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Localisation of Bgl2p upon antifungal drug treatment in Candida albicans
International Journal of Antimicrobial Agents 33 (2009) 143–148
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Localisation of Bgl2p upon antifungal drug treatment in Candida albicans Letizia Angiolella a,∗ , Alberto Vitali b , Annarita Stringaro c , Giuseppina Mignogna d , Bruno Maras d , Mariantonietta Bonito a , Marisa Colone c , Anna Teresa Palamara a , Antonio Cassone e a
Department of Public Health Sciences ‘G. Sanarelli’, University of Rome ‘La Sapienza’, Piazzale Aldo Moro, 00161 Rome, Italy Institute of Chemistry Molecular Recognition, C.N.R., c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy c Departments of Technology and Health, Istituto Superiore di Sanità, Rome, Italy d Department of Biochemical Sciences ‘A. Rossi Fanelli’, University of Rome ‘La Sapienza’, Rome, Italy e Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 23 June 2008 Accepted 7 August 2008 Keywords: Candida albicans Cell wall proteins Bgl2p Glucanosyltransferase
a b s t r a c t Several proteins are covalently bound to the cell wall glucan (glucan-associated proteins (GAPs)) in Candida albicans and different drugs may cause their modulation. Proteomic analysis is a suitable approach to study differential GAP patterns between control and drug-treated cells. Since antimycotics induce variation in GAP content, we investigated the effect of a sublethal dose of micafungin and observed a clear increase in Bgl2p, an enzyme with glucanosyltransferase activity, with respect to a general decrease in cell wall protein content. Immunoelectron microscopy using mouse antiserum confirmed this increase of Bgl2p on the outer cell wall but also revealed a dramatic increase in the immature Bgl2p isoform in the cytoplasm of drug-treated cells. Since this increased expression of Bgl2p is clearly dependent upon micafungin treatment, this enzyme appears to be one of the survival strategies of C. albicans and thus could be considered the molecular basis of antifungal resistance and also as a potential valuable candidate for future vaccine development. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction The cell wall of the opportunistic human pathogen Candida albicans is a dynamic structure, essentially composed of branched -glucan (1–3 and 1–6 linkages), chitin and mannoproteins [1,2]. -Glucan accounts for >50% of cell wall dry weight and confers physical strength and cell wall shape. Mannoproteins represent 30–40% of the cell wall and determine the cell surface properties, enabling C. albicans cells to adhere to host tissues [3] and stimulating the host immune response [4]. Together, mannoproteins and glucans form the cell wall skeleton in which several proteins are embedded, more or less tightly bound to the polysaccharides. Some of these proteins can only be released from the cell wall by enzymatic degradation of -glucan with -glucanase [5,6]. Representative glucan-associated proteins (GAPs) are connected with glucan metabolism and can be secreted both extracellularly and into the periplasmic space. Their putative physiological roles include: localised breakdown of -glucan for cell wall expansion; mobilisation of glucan for energetic purposes; and hydrolysis of exogenous material taken up as a nutrient [7]. Since cell wall growth
∗ Corresponding author. Fax: +39 06 446 8625. E-mail address: [email protected] (L. Angiolella).
requires continuous remodelling of the polysaccharides structure, it has been suggested that some glucanases may, within the wall, catalyse transglycosylation rather than hydrolytic reactions [8]. They can therefore contribute, along with hydrolases, to rearrangement and assembly of cell wall glucan to create a strong but flexible macromolecular network [9]. In previous works [6,10], we observed that one of the most abundant GAPs, the 34 kDa product of the BGL2 gene, was markedly modulated during treatment with fluconazole and cilofungin, an antimycotic belonging to the echinocandin class. On the basis of a C. albicans mutant phenotype [11], some authors hypothesised that the Bgl2 protein may be a glucan-branching enzyme and thus be responsible for transformation of the initial linear (1–3) glucan into the branched (1–3) (1–6) glucan [12], which is thought to confer its characteristic strength to the cell wall [13]. This glucanosyltransferase activity was further demonstrated using the purified enzyme [12]. In this paper, we have determined the expression and localisation of Bgl2 protein of the cell wall and in the cytosol of C. albicans following treatment with a subinhibitory dose of micafungin, a new antimycotic belonging to the echinocandin class of antifungal agents [14]. The rationale for our choice of this antimycotic resides in the mechanism of action of micafungin since it is known that it selectively inhibits the expression of (1–3) glucan synthase and
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thus affects the molecular structure of the cell wall. Our finding that micafungin increases Bgl2p expression demonstrates the role of this protein in the antimycotic response and may prove useful to ‘unprime’ drug resistance and to design new vaccines against C. albicans. 2. Materials and methods 2.1. Strain and growth conditions To assess the influence of micafungin (Fujisaka Pharmaceutical, Osawa, Japan) on GAP composition, C. albicans strain CO23 was cultured in yeast nitrogen base (YNB) medium supplemented with 1% (w/v) glucose and 0.8 M mannitol as an osmotic stabiliser at 28 ◦ C for 24 h. To recover a sufficient amount of cell wall material and cytoplasm for analysis, the fungus inoculum size was 107 cells/mL. The drug was added at a concentration of 0.01 g/mL, which caused only a partial inhibition of growth; under these conditions the final optical density (OD) at 560 nm of the culture reached a value of 1.2–1.4. 2.2. Cell wall preparation and GAP extraction Preparation of cytoplasm and cell wall was carried out as described previously [10]. Briefly, clean cell walls were resuspended in 50 mM Tris–HCl buffer (pH 6.8) containing 50 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 units/mL of a purified (1–3)-glucanase (Zymoliase 100T) (Seigako, Kogyo Co. Ltd., Tokyo, Japan). Following incubation at 37 ◦ C, the digested walls were centrifuged and the solubilised proteins were precipitated by addition of absolute ethanol. The proteins in this precipitate will be referred to as glucan-associated proteins (GAPs). 2.3. Two-dimensional (2D) electrophoresis Before electrophoresis, an aliquot of 30 g from each GAP sample was desalted using VivaspinTM 10,000 MWCO PES (Sartorius, Goettingen, Germany), lyophilised and then re-dissolved in 185 L of a rehydration buffer containing 8 M urea, 2% w/v CHAPS, 50 mM DTT and 0.2% v/v Bio-LytesTM (pH range 3–10). Each sample was loaded by passive rehydration on an 11 cm pre-casted Immobiline strip with a linear pH 3–10 gradient. All isoelectric focusing (IEF) was carried out on a Protean® IEF Cell. After the IEF separation was completed, the strips were first soaked for 30 min in 130 mM DTT and then treated for 30 min with 135 mM iodoacetamide, in the equilibration buffer (50 mM Tris (pH 8.8), 6 M urea, 20% v/v glycerol, 2% w/v sodium dodecyl sulphate (SDS)). The second dimension was carried out on pre-casted 4–12% BisTris gels (13 × 8.5 cm) using a Criterion apparatus. Electrophoretic analyses were developed in NuPAGE® MES SDS Running Buffer (Invitrogen, Paisley, UK) at 200 V constant voltage. Gels were stained using Bio-Safe Coomassie Blue G-250. All electrophoresis chemicals and apparatus were obtained from Bio-Rad (Hercules, CA), unless otherwise indicated. After de-staining, gels were digitalised using a computing densitometer (GS-710 Imaging Densitometer; Bio-Rad) with a pixel size of 42.3 × 42.3 m and were analysed with PDQuestTM image analysis system version 7.2.0 (Bio-Rad). The computer analysis allowed automatic detection and quantification of protein spots. For each relevant proteic band, the OD was determined. The total OD was considered as the arithmetic sum of each OD. Relative amount of each protein is calculated as the ratio of each OD to the total OD.
