging issue. In fact, it is next to impossible to entirely remove the EM from a given biofilm, because some of the EM fraction may remain bound to the microbial cells, and because the isolation procedure may damage sessile cells, causing intracellular material to leak into the matrix. There is no universal EM isolation method as the extraction procedure has to be adapted to the specific type of biofilm under investigation. Centrifugation, filtration, heating, blending, sonication and treatment with complexing agents, ion-exchange resins and sodium hydroxide have been reported for extraction/isolation of EM in bacterial (Frølund et al. 1996; Nielsen and Jahn 1999) and fungal (Baillie and Douglas 2000; Thomas et al. 2006; Zarnowski et al. 2014) biofilms. It must be stressed that some of the methods indicated above for EM isolation almost certainly lead to contamination with cytoplasmic components. Consequently, the use of putative cytosolic enzymes as specific markers for EM purity, as suggested by some authors (Wingender et al. 2001), must be taken with some caution. First, lysis of cells within the biofilm structure occurs to a certain extent during biofilm formation and maturation. Second, in yeasts such as Saccharomyces cerevisiae and C. albicans strong evidence exists in support of the existence of non-canonical secretion pathways that could be responsible for the localization outside de plasma membrane of several otherwise considered cytosolic proteins such as glycolytic enzymes, chaperones, translation factors and others (Monteoliva et al. 2002; Nombela, Gil and Chaffin 2006; Chaffin 2008; Gil-Bona et al. 2015; Gómez-Molero et al. 2016). Third, several studies have revealed that fungal pathogens (including C. albicans) produce extracellular vesicles, similar to the well-described mammalian exosomes, that carry intracellular macromolecular components of great biological importance through the cell wall (Wolf and Casadevall 2014; Vargas et al. 2015; Nimrichter et al. 2016). Non-conventional secretion pathway and exosomes may be responsible, at least partly, for the presence of a large variety of cytosolic components in the EM of C. albicans biofilms. In the present work, we have employed 60% ethanol (v/v) to release EM polypeptidic components from mature (48 h old) biofilms formed by different C. albicans strains for subsequent proteomic analysis, because a solubilizing agent much gentle than other chemicals such as 2% (wt/v) sodium dodecyl sulfate is currently used to extract cell wall proteins from C. albicans (Chaffin et al. 1998; Chaffin 2008). Although it is well known that distinct polypeptide species are insoluble in EtOH, it can be expected that this agent is able to efficiently solubilize most protein components present in the highly hydrophilic environment of the biofilm EM. Four single null mutant C. albicans strains for cell-wall-related genes were studied in this work. Two mutants were for genes acting at the level of N-glycosylation. One of these genes, ALG5, codes for a glucosyltransferase which transfers glucose to dolichol phosphate for a later transference of glucose to the oligosaccharide nucleus by Alg6p, Alg10p and Alg8p in the lumen of the endoplasmic reticulum (ER). These three glucose residues are later on eliminated (this modification is quite important due to the fact that it is involved in quality control of glycoproteins in the ER; Aebi et al. 2010; Orlean 2012). The second gene, MNN9, acts at the level of the Golgi apparatus and codes for a protein that contributes mostly to the synthesis of the α-1,6 mannose backbone of the outer core in yeasts (Durán 2008; Cívicos 2009). The other two single null mutants were in genes that code for protein species containing an eight-cysteine domain referred as common in several fungal extracellular membrane (CFEM), respectively, PGA10 (also designated as RBT51, coding for a predicted glycosylphosphatidylinositol-anchored protein; [De Groot, Hellingwerf and Klis 2003; Weissman and Kornitzer 2004]) and CSA1 (encoding a mycelial surface antigen; [Lamarre, Deslauriers and Bourbonnais 2000]). Fungal proteins containing the CFEM domain may function as cell-surface receptors or signal transducers, or as adhesion molecules in host–pathogen interactions (Kulkarni, Kelkar and Dean 2003). The CAI4-URA3 reference strain was used as a control. The results obtained in the present communication were compared with proteomic data reported by other authors on the characterization of the secretome of C. albicans (Sorgo et al. 2010) and identification of protein moieties present and/or bound to the surface of sessile cells and/or EM of C. albicans biofilms (Thomas et al. 2006; Martínez-Gomáriz et al. 2009; López-Ribot 2014; Zarnowski et al. 2014). Our results show that although impaired biofilm formation can be observed in all the four different classes of cell wall mutants studied, this must be attributed to defects in aggregation and adhesion to the substrate due to abnormal cell wall structure and/or functionality and not to alterations of the EM composition itself. MATERIALS AND METHODS Strains and growth conditions The Candida albicans strains used are listed in Table 1. Cells were routinely grown in YPD (2% glucose, 1% yeast extract, 2% Bacto peptone [Difco]) or YNB (0.67% yeast nitrogen base without amino acids, 2% glucose) media at 28°C with shaking (100 rpm). Media were supplemented with uridine (25 μg ml−1) when required. Table 1. Characteristics of C. albicans strains used in this work. Strain  Genotype  Parental strain  Reference  SC5314    Wild type  Gillum, Tsay and Kirsch (1984)  CAI4  ura3Δ::λimm434/ura3Δ::λimm434  SC5314  Fonzi and Irwin (1993)  CAI4-URA3  ura3Δ::λimm434/ura3Δ::λimm434, RP10::URA3  CAI4  This worka  PGA10  ura3Δ::λimm434/ura3Δ::λimm434, pga10Δ::hisG/pga10::hisG RP10::URA3  CA3  Pérez et al. (2006, 2011)  CSA1  ura3Δ::λimm434/ura3Δ::λimm434, csa1Δ::hisG/csa1Δ::hisG-URA3-hisG  CAI4  Braun et al. (2000)  ALG5  Δura3::imm434/ ura3Δ::λimm434 Δalg5::hisG/Δalg5::hisG  CAI4  Durán (2008)  MNN9  ura3Δ::λimm434/ura3Δ::λimm434 Δmnn9::hisG/Δmnn9::hisG  CAI4  Cívicos (2009)  Strain  Genotype  Parental strain  Reference  SC5314    Wild type  Gillum, Tsay and Kirsch (1984)  CAI4  ura3Δ::λimm434/ura3Δ::λimm434  SC5314  Fonzi and Irwin (1993)  CAI4-URA3  ura3Δ::λimm434/ura3Δ::λimm434, RP10::URA3  CAI4  This worka  PGA10  ura3Δ::λimm434/ura3Δ::λimm434, pga10Δ::hisG/pga10::hisG RP10::URA3  CA3  Pérez et al. (2006, 2011)  CSA1  ura3Δ::λimm434/ura3Δ::λimm434, csa1Δ::hisG/csa1Δ::hisG-URA3-hisG  CAI4  Braun et al. (2000)  ALG5  Δura3::imm434/ ura3Δ::λimm434 Δalg5::hisG/Δalg5::hisG  CAI4  Durán (2008)  MNN9  ura3Δ::λimm434/ura3Δ::λimm434 Δmnn9::hisG/Δmnn9::hisG  CAI4  Cívicos (2009)  a CAI4-URA3 strain was kindly provided by Dr. Gwyneth Bertram, School of Medical Science, University of Aberdeen. View Large Table 1. Characteristics of C. albicans strains used in this work. Strain  Genotype  Parental strain  Reference  SC5314    Wild type  Gillum, Tsay and Kirsch (1984)  CAI4  ura3Δ::λimm434/ura3Δ::λimm434  SC5314  Fonzi and Irwin (1993)  CAI4-URA3  ura3Δ::λimm434/ura3Δ::λimm434, RP10::URA3  CAI4  This worka  PGA10  ura3Δ::λimm434/ura3Δ::λimm434, pga10Δ::hisG/pga10::hisG RP10::URA3  CA3  Pérez et al. (2006, 2011)  CSA1  ura3Δ::λimm434/ura3Δ::λimm434, csa1Δ::hisG/csa1Δ::hisG-URA3-hisG  CAI4  Braun et al. (2000)  ALG5  Δura3::imm434/ ura3Δ::λimm434 Δalg5::hisG/Δalg5::hisG  CAI4  Durán (2008)  MNN9  ura3Δ::λimm434/ura3Δ::λimm434 Δmnn9::hisG/Δmnn9::hisG  CAI4  Cívicos (2009)  Strain  Genotype  Parental strain  Reference  SC5314    Wild type  Gillum, Tsay and Kirsch (1984)  CAI4  ura3Δ::λimm434/ura3Δ::λimm434  SC5314  Fonzi and Irwin (1993)  CAI4-URA3  ura3Δ::λimm434/ura3Δ::λimm434, RP10::URA3  CAI4  This worka  PGA10  ura3Δ::λimm434/ura3Δ::λimm434, pga10Δ::hisG/pga10::hisG RP10::URA3  CA3  Pérez et al. (2006, 2011)  CSA1  ura3Δ::λimm434/ura3Δ::λimm434, csa1Δ::hisG/csa1Δ::hisG-URA3-hisG  CAI4  Braun et al. (2000)  ALG5  Δura3::imm434/ ura3Δ::λimm434 Δalg5::hisG/Δalg5::hisG  CAI4  Durán (2008)  MNN9  ura3Δ::λimm434/ura3Δ::λimm434 Δmnn9::hisG/Δmnn9::hisG  CAI4  Cívicos (2009)  a CAI4-URA3 strain was kindly provided by Dr. Gwyneth Bertram, School of Medical Science, University of Aberdeen. View Large Biofilm formation Formation of biofilms of the different C. albicans strains was assessed by using the procedure described elsewhere (Ramage et al. 2001; Pérez et al. 2006; Blanes 2012). Briefly, cells were grown overnight in an orbital shaker in YPD medium, harvested and washed in sterile 10 mM phosphate-buffered saline (PBS), pH 7.4. Cells were suspended in RPMI-1640 medium supplemented with L-glutamine and buffered with HEPES (Sigma Chemical Co., St. Louis, MO, USA) to a final concentration of 1 × 106 cells ml−1. Biofilms were formed by pipetting 100 μl of the standardized cell suspensions into wells of commercially available polystyrene presterilized, 12-wells microtiter plates. To harvest large amounts of biofilms, 500 ml flat bottom cell tissue flasks (Nalge Nunc International, Denmark) containing 50 ml of RPMI-1640 medium were inoculated with 5 ml of the standardized cell suspensions. Plates and flasks were incubated at 37°C with gentle shaking (65–70 rpm). Kinetics of biofilm formation was determined by assessing the reduction of the XTT reagent (2,3-bis[2-methoxy-4-nitro-5-sulfo-phenyl]-2H-tetrazolium-5-carboxanilide); visualization of biofilms formed on the bottom of the wells of the microtiter plates was done by staining with 200 μl of 0.5% crystal violet solution for 15 min at room temperature (Ramage et al. 2001; Pérez et al. 2006; Blanes 2012). After incubation spent RPMI-1640 medium was removed from the bottles and biofilms grown on the surface of the flasks were detached with the help of a cell scraper. The biomass of biofilms was collected, washed twice with sterile distilled water and processed for extraction of putative biofilm EM proteins as described below. Protein solubilization Mature (48 h of incubation) biofilms formed on cell tissue culture flasks as described in the previous section were weighted after washing twice with sterile distilled water. Biomass was resuspended in a volume of a 60% (v/v) dilution in distilled water of EtOH equivalent to the wet weight of biofilm biomass to be processed. The resulting suspension was incubated at room temperature for 1 h with gentle shaking (∼70 rpm) followed by centrifugation at 4°C (4.000 × g, 10 min) to remove the extracted biofilms and the supernatant (EtOH extract) was concentrated by freeze-drying. The lyophilized material was resuspended in a small volume of distilled water, divided into aliquots and stored frozen (–85°C) until further analysis. For proteomic analysis experiments (see below), large batches of biofilm biomass formed by each of the different C. albicans strains examined were processed (usually biofilms formed in 20 flasks for each strain), due to the low extracting ability exhibited by EtOH (unpublished observations). Protein concentration in the different EtOH extracts was determined by the method of Bradford (1976) using bovine serum albumin as standard. Two-dimensional electrophoresis Protein samples (300 μg) were precipitated using 2-D Clean-Up Kit (GE Healthcare, Madrid, Spain) following the manufacturer's instructions, solubilized in 340 μl of rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% carrier ampholytes pH 3–11 [IPG buffer, GE Healthcare, Madrid, Spain], 50 mM DTT, 0.002% bromophenol blue) and applied onto the Immobiline DryStrips (pH 3–11 NL, 18 cm, GE Healthcare, Madrid, Spain). Isoelectric focusing was performed using an IPGphor apparatus (GE Healthcare, Madrid, Spain). The gels were actively rehydrated at 30–60 V for 12 h. Focusing started at 500 V and the voltage was gradually increased to 8000 V and kept constant for a further 6 h. Immediately after being focused, IPG strips were equilibrated for 15 min in 1% DTT (w/v) in equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% glycerol [v/v], 2% SDS [w/v], 0.01% bromophenol blue [w/v]), followed by another 15 min in 2.5% (w/v) iodoacetamide in the same buffer. The second-dimensional separation was performed in 12% SDS-polyacrylamide gels. The gels (180 × 200 × 1.5 mm) were run at 40 mA per gel in an SE 600 Ruby apparatus (GE Healthcare, Madrid, Spain). Mr values were estimated using SDS-PAGE standard (Bio-Rad). Staining was carried out with SYPRO Ruby Protein Gel Stain (Sigma‐Aldrich, St Louis, MO) as follows: gel was fixed in 50% methanol, 7% acetic acid for 30 min, incubated overnight in SYPRO Ruby staining solution, washed twice with 10% methanol, 7% acetic acid for 30 min and finally, washed twice with water for 10 min. Image acquisition and data analysis and identification of proteins by MS were performed exactly as described previously (Hodurova et al. 2012). The searches for peptide mass fingerprints and tandem MS spectra were performed in the Candida Genome (http://www.candidagenome.org) and the Génolevures databases (http://www.genolevures.org) databases. Search parameters were set as follows: carbamidomethylated cysteins as fixed modification and oxidized methionine's as variable one, peptide mass tolerance set to 50 ppm and one missed cleavage allowed. In all protein identifications, the probability scores were greater than the minimum score fixed by MASCOT as significant with a P-value below 0.05. RESULTS Biofilm formation by Candida albicans strains The biofilm-forming ability of C. albicans null mutants for PGA10 and CSA1 genes that code for protein species containing the so-called CFEM domain was previously examined at the morphological level by our group using as control the C. albicans CAI4-URA3 reference strain (Pérez et al. 2006). Both null mutants formed fragile biofilms that appeared partially split and weakly attached to the substratum (Pérez et al. 2006). Ability of C. albicans null mutants for ALG5 and MNN9 genes was assessed in this work using as control the CAI4-URA3 strain. The kinetics of adherence and subsequent biofilm formation by the two C. albicans mutants on the surface of the polystyrene wells over 48 h, as determined by the colorimetric XTT-reduction assay (see the ‘Materials and Methods’ section), is illustrated in Fig. 1. Initially, the mutant strains appeared to form biofilm at the same rate as the CAI4-URA3 control strain, but after 8 h under biofilm-forming conditions the mnn9Δ and alg5Δ strains exhibited a decrease in XTT absorbance (Fig. 1a) and the absence of a consolidated biofilm was also evident in both cases. Similar to C. albicans null mutants for genes coding proteins containing the CFEM domain, the mnn9Δ and alg5Δ strains also formed fragile biofilms, weakly adhered to the bottom of the polystyrene wells (Fig. 1b). Light microscopy observations performed in parallel confirmed the poor adherence of biofilms formed by null mutant cells (data not shown). Overall, results reported in this work and in previous communications from our group (Pérez et al. 2006, 2011; Blanes 2012) support the contention that disruption of PGA10, CSA1, MNN9 and ALG5 genes induced a cascade of pleiotropic effects causing several important phenotypic alterations, among them the inability to form normal biofilms. Figure 1. View largeDownload slide (a) Kinetics of C. albicans biofilms formation by the reference CAI4-URA3 (open diamonds) and mnn9Δ (filled circles) and alg5Δ (filled traingles) strains in microtitre plates, as determined by colorimetric readings using the XTT-reduction assay. Results shown are the mean values of eight replicates. Experiments were repeated several times with similar results. (b) Adherence of biofilms formed by CAI4-URA3 (1) and mnn9Δ (2) and alg5Δ (3) strains to the polystyrene surface of cell culture plates before (upper row) and after (lower row) staining with crystal violet followed by washing with PBS buffer. Figure 1. View largeDownload slide (a) Kinetics of C. albicans biofilms formation by the reference CAI4-URA3 (open diamonds) and mnn9Δ (filled circles) and alg5Δ (filled traingles) strains in microtitre plates, as determined by colorimetric readings using the XTT-reduction assay. Results shown are the mean values of eight replicates. Experiments were repeated several times with similar results. (b) Adherence of biofilms formed by CAI4-URA3 (1) and mnn9Δ (2) and alg5Δ (3) strains to the polystyrene surface of cell culture plates before (upper row) and after (lower row) staining with crystal violet followed by washing with PBS buffer. Analysis of Candida albicans CAI4-URA3 biofilm proteome by 2-DE and MALDI-TOF In the present work, we have optimized 2-DE protocols for C. albicans biofilm proteome leading to highly reproducible 2-DE protein patterns. Using IPG strips of pH 3–7 range and 12% SDS-polyacrylamide gel, we were able to resolve and detect about 276 spots in the CAI4-URA3 strain (Fig. 2), corresponding to soluble proteins with molecular mass from 10 to 94 kDa and pIs from 4 to 7. A total of 116 protein spots with expected enough protein content to guarantee an accurate identification were excised from the 2-DE gel and subjected to MALDI-TOF MS analysis. A total of 72 proteins and 45 isoforms were unambiguously identified in the EtOH extracts from C. albicans CAI4-URA3 biofilms, which are listed in Table 2a and b. The corresponding spots are indicated by the same identifying numbers appearing in Table 2a and b, in the 2-DE map (Fig. 2). Moreover, analysis of EtOH extracts from biofilms produced by the C. albicans mutants for cell-wall-related genes (mostly those from biofilms formed by the alg5Δ strain) revealed 14 additional species not present in the extracts from the CAI4-URA3 reference strain (Table 3), so that a total of 86 putative EM proteins from Candida biofilms have been identified in this paper. Figure 2. View largeDownload slide 2-DE proteome map of the EtOH extract from biofilms of C. albicans CAI4-URA3 strain. Proteins were separated by 2-DE using 18 cm DryStrips with the 3–7 (nonlinear) pH range and 12% linear SDS-PAGE gels, and stained with SYPRO Ruby. Proteins identified by MALDI-TOF mass spectrometry are numbered according to data shown in Tables 2a and 2b. Figure 2. View largeDownload slide 2-DE proteome map of the EtOH extract from biofilms of C. albicans CAI4-URA3 strain. Proteins were separated by 2-DE using 18 cm DryStrips with the 3–7 (nonlinear) pH range and 12% linear SDS-PAGE gels, and stained with SYPRO Ruby. Proteins identified by MALDI-TOF mass spectrometry are numbered according to data shown in Tables 2a and 2b. Table 2a. Proteins identified in the EtOH extracts from biofilms formed by the reference CAI4-URA3 strain of C. albicans. Spot  