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Characterization of Candida albicans antigenic determinants by two-dimensional polyacrylamide gel electrophoresis and enhanced chemiluminescence Pedro Luis Barea a , Esther Calvo a , Joseè Antonio Rodriguez a , Aitor Rementeria b , Roberto Calcedo a , Mar|èa Jesuès Sevilla a , Joseè Pontoèn c , Fernando Luis Hernando a; * a
Departamento de Inmunolog|èa, Microbiolog|èa y Parasitolog|èa, Facultad de Ciencias, Universidad del Pais Vasco, Bilbao, Spain b Facultad de Farmacia, Universidad del Pais Vasco, Bilbao, Spain c Facultad de Medicina y Odontologia, Universidad del Pais Vasco, Bilbao, Spain Received 29 October 1998; received in revised form 10 December 1998; accepted 14 December 1998
Abstract The use of a two-dimensional polyacrylamide gel electrophoresis joined with Western blotting allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of Candida albicans blastoconidia. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and noninfected individuals showed that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of antibodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a different group of antigens. The technique used resolved the isoforms of several antigens including enolase. It is concluded that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen. z 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Candida albicans; Antigens ; 2D SDS-PAGE; Western blot; Chemiluminescence
1. Introduction Detection of Candida antigens in sera or the antibody response produced against those antigens have been extensively studied in the hope of identifying
* Corresponding author. Apartado 644, Universidad del Pa|ès Vasco, 48080 Bilbao, Vizcaya, Spain. Tel.: +34 (4) 601-2000, ext. 5407; Fax: +34 (4) 464-8500; E-mail:
[email protected]
serological markers of invasive candidiasis [1,2], a disease of increasing importance and di¤cult diagnosis. However, no single serological test developed to date is completely reliable. The antigenic composition of Candida albicans appears to be very complex and it includes proteins, polysaccharides and glycoproteins [3]. Identi¢cation of immunodominant antigens or those antigens expressed exclusively in a pathogenic situation may be of relevance for serodiagnosis of invasive candidiasis
0928-8244 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 9 2 8 - 8 2 4 4 ( 9 8 ) 0 0 1 5 4 - 0
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[4,5]. However, this identi¢cation is usually complicated by the complexity of the antigenic extracts used, since even using techniques, such SDS-polyacrylamide gel electrophoresis and Western blotting, the antigens detected may represent groups of antigens with di¡erent isoelectric points (pI) and the same molecular weight. The two-dimensional gel electrophoresis protein separation method provides a powerful technique for protein research. This technique developed by O'Farrell [6] was used by Manning and Mitchell [7] in an attempt to obtain information about the C. albicans yeast and germ tube speci¢c proteins. In that study, the assignment and matching of spots was di¤cult because of lack of speci¢c software. Up to date, few studies have been made with this technique in Candida. Brown and Cha¤n [8] observed ¢ve yeast speci¢c proteins and two proteins whose synthesis was reduced in the mycelial phase but found no mycelium speci¢c proteins. Burnie et al. [9] studied the antigenic di¡erences between yeast and mycelial extracts and found one yeast speci¢c mannoprotein, but no mycelial speci¢c antigens. Ahrens et al. [10] and Swoboda et al. [11] found some speci¢cally synthesized polypeptides when cells were shifted from 25 to 37³C and Finney [12] found one speci¢c hyphae and bud associated polypeptide in a [35 S]methionine-labeled extract. Recently, Niimi et al. [13] studied the initiation of morphogenesis in Candida by two-dimensional polyacrylamide gel electrophoresis (2D PAGE) showing that the changes were mainly produced by variations in pH or carbon source. In this study, we describe a procedure that combines the high resolution of 2D PAGE with linear pH gradient to separate Candida proteins and Western blotting coupled to enhanced chemiluminescence (ECL) to identify the antigens eliciting antibody responses during invasive candidiasis in humans and mice.