2.4. Protein identification Selected spots were manually excised from gel and proteolysis was achieved using the In-gel Digest96 KitTM (Millipore, Bedford, MA) with 15 L of trypsin provided by the kit manufacturer (ca. 11 g/mL in 25 mM ammonium bicarbonate) at 37 ◦ C for 3 h. After digestion, in-gel tryptic peptide re-suspension was performed by incubation of each gel piece in 130 L of a 0.2% trifluoroacetic acid (TFA) aqueous solution for 30 min at room temperature. Finally, tryptic peptides were eluted from the microcolumns to a matrix-assisted laser desorption/ionisation (MALDI) target plate with 1.3 L of a solution of ␣-cyano-4-hydroxy-trans-cinnamic acid matrix (2 mg/mL) in 70% acetonitrile containing 0.1% TFA. MALDItime-of flight (ToF) analyses were performed in a Voyager-DETM STR instrument (Applied Biosystems, Framingham, MA) equipped with a 337 nm nitrogen laser and operating in reflector mode. Peptide mass fingerprints were used to search for protein candidates in a fungi protein database at the National Center for Biotechnology Information (NCBI) using the MASCOT software program. 2.5. Enzyme purification A 2 mL GAP preparation was loaded onto a 10 mL Hi-Trap desalting column (Amersham, Piscataway, NJ) equilibrated with a 10 mM sodium acetate buffer (pH 5.5). The column was eluted at a flow rate of 0.8 mL/min and 0.5 mL fractions were collected. Fractions containing Bgl2p were pooled and then loaded onto a concanavalin A (Con A)–Sepharose column equilibrated with sodium acetate buffer (10 mM, pH 5.5). The column was washed with two volumes of buffer before being eluted with a 5 M ␣-dmannopyranoside solution. The eluted sample was dialysed against a sodium acetate buffer (10 mM, pH 6) and then loaded onto a DEAE Sepharose Fast Flow column (12 × 1.5 cm) equilibrated with the same buffer and the column was eluted with a linear NaCl gradient (0–0.5 M) at a 1 mL/min flow rate. The final purification was achieved using 1 mL Mono Source Q column (Amersham) equilibrated with sodium acetate buffer (20 mM, pH 5); the column was eluted with a linear NaCl gradient (0–0.5 M). SDS–polyacrylamide gel electrophoresis (PAGE) analyses were all performed using 12% gels under denaturing conditions. Low molecular mass standard proteins (Bio-Rad) were used for molecular mass determination. Colloidal silver blue [15] and Periodic acid-Schiff (PAS) staining method was employed to detect the proteins. 2.6. Antiserum preparation Antibodies against the purified Bgl2 protein were obtained by immunisation of 10 female 8–10-month-old BALB/c mice. For immunisation, three peritoneal injections of the protein suspension were inoculated in mice at weekly intervals. After 28 days, blood samples were taken from the animals and serum was harvested. This immune serum recognised only the Bgl2p band in a SDS–PAGE of the whole GAP preparation of the cell wall. 2.7. Western blotting analyses Western blotting was performed in a Tris-glycine buffer (25 mM, pH 8.5, 20% v/v methanol) with a constant voltage of 100 V for 1 h. Following electrotransfer, the nitrocellulose membrane was washed with 0.05% Tween 20 in phosphate-buffered saline (PBS) buffer and then incubated with a 10% albumin solution for 1 h. Incubation with polyclonal serum (1:5000) was performed overnight at 37 ◦ C. After washing with 0.05% Tween 20 in PBS buffer, an IgG antimouse antibody conjugated with alkaline phosphatase was used
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(1:15 000) and left to react for 1 h at 37 ◦ C. The band of interest was detected with staining based on nitro blue tetrazolium (NBT) (Sigma–Aldrich, St Louis, MO) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma). 2.8. Enzyme assay Glucanosyltransferase activity was assayed by MALDI mass spectrometry analysis [16] using laminarioligosaccharides as standards. A suitable amount of enzyme (1 g) was mixed with a 2 mM solution of laminariopentaose dissolved in sodium acetate buffer (10 mM, pH 5.5) and left for 4 h at 30 ◦ C. Aliquots (1 L) of the reaction mixture were taken at intervals of 30 min, mixed with a 0.2% TFA solution and deposited on a stainless MALDI target. Soon after, 0.5 L of a 2,5-dihydroxybenzoic acid (DHB) solution (12 mg/mL dissolved in 80% methanol) were deposited on the sample and left to crystallise at room temperature. The MALDI mass spectra were measured in reflector mode using an Autoflex (Bruker Daltonics, Bremen, Germany) ToF mass spectrometer. The spectra were recorded in the positive ion mode using a nitrogen laser with an emission wavelength at 337 nm. Laminarioligosaccharides were obtained from laminarin hydrolysis with 50% TFA at 100 ◦ C as reported [17], whilst pure laminariopentaose was purchased from Associates of Cape Cod, Inc. (Falmouth, MA). Exoglucanase activity was determined in a standard assay with 12 mM p-nitrophenol glucopyranoside (pNPG) in 25 mM sodium acetate buffer (pH 6.0). Assay mixtures (200 L) were incubated for 40 min at 35 ◦ C. The reaction was stopped by adding 80 L of 1 M Na2 CO3 and p-nitrophenol formation was determined at 414 nm. 2.9. Sequence analysis Sequence analysis of the purified protein from the cell wall was obtained using a Procise® instrument (Applied Biosystems). Sequence analysis of the cytosolic form of Bgl2p was obtained after partial purification of the cytosolic extract of micafungintreated cells. Preliminary anionic exchange chromatography of the cellular extract was performed with a 1 mL Resource–Q (Amersham) column equilibrated with Tris–HCl (25 mM, pH 8.0) buffer. The acidic fraction, eluted with 1 mL of 0.5 M NaCl in Tris–HCl buffer, was subjected to Western blotting employing the polyclonal serum preparation. A Coomassie-stained band corresponding to the antibody-reactive band around 34 kDa was subjected to automated Edman degradation using a Procise® instrument.
Fig. 1. Two-dimensional electrophoresis of glucan-associated proteins (GAPs) from Candida albicans CO23 strain. Proteins identified by peptide mass fingerprint are indicated. ZymoliaseTM (Zym), utilised to release GAPs from the Candida cell wall, is a mixture of two different proteins indicated in the gel by oval shapes. GPM1, phosphoglycerate mutase isoforms; Bgl2p, glucanosyltransferase.