Common  Systematic        numbera  nameb  namec  IPd  Scoree  Biological function  1  Met6p  CR_01620C  5.5  528  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  3  Aco1p  CE_08210C  5.9  137  Aconitase  4  Glx3p  C3_02610C  4.6  235  Glutathione-independent glyoxalase  5  Tkl1p  C1_08320W  5.5  265  Transcetolase  6  Pdc11p  C4_06570C  5.1  216  Piruvate decarboxilase  9  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  11  Dug1p  C5_04300C  4.8  105  Cys-Gly metalopeptidase  12  Gnd1p  C1_13860C  5.7  283  6-Phosphogluconate dehydrogenase  13  Eno1p  C1_08500C  5.1  348  Enolase 1  15  Sah1p  C5_04270C  5.2  251  Adenosyl homocysteinase  19  Fdh1p  CR_05170C  5.6  305  Formate dehydrogenase  20  Adh1p  C5_05050W  5.6  330  Alcohol dehydrogenase 1  22  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  24  Ado1p  C6_03080C  4.7  335  Adenosine kinase  25  Tal1p  CR_03720W  4.6  387  Transaldolase  26  Car1p  C5_04490C  4.8  215  Arginase  27  Ipp1p  C2_08810C  4.8  385  Inorganic pyrophosphatase  28  Grp2p  C5_02860C  5.5  317  Putative oxidoreductase  31  Pmu1p  C3_06920W  5.1  263  Protein similar to phosphomutase  32  Mdh1p  C4_01900C  5.2  206  Malate dehydrogenase  33  Trr1p  C5_02710W  5.4  295  Thioredoxin reductase (NADPH)  34  NAf  C5_01230C  5.4  236  Similar to an aldose 1-epimerase-related protein  36  Gcy1p  C3_07340W  6.5  294  Aldo/keto reductase  37  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  38  NAf  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  39  Sol3p  CR_06700C  5.1  271  Putative 6-phosphogluconolactonase  42  Pmm1p  C1_02480W  5.2  209  Phosphomannomutase  43  Tpi1p  C3_07440W  5.8  326  Triose-phosphate isomerase  45  Rib3p  C1_12360C  5.1  189  3,4-Dihydroxy-2-butanone 4-phosphate  47  Guk1p  C5_03790W  5.5  272  Putative guanylate kinase  48  NAf  C1_03510C  4.7  82  Protein of unknown function  49  Cpr3p  C2_02320C  5.1  184  Putative peptidyl-prolyl cis-trans isomerase  50  Rbp1p  C7_02570C  5.5  120  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  51  Cyp5p  C3_06360C  4.3  292  Putative peptidyl-prolyl cis-trans isomerase  52  Pst3p  CR_05390W  5.9  211  Putative flavodoxin  54  Cyp1p  C7_02380C  6.5  265  Peptidyl-prolyl cis-trans isomerase  57  Pfy1p  C1_08030W  5.1  195  Profilin  59  Ttr1p  C1_00490C  5.5  102  Putative glutaredoxin  60  Trx1p  CR_10350C  4.7  164  Thioredoxin  61  Apt1p  C2_01430W  5.1  269  Adenine phosphoribosyltransferase  62  Ynk1p  C5_02890W  6.0  187  Nucleoside diphosphate kinase  63  Vma7p  C2_04150C  4.6  122  Putative subunit of the V-ATPase complex  64  Dog1p  C6_01900C  4.8  250  Putative 2-deoxyglucose-6-phosphatase  66  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  68  Ade8p  C2_03090C  6.6  204  Putative phosphoribosylglycinamide formyl-transferase  69  Mms2p  C1_12650C  5.3  186  Ubiquitin conjugating enzyme  70  Uga1p  C2_04190C  5.7  201  Putative GABA transaminase  72  Pgi1p  CR_06340C  6.1  338  Glucose-6-phosphate isomerase  73  Leu2p  C7_00400W  4.9  115  Isopropyl malate dehydrogenase  74  Ssc1p  C2_07380W  5.8  252  Heat shock protein  75  Hsp70p  C1_13480W  4.5-4.7  187  Putative Hsp70p family chaperone    Ssa2p  C1_04300C  4.5-4.7  123  Putative Hsp70p family chaperone  76  Adh2p  C1_08330C  5.8  86  Alcohol dehydrogenase 2  77  Rps3p  CR_04810W  4.7  79  Ribosomal protein S3  78  Ifr2p  CR_03280W  6.0  260  Putative Zinc-binding dehydrogenase  79  Hbr2p  C6_04220C  6.1  173  Putative alanine glyoxylate aminotransferase  80  Nit3p  C1_10700C  5.8  212  Putative nitrilase  81  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  82  Pnc1p  C7_03520W  5.1  94  Putative nicotinamidase  83  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  84  Hpt1p  C2_02740C  4.8  179  Putative hypoxanthine-guanine phosphoribosyltransferase  85  Ubc1p  C6_04280W  4.9  184  Ortholog(s) have ubiquitin-protein transferase activity  86  Ura5p  CR_01650W  5.5  179  Putative orotate phosphoribosyl transferase  87  NAf  C2_09600C  4.3  149  Protein of unknown function  88  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  89  NAf  CR_09240C  4.4  189  Protein of unknown function  95  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  97  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  103  Vps21p  CR_08060C  NDg  187  Protein with GTPase activity  108  NAf  C2_07630C  NDg  196  Protein of unknown function  113  NAf  C2_05550W  NDg  237  Predicted cytochrome b5-like heme/steroid binding domain  114  Ofr1p  C1_08060W  6.7  NAf  Protein of unknown function  Spot  Common  Systematic        numbera  nameb  namec  IPd  Scoree  Biological function  1  Met6p  CR_01620C  5.5  528  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  3  Aco1p  CE_08210C  5.9  137  Aconitase  4  Glx3p  C3_02610C  4.6  235  Glutathione-independent glyoxalase  5  Tkl1p  C1_08320W  5.5  265  Transcetolase  6  Pdc11p  C4_06570C  5.1  216  Piruvate decarboxilase  9  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  11  Dug1p  C5_04300C  4.8  105  Cys-Gly metalopeptidase  12  Gnd1p  C1_13860C  5.7  283  6-Phosphogluconate dehydrogenase  13  Eno1p  C1_08500C  5.1  348  Enolase 1  15  Sah1p  C5_04270C  5.2  251  Adenosyl homocysteinase  19  Fdh1p  CR_05170C  5.6  305  Formate dehydrogenase  20  Adh1p  C5_05050W  5.6  330  Alcohol dehydrogenase 1  22  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  24  Ado1p  C6_03080C  4.7  335  Adenosine kinase  25  Tal1p  CR_03720W  4.6  387  Transaldolase  26  Car1p  C5_04490C  4.8  215  Arginase  27  Ipp1p  C2_08810C  4.8  385  Inorganic pyrophosphatase  28  Grp2p  C5_02860C  5.5  317  Putative oxidoreductase  31  Pmu1p  C3_06920W  5.1  263  Protein similar to phosphomutase  32  Mdh1p  C4_01900C  5.2  206  Malate dehydrogenase  33  Trr1p  C5_02710W  5.4  295  Thioredoxin reductase (NADPH)  34  NAf  C5_01230C  5.4  236  Similar to an aldose 1-epimerase-related protein  36  Gcy1p  C3_07340W  6.5  294  Aldo/keto reductase  37  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  38  NAf  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  39  Sol3p  CR_06700C  5.1  271  Putative 6-phosphogluconolactonase  42  Pmm1p  C1_02480W  5.2  209  Phosphomannomutase  43  Tpi1p  C3_07440W  5.8  326  Triose-phosphate isomerase  45  Rib3p  C1_12360C  5.1  189  3,4-Dihydroxy-2-butanone 4-phosphate  47  Guk1p  C5_03790W  5.5  272  Putative guanylate kinase  48  NAf  C1_03510C  4.7  82  Protein of unknown function  49  Cpr3p  C2_02320C  5.1  184  Putative peptidyl-prolyl cis-trans isomerase  50  Rbp1p  C7_02570C  5.5  120  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  51  Cyp5p  C3_06360C  4.3  292  Putative peptidyl-prolyl cis-trans isomerase  52  Pst3p  CR_05390W  5.9  211  Putative flavodoxin  54  Cyp1p  C7_02380C  6.5  265  Peptidyl-prolyl cis-trans isomerase  57  Pfy1p  C1_08030W  5.1  195  Profilin  59  Ttr1p  C1_00490C  5.5  102  Putative glutaredoxin  60  Trx1p  CR_10350C  4.7  164  Thioredoxin  61  Apt1p  C2_01430W  5.1  269  Adenine phosphoribosyltransferase  62  Ynk1p  C5_02890W  6.0  187  Nucleoside diphosphate kinase  63  Vma7p  C2_04150C  4.6  122  Putative subunit of the V-ATPase complex  64  Dog1p  C6_01900C  4.8  250  Putative 2-deoxyglucose-6-phosphatase  66  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  68  Ade8p  C2_03090C  6.6  204  Putative phosphoribosylglycinamide formyl-transferase  69  Mms2p  C1_12650C  5.3  186  Ubiquitin conjugating enzyme  70  Uga1p  C2_04190C  5.7  201  Putative GABA transaminase  72  Pgi1p  CR_06340C  6.1  338  Glucose-6-phosphate isomerase  73  Leu2p  C7_00400W  4.9  115  Isopropyl malate dehydrogenase  74  Ssc1p  C2_07380W  5.8  252  Heat shock protein  75  Hsp70p  C1_13480W  4.5-4.7  187  Putative Hsp70p family chaperone    Ssa2p  C1_04300C  4.5-4.7  123  Putative Hsp70p family chaperone  76  Adh2p  C1_08330C  5.8  86  Alcohol dehydrogenase 2  77  Rps3p  CR_04810W  4.7  79  Ribosomal protein S3  78  Ifr2p  CR_03280W  6.0  260  Putative Zinc-binding dehydrogenase  79  Hbr2p  C6_04220C  6.1  173  Putative alanine glyoxylate aminotransferase  80  Nit3p  C1_10700C  5.8  212  Putative nitrilase  81  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  82  Pnc1p  C7_03520W  5.1  94  Putative nicotinamidase  83  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  84  Hpt1p  C2_02740C  4.8  179  Putative hypoxanthine-guanine phosphoribosyltransferase  85  Ubc1p  C6_04280W  4.9  184  Ortholog(s) have ubiquitin-protein transferase activity  86  Ura5p  CR_01650W  5.5  179  Putative orotate phosphoribosyl transferase  87  NAf  C2_09600C  4.3  149  Protein of unknown function  88  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  89  NAf  CR_09240C  4.4  189  Protein of unknown function  95  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  97  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  103  Vps21p  CR_08060C  NDg  187  Protein with GTPase activity  108  NAf  C2_07630C  NDg  196  Protein of unknown function  113  NAf  C2_05550W  NDg  237  Predicted cytochrome b5-like heme/steroid binding domain  114  Ofr1p  C1_08060W  6.7  NAf  Protein of unknown function  aSee Fig. 2; b,cSwissProt/Candida GenomeDB; dIsoelectric point; eProbability of identification value (higher figures indicate higher statistic reliability).fNot available. gNot determined. View Large Table 2a. Proteins identified in the EtOH extracts from