2. Materials and methods 2.1. Microorganism and culture conditions Candida albicans VW32 (kindly supplied by D. Poulain) was used in all the experiments. Cells
were grown at 24³C in Sabouraud dextrose agar during 24 h and then in Sabouraud dextrose broth at 24³C for 24 h in a rotary shaker (120 rpm). Harvested cells were washed twice and resuspended in 50 mM phosphate-bu¡ered saline (PBS). 2.2. Antigen extraction Suspensions of 108 cells ml31 in distilled water with 2% L-mercaptoethanol were sonicated in an ice bath at intervals of 20 s for 5 min at 60 W and centrifuged (20 min, 11 000Ug). The supernatant was mixed in a solution of 4 vols. of 10% (w/v) of trichloroacetic acid (TCA) in acetone with 0.07% (v/ v) L-mercaptoethanol and proteins were allowed to precipitate for 45 min at 320³C. After centrifugation at 11 000Ug for 20 min, the pellet was dissolved in 0.5 ml sample bu¡er (9 M urea, 2% L-mercaptoethanol, 2% Pharmalyte 3^10 and 2% NP40). Total protein was determined by the Bradford method [14]. 2.3. Sera Ten mice were injected intravenously with 1U105 blastoconidia. Blood was obtained after 30 days of infection. The infected mice were recovering from the infection since this sublethal dose of C. albicans causes a 80% of mortality at day 20. Blood from non-infected animals was used to obtain control antisera. Sera from patients with invasive infection by C. albicans and from patients without evidence of invasive candidiasis were also used. 2.4. Anti-enolase monoclonal antibody A monoclonal antibody against C. albicans enolase was used in order to identify the antigens on the protein pro¢le. Brie£y, a hybridoma cell line A2C7 (ATCC HB-8397) that secretes a mouse monoclonal antibody (IgG1 U) was suspended in an appropriate volume of Dulbecco's modi¢ed Eagle's medium with 10% fetal bovine serum (FBS) and thawed at 37³C in a water bath. The culture medium was changed 24 h after thawing to remove the protective freezing additive and monoclonal antibodies were produced after clonal selection of cells incubated in RPMI 1640 medium with 10% FBS for 7 days at 37³C in CO2 atmosphere.
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2.5. IEF and 2D SDS-PAGE Isoelectric focusing (IEF) was carried out in immobilized linear pH 3^10 gradient (IPG gels, Pharmacia) by a modi¢cation of the method described by Goërg et al. [15]. Gels were loaded with 600 Wg of protein in sample bu¡er containing 2% Pharmalyte pH 3^10 were loaded. A 500-V tension was applied for 2 h and then increased to 3500 V up to obtain 55 kV h31 at 20³C. The IPG strips were equilibrated in 10 ml of 0.05 M Tris-HCl bu¡er, pH 6.8, containing 6 M urea, 1% (w/v) SDS, 30% (w/v) glycerol and 2% L-mercaptoethanol. After 15 min, the procedure was repeated with another 10 ml equilibration bu¡er containing 240 mM iodoacetamide and 0.5% (w/v) Bromophenol blue solution. For the second dimension, IPG gels were transferred to an 8^18% gradient polyacrylamide gel (Excel-gel, Pharmacia) and performed at 20 mA, 600 V and 10 W at 15³C. 2.6. Western blotting The proteins separated in the 2D PAGE gels were electrotransferred to an Immobilon P membrane (Millipore) using a semidry transfer system with 400 mA for 2 h. The membrane was treated with blocking solution containing 1% non-fat skim milk powder in PBS-T (0.5% (w/v) Tween 20 in 50 mM PBS). After three washing steps in PBS the blots were incubated for 1 h at 37³C with the antisera (mice antisera diluted 1/2000 in PBS, antisera from patients without invasive candidiasis diluted 1/2500 and antisera from patients with invasive candidiasis diluted 1/5000) and washed three times. After washing, the blots were incubated with goat anti-mouse polyvalent Ig-HRP and anti-human IgG-HRP (Sigma) respectively, diluted 1/2500. Blots incubated with monoclonal antibody A2C7 were incubated with a goat anti-mouse IgG-HRP (Sigma) diluted 1/2500 as secondary antibody. Antigen^antibody reactions were observed after detection of peroxidase activity by chemiluminescence (ECL, Amersham) according to the manufacturer's instructions. Finally, the blot was wrapped in plastic and exposed to X-ray ¢lm (Curix RP-2, Kodak). X-ray ¢lms and silver stained gels were analyzed with the BioImage 50S system and the 2D Protein Analyzer software (Millipore) in a Sparc 4 station
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(SUN Mycrosystems). Spot intensity was measured as an integrated intensity (II) representing the spot optical density minus background optical density and integrated with the spot area, resolving the possible problem due to overexposure of the blots with the luminescence reactives. Each gel was reproduced at least ¢ve times and di¡erences in individual quanti¢cation of proteins were lower than 10%.