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2.10. Immunolabelling of cryosections Candida albicans samples were fixed with 4% paraformaldehyde plus 0.5% glutaraldehyde in PBS (pH 7.4) for 2 h at 4 ◦ C, washed in PBS and then infiltrated with 2.3 M sucrose in PBS for 3 h at 4 ◦ C, frozen in liquid N2 and cryosectioned following the method of Tokuyasu [18]. Ultrathin cryosections, obtained using Leica Ultracut UCT device (Leica Microsystems, Wien, Austria), were collected using sucrose and methylcellulose and incubated overnight at 4 ◦ C with the specific polyclonal antibody anti-Bgl2p (1:20 diluted) and then with goat anti-mouse–10 nm gold conjugates (1:10 diluted) (Sigma–Aldrich, Milan, Italy). Finally, ultrathin cryosections were stained with a 2% methylcellulose and 0.4% uranyl acetate solution. Samples were examined with a Philips 208 transmission electron microscope (FEI Company, Eindhoven, The Netherlands). 3. Results 3.1. Increase in Bgl2p after micafungin treatment To assess Bgl2p quantitative variation in C. albicans cell wall upon treatment with micafungin, 2D electrophoretic analyses of GAP from control and drug-treated cells was performed, followed by identification of protein spots by peptide mass fingerprinting with MALDI-ToF. These analyses allowed the identification of major GAPs as: isoforms of glycolytic phosphoglyceromutase 1, differing in isoelectric point and/or molecular weight; Bgl2p; and LDG7, a protein belonging to a family of related proteins whose function is still unknown (Fig. 1). The 2D gels were analysed by a computing densitometer. This allowed the relative amount of each protein band to be determined. Total protein quantity of each gel was determined by adding the OD of all considered spots. This analysis helped to determine the relative amount of the same spot in the 2D gel of the control and treated samples, which was expressed as a percentage with respect to the total OD. The result shows a marked variation in the intensity of protein spots upon treatment with micafungin (Fig. 2A). In the context of a general decrease in total protein content, an increase in the relative quantity of the Bgl2p band with respect to the total protein content (16 ± 0.3% vs. 9.6 ± 0.4% for the control cells) may be easily seen when comparing the two 2D GAP panels of control and micafungintreated samples (Fig. 2B). 3.2. Protein purification and enzymatic activity Increased expression of Bgl2p under antifungal treatment led us to purify the protein for structural and functional study. The enzyme is glycosylated as revealed by a reaction with PAS-Schiff staining after SDS–PAGE as well as by the high affinity binding to the ConA resin, confirming the previously obtained results from a ConA–digoxigenin immunoblotting reaction [10]. The observation that Bgl2p is a glycoprotein is in agreement with the data obtained for the homologous proteins BGT1 [17] and BGT2 [19] from Aspergillus fumigatus and BGL2 from Saccharomyces cerevisiae [20,21]. Sequence analysis gave the result MGDLAFN, corresponding to the sequence reported in the Swiss Prot databank (entry P 43070) after the excision of the signal peptide. To obtain a direct demonstration of the glucanolytic and/or glucanosyltranferase activity of Bgl2p, purified enzyme was assayed against two different substrates, pNPG and laminarin oligosaccharides. pNPG is the standard substrate employed to detect exoglucanase activity, whilst laminarin oligosaccharides allow the detection of endoglycolysis followed by a transfer reaction.
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Fig. 2. (A) Two-dimensional (“D) electrophoresis of glucan-associated proteins (GAPs) from Candida albicans CO23 strain treated with micafungin (FK). (B) Quantitative analysis of Bgl2p in C. albicans CO23 and C. albicans CO23 treated with micafungin (CO23+FK) 2D electrophoreses. Relative quantity of the considered protein spots is expressed as %optical density (OD) of the total intensity of all spots (see Section 2.3).
The products obtained by the enzymatic activity were identified by mass spectrometry analysis using a MALDI-ToF apparatus. The enzymatic reaction using pNPG as substrate failed to produce any free glucose, confirming no exoglucanase activity of Bgl2p. However glucanolytic activity was confirmed with laminarin as the substrate. The enzyme activity was not affected by the exoglucanase inhibitor ␦-gluconolactone, confirming the peculiar glucanosyltransferase activity of our purified enzyme preparation.
Fig. 3. Electron micrographs of Candida albicans cells showing localisation of Bgl2p on (A) control cells and (B) micafungin-treated cells after the reaction with the specific polyclonal anti-Bgl2p antibody and revealed by goat anti-mouse–10 nm gold conjugates.
3.3. Localisation of Bgl2p
4. Discussion
Localisation of the Bgl2p in control and micafungin-treated cells was analysed on gold–antibody immunolabelled cells after glutaraldehyde fixation. This method of preparation avoids the use of osmium tetroxide and uranyl acetate and allows good cell preservation, although it reduces the visibility of the electron transparent layers of the cell wall. Immunolabelling was carried out using a polyclonal antibody against the Bgl2p purified from the fungal cell as primary antibody and a gold conjugate antibody as the secondary one. Observation of the immunolabelled sections of control cells revealed the presence of Bgl2p spread throughout all layers of the cell wall and partially inside the cytoplasm (Fig. 3A). Micafungintreated cells showed a remarkable increase in labelling, not only in the cell wall but also in the cytoplasmic compartment (Fig. 3B). 3.4. Cytosolic Bgl2p
The C. albicans cell wall is a complex system of polysaccharides and proteins functionally linked together to shape the cells and to maintain the cell wall architecture in an efficient metabolic structure. GAPs are proteins covalently linked to glucan that play an important role in cell wall metabolism and in particular in adaptive changes required by the presence of intracellular and/or environmental stressing agents [22,23]. Antimycotics constitute one of the most stressing factors and thus they exert a rather marked influence on the GAP pattern in the C. albicans cell wall. Bgl2p is one of the most abundant cell wall proteins [20]. It was characterised as an endo-1,3--glucanase and glucanosyltransferase that introduces -1,6-linkages in 1,3--glucan chains [12,20] and thus participates in cross-linking of the glucan component of the cell wall [4]. Deletion of BGL2 does not obviously affect cell morphology, whereas its overexpression is harmful for cell viability [20]. We have previously demonstrated that Bgl2p was induced by the
The increased labelling in the cytosol of the drug-treated cells indicated that micafungin elicited the expression of Bgl2p. Enzymatic activity assayed on the cytosolic preparation failed to reveal the presence of active enzyme. Cytosolic Bgl2p was partially purified and identified by Western blot analysis. Automated Edman degradation of the pertinent band gave the sequence MQIKFLTT, which matched the initial residues of the BGL2 gene product reported in the Swiss Prot databank. This indicates that the cytosolic Bgl2p isoform still contains the signal peptide and confirms
Fig. 4. DNA-deduced sequence of the first 40 amino acid residues of the BGL2 gene. Signal peptides (residues 1–18) are reported in bold. The (a) arrow encompasses the sequence obtained from the analysis of cytosolic Bgl2p, whilst the (b) arrow indicates the sequence from cell wall Bgl2p.
the presence of Bgl2p as an unprocessed form in the cytoplasm of treated cells (Fig. 4). The presence of Bgl2p in the immature form could explain the lack of enzymatic activity.