biofilms formed by the reference CAI4-URA3 strain of C. albicans. Spot  Common  Systematic        numbera  nameb  namec  IPd  Scoree  Biological function  1  Met6p  CR_01620C  5.5  528  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  3  Aco1p  CE_08210C  5.9  137  Aconitase  4  Glx3p  C3_02610C  4.6  235  Glutathione-independent glyoxalase  5  Tkl1p  C1_08320W  5.5  265  Transcetolase  6  Pdc11p  C4_06570C  5.1  216  Piruvate decarboxilase  9  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  11  Dug1p  C5_04300C  4.8  105  Cys-Gly metalopeptidase  12  Gnd1p  C1_13860C  5.7  283  6-Phosphogluconate dehydrogenase  13  Eno1p  C1_08500C  5.1  348  Enolase 1  15  Sah1p  C5_04270C  5.2  251  Adenosyl homocysteinase  19  Fdh1p  CR_05170C  5.6  305  Formate dehydrogenase  20  Adh1p  C5_05050W  5.6  330  Alcohol dehydrogenase 1  22  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  24  Ado1p  C6_03080C  4.7  335  Adenosine kinase  25  Tal1p  CR_03720W  4.6  387  Transaldolase  26  Car1p  C5_04490C  4.8  215  Arginase  27  Ipp1p  C2_08810C  4.8  385  Inorganic pyrophosphatase  28  Grp2p  C5_02860C  5.5  317  Putative oxidoreductase  31  Pmu1p  C3_06920W  5.1  263  Protein similar to phosphomutase  32  Mdh1p  C4_01900C  5.2  206  Malate dehydrogenase  33  Trr1p  C5_02710W  5.4  295  Thioredoxin reductase (NADPH)  34  NAf  C5_01230C  5.4  236  Similar to an aldose 1-epimerase-related protein  36  Gcy1p  C3_07340W  6.5  294  Aldo/keto reductase  37  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  38  NAf  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  39  Sol3p  CR_06700C  5.1  271  Putative 6-phosphogluconolactonase  42  Pmm1p  C1_02480W  5.2  209  Phosphomannomutase  43  Tpi1p  C3_07440W  5.8  326  Triose-phosphate isomerase  45  Rib3p  C1_12360C  5.1  189  3,4-Dihydroxy-2-butanone 4-phosphate  47  Guk1p  C5_03790W  5.5  272  Putative guanylate kinase  48  NAf  C1_03510C  4.7  82  Protein of unknown function  49  Cpr3p  C2_02320C  5.1  184  Putative peptidyl-prolyl cis-trans isomerase  50  Rbp1p  C7_02570C  5.5  120  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  51  Cyp5p  C3_06360C  4.3  292  Putative peptidyl-prolyl cis-trans isomerase  52  Pst3p  CR_05390W  5.9  211  Putative flavodoxin  54  Cyp1p  C7_02380C  6.5  265  Peptidyl-prolyl cis-trans isomerase  57  Pfy1p  C1_08030W  5.1  195  Profilin  59  Ttr1p  C1_00490C  5.5  102  Putative glutaredoxin  60  Trx1p  CR_10350C  4.7  164  Thioredoxin  61  Apt1p  C2_01430W  5.1  269  Adenine phosphoribosyltransferase  62  Ynk1p  C5_02890W  6.0  187  Nucleoside diphosphate kinase  63  Vma7p  C2_04150C  4.6  122  Putative subunit of the V-ATPase complex  64  Dog1p  C6_01900C  4.8  250  Putative 2-deoxyglucose-6-phosphatase  66  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  68  Ade8p  C2_03090C  6.6  204  Putative phosphoribosylglycinamide formyl-transferase  69  Mms2p  C1_12650C  5.3  186  Ubiquitin conjugating enzyme  70  Uga1p  C2_04190C  5.7  201  Putative GABA transaminase  72  Pgi1p  CR_06340C  6.1  338  Glucose-6-phosphate isomerase  73  Leu2p  C7_00400W  4.9  115  Isopropyl malate dehydrogenase  74  Ssc1p  C2_07380W  5.8  252  Heat shock protein  75  Hsp70p  C1_13480W  4.5-4.7  187  Putative Hsp70p family chaperone    Ssa2p  C1_04300C  4.5-4.7  123  Putative Hsp70p family chaperone  76  Adh2p  C1_08330C  5.8  86  Alcohol dehydrogenase 2  77  Rps3p  CR_04810W  4.7  79  Ribosomal protein S3  78  Ifr2p  CR_03280W  6.0  260  Putative Zinc-binding dehydrogenase  79  Hbr2p  C6_04220C  6.1  173  Putative alanine glyoxylate aminotransferase  80  Nit3p  C1_10700C  5.8  212  Putative nitrilase  81  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  82  Pnc1p  C7_03520W  5.1  94  Putative nicotinamidase  83  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  84  Hpt1p  C2_02740C  4.8  179  Putative hypoxanthine-guanine phosphoribosyltransferase  85  Ubc1p  C6_04280W  4.9  184  Ortholog(s) have ubiquitin-protein transferase activity  86  Ura5p  CR_01650W  5.5  179  Putative orotate phosphoribosyl transferase  87  NAf  C2_09600C  4.3  149  Protein of unknown function  88  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  89  NAf  CR_09240C  4.4  189  Protein of unknown function  95  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  97  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  103  Vps21p  CR_08060C  NDg  187  Protein with GTPase activity  108  NAf  C2_07630C  NDg  196  Protein of unknown function  113  NAf  C2_05550W  NDg  237  Predicted cytochrome b5-like heme/steroid binding domain  114  Ofr1p  C1_08060W  6.7  NAf  Protein of unknown function  Spot  Common  Systematic        numbera  nameb  namec  IPd  Scoree  Biological function  1  Met6p  CR_01620C  5.5  528  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  3  Aco1p  CE_08210C  5.9  137  Aconitase  4  Glx3p  C3_02610C  4.6  235  Glutathione-independent glyoxalase  5  Tkl1p  C1_08320W  5.5  265  Transcetolase  6  Pdc11p  C4_06570C  5.1  216  Piruvate decarboxilase  9  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  11  Dug1p  C5_04300C  4.8  105  Cys-Gly metalopeptidase  12  Gnd1p  C1_13860C  5.7  283  6-Phosphogluconate dehydrogenase  13  Eno1p  C1_08500C  5.1  348  Enolase 1  15  Sah1p  C5_04270C  5.2  251  Adenosyl homocysteinase  19  Fdh1p  CR_05170C  5.6  305  Formate dehydrogenase  20  Adh1p  C5_05050W  5.6  330  Alcohol dehydrogenase 1  22  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  24  Ado1p  C6_03080C  4.7  335  Adenosine kinase  25  Tal1p  CR_03720W  4.6  387  Transaldolase  26  Car1p  C5_04490C  4.8  215  Arginase  27  Ipp1p  C2_08810C  4.8  385  Inorganic pyrophosphatase  28  Grp2p  C5_02860C  5.5  317  Putative oxidoreductase  31  Pmu1p  C3_06920W  5.1  263  Protein similar to phosphomutase  32  Mdh1p  C4_01900C  5.2  206  Malate dehydrogenase  33  Trr1p  C5_02710W  5.4  295  Thioredoxin reductase (NADPH)  34  NAf  C5_01230C  5.4  236  Similar to an aldose 1-epimerase-related protein  36  Gcy1p  C3_07340W  6.5  294  Aldo/keto reductase  37  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  38  NAf  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  39  Sol3p  CR_06700C  5.1  271  Putative 6-phosphogluconolactonase  42  Pmm1p  C1_02480W  5.2  209  Phosphomannomutase  43  Tpi1p  C3_07440W  5.8  326  Triose-phosphate isomerase  45  Rib3p  C1_12360C  5.1  189  3,4-Dihydroxy-2-butanone 4-phosphate  47  Guk1p  C5_03790W  5.5  272  Putative guanylate kinase  48  NAf  C1_03510C  4.7  82  Protein of unknown function  49  Cpr3p  C2_02320C  5.1  184  Putative peptidyl-prolyl cis-trans isomerase  50  Rbp1p  C7_02570C  5.5  120  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  51  Cyp5p  C3_06360C  4.3  292  Putative peptidyl-prolyl cis-trans isomerase  52  Pst3p  CR_05390W  5.9  211  Putative flavodoxin  54  Cyp1p  C7_02380C  6.5  265  Peptidyl-prolyl cis-trans isomerase  57  Pfy1p  C1_08030W  5.1  195  Profilin  59  Ttr1p  C1_00490C  5.5  102  Putative glutaredoxin  60  Trx1p  CR_10350C  4.7  164  Thioredoxin  61  Apt1p  C2_01430W  5.1  269  Adenine phosphoribosyltransferase  62  Ynk1p  C5_02890W  6.0  187  Nucleoside diphosphate kinase  63  Vma7p  C2_04150C  4.6  122  Putative subunit of the V-ATPase complex  64  Dog1p  C6_01900C  4.8  250  Putative 2-deoxyglucose-6-phosphatase  66  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  68  Ade8p  C2_03090C  6.6  204  Putative phosphoribosylglycinamide formyl-transferase  69  Mms2p  C1_12650C  5.3  186  Ubiquitin conjugating enzyme  70  Uga1p  C2_04190C  5.7  201  Putative GABA transaminase  72  Pgi1p  CR_06340C  6.1  338  Glucose-6-phosphate isomerase  73  Leu2p  C7_00400W  4.9  115  Isopropyl malate dehydrogenase  74  Ssc1p  C2_07380W  5.8  252  Heat shock protein  75  Hsp70p  C1_13480W  4.5-4.7  187  Putative Hsp70p family chaperone    Ssa2p  C1_04300C  4.5-4.7  123  Putative Hsp70p family chaperone  76  Adh2p  C1_08330C  5.8  86  Alcohol dehydrogenase 2  77  Rps3p  CR_04810W  4.7  79  Ribosomal protein S3  78  Ifr2p  CR_03280W  6.0  260  Putative Zinc-binding dehydrogenase  79  Hbr2p  C6_04220C  6.1  173  Putative alanine glyoxylate aminotransferase  80  Nit3p  C1_10700C  5.8  212  Putative nitrilase  81  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  82  Pnc1p  C7_03520W  5.1  94  Putative nicotinamidase  83  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  84  Hpt1p  C2_02740C  4.8  179  Putative hypoxanthine-guanine phosphoribosyltransferase  85  Ubc1p  C6_04280W  4.9  184  Ortholog(s) have ubiquitin-protein transferase activity  86  Ura5p  CR_01650W  5.5  179  Putative orotate phosphoribosyl transferase  87  NAf  C2_09600C  4.3  149  Protein of unknown function  88  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  89  NAf  CR_09240C  4.4  189  Protein of unknown function  95  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  97  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  103  Vps21p  CR_08060C  NDg  187  Protein with GTPase activity  108  NAf  C2_07630C  NDg  196  Protein of unknown function  113  NAf  C2_05550W  NDg  237  Predicted cytochrome b5-like heme/steroid binding domain  114  Ofr1p  C1_08060W  6.7  NAf  Protein of unknown function  aSee Fig. 2; b,cSwissProt/Candida GenomeDB; dIsoelectric point; eProbability of identification value (higher figures indicate higher statistic reliability).fNot available. gNot determined. View