3. Results About 600 spots were discriminated by 2D PAGE in blastoconidia extracts of C. albicans. Prominent proteins were resolved as families showing the same molecular weight (MW), but di¡erent pI (Fig. 1). Characteristic regions and spots marked as P followed by the approximate MW number (exact MW numbers are noted in Table 1) were shown in 20^30, 37^40 and 45^50 kDa ranges. Usually, the regions contain more than 30 peptides on each with pI between 4.5 and 7. In the 80-kDa range, there were 10 peptides with a pI ranging from 5.5 to 6.5. IPG gels with pH linear gradients and SDS-PAGE allowed a very good reproducibility and only minor changes in the spot position. The antigenic reactivity of the extract was studied by Western blotting with a panel of sera from mice and humans with or without Candida infections. Variations in the antigen^antibody reactivity were found in some protein families and were measured as variations in the II of each spot. We ¢rst analyzed the reactivity of the antibodies present in sera from non-infected and C. albicansinfected mice with antigens present in the blastoconidia extract. Fifty-two spots were revealed by the antibodies present in sera from non-infected mice (Fig. 2a) and 34 spots were shown by the antibodies present in sera from infected mice (Fig. 2b). When the reactivity of the antibodies from both antisera was compared, twenty-four spots were speci¢cally stained by antibodies present in the sera from noninfected mice. Only the ten most signi¢cant spots are referenced in Table 1, showing antigens P26 and P18 (with 26 and 18 kDa and pI 6.7 and 6.2) the highest reactivity among them. Six spots with a low II were speci¢cally recognized by antibodies present in the sera from infected mice. The three of them showing
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Fig. 1. Silver-stained two-dimensional SDS-PAGE separation of a Candida albicans protein extract. Spots are referenced as P followed by the MW number.
a signi¢cant intensity were marked in Fig. 2b (arrowhead) and are listed in Table 1. Twenty-eight spots were revealed by antibodies present in both sera. However, di¡erences in II between the spots stained by antibodies present in both sera were observed (Table 1). Thus, some spots showed more reactivity with antibodies present in the sera from infected mice than with antibodies from sera from non-infected mice, while in other spots, the opposite was observed (Table 1). The antigens showing the highest increase in reactivity with antibodies in sera from infected mice had a MW of 36.2 (P37-3), 48 (P48) and 80 kDa (P80-2) and pI 6.7, 6.0 and 6.1, respectively. The highest decrease in II among sera from non-infected mice was observed in the region of 56 and 65 kDa and pI 5.7 (Table 1).
The use of 2D SDS-PAGE and Western blotting allowed us to detect antibodies to isoforms of the same antigen. This was the case of antigens of 80, 56, 48 and 37 kDa which showed di¡erent isoforms with small variations in their pI (Table 1). The variations in reactivity of the 80 kDa antigen seemed to be related to the pI of the antigens, since the highest reactivity was observed in the isoforms with acidic pI, while those with basic pI lost their reactivity with antibodies in sera from infected mice (Table 1). Similar changes were observed on the P37-3 and P48 peptide groups where intensity of some spots increased three times after infection The same type of study was performed with sera from individuals without candidiasis and from patients with invasive candidiasis. Immunoblots per-
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with a very low intensity were speci¢cally stained by antibodies present in sera from non-infected individuals. Twelve spots were revealed only by the antibodies present in sera from patients with invasive candidiasis (Fig. 3a,b). Four antigens reacted with antibodies present in sera from non-infected individuals or from patients with invasive candidiasis. As with the mice sera, di¡erences in the reactivity of the antibodies present in both sera with these antigens were also observed. An antigen of PM48 kDa and a pI of 6.2 showed an increase in the II when its reactivity with the antibodies present in sera from noninfected individuals was compared to that shown by the antibodies present in sera from patients with in-