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presence of subinhibitory doses of both fluconazole and cilofungin. This indicates that, independently on the drugs utilised and their mechanism of antifungal action, an increase of Bgl2p expression is a common response of C. albicans [6,10]. In this study, we confirmed by 2D electrophoretic analyses the change of GAPs in C. albicans upon treatment with micafungin by comparing the 2D GAP maps obtained for treated and untreated organisms (Fig. 1). Although proteomic analysis of cell wall proteins has been reported [24–26], no previous 2D map was available for GAPs of C. albicans following treatment with an antifungal. When comparing the map of C. albicans treated with micafungin with the untreated control, a general decrease of major spots was observed with the exception of Bgl2p (Fig. 2A), demonstrating and confirming the role of this protein in the response to antimycotic drugs. A possible explanation of Bgl2p modulation may reside in cell wall compensatory mechanisms responsible for remodelling the cell wall. These mechanisms come into play to prevent cell lysis whenever its integrity is compromised by environmental stresses, by cell wall-perturbing compounds or in cell wall mutants [27–30]. Localisation of Bgl2p on the cell wall structure was analysed by immunogold experiments in control and micafungin-treated cells. The number of gold particles in the cell wall of the treated cells was higher than that in the control cells (Fig. 3). Furthermore, this noticeable increase of Bgl2p after micafungin treatment was not limited to the cell wall but also involved the cytoplasmic compartment, which appeared to be strongly labelled (Fig. 3B). It has been demonstrated that a defect in Bgl2p transport to the cell wall might be accompanied by the intracellular accumulation of this protein [31]. This does not appear to be the case in C. albicans treated with micafungin since the amount of Bgl2p on the cell wall is similar or even increased with respect to control cells and thus the increase in the cytosol may be reasonably explained by a more active translation of the BGL2 gene. Bgl2p in the cytoplasm is present in the unprocessed form as demonstrated by direct sequence analysis, in contrast to the sequence obtained by Edman degradation analysis of the purified cell wall isoform that lacks the first 18 residues with respect to the genomic-derived sequence (Fig. 4). These data confirm the presence of a signal peptide that is excised upon translocation of the protein from the cytoplasm to the cell wall and this is in agreement with the secretion pathway of a cell wall-localised protein. The structural difference in the two isoforms may explain the lack of glucanosyltransferase activity in the cytosolic compartment and thus assign the catalytic activity only to the mature form. Taking the data obtained from 2D maps and localisation by immunogold together, it is possible to conclude that after 24 h of sublethal micafungin treatment, a general decrease of GAPs occurs indicating the cytotoxic effect of the drug. However, an acute, likely compensatory, response of the organism takes place represented by a marked stimulation of Bgl2p expression. In this context, possible roles of Bgl2 enzyme in cell wall maintenance upon drug treatment could be assumed in terms of repair, cross-linking and incorporation of newly synthesised chains of -1,3 glucan into the previously existing cell wall structure. Although further studies are necessary to validate this hypothesis, it is of some interest that the phenotype arising after disruption of the BGL2 gene shows a decrease in the resistance of C. albicans to the antifungal nikkomycin Z [11]. Analysis of the fungal cell wall continues to be an active and exciting area of research. Changes in the biochemical and physical properties of the wall as a result of this remodelling have implications for interactions of fungi with their environments. In fungal pathogens the cell wall constitutes the interface with the host, and cell wall proteins of C. albicans are important targets for both humoral and cell-mediated immune responses. Antibodies against
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other fungal cell wall proteins and extracellular -glucosidases are largely used in the serodiagnosis of histoplasmosis [32]. Vaccines eliciting antibodies and/or cell-mediated immunity against Bgl2p [33], Camp65/SCw1 [34] and the adhesins Als1 and Als3 [35] have been protective in animal model of candidiasis. In this context, Bgl2p should also be considered as a potential vaccine candidate against candidiasis. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Shepherd MG. Cell envelope of Candida albicans. Crit Rev Microbiol 1987;15:7–25. [2] Fleet GH. Cell walls. In: Rose AH, Harrison JS, editors. The yeasts, vol. 4. London, UK: Academic Press; 1991. [3] Jeng HW, Holmes AR, Cannon D. Characterization of two Candida albicans surface mannoprotein adhesins that bind immobilized saliva components. Med Mycol 2005;43:209–17. [4] Chaffin WL, Lopez-Ribot JL, Casanova M, Gozalbo D, Martinez JP. Cell wall and secreted proteins of Candida albicans: identification, functions and expression. Microbiol Mol Biol Rev 1998;62:130–80. [5] Kapteyn JC, Van Den Ende H, Klis FM. The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1999;1426:373– 83. [6] Angiolella L, Micocci MM, D’Alessio S, Girolamo A, Maras B, Cassone A. Identification of major glucan-associated cell wall proteins of Candida albicans and their role in fluconazole resistance. Antimicrob Agents Chemother 2002;46:1688–94. [7] Nombela C, Molina M, Cenamor R, Sanchez M. Yeast beta-glucanases: a complex system of secreted enzymes. Microbiol Sci 1988;5:328–32. [8] De Nobel H, Kliss FM. Organization and construction of the yeast cell wall. In: Sturgeon R, editor. Advances in macromolecular carbohydrate research, vol. 2. Amsterdam, The Netherlands: Elsevier Science; 2003. [9] Stubbs HJ, Brusch DJ, Emerson JB, Sullivan PA. Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans. Eur J Biochem 1999;263:889–95. [10] Angiolella L, Facchin M, Stringaro A, Maras B, Simonetti N, Cassone A. Identification of glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J Infect Dis 1996;173:684– 90. [11] Sarthy A, McGonial T, Coen M, Frost DJ, Meulbroek JA, Goldman RC. Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-betaglucosyltransferase. Microbiology 1997;143:367–76. [12] Hartland RP, Emerson GW, Sullivan PA. A secreted beta-glucan-branching enzyme from Candida albicans. Proc Biol Sci 1991;246:155–60. [13] Goldman RC, Sullivan PA, Zakula D, Capobianco JO. Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. Eur J Biochem 1995;227:372–8. [14] Chandrasekar PH, Sobel JO. Micafungin: a new echinocandin. Clin Infect Dis 2006;42:1171–8. [15] Candiano G, Bruschi M, Musante L, Santucci GM, Ghiggheri B, Carnemolla P, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteomic analysis. Electrophoresis 2002;25:1327–33. [16] Chizhov AO, Dell A, Morris HR, Reason AJ, Haslam RA, McDowell OS, et al. Structural analysis of laminarans by MALDI and FAB mass spectrometry. Carbohydr Res 1998;310:203–10. [17] Hartland RP, Fontaine T, Debeaupuis JP, Simenel C, Delepierre M, Latge JP. A novel beta-(1-3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J Biol Chem 1996;271:26843–9. [18] Tokuyasu KT. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–65. [19] Mouyna I, Hartland RP, Fontaine T, Diaquin M, Simenel C, Delepierre M, et al. A 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p. Microbiology 1998;144:3171– 80. [20] Mrsa V, Klebl F, Tanner W. Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase. J Bacteriol 1993;175:2102–6. [21] Cappellaro C, Mrsa V, Tanner W. New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating. J Bacteriol 1998;180:5030–7. [22] Kapteyn JC, Hoyer LL, Hecht JE, Muller WH, Andel A, Verkleij AJ, et al. The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 2000;35:601–11. [23] Aguilar-Uscanga B, Francois JM. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Lett Appl Microbiol 2003;37:268–74. [24] De Groot WJ, de Boet AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, et al. Proteomic analysis of Candida albicans cell walls reveals covalently
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International Journal of Antimicrobial Agents journal homepage: http://www.elsevier.com/locate/ijantimicag
Localisation of Bgl2p upon antifungal drug treatment in Candida albicans Letizia Angiolella a,∗ , Alberto Vitali b , Annarita Stringaro c , Giuseppina Mignogna d , Bruno Maras d , Mariantonietta Bonito a , Marisa Colone c , Anna Teresa Palamara a , Antonio Cassone e a
Department of Public Health Sciences ‘G. Sanarelli’, University of Rome ‘La Sapienza’, Piazzale Aldo Moro, 00161 Rome, Italy Institute of Chemistry Molecular Recognition, C.N.R., c/o Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy c Departments of Technology and Health, Istituto Superiore di Sanità, Rome, Italy d Department of Biochemical Sciences ‘A. Rossi Fanelli’, University of Rome ‘La Sapienza’, Rome, Italy e Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy b
a r t i c l e
i n f o
Article history: Received 23 June 2008 Accepted 7 August 2008 Keywords: Candida albicans Cell wall proteins Bgl2p Glucanosyltransferase
a b s t r a c t Several proteins are covalently bound to the cell wall glucan (glucan-associated proteins (GAPs)) in Candida albicans and different drugs may cause their modulation. Proteomic analysis is a suitable approach to study differential GAP patterns between control and drug-treated cells. Since antimycotics induce variation in GAP content, we investigated the effect of a sublethal dose of micafungin and observed a clear increase in Bgl2p, an enzyme with glucanosyltransferase activity, with respect to a general decrease in cell wall protein content. Immunoelectron microscopy using mouse antiserum confirmed this increase of Bgl2p on the outer cell wall but also revealed a dramatic increase in the immature Bgl2p isoform in the cytoplasm of drug-treated cells. Since this increased expression of Bgl2p is clearly dependent upon micafungin treatment, this enzyme appears to be one of the survival strategies of C. albicans and thus could be considered the molecular basis of antifungal resistance and also as a potential valuable candidate for future vaccine development. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.