Large Table 2b. Isoforms of proteins identified in the EtOH extracts from biofilms formed by the reference CAI4-URA3 strain of C. albicans. Spot  Common  Systematic      Biological function  numbera  nameb  namec  IPd  Scoree    2  Met6p  CR_01620C  5.5  340  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  7  Pdc11p  C4_06570C  5.1  157  Piruvate decarboxilase  8  Pdc11p  C4_06570C  5.1  118  Piruvate decarboxilase  10  Glx3p  C3_02610C  4.6  105  Glutathione-independent glyoxalase  14  Eno1p  C1_08500C  5.1  172  Enolase 1  16  Eno1p  C1_08500C  5.4  375  Enolase 1  17  Eno1p  C1_08500C  5.5  299  Enolase 1  18  Eno1p  C1_08500C  5.5  259  Enolase 1  21  Adh1p  C5_05050W  5.8  236  Alcohol dehydrogenase 1  23  Pgk1p  C6_00750C  6.0  350  Phosphoglycerate kinase  29  Grp2p  C5_02860C  5.6  279  Putative oxidoreductase  30  Grp2p  C5_02860C  5.9  324  Putative oxidoreductase  35  Mdh1p  C4_01900C  5.5  312  Malate dehydrogenase  40  Glx3p  C3_02610C  4.6  255  Glutathione-independent glyoxalase  41  Glx3p  C3_02610C  4.6  161  Glutathione-independent glyoxalase  44  Glx3p  C3_02610C  4.7  175  Glutathione-independent glyoxalase  46  Rib3p  C1_12360C  5.4  160  3,4-Dihydroxy-2-butanone 4-phosphate  53  Rbp1p  C7_02570C  6.5  156  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  55  Cyp1p  C7_02380C  6.9  123  Peptidyl-prolyl cis-trans isomerase  56  Cyp1p  C7_02380C  5.9  108  Peptidyl-prolyl cis-trans isomerase  58  Pfy1p  C1_08030W  5.1  195  Profilin  65  Dog1p  C6_01900C  4.9  215  Putative 2-deoxyglucose-6-phosphatase  67  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  71  Pgi1p  CR_06340C  6.0  228  Glucose-6-phosphate isomerase  90  NAf  CR_09240C  4.5  160  Protein of unknown function  91  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  92  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  93  Ado1p  C6_03080C  NDg  324  Adenosine kinase  94  Ado1p  C6_03080C  NDg  217  Adenosine kinase  96  Sol3p  CR_06700C  NDg  223  Putative 6-phosphogluconolactonase  98  Glx3p  C3_02610C  NDg  205  Glutathione-independent glyoxalase  99  Tpi1p  C3_07440W  NDg  216  Triose-phosphate isomerase  100  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  101  Glx3p  C3_02610C  NDg  152  Glutathione-independent glyoxalase  102  Pfy1p  C1_08030W  NDg  181  Profilin  104  Cpr3p  C2_02320C  NDg  155  Putative peptidyl-prolyl cis-trans isomerase  105  Cpr3p  C2_02320C  NDg  173  Putative peptidyl-prolyl cis-trans isomerase  106  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  107  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  109  Cyp1p  C7_02380C  NDg  130  Peptidyl-prolyl cis-trans isomerase  110  Ynk1p  C5_02890W  NDg  178  Nucleoside diphosphate kinase  111  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  112  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  115  Ntf2p  C1_10100C  NDg  185  Putative nuclear envelope protein  116  Pfy1p  C1_08030W  NDg  168  Profilin  Spot  Common  Systematic      Biological function  numbera  nameb  namec  IPd  Scoree    2  Met6p  CR_01620C  5.5  340  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  7  Pdc11p  C4_06570C  5.1  157  Piruvate decarboxilase  8  Pdc11p  C4_06570C  5.1  118  Piruvate decarboxilase  10  Glx3p  C3_02610C  4.6  105  Glutathione-independent glyoxalase  14  Eno1p  C1_08500C  5.1  172  Enolase 1  16  Eno1p  C1_08500C  5.4  375  Enolase 1  17  Eno1p  C1_08500C  5.5  299  Enolase 1  18  Eno1p  C1_08500C  5.5  259  Enolase 1  21  Adh1p  C5_05050W  5.8  236  Alcohol dehydrogenase 1  23  Pgk1p  C6_00750C  6.0  350  Phosphoglycerate kinase  29  Grp2p  C5_02860C  5.6  279  Putative oxidoreductase  30  Grp2p  C5_02860C  5.9  324  Putative oxidoreductase  35  Mdh1p  C4_01900C  5.5  312  Malate dehydrogenase  40  Glx3p  C3_02610C  4.6  255  Glutathione-independent glyoxalase  41  Glx3p  C3_02610C  4.6  161  Glutathione-independent glyoxalase  44  Glx3p  C3_02610C  4.7  175  Glutathione-independent glyoxalase  46  Rib3p  C1_12360C  5.4  160  3,4-Dihydroxy-2-butanone 4-phosphate  53  Rbp1p  C7_02570C  6.5  156  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  55  Cyp1p  C7_02380C  6.9  123  Peptidyl-prolyl cis-trans isomerase  56  Cyp1p  C7_02380C  5.9  108  Peptidyl-prolyl cis-trans isomerase  58  Pfy1p  C1_08030W  5.1  195  Profilin  65  Dog1p  C6_01900C  4.9  215  Putative 2-deoxyglucose-6-phosphatase  67  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  71  Pgi1p  CR_06340C  6.0  228  Glucose-6-phosphate isomerase  90  NAf  CR_09240C  4.5  160  Protein of unknown function  91  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  92  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  93  Ado1p  C6_03080C  NDg  324  Adenosine kinase  94  Ado1p  C6_03080C  NDg  217  Adenosine kinase  96  Sol3p  CR_06700C  NDg  223  Putative 6-phosphogluconolactonase  98  Glx3p  C3_02610C  NDg  205  Glutathione-independent glyoxalase  99  Tpi1p  C3_07440W  NDg  216  Triose-phosphate isomerase  100  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  101  Glx3p  C3_02610C  NDg  152  Glutathione-independent glyoxalase  102  Pfy1p  C1_08030W  NDg  181  Profilin  104  Cpr3p  C2_02320C  NDg  155  Putative peptidyl-prolyl cis-trans isomerase  105  Cpr3p  C2_02320C  NDg  173  Putative peptidyl-prolyl cis-trans isomerase  106  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  107  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  109  Cyp1p  C7_02380C  NDg  130  Peptidyl-prolyl cis-trans isomerase  110  Ynk1p  C5_02890W  NDg  178  Nucleoside diphosphate kinase  111  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  112  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  115  Ntf2p  C1_10100C  NDg  185  Putative nuclear envelope protein  116  Pfy1p  C1_08030W  NDg  168  Profilin  aSee Fig. 2; b,cSwissProt/Candida GenomeDB; dIsoelectric point; eProbability of identification value (higher figures indicate higher statistic reliability). fNot available. gNot determined. View Large Table 2b. Isoforms of proteins identified in the EtOH extracts from biofilms formed by the reference CAI4-URA3 strain of C. albicans. Spot  Common  Systematic      Biological function  numbera  nameb  namec  IPd  Scoree    2  Met6p  CR_01620C  5.5  340  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  7  Pdc11p  C4_06570C  5.1  157  Piruvate decarboxilase  8  Pdc11p  C4_06570C  5.1  118  Piruvate decarboxilase  10  Glx3p  C3_02610C  4.6  105  Glutathione-independent glyoxalase  14  Eno1p  C1_08500C  5.1  172  Enolase 1  16  Eno1p  C1_08500C  5.4  375  Enolase 1  17  Eno1p  C1_08500C  5.5  299  Enolase 1  18  Eno1p  C1_08500C  5.5  259  Enolase 1  21  Adh1p  C5_05050W  5.8  236  Alcohol dehydrogenase 1  23  Pgk1p  C6_00750C  6.0  350  Phosphoglycerate kinase  29  Grp2p  C5_02860C  5.6  279  Putative oxidoreductase  30  Grp2p  C5_02860C  5.9  324  Putative oxidoreductase  35  Mdh1p  C4_01900C  5.5  312  Malate dehydrogenase  40  Glx3p  C3_02610C  4.6  255  Glutathione-independent glyoxalase  41  Glx3p  C3_02610C  4.6  161  Glutathione-independent glyoxalase  44  Glx3p  C3_02610C  4.7  175  Glutathione-independent glyoxalase  46  Rib3p  C1_12360C  5.4  160  3,4-Dihydroxy-2-butanone 4-phosphate  53  Rbp1p  C7_02570C  6.5  156  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  55  Cyp1p  C7_02380C  6.9  123  Peptidyl-prolyl cis-trans isomerase  56  Cyp1p  C7_02380C  5.9  108  Peptidyl-prolyl cis-trans isomerase  58  Pfy1p  C1_08030W  5.1  195  Profilin  65  Dog1p  C6_01900C  4.9  215  Putative 2-deoxyglucose-6-phosphatase  67  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  71  Pgi1p  CR_06340C  6.0  228  Glucose-6-phosphate isomerase  90  NAf  CR_09240C  4.5  160  Protein of unknown function  91  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  92  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  93  Ado1p  C6_03080C  NDg  324  Adenosine kinase  94  Ado1p  C6_03080C  NDg  217  Adenosine kinase  96  Sol3p  CR_06700C  NDg  223  Putative 6-phosphogluconolactonase  98  Glx3p  C3_02610C  NDg  205  Glutathione-independent glyoxalase  99  Tpi1p  C3_07440W  NDg  216  Triose-phosphate isomerase  100  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  101  Glx3p  C3_02610C  NDg  152  Glutathione-independent glyoxalase  102  Pfy1p  C1_08030W  NDg  181  Profilin  104  Cpr3p  C2_02320C  NDg  155  Putative peptidyl-prolyl cis-trans isomerase  105  Cpr3p  C2_02320C  NDg  173  Putative peptidyl-prolyl cis-trans isomerase  106  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  107  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  109  Cyp1p  C7_02380C  NDg  130  Peptidyl-prolyl cis-trans isomerase  110  Ynk1p  C5_02890W  NDg  178  Nucleoside diphosphate kinase  111  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  112  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  115  Ntf2p  C1_10100C  NDg  185  Putative nuclear envelope protein  116  Pfy1p  C1_08030W  NDg  168  Profilin  Spot  Common  Systematic      Biological function  numbera  nameb  namec  IPd  Scoree    2  Met6p  CR_01620C  5.5  340  Essential 5-methyltetrahydro-pteroyltriglutamate homocysteine methyltransferase  7  Pdc11p  C4_06570C  5.1  157  Piruvate decarboxilase  8  Pdc11p  C4_06570C  5.1  118  Piruvate decarboxilase  10  Glx3p  C3_02610C  4.6  105  Glutathione-independent glyoxalase  14  Eno1p  C1_08500C  5.1  172  Enolase 1  16  