formed with the same dilution of the serum (1/5000) showed the greatest di¡erences because a very low level of reactivity was detected in sera from healthy persons. To increase the reactivity, these sera were only diluted 1/2500. However, the sera from patients with invasive candidiasis were diluted 1/5000 due to their high reactivity. Twelve spots were stained by the antibodies present in sera from individuals without candidiasis (Fig. 3a) and 16 spots were revealed by the antibodies present in sera from patients with invasive candidiasis (Fig. 3b). Among the latter, the most reactive antigens showed MW of 37, 48, 50, and 65 kDa and pI between 5.6 and 6.2 (Table 2). After comparing the reactivity of both sera, 8 spots
Table 1 Signi¢cative spots and integrated intensity (II) found in 2D ECL Western blotting using sera from mice infected with C. albicans (Infection) and normal controls (Control) Spot
Speci¢city
ControlCInfection
II Control
II Infection
MW
pI
P80-1 P80-1 P80-1 P50-1 P50-2 P37 P26 P23 P18 P35 P56 P56 P65 P65 P65 P56 P56 P56 P56
Control Control Control Control Control Control Control Control Control Control
Lost Lost Lost Lost Lost Lost Lost Lost Lost Lost Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease
192 482 256 921 424 480 6224 394 1887 642 90 567 441 294 121 147 307 929 785
36 139 314 106 115 74 166 64 97
80 80.3 80.6 49.6 48.8 37.8 26.5 23 18 35 56 55 62.8 65.2 66.5 56.5 56.1 57 56.3
6.46 6.5 6.6 6.2 6.8 6.8 6.6 6.2 6.2 6.2 5.1 4.9 5.7 5.6 5.5 5.5 5.6 5.7 5.8
P55 P37-2 P21 P80-2 P80-2 P80-2 P80-2 P48 P48 P48 P37-3 P37-3 P37-3
Infection Infection Infection
784 726 176 87 84 299 396 652 1087 316
125 182 274 1142 1539 283 156 144 543 1164 760 1533 1212
55 38.5 21 80 79.4 82.2 80 48 47.5 49.9 36 35.5 36.2
5.9 6.9 6.7 6.0 6.1 6.0 5.8 6.1 6 5.9 6.5 6.5 6.7
Expression Expression Expression Increase Increase Increase Increase Increase Increase Increase Increase Increase Increase
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Fig. 2. Two-dimensional Western blot of protein extract against a non-infected mouse serum (a) and a infected mouse serum (b) revealed by enhanced chemiluminescence. Arrowheads represent speci¢c spots and arrows represent spots whose integrated intensity decreased (a) or increased (b) after transition to infection.
vasive candidiasis. The more relevant antigens were referenced in Table 2. Similar results were found with sera from di¡erent patients. A strong reaction in the acidic high MW region was found speci¢cally in sera from patients with invasive candidiasis, while no reaction was observed when sera from non-infected individuals were used, even when these sera were concentrated twice (Fig. 3a,b). This region showed a polydispersed reaction ranging between 60 and 150 kDa and within a 4^5 pI range. When the reactivity of the sera from the mice and humans infected with C. albicans studied was compared, di¡erences in the antigenic reactivity were observed. However, the presence of antibodies against an immunodominant antigen of 48 kDa was detected in the two models of candidiasis studied. To verify if that antigen was enolase, the reactivity of a mono-
clonal antibody against C. albicans enolase was studied by Western blotting against the blastoconidia extract. The monoclonal antibody reacted with ¢ve peptides in the 48-kDa region with a pI ranging between 5.9 and 6.4 (Fig. 4). A low reactivity was also observed with peptides of 30^40 kDa and pI between 5.2 and 6.5. The molecular weight of the peptides was coincident with that referenced by manufacturer and by Walsh et al. [2] for the C. albicans enolase.
4. Discussion The antigenic composition of C. albicans is very complex and therefore, the analysis of antigenic extracts from this fungus will be bene¢ted by the use of highly resolutive techniques, such as 2D SDS-PAGE. In our study, this technique resolved about 600 pro-
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Fig. 2 (continued).