1. Introduction The cell wall of the opportunistic human pathogen Candida albicans is a dynamic structure, essentially composed of branched -glucan (1–3 and 1–6 linkages), chitin and mannoproteins [1,2]. -Glucan accounts for >50% of cell wall dry weight and confers physical strength and cell wall shape. Mannoproteins represent 30–40% of the cell wall and determine the cell surface properties, enabling C. albicans cells to adhere to host tissues [3] and stimulating the host immune response [4]. Together, mannoproteins and glucans form the cell wall skeleton in which several proteins are embedded, more or less tightly bound to the polysaccharides. Some of these proteins can only be released from the cell wall by enzymatic degradation of -glucan with -glucanase [5,6]. Representative glucan-associated proteins (GAPs) are connected with glucan metabolism and can be secreted both extracellularly and into the periplasmic space. Their putative physiological roles include: localised breakdown of -glucan for cell wall expansion; mobilisation of glucan for energetic purposes; and hydrolysis of exogenous material taken up as a nutrient [7]. Since cell wall growth
∗ Corresponding author. Fax: +39 06 446 8625. E-mail address: [email protected] (L. Angiolella).
requires continuous remodelling of the polysaccharides structure, it has been suggested that some glucanases may, within the wall, catalyse transglycosylation rather than hydrolytic reactions [8]. They can therefore contribute, along with hydrolases, to rearrangement and assembly of cell wall glucan to create a strong but flexible macromolecular network [9]. In previous works [6,10], we observed that one of the most abundant GAPs, the 34 kDa product of the BGL2 gene, was markedly modulated during treatment with fluconazole and cilofungin, an antimycotic belonging to the echinocandin class. On the basis of a C. albicans mutant phenotype [11], some authors hypothesised that the Bgl2 protein may be a glucan-branching enzyme and thus be responsible for transformation of the initial linear (1–3) glucan into the branched (1–3) (1–6) glucan [12], which is thought to confer its characteristic strength to the cell wall [13]. This glucanosyltransferase activity was further demonstrated using the purified enzyme [12]. In this paper, we have determined the expression and localisation of Bgl2 protein of the cell wall and in the cytosol of C. albicans following treatment with a subinhibitory dose of micafungin, a new antimycotic belonging to the echinocandin class of antifungal agents [14]. The rationale for our choice of this antimycotic resides in the mechanism of action of micafungin since it is known that it selectively inhibits the expression of (1–3) glucan synthase and
0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.08.021
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thus affects the molecular structure of the cell wall. Our finding that micafungin increases Bgl2p expression demonstrates the role of this protein in the antimycotic response and may prove useful to ‘unprime’ drug resistance and to design new vaccines against C. albicans. 2. Materials and methods 2.1. Strain and growth conditions To assess the influence of micafungin (Fujisaka Pharmaceutical, Osawa, Japan) on GAP composition, C. albicans strain CO23 was cultured in yeast nitrogen base (YNB) medium supplemented with 1% (w/v) glucose and 0.8 M mannitol as an osmotic stabiliser at 28 ◦ C for 24 h. To recover a sufficient amount of cell wall material and cytoplasm for analysis, the fungus inoculum size was 107 cells/mL. The drug was added at a concentration of 0.01 g/mL, which caused only a partial inhibition of growth; under these conditions the final optical density (OD) at 560 nm of the culture reached a value of 1.2–1.4. 2.2. Cell wall preparation and GAP extraction Preparation of cytoplasm and cell wall was carried out as described previously [10]. Briefly, clean cell walls were resuspended in 50 mM Tris–HCl buffer (pH 6.8) containing 50 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF) and 20 units/mL of a purified (1–3)-glucanase (Zymoliase 100T) (Seigako, Kogyo Co. Ltd., Tokyo, Japan). Following incubation at 37 ◦ C, the digested walls were centrifuged and the solubilised proteins were precipitated by addition of absolute ethanol. The proteins in this precipitate will be referred to as glucan-associated proteins (GAPs). 2.3. Two-dimensional (2D) electrophoresis Before electrophoresis, an aliquot of 30 g from each GAP sample was desalted using VivaspinTM 10,000 MWCO PES (Sartorius, Goettingen, Germany), lyophilised and then re-dissolved in 185 L of a rehydration buffer containing 8 M urea, 2% w/v CHAPS, 50 mM DTT and 0.2% v/v Bio-LytesTM (pH range 3–10). Each sample was loaded by passive rehydration on an 11 cm pre-casted Immobiline strip with a linear pH 3–10 gradient. All isoelectric focusing (IEF) was carried out on a Protean® IEF Cell. After the IEF separation was completed, the strips were first soaked for 30 min in 130 mM DTT and then treated for 30 min with 135 mM iodoacetamide, in the equilibration buffer (50 mM Tris (pH 8.8), 6 M urea, 20% v/v glycerol, 2% w/v sodium dodecyl sulphate (SDS)). The second dimension was carried out on pre-casted 4–12% BisTris gels (13 × 8.5 cm) using a Criterion apparatus. Electrophoretic analyses were developed in NuPAGE® MES SDS Running Buffer (Invitrogen, Paisley, UK) at 200 V constant voltage. Gels were stained using Bio-Safe Coomassie Blue G-250. All electrophoresis chemicals and apparatus were obtained from Bio-Rad (Hercules, CA), unless otherwise indicated. After de-staining, gels were digitalised using a computing densitometer (GS-710 Imaging Densitometer; Bio-Rad) with a pixel size of 42.3 × 42.3 m and were analysed with PDQuestTM image analysis system version 7.2.0 (Bio-Rad). The computer analysis allowed automatic detection and quantification of protein spots. For each relevant proteic band, the OD was determined. The total OD was considered as the arithmetic sum of each OD. Relative amount of each protein is calculated as the ratio of each OD to the total OD.