Eno1p  C1_08500C  5.4  375  Enolase 1  17  Eno1p  C1_08500C  5.5  299  Enolase 1  18  Eno1p  C1_08500C  5.5  259  Enolase 1  21  Adh1p  C5_05050W  5.8  236  Alcohol dehydrogenase 1  23  Pgk1p  C6_00750C  6.0  350  Phosphoglycerate kinase  29  Grp2p  C5_02860C  5.6  279  Putative oxidoreductase  30  Grp2p  C5_02860C  5.9  324  Putative oxidoreductase  35  Mdh1p  C4_01900C  5.5  312  Malate dehydrogenase  40  Glx3p  C3_02610C  4.6  255  Glutathione-independent glyoxalase  41  Glx3p  C3_02610C  4.6  161  Glutathione-independent glyoxalase  44  Glx3p  C3_02610C  4.7  175  Glutathione-independent glyoxalase  46  Rib3p  C1_12360C  5.4  160  3,4-Dihydroxy-2-butanone 4-phosphate  53  Rbp1p  C7_02570C  6.5  156  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  55  Cyp1p  C7_02380C  6.9  123  Peptidyl-prolyl cis-trans isomerase  56  Cyp1p  C7_02380C  5.9  108  Peptidyl-prolyl cis-trans isomerase  58  Pfy1p  C1_08030W  5.1  195  Profilin  65  Dog1p  C6_01900C  4.9  215  Putative 2-deoxyglucose-6-phosphatase  67  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  71  Pgi1p  CR_06340C  6.0  228  Glucose-6-phosphate isomerase  90  NAf  CR_09240C  4.5  160  Protein of unknown function  91  Pdc11p  C4_06570C  5.2  319  Piruvate decarboxilase  92  Thr4p  C5_02270W  5.0  153  Putative threonine synthase  93  Ado1p  C6_03080C  NDg  324  Adenosine kinase  94  Ado1p  C6_03080C  NDg  217  Adenosine kinase  96  Sol3p  CR_06700C  NDg  223  Putative 6-phosphogluconolactonase  98  Glx3p  C3_02610C  NDg  205  Glutathione-independent glyoxalase  99  Tpi1p  C3_07440W  NDg  216  Triose-phosphate isomerase  100  Gpm1p  C2_03270W  NDg  303  Phosphoglycerate mutase  101  Glx3p  C3_02610C  NDg  152  Glutathione-independent glyoxalase  102  Pfy1p  C1_08030W  NDg  181  Profilin  104  Cpr3p  C2_02320C  NDg  155  Putative peptidyl-prolyl cis-trans isomerase  105  Cpr3p  C2_02320C  NDg  173  Putative peptidyl-prolyl cis-trans isomerase  106  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  107  Rbp1p  C7_02570C  NDg  141  Peptidyl-prolyl cis-trans isomerase; rapamycin-binding protein  109  Cyp1p  C7_02380C  NDg  130  Peptidyl-prolyl cis-trans isomerase  110  Ynk1p  C5_02890W  NDg  178  Nucleoside diphosphate kinase  111  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  112  Glx3p  C3_02610C  NDg  218  Glutathione-independent glyoxalase  115  Ntf2p  C1_10100C  NDg  185  Putative nuclear envelope protein  116  Pfy1p  C1_08030W  NDg  168  Profilin  aSee Fig. 2; b,cSwissProt/Candida GenomeDB; dIsoelectric point; eProbability of identification value (higher figures indicate higher statistic reliability). fNot available. gNot determined. View Large Table 3. Characteristics of some protein species that were found to be specifically present or absent in the EtOH extracts from biofilms formed by the reference (CAI4-URA3) and null mutant (pga10Δ, csa1Δ, mnn9Δ and alg5Δ) strains of C. albicans. Common  Systematic      Biological function            namea  nameb  IPc  Scored    CAI4-URA3  pga10Δ  csa1Δ  mnn9Δ  alg5Δ  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  +  +  +  +  –  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  +  +  +  +  –  NAe  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  +  +  +  +  –  NAe  CR_09670C  6.2  262  Putative esterase  –  –  –  +  –  NAe  C3_06860C  6.3  284  Putative xylose and arabinose reductase  –  –  –  +  +  Ham1p  C5_03860W  5.2  180  Putative deoxyribonucleoside triphosphate pyrophosphohydrolase  –  +  +  +  –  Hnt1p  C1_10780C  6.2  135  Protein with similarity to protein kinase C inhibitor-I  –  +  +  +  +  NAe  C1_01400C  6.3  156  Ortholog(s) have sphingosine-1-phosphate phosphatase activity  –  +  +  +  +  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  +  +  –  +  –  Pst2p  C2_08640C  5,5  268  Putative NADH:quinone oxidoreductase  –  –  +  +  +  Mcr1p  C6_02040W  6.9  399  NADH-cytochrome-b5 reductase  –  –  +  –  +  Ykt6p  C1_02860C  5.1  113  Putative protein of the vacuolar SNARE Complex  –  +  +  +  +  NAe  C4_02200C  6.5  120  Ortholog of C. dubliniensis CD36  –  +  +  +  +  Apr1p  C2_07400C  4.2  183  Vacuolar aspartic proteinase  –  –  –  –  +  Ssc1p  C2_07380W  5.8  252  Heat shock protein  +  +  –  +  +  Aat1p  C2_05250C  6.7  329  Aspartate aminotransferase  –  –  –  –  +  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  +  +  +  –  +  NAe  C3_06860C  6.2  228  Putative xylose and arabinose reductase  –  –  –  –  +  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  +  –  –  –  +  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  +  +  –  –  –  Apt1p  C2_01430W  5.4  182  Adenine phosphoribosyltransferase  +  +  –  +  +  Sod3p  C7_00110W  6.5  154  Cytosolic manganese containing  –  +  +  –  +  Rdi1p  C3_05000W  4.8  328  Putative rho GDP dissociation inhibitor  –  +  –  +  +  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  +  +  –  +  +  Common  Systematic      Biological function            namea  nameb  IPc  Scored    CAI4-URA3  pga10Δ  csa1Δ  mnn9Δ  alg5Δ  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  +  +  +  +  –  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  +  +  +  +  –  NAe  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  +  +  +  +  –  NAe  CR_09670C  6.2  262  Putative esterase  –  –  –  +  –  NAe  C3_06860C  6.3  284  Putative xylose and arabinose reductase  –  –  –  +  +  Ham1p  C5_03860W  5.2  180  Putative deoxyribonucleoside triphosphate pyrophosphohydrolase  –  +  +  +  –  Hnt1p  C1_10780C  6.2  135  Protein with similarity to protein kinase C inhibitor-I  –  +  +  +  +  NAe  C1_01400C  6.3  156  Ortholog(s) have sphingosine-1-phosphate phosphatase activity  –  +  +  +  +  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  +  +  –  +  –  Pst2p  C2_08640C  5,5  268  Putative NADH:quinone oxidoreductase  –  –  +  +  +  Mcr1p  C6_02040W  6.9  399  NADH-cytochrome-b5 reductase  –  –  +  –  +  Ykt6p  C1_02860C  5.1  113  Putative protein of the vacuolar SNARE Complex  –  +  +  +  +  NAe  C4_02200C  6.5  120  Ortholog of C. dubliniensis CD36  –  +  +  +  +  Apr1p  C2_07400C  4.2  183  Vacuolar aspartic proteinase  –  –  –  –  +  Ssc1p  C2_07380W  5.8  252  Heat shock protein  +  +  –  +  +  Aat1p  C2_05250C  6.7  329  Aspartate aminotransferase  –  –  –  –  +  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  +  +  +  –  +  NAe  C3_06860C  6.2  228  Putative xylose and arabinose reductase  –  –  –  –  +  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  +  –  –  –  +  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  +  +  –  –  –  Apt1p  C2_01430W  5.4  182  Adenine phosphoribosyltransferase  +  +  –  +  +  Sod3p  C7_00110W  6.5  154  Cytosolic manganese containing  –  +  +  –  +  Rdi1p  C3_05000W  4.8  328  Putative rho GDP dissociation inhibitor  –  +  –  +  +  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  +  +  –  +  +  a,bSwissProt/Candida GenomeDB. cIsoelectric focusing point. dProbability of identification value (higher figures indicate higher statistic reliability).eNot available. View Large Table 3. Characteristics of some protein species that were found to be specifically present or absent in the EtOH extracts from biofilms formed by the reference (CAI4-URA3) and null mutant (pga10Δ, csa1Δ, mnn9Δ and alg5Δ) strains of C. albicans. Common  Systematic      Biological function            namea  nameb  IPc  Scored    CAI4-URA3  pga10Δ  csa1Δ  mnn9Δ  alg5Δ  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  +  +  +  +  –  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  +  +  +  +  –  NAe  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  +  +  +  +  –  NAe  CR_09670C  6.2  262  Putative esterase  –  –  –  +  –  NAe  C3_06860C  6.3  284  Putative xylose and arabinose reductase  –  –  –  +  +  Ham1p  C5_03860W  5.2  180  Putative deoxyribonucleoside triphosphate pyrophosphohydrolase  –  +  +  +  –  Hnt1p  C1_10780C  6.2  135  Protein with similarity to protein kinase C inhibitor-I  –  +  +  +  +  NAe  C1_01400C  6.3  156  Ortholog(s) have sphingosine-1-phosphate phosphatase activity  –  +  +  +  +  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  +  +  –  +  –  Pst2p  C2_08640C  5,5  268  Putative NADH:quinone oxidoreductase  –  –  +  +  +  Mcr1p  C6_02040W  6.9  399  NADH-cytochrome-b5 reductase  –  –  +  –  +  Ykt6p  C1_02860C  5.1  113  Putative protein of the vacuolar SNARE Complex  –  +  +  +  +  NAe  C4_02200C  6.5  120  Ortholog of C. dubliniensis CD36  –  +  +  +  +  Apr1p  C2_07400C  4.2  183  Vacuolar aspartic proteinase  –  –  –  –  +  Ssc1p  C2_07380W  5.8  252  Heat shock protein  +  +  –  +  +  Aat1p  C2_05250C  6.7  329  Aspartate aminotransferase  –  –  –  –  +  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  +  +  +  –  +  NAe  C3_06860C  6.2  228  Putative xylose and arabinose reductase  –  –  –  –  +  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  +  –  –  –  +  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  +  +  –  –  –  Apt1p  C2_01430W  5.4  182  Adenine phosphoribosyltransferase  +  +  –  +  +  Sod3p  C7_00110W  6.5  154  Cytosolic manganese containing  –  +  +  –  +  Rdi1p  C3_05000W  4.8  328  Putative rho GDP dissociation inhibitor  –  +  –  +  +  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  +  +  –  +  +  Common  Systematic      Biological function            namea  nameb  IPc  Scored    CAI4-URA3  pga10Δ  csa1Δ  mnn9Δ  alg5Δ  Pgk1p  C6_00750C  5.9  283  Phosphoglycerate kinase  +  +  +  +  –  Cip1p  C6_01070C  4.6  149  Possible oxidoreductase  +  +  +  +  –  NAe  CR_08050C  5.1  214  Putative thiamine biosynthesis enzyme  +  +  +  +  –  NAe  CR_09670C  6.2  262  Putative esterase  –  –  –  +  –  NAe  C3_06860C  6.3  284  Putative xylose and arabinose reductase  –  –  –  +  +  Ham1p  C5_03860W  5.2  180  Putative deoxyribonucleoside triphosphate pyrophosphohydrolase  –  +  +  +  –  Hnt1p  C1_10780C  6.2  135  Protein with similarity to protein kinase C inhibitor-I  –  +  +  +  +  NAe  C1_01400C  6.3  156  Ortholog(s) have sphingosine-1-phosphate phosphatase activity  –  +  +  +  +  Tma19p  CR_00860C  4.1  140  Uncharacterized cell wall protein, ortholog of S. cerevisiae Tma19p  +  +  –  +  –  Pst2p  C2_08640C  5,5  268  Putative NADH:quinone oxidoreductase  –  –  +  +  +  Mcr1p  C6_02040W  6.9  399  NADH-cytochrome-b5 reductase  –  –  +  –  +  Ykt6p  C1_02860C  5.1  113  Putative protein of the vacuolar SNARE Complex  –  +  +  +  +  NAe  C4_02200C  6.5  120  Ortholog of C. dubliniensis CD36  –  +  +  +  +  Apr1p  C2_07400C  4.2  183  Vacuolar aspartic proteinase  –  –  –  –  +  Ssc1p  C2_07380W  5.8  252  Heat shock protein  +  +  –  +  +  Aat1p  C2_05250C  6.7  329  Aspartate aminotransferase  –  –  –  –  +  Hem13p  C3_04060C  5.8  159  Coproporphyrinogen III oxidase  +  +  +  –  +  NAe  C3_06860C  6.2  228  Putative xylose and arabinose reductase  –  –  –  –  +  Tdh3p  C3_06870W  6.7  251  NAD-linked glyceraldehyde-3-phosphate dehydrogenase  +  –  –  –  +  Ura3p  C3_01350C  5.3  212  Orotidine-5′-phosphate decarboxylase  +  +  –  –  –  Apt1p  C2_01430W  5.4  182  Adenine phosphoribosyltransferase  +  +  –  +  +  Sod3p  C7_00110W  6.5  154  Cytosolic manganese containing  –  +  +  –  +  Rdi1p  C3_05000W  4.8  328  Putative rho GDP dissociation inhibitor  –  +  –  +  +  Ypt1p  C1_03500W  4.6  122  Functional homolog of S. cerevisiae Ypt1p  +  +  –  +  +  a,bSwissProt/Candida GenomeDB. cIsoelectric focusing point. dProbability of identification value (higher figures indicate higher statistic reliability).eNot available. View Large The 86 identified proteins were functionally classified based on the gene ontology annotation in the CADB or Génolevures, as well as assigned to metabolic pathways or biological process using the GO Term Finder and UniProt databases (Fig. 3; Supplementary data 1, Supporting Information). Seneviratne et al. (2008) reported that sessile (biofilm) C. albicans cells differentially expressed some proteins with antioxidant activity (Ahp1p and Tsa1p), and concluded that the biofilm extracellular environment, i.e. the EM, displays a very low content of reactive oxygen species. Results reported here are somehow in line with these observations since 19.7% of proteins identified in the EtOH biofilm extracts belong to the category GO:0016491 which includes protein species with oxidoreductase activity (Fig. 3; Supplementary data 1), although Ahp1p and Tsa1p species were not identified in the present work. Figure 3. View largeDownload slide Proteins identified in the 2-DE proteome map of the EM EtOH-extract from biofilms of C. albicans CAI4-URA3 strain grouped according to their GO categories (listed in Supplementary data 1, Supporting Information). Figure 3. View largeDownload slide Proteins identified in the 2-DE proteome map of the EM EtOH-extract from biofilms of C. albicans CAI4-URA3 strain grouped according to their GO categories (listed in Supplementary data 1, Supporting Information). According to the criterion reported by Kyte and Doolittle (1982), more than 96% of the 86 identified polypeptides displayed negative hydropathy indexes (Supplementary data 2, Supporting Information). Since biofilms can actually be considered as hydrogels, the presence of a vast majority of polypeptides with negative hydropathy indexes is consistent with the highly hydrophilic environment of the biofilm EM. Prediction of the presence of signal peptide following analysis of the corresponding amino acid sequences in the Signal IP 3.0 Server database revealed that 82 of the 86 proteins identified in the EtOH extracts from biofilms lack signal peptide (Supplementary data 3, Supporting Information). The presence of such putative cytosolic polypeptides in the biofilm EM is odd yet, as above mentioned (see the ‘Introduction’ section) strong evidence exist in support of the existence of non-classical secretory pathways in Saccharomyces cerevisiae and C. albicans that could be responsible, at least partly, for the presence of a large variety of cytosolic components in the EM of C. albicans biofilms (Monteoliva et al. 2002; Nombela, Gil and Chaffin 2006; Chaffin 2008; Gil-Bona et al. 2015; Gómez-Molero et al. 2016). This type of proteins may represent ‘moonlighting’ (multifunctional) species, displaying different functions depending on their subcellular location. It has been recently suggested that an association may somehow exists between these moonlighting proteins and several virulence factors, including oxidative stress response, adhesion of fungal cells to host tissues and biofilm formation in C. albicans (Serrano-Fujarte, López-Romero and Cuéllar-Cruz 2016). A unique feature of the proteome approach performed by 2-DE is the capacity to detect post-translational protein modifications. In the present analysis, up to 45 cases of redundancy (i.e. the same protein identified in several spots) have been found (Table 2b), which may represent isoforms, suggesting different types of post-translational modifications such as glycosylation, phosphorylation or proteolytic processing (Table 2b). Thus, Glx3p (a glutathione-independent glyoxalase) has been identified in eight spots. Eno1p (enolase) was detected in five spots and for Pdc11p (pyruvate decarboxylase) four spots were found. Up to 10 other proteins have been identified in two different spots (Table 2a and b). Similar results have been reported by Martínez-Gomáriz et al. (2009) where several polypeptide species (i.e. Fba1p, Met6p, Cof1p and Pdc11p) were simultaneously detected in distinct spots in the 2-D electrophoregrams. Changes in the biofilm proteome of Candida albicans mutants for cell-wall-related genes Visual inspection of 2-D electrophoregrams from EtOH extracts of biofilms formed by C. albicans mutant strains for cell-wall-related genes revealed a closely similar and highly reproducible protein spot pattern in all cases (Fig. 4). From the 86 polypeptide species identified, 72 to 82 of them (depending on the strain considered) were consistently found in all electrophoregrams, although some qualitative differences were observed (Table 3). Thus, 8 proteins (CR_09670C, C3_06860C, Pst2p, Mcr1p, Aat1p, Apr1p, C3_06860C and Tdh3p) were not detected in the extracts from pga10Δ mutant, up to 12 species (CR_09670C, C3_06860C, Tma19p, Apr1p, Ssc1p, Aat1p, C3_06860C, Tdh3p, Apt1p, Rdi1p, Ura3p and Ypt1p) were absent in the case of biofilm extracts from csa1Δ strain, as well as 8 (Mcr1p, Apr1p, Aat1p, Hem13p, C3_06860C, Tdh3p, Sod3p and Ura3p) and 7 proteins (Pgk1p, Cip1p, CR_08050C, CR_09670C, Ham1p, Tma19p and Ura3p) do not appear in the 2-D electrophoregrams of EtOH biofilm samples from mnn9Δ and alg5Δ mutants, respectively, although 3 species (Apr1p, Aat1p, C3_06860C) were exclusively detected in the latter strain (Table 3). Up to 14 proteins were absent (CR_09670C, C3_06860C, Ham1p, Hnt1p, C1_01400C, Pst2p, Mcr1p, Ykt6p, C4_02200C, Apr1p, Aat1p, C3_06860C, Sod3p and Rdi1p) in the biofilm extracts from the reference CAI4-URA3 strain (Table 3). Figure 4. View largeDownload slide 2-DE maps of the EtOH extracts from biofilms formed by different null mutant strains of C. albicans for cell wall-related genes (PGA10, CSA1, MNN9 and ALG5). Proteins were separated by 2-DE using 18 cm DryStrips with the 3–7 (nonlinear) pH range and 12% linear SDS-PAGE gels, and stained with SYPRO Ruby. Figure 4. View largeDownload slide 2-DE maps of the EtOH extracts from biofilms formed by different null mutant strains of C. albicans for cell wall-related genes (PGA10, CSA1, MNN9 and ALG5). Proteins were separated by 2-DE using 18 cm DryStrips with the 3–7 (nonlinear) pH range and 12% linear SDS-PAGE gels, and stained with SYPRO Ruby. A potential role in the formation, development and/or maintenance of the biofilm structure in C. albicans has been suggested for the proteins encoded by PGA10 and CSA1 genes (Pérez et al. 2006). However, the proteomic analysis of EtOH extracts obtained from mature (48 h) biofilms did not reveal the presence of the corresponding gene products in the different samples analyzed (see below), even in the case of biofilm EtOH extracts from the CAI4-URA3 reference strain that, as above stated, formed strong biofilms, firmly attached to the inner surface of polystyrene culture flasks. Proteins containing the CFEM domain such as Pga10p and Csa1p may function as cell-surface receptors or signal transducers, or as adhesion molecules in host–pathogen interactions (Kulkarni, Kelkar and Dean 2003). Hence, it can be hypothesized that PGA10 and CSA1 gene products could be present in the extracellular environment (the biofilm EM) during the initial stages of biofilm formation, during which cell–cell and cell–substrate interaction/adhesion processes play an essential role, experiencing