2.4. Protein identification Selected spots were manually excised from gel and proteolysis was achieved using the In-gel Digest96 KitTM (Millipore, Bedford, MA) with 15 L of trypsin provided by the kit manufacturer (ca. 11 g/mL in 25 mM ammonium bicarbonate) at 37 ◦ C for 3 h. After digestion, in-gel tryptic peptide re-suspension was performed by incubation of each gel piece in 130 L of a 0.2% trifluoroacetic acid (TFA) aqueous solution for 30 min at room temperature. Finally, tryptic peptides were eluted from the microcolumns to a matrix-assisted laser desorption/ionisation (MALDI) target plate with 1.3 L of a solution of ␣-cyano-4-hydroxy-trans-cinnamic acid matrix (2 mg/mL) in 70% acetonitrile containing 0.1% TFA. MALDItime-of flight (ToF) analyses were performed in a Voyager-DETM STR instrument (Applied Biosystems, Framingham, MA) equipped with a 337 nm nitrogen laser and operating in reflector mode. Peptide mass fingerprints were used to search for protein candidates in a fungi protein database at the National Center for Biotechnology Information (NCBI) using the MASCOT software program. 2.5. Enzyme purification A 2 mL GAP preparation was loaded onto a 10 mL Hi-Trap desalting column (Amersham, Piscataway, NJ) equilibrated with a 10 mM sodium acetate buffer (pH 5.5). The column was eluted at a flow rate of 0.8 mL/min and 0.5 mL fractions were collected. Fractions containing Bgl2p were pooled and then loaded onto a concanavalin A (Con A)–Sepharose column equilibrated with sodium acetate buffer (10 mM, pH 5.5). The column was washed with two volumes of buffer before being eluted with a 5 M ␣-dmannopyranoside solution. The eluted sample was dialysed against a sodium acetate buffer (10 mM, pH 6) and then loaded onto a DEAE Sepharose Fast Flow column (12 × 1.5 cm) equilibrated with the same buffer and the column was eluted with a linear NaCl gradient (0–0.5 M) at a 1 mL/min flow rate. The final purification was achieved using 1 mL Mono Source Q column (Amersham) equilibrated with sodium acetate buffer (20 mM, pH 5); the column was eluted with a linear NaCl gradient (0–0.5 M). SDS–polyacrylamide gel electrophoresis (PAGE) analyses were all performed using 12% gels under denaturing conditions. Low molecular mass standard proteins (Bio-Rad) were used for molecular mass determination. Colloidal silver blue [15] and Periodic acid-Schiff (PAS) staining method was employed to detect the proteins. 2.6. Antiserum preparation Antibodies against the purified Bgl2 protein were obtained by immunisation of 10 female 8–10-month-old BALB/c mice. For immunisation, three peritoneal injections of the protein suspension were inoculated in mice at weekly intervals. After 28 days, blood samples were taken from the animals and serum was harvested. This immune serum recognised only the Bgl2p band in a SDS–PAGE of the whole GAP preparation of the cell wall. 2.7. Western blotting analyses Western blotting was performed in a Tris-glycine buffer (25 mM, pH 8.5, 20% v/v methanol) with a constant voltage of 100 V for 1 h. Following electrotransfer, the nitrocellulose membrane was washed with 0.05% Tween 20 in phosphate-buffered saline (PBS) buffer and then incubated with a 10% albumin solution for 1 h. Incubation with polyclonal serum (1:5000) was performed overnight at 37 ◦ C. After washing with 0.05% Tween 20 in PBS buffer, an IgG antimouse antibody conjugated with alkaline phosphatase was used
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(1:15 000) and left to react for 1 h at 37 ◦ C. The band of interest was detected with staining based on nitro blue tetrazolium (NBT) (Sigma–Aldrich, St Louis, MO) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (Sigma). 2.8. Enzyme assay Glucanosyltransferase activity was assayed by MALDI mass spectrometry analysis [16] using laminarioligosaccharides as standards. A suitable amount of enzyme (1 g) was mixed with a 2 mM solution of laminariopentaose dissolved in sodium acetate buffer (10 mM, pH 5.5) and left for 4 h at 30 ◦ C. Aliquots (1 L) of the reaction mixture were taken at intervals of 30 min, mixed with a 0.2% TFA solution and deposited on a stainless MALDI target. Soon after, 0.5 L of a 2,5-dihydroxybenzoic acid (DHB) solution (12 mg/mL dissolved in 80% methanol) were deposited on the sample and left to crystallise at room temperature. The MALDI mass spectra were measured in reflector mode using an Autoflex (Bruker Daltonics, Bremen, Germany) ToF mass spectrometer. The spectra were recorded in the positive ion mode using a nitrogen laser with an emission wavelength at 337 nm. Laminarioligosaccharides were obtained from laminarin hydrolysis with 50% TFA at 100 ◦ C as reported [17], whilst pure laminariopentaose was purchased from Associates of Cape Cod, Inc. (Falmouth, MA). Exoglucanase activity was determined in a standard assay with 12 mM p-nitrophenol glucopyranoside (pNPG) in 25 mM sodium acetate buffer (pH 6.0). Assay mixtures (200 L) were incubated for 40 min at 35 ◦ C. The reaction was stopped by adding 80 L of 1 M Na2 CO3 and p-nitrophenol formation was determined at 414 nm. 2.9. Sequence analysis Sequence analysis of the purified protein from the cell wall was obtained using a Procise® instrument (Applied Biosystems). Sequence analysis of the cytosolic form of Bgl2p was obtained after partial purification of the cytosolic extract of micafungintreated cells. Preliminary anionic exchange chromatography of the cellular extract was performed with a 1 mL Resource–Q (Amersham) column equilibrated with Tris–HCl (25 mM, pH 8.0) buffer. The acidic fraction, eluted with 1 mL of 0.5 M NaCl in Tris–HCl buffer, was subjected to Western blotting employing the polyclonal serum preparation. A Coomassie-stained band corresponding to the antibody-reactive band around 34 kDa was subjected to automated Edman degradation using a Procise® instrument.
Fig. 1. Two-dimensional electrophoresis of glucan-associated proteins (GAPs) from Candida albicans CO23 strain. Proteins identified by peptide mass fingerprint are indicated. ZymoliaseTM (Zym), utilised to release GAPs from the Candida cell wall, is a mixture of two different proteins indicated in the gel by oval shapes. GPM1, phosphoglycerate mutase isoforms; Bgl2p, glucanosyltransferase.