a progressive degradation and disappearance whenever maturation and consolidation of the biofilm structure occurs. This contention is in line with the hypothesis that a two-tier system for cohesive and adhesive interactions may exists in C. albicans biofilms, one mediated by surface adhesins in the cell wall and a second one provided by the EM itself (López-Ribot 2014). DISCUSSION It has been suggested by some authors that a close relationship exists between the material excreted to the exocellular medium by planktonic cells of Candida albicans and the polypeptide component of the EM of biofilms formed by this fungal species (Thomas et al. 2006). Sorgo et al. (2010) performed a comprehensive characterization of the C. albicans ‘secretome’ (proteins present in the spent liquid culture medium) under different growing conditions by means of tandem liquid chromatography and mass spectrometry, identifying 79 different species including 44 secretory proteins and 28 cytosolic proteins that, contrary to secretory proteins, lack signal peptide. Although spontaneous cell lysis may account for the presence of putative cytosolic proteins in the exocellular environment (i.e. the spent culture medium or the biofilm EM), these proteins could also reach a location outside the plasma membrane through non-canonical secretory pathways and/or exosomes (Monteoliva et al. 2002; Nombela, Gil and Chaffin 2006; Chaffin 2008; Wolf and Casadevall 2014; Gil-Bona et al. 2015; Vargas et al. 2015; Gómez-Molero et al. 2016; Nimrichter et al. 2016). Only a partial coincidence was found between our results, a superoxide dismutase coded by SOD3 gene, and those reported by Sorgo et al. (2010), two superoxide dismutase species, coded by SOD4 and SOD5 genes, bound to the C. albicans cell wall structure through a GPI motif. The different type biological sample used as starting material used in each case (ultrafiltration concentrates of the spent culture medium [Sorgo et al. 2010] and whole biofilms [this work]) may account for this low coincidence of results. In any case, the fact that the qualitative polypeptide patterns of cytosolic proteins found in the C. albicans ‘secretome’ (Sorgo et al. 2010), and those detected in the EtOH extracts from biofilms (this work) are completely different but very repetitive in each case, suggests that every set of putative cytosolic proteins reach its final destination, i.e. the spent culture medium where planktonic cells have grown or the EM of biofilms (sessile cells), by distinct but highly regulated processes. The DiGE (difference gel electrophoresis) technique has been used by Martínez-Gomáriz et al. (2009) to characterize the patterns of cytosolic proteins and non-covalently linked wall proteins, both in sessile cells (present in biofilms) and planktonic cells (yeasts and hyphae) growing in liquid medium. A total of 114 and 85 protein spots were analyzed in the electroforegrams of cytosolic and cell-surface-bound proteins, respectively. From the 36 polypeptides identified by Martínez-Gomáriz et al. (2009) in the subproteome of surface-bound proteins of sessile (biofilm resident) cells, 16 of them (Met6p, Pdc11p, Gnd1p, Eno1p, Leu2p, Pgk1p, Adh1p, Tal1p, Ado1p, Ipp1p, Grp2p, Tpi1p, Sol3p, Cpr3p, Rbp1p and IPF17186 [Glx3p]) were also detected in the EtOH extracts from biofilms analyzed in this paper (Tables 2a and 3), which represent a significant percentage (44.4%) of coincidence between their results and ours. On the other hand, Zarnowski et al. (2014) have recently identified, also using proteomic techniques, a total of 565 different proteins in the EM of C. albicans biofilms, including a few predicted to form part of the secretome, but also many secretion signal-less proteins, which suggests, once again, a non-canonical secretion pathway, release via exosomal vesicles, and/or the accumulation of proteins after cell death ((Monteoliva et al. 2002; Nombela, Gil and Chaffin 2006; Chaffin 2008; López-Ribot 2014; Wolf and Casadevall 2014; Gil-Bona et al. 2015; Vargas et al. 2015; Gómez-Molero et al. 2016; Nimrichter et al. 2016). From the 565 polypeptides identified by Zarnowski et al. (2014) in the EM subproteome, 21 of them (Thr4p, Gnd1/Dor14p, Fdh1p, Pgk1p, Tal1p, Car1p, Mdh1p, Trr1p, Sol3p, Tpi1p, Ynk1p, Vma7p, Ykt6p, Hsp70p, Ssa2p, Aat1p, Rps3p, Hem13p, Tdh3p and Ubc1p) were also detected in EtOH extracts from biofilms analyzed in this paper (Tables 2a and 3), which represent a low percentage (3.71%) of coincidence between their results and ours. The difference between the number of proteins associated with C. albicans biofilms identified in this work and in the Martínez-Gomáriz et al. (2009) paper could be due to the extracting reagents (β-ME in ammonium carbonate buffer [Martínez-Gomáriz et al. 2009] vs EtOH [this work]) and the starting amount of biological material subjected to extraction procedures in each case. Similarly, several factors may account for the strikingly low number of common singular proteins identified here and in the Zarnowski et al. (2014) report. First, different C. albicans strains used in each case. Second, the experimental conditions employed to generate biofilms (a rolling bottle system that possibly generate biofilms displaying a high metabolic activity [Zarnowski et al. 2014] vs flat bottom cell tissue flasks [this work]). Third, the extraction procedure applied to solubilize EM proteins (sonication of the entire biofilm material [Zarnowski et al. 2014] vs treatment of the biofilm mass with 60% EtOH [this work]). And, fourth the starting amount of biological samples subjected to the extraction procedure in each case; in this context, a sample corresponding to a biofilm area of 59.5 m2 was collected for sonication treatment to release putative biofilm EM components in the Zarnowski et al. (2014) paper, whereas the sample extracted with ethanol in this work corresponded to a biofilm area of only about 0.5 m2 (biofilms collected from 20 flat bottom cell tissue flasks for each individual experiment; see the ‘Materials and Methods’ section). Taking into account that we were able to visualize in the electrophoretic gels about 276 protein spots and to identify 131 polypeptides by proteomic analysis, it seems that 60% EtOH is able to extract proteins more efficiently than the β-ME/CO3(NH4)2 agent used by Martínez-Gomáriz et al. (2009) in a highly hydrophilic environment such as the biofilm EM is, being thus able to solubilize less abundant proteins from the starting biofilm batches employed in this work, that contained by far much less biofilm mass than samples processed by Zarnowski et al. (2014). In any case, detection of a number of common polypeptides in all these experimental approaches suggests that these proteins may play essential functions in the biology of C. albicans biofilms. Once again, Adh1p, a protein consistently detected in all instances, is a clear example in this context, as this enzyme is able to regulate biofilm formation in C. albicans through an ethanol-dependent mechanism (Mukherjee et al. 2006). Somewhat surprisingly, Adh1p was not detected by Zarnowski et al. (2014) in the biofilm EM material released by sonication. Overall, our results show that a common and consistent EM biofilm profile appeared with a reference strain and four mutants affected in cell wall biogenesis in our experimental conditions, suggesting that cell wall formation plays an essential role in biofilm formation. Only minor changes are produced both in the protein composition and in the protein abundance of C. albicans null mutants for cell-wall-related genes. In addition, we also provided a reproducible system for analyzing any kind of interactions in a reproducible way. SUPPLEMENTARY DATA Supplementary Data. We acknowledge D. Kornitzer (Haifa, Israel) and A.D. Johnson (San Francisco, CA, USA) for the kind gift of the mutant strains for the PGA10 (RBT51) and CSA1 genes. FUNDING This work was supported in part by grants , , Spain and , , Valencia, Spain (to J.P.M.), and grant from , Spain (to E.V.). AUTHORS CONTRIBUTIONS JPM and AD were responsible for the design and supervision of the study, performed data analysis and wrote the manuscript. RB, AM and MC performed the experiments and contributed to data analysis. EV provided financial support and technical advice for some experiments. All authors read and approved the final manuscript. Conflict of interest.None declared. REFERENCES Aebi M Bernasconi R Clerc S et al.   N-glycan structures: recognition and processing in the ER Trends Biochem Sci  2010 35 74 82 Google Scholar CrossRef Search ADS PubMed  Al-Fattani MA Douglas LJ Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance J Med Microbiol  2006 55 999 1008 Google Scholar CrossRef Search ADS PubMed  Baillie GS Douglas LJ Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents J Antimicrob Chemoth  2000 46 397 403 Google Scholar CrossRef Search ADS   Blanes R. 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TI - Null mutants of Candida albicans for cell-wall-related genes form fragile biofilms that display an almost identical extracellular matrix proteome
JO - FEMS Yeast Research
DO - 10.1093/femsyr/fow075
DA - 2016-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/null-mutants-of-candida-albicans-for-cell-wall-related-genes-form-BI40uXPO0Y
SP - fow075
VL - 16
IS - 7
DP - DeepDyve
ER -