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2.10. Immunolabelling of cryosections Candida albicans samples were fixed with 4% paraformaldehyde plus 0.5% glutaraldehyde in PBS (pH 7.4) for 2 h at 4 ◦ C, washed in PBS and then infiltrated with 2.3 M sucrose in PBS for 3 h at 4 ◦ C, frozen in liquid N2 and cryosectioned following the method of Tokuyasu [18]. Ultrathin cryosections, obtained using Leica Ultracut UCT device (Leica Microsystems, Wien, Austria), were collected using sucrose and methylcellulose and incubated overnight at 4 ◦ C with the specific polyclonal antibody anti-Bgl2p (1:20 diluted) and then with goat anti-mouse–10 nm gold conjugates (1:10 diluted) (Sigma–Aldrich, Milan, Italy). Finally, ultrathin cryosections were stained with a 2% methylcellulose and 0.4% uranyl acetate solution. Samples were examined with a Philips 208 transmission electron microscope (FEI Company, Eindhoven, The Netherlands). 3. Results 3.1. Increase in Bgl2p after micafungin treatment To assess Bgl2p quantitative variation in C. albicans cell wall upon treatment with micafungin, 2D electrophoretic analyses of GAP from control and drug-treated cells was performed, followed by identification of protein spots by peptide mass fingerprinting with MALDI-ToF. These analyses allowed the identification of major GAPs as: isoforms of glycolytic phosphoglyceromutase 1, differing in isoelectric point and/or molecular weight; Bgl2p; and LDG7, a protein belonging to a family of related proteins whose function is still unknown (Fig. 1). The 2D gels were analysed by a computing densitometer. This allowed the relative amount of each protein band to be determined. Total protein quantity of each gel was determined by adding the OD of all considered spots. This analysis helped to determine the relative amount of the same spot in the 2D gel of the control and treated samples, which was expressed as a percentage with respect to the total OD. The result shows a marked variation in the intensity of protein spots upon treatment with micafungin (Fig. 2A). In the context of a general decrease in total protein content, an increase in the relative quantity of the Bgl2p band with respect to the total protein content (16 ± 0.3% vs. 9.6 ± 0.4% for the control cells) may be easily seen when comparing the two 2D GAP panels of control and micafungintreated samples (Fig. 2B). 3.2. Protein purification and enzymatic activity Increased expression of Bgl2p under antifungal treatment led us to purify the protein for structural and functional study. The enzyme is glycosylated as revealed by a reaction with PAS-Schiff staining after SDS–PAGE as well as by the high affinity binding to the ConA resin, confirming the previously obtained results from a ConA–digoxigenin immunoblotting reaction [10]. The observation that Bgl2p is a glycoprotein is in agreement with the data obtained for the homologous proteins BGT1 [17] and BGT2 [19] from Aspergillus fumigatus and BGL2 from Saccharomyces cerevisiae [20,21]. Sequence analysis gave the result MGDLAFN, corresponding to the sequence reported in the Swiss Prot databank (entry P 43070) after the excision of the signal peptide. To obtain a direct demonstration of the glucanolytic and/or glucanosyltranferase activity of Bgl2p, purified enzyme was assayed against two different substrates, pNPG and laminarin oligosaccharides. pNPG is the standard substrate employed to detect exoglucanase activity, whilst laminarin oligosaccharides allow the detection of endoglycolysis followed by a transfer reaction.
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Fig. 2. (A) Two-dimensional (“D) electrophoresis of glucan-associated proteins (GAPs) from Candida albicans CO23 strain treated with micafungin (FK). (B) Quantitative analysis of Bgl2p in C. albicans CO23 and C. albicans CO23 treated with micafungin (CO23+FK) 2D electrophoreses. Relative quantity of the considered protein spots is expressed as %optical density (OD) of the total intensity of all spots (see Section 2.3).
The products obtained by the enzymatic activity were identified by mass spectrometry analysis using a MALDI-ToF apparatus. The enzymatic reaction using pNPG as substrate failed to produce any free glucose, confirming no exoglucanase activity of Bgl2p. However glucanolytic activity was confirmed with laminarin as the substrate. The enzyme activity was not affected by the exoglucanase inhibitor ␦-gluconolactone, confirming the peculiar glucanosyltransferase activity of our purified enzyme preparation.
Fig. 3. Electron micrographs of Candida albicans cells showing localisation of Bgl2p on (A) control cells and (B) micafungin-treated cells after the reaction with the specific polyclonal anti-Bgl2p antibody and revealed by goat anti-mouse–10 nm gold conjugates.
3.3. Localisation of Bgl2p
4. Discussion
Localisation of the Bgl2p in control and micafungin-treated cells was analysed on gold–antibody immunolabelled cells after glutaraldehyde fixation. This method of preparation avoids the use of osmium tetroxide and uranyl acetate and allows good cell preservation, although it reduces the visibility of the electron transparent layers of the cell wall. Immunolabelling was carried out using a polyclonal antibody against the Bgl2p purified from the fungal cell as primary antibody and a gold conjugate antibody as the secondary one. Observation of the immunolabelled sections of control cells revealed the presence of Bgl2p spread throughout all layers of the cell wall and partially inside the cytoplasm (Fig. 3A). Micafungintreated cells showed a remarkable increase in labelling, not only in the cell wall but also in the cytoplasmic compartment (Fig. 3B). 3.4. Cytosolic Bgl2p
The C. albicans cell wall is a complex system of polysaccharides and proteins functionally linked together to shape the cells and to maintain the cell wall architecture in an efficient metabolic structure. GAPs are proteins covalently linked to glucan that play an important role in cell wall metabolism and in particular in adaptive changes required by the presence of intracellular and/or environmental stressing agents [22,23]. Antimycotics constitute one of the most stressing factors and thus they exert a rather marked influence on the GAP pattern in the C. albicans cell wall. Bgl2p is one of the most abundant cell wall proteins [20]. It was characterised as an endo-1,3--glucanase and glucanosyltransferase that introduces -1,6-linkages in 1,3--glucan chains [12,20] and thus participates in cross-linking of the glucan component of the cell wall [4]. Deletion of BGL2 does not obviously affect cell morphology, whereas its overexpression is harmful for cell viability [20]. We have previously demonstrated that Bgl2p was induced by the
The increased labelling in the cytosol of the drug-treated cells indicated that micafungin elicited the expression of Bgl2p. Enzymatic activity assayed on the cytosolic preparation failed to reveal the presence of active enzyme. Cytosolic Bgl2p was partially purified and identified by Western blot analysis. Automated Edman degradation of the pertinent band gave the sequence MQIKFLTT, which matched the initial residues of the BGL2 gene product reported in the Swiss Prot databank. This indicates that the cytosolic Bgl2p isoform still contains the signal peptide and confirms
Fig. 4. DNA-deduced sequence of the first 40 amino acid residues of the BGL2 gene. Signal peptides (residues 1–18) are reported in bold. The (a) arrow encompasses the sequence obtained from the analysis of cytosolic Bgl2p, whilst the (b) arrow indicates the sequence from cell wall Bgl2p.
the presence of Bgl2p as an unprocessed form in the cytoplasm of treated cells (Fig. 4). The presence of Bgl2p in the immature form could explain the lack of enzymatic activity.
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presence of subinhibitory doses of both fluconazole and cilofungin. This indicates that, independently on the drugs utilised and their mechanism of antifungal action, an increase of Bgl2p expression is a common response of C. albicans [6,10]. In this study, we confirmed by 2D electrophoretic analyses the change of GAPs in C. albicans upon treatment with micafungin by comparing the 2D GAP maps obtained for treated and untreated organisms (Fig. 1). Although proteomic analysis of cell wall proteins has been reported [24–26], no previous 2D map was available for GAPs of C. albicans following treatment with an antifungal. When comparing the map of C. albicans treated with micafungin with the untreated control, a general decrease of major spots was observed with the exception of Bgl2p (Fig. 2A), demonstrating and confirming the role of this protein in the response to antimycotic drugs. A possible explanation of Bgl2p modulation may reside in cell wall compensatory mechanisms responsible for remodelling the cell wall. These mechanisms come into play to prevent cell lysis whenever its integrity is compromised by environmental stresses, by cell wall-perturbing compounds or in cell wall mutants [27–30]. Localisation of Bgl2p on the cell wall structure was analysed by immunogold experiments in control and micafungin-treated cells. The number of gold particles in the cell wall of the treated cells was higher than that in the control cells (Fig. 3). Furthermore, this noticeable increase of Bgl2p after micafungin treatment was not limited to the cell wall but also involved the cytoplasmic compartment, which appeared to be strongly labelled (Fig. 3B). It has been demonstrated that a defect in Bgl2p transport to the cell wall might be accompanied by the intracellular accumulation of this protein [31]. This does not appear to be the case in C. albicans treated with micafungin since the amount of Bgl2p on the cell wall is similar or even increased with respect to control cells and thus the increase in the cytosol may be reasonably explained by a more active translation of the BGL2 gene. Bgl2p in the cytoplasm is present in the unprocessed form as demonstrated by direct sequence analysis, in contrast to the sequence obtained by Edman degradation analysis of the purified cell wall isoform that lacks the first 18 residues with respect to the genomic-derived sequence (Fig. 4). These data confirm the presence of a signal peptide that is excised upon translocation of the protein from the cytoplasm to the cell wall and this is in agreement with the secretion pathway of a cell wall-localised protein. The structural difference in the two isoforms may explain the lack of glucanosyltransferase activity in the cytosolic compartment and thus assign the catalytic activity only to the mature form. Taking the data obtained from 2D maps and localisation by immunogold together, it is possible to conclude that after 24 h of sublethal micafungin treatment, a general decrease of GAPs occurs indicating the cytotoxic effect of the drug. However, an acute, likely compensatory, response of the organism takes place represented by a marked stimulation of Bgl2p expression. In this context, possible roles of Bgl2 enzyme in cell wall maintenance upon drug treatment could be assumed in terms of repair, cross-linking and incorporation of newly synthesised chains of -1,3 glucan into the previously existing cell wall structure. Although further studies are necessary to validate this hypothesis, it is of some interest that the phenotype arising after disruption of the BGL2 gene shows a decrease in the resistance of C. albicans to the antifungal nikkomycin Z [11]. Analysis of the fungal cell wall continues to be an active and exciting area of research. Changes in the biochemical and physical properties of the wall as a result of this remodelling have implications for interactions of fungi with their environments. In fungal pathogens the cell wall constitutes the interface with the host, and cell wall proteins of C. albicans are important targets for both humoral and cell-mediated immune responses. Antibodies against
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other fungal cell wall proteins and extracellular -glucosidases are largely used in the serodiagnosis of histoplasmosis [32]. Vaccines eliciting antibodies and/or cell-mediated immunity against Bgl2p [33], Camp65/SCw1 [34] and the adhesins Als1 and Als3 [35] have been protective in animal model of candidiasis. In this context, Bgl2p should also be considered as a potential vaccine candidate against candidiasis. Funding: No funding sources. Competing interests: None declared. Ethical approval: Not required. References [1] Shepherd MG. Cell envelope of Candida albicans. Crit Rev Microbiol 1987;15:7–25. [2] Fleet GH. Cell walls. In: Rose AH, Harrison JS, editors. The yeasts, vol. 4. London, UK: Academic Press; 1991. [3] Jeng HW, Holmes AR, Cannon D. Characterization of two Candida albicans surface mannoprotein adhesins that bind immobilized saliva components. Med Mycol 2005;43:209–17. [4] Chaffin WL, Lopez-Ribot JL, Casanova M, Gozalbo D, Martinez JP. Cell wall and secreted proteins of Candida albicans: identification, functions and expression. Microbiol Mol Biol Rev 1998;62:130–80. [5] Kapteyn JC, Van Den Ende H, Klis FM. The contribution of cell wall proteins to the organization of the yeast cell wall. Biochim Biophys Acta 1999;1426:373– 83. [6] Angiolella L, Micocci MM, D’Alessio S, Girolamo A, Maras B, Cassone A. Identification of major glucan-associated cell wall proteins of Candida albicans and their role in fluconazole resistance. Antimicrob Agents Chemother 2002;46:1688–94. [7] Nombela C, Molina M, Cenamor R, Sanchez M. Yeast beta-glucanases: a complex system of secreted enzymes. Microbiol Sci 1988;5:328–32. [8] De Nobel H, Kliss FM. Organization and construction of the yeast cell wall. In: Sturgeon R, editor. Advances in macromolecular carbohydrate research, vol. 2. Amsterdam, The Netherlands: Elsevier Science; 2003. [9] Stubbs HJ, Brusch DJ, Emerson JB, Sullivan PA. Hydrolase and transferase activities of the beta-1,3-exoglucanase of Candida albicans. Eur J Biochem 1999;263:889–95. [10] Angiolella L, Facchin M, Stringaro A, Maras B, Simonetti N, Cassone A. Identification of glucan-associated enolase as a main cell wall protein of Candida albicans and an indirect target of lipopeptide antimycotics. J Infect Dis 1996;173:684– 90. [11] Sarthy A, McGonial T, Coen M, Frost DJ, Meulbroek JA, Goldman RC. Phenotype in Candida albicans of a disruption of the BGL2 gene encoding a 1,3-betaglucosyltransferase. Microbiology 1997;143:367–76. [12] Hartland RP, Emerson GW, Sullivan PA. A secreted beta-glucan-branching enzyme from Candida albicans. Proc Biol Sci 1991;246:155–60. [13] Goldman RC, Sullivan PA, Zakula D, Capobianco JO. Kinetics of beta-1,3 glucan interaction at the donor and acceptor sites of the fungal glucosyltransferase encoded by the BGL2 gene. Eur J Biochem 1995;227:372–8. [14] Chandrasekar PH, Sobel JO. Micafungin: a new echinocandin. Clin Infect Dis 2006;42:1171–8. [15] Candiano G, Bruschi M, Musante L, Santucci GM, Ghiggheri B, Carnemolla P, et al. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteomic analysis. Electrophoresis 2002;25:1327–33. [16] Chizhov AO, Dell A, Morris HR, Reason AJ, Haslam RA, McDowell OS, et al. Structural analysis of laminarans by MALDI and FAB mass spectrometry. Carbohydr Res 1998;310:203–10. [17] Hartland RP, Fontaine T, Debeaupuis JP, Simenel C, Delepierre M, Latge JP. A novel beta-(1-3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J Biol Chem 1996;271:26843–9. [18] Tokuyasu KT. A technique for ultracryotomy of cell suspensions and tissues. J Cell Biol 1973;57:551–65. [19] Mouyna I, Hartland RP, Fontaine T, Diaquin M, Simenel C, Delepierre M, et al. A 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus is a homologue of the yeast Bgl2p. Microbiology 1998;144:3171– 80. [20] Mrsa V, Klebl F, Tanner W. Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase. J Bacteriol 1993;175:2102–6. [21] Cappellaro C, Mrsa V, Tanner W. New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating. J Bacteriol 1998;180:5030–7. [22] Kapteyn JC, Hoyer LL, Hecht JE, Muller WH, Andel A, Verkleij AJ, et al. The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol Microbiol 2000;35:601–11. [23] Aguilar-Uscanga B, Francois JM. A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Lett Appl Microbiol 2003;37:268–74. [24] De Groot WJ, de Boet AD, Cunningham J, Dekker HL, de Jong L, Hellingwerf KJ, et al. Proteomic analysis of Candida albicans cell walls reveals covalently
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