teins in the extract with pI ranging from 5.0 to 7.0, which are in agreement with the pI described by Manning and Mitchell [7] for cytoplasmic proteins of C. albicans or those described by Niimi et al. [13] for C. albicans soluble proteins. The use of 2D SDS-PAGE in combination with Western blotting and ECL allowed us to investigate the reactivities of antibodies present in sera from mice and humans to antigens of C. albicans blastoconidia improving the autoradiography resolution [16] and with reproducible results by using immobilized linear pH gradient in the ¢rst dimension [15]. The analysis of the antibody response in the two models studied and the comparison between the antibody response in infected and non-infected individuals has shown that the infection by C. albicans produces changes in the antibody response which may be of relevance in the serodiagnosis of invasive candidiasis. These changes include the induction of anti-
bodies against new antigens, the disappearance of antibodies against a group of antigens and variations in the reactivity of antibodies directed to a di¡erent group of antigens. From a diagnostic point of view, the most relevant antibodies to be detected in a patient with invasive candidiasis would be those absent in sera from noninfected individuals or those whose reactivity increases in sera from patients with invasive candidiasis. In this study, these potentially useful antibodies reacted with antigens of 80, 65, 48, 37 and 45 kDa. With the exception of an antigen of 48 kDa and a pI of 6.2, which showed a low reactivity with antibodies present in sera from uninfected individuals, but increased its reactivity with antibodies present in sera from patients with candidiasis, the rest of them were speci¢cally recognized by antibodies present in sera from patients with invasive candidiasis. Interestingly, some of these antigens were also considered to be of
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Fig. 3. Two-dimensional Western blot of protein extract against a control human serum diluted 1/2500 (a) and a serum from a patient with invasive candidiasis diluted 1/5000 (b) revealed by enhanced chemiluminescence. Arrowheads and arrows represent spots as above.
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potential diagnostic relevance when the antibody response in sera from infected mice was analyzed. In fact, antigens of 80, 48 and 37 kDa showed an increase in reactivity with antibodies present in sera from infected mice. The fact that the same antigens are detected by antibodies from the two models of infection studied seems to reinforce its diagnostic utility. Antibodies to antigens of MW similar to those described in our study have been detected in other studies. Di¡erent groups have identi¢ed immunodominant cytoplasmic antigens of 45^54 kDa [17^ 21] or related to important receptors as laminin and ¢brinogen [22] whose MW (37 and 58 kDa) are in the region of our system characterized by a high number of reactive peptides. Among these antigens, two of them have been extensively characterized. Matthews et al. ([23^25]) identi¢ed an antigen of 47 kDa which shows homology to hsp90 of Saccharomyces cerevisiae and which has shown diagnostic and therapeutic potential in systemic candidiasis. Strockbine et al. [20,26], Sundstrom and Aliaga [27] and Brett Mason et al. [28] have identi¢ed and cloned an immunodominant antigen of 48 kDa which elicits a strong antibody response in patients with invasive candidiasis. The polydispersed acidic high molecular weight region seems to show a diagnostic value due to the di¡erential response found even when the patient
sera was diluted twice. Similar results were obtained with di¡erent sera and a study to assess its usefulness in the serodiagnosis of invasive candidiasis is in progress. By employing monoclonal antibodies and extensive solubilization methods, this region has been shown to contain mannoprotein material [29,30], but the diagnostic value was not clear in one dimensional Western blot [18]. We could con¢rm by 2D that this region reacted with a ConA^HRP conjugate showing the mannoprotein nature of these antigens (data not shown). Identi¢cation of a protein in a complex antigenic extract on the sole basis of its MW is di¤cult and sometimes the same antigen may have small di¡erences in the MW in di¡erent studies. The di¡erences observed in the MW may be related to di¡erences in the calculation of the MW or to the presence of several antigens with the same MW. As an aid in the identi¢cation of an immunodominant antigen of C. albicans, we used a monoclonal antibody against C. albicans enolase. The reactivity of this antibody allowed us the identi¢cation of di¡erent isoforms of the antigen which showed di¡erences in reactivity with antibodies in sera from patients and mice with candidiasis. This phenomenon was also observed in antigens of 80 and 37 kDa which showed di¡erent isoforms with the same MW, variations in their pI and di¡erences
Table 2 Signi¢cative spots and integrated intensity (II) found in 2D ECL Western blotting using sera from patients with invasive candidiasis (Infection) and individuals without Candida infection (Control) Spot
Speci¢city
ControlCInfection
II Control
P60H P60H P60H P60H
Control Control Control Control
Lost Lost Lost Lost
341.6 244.2 416.3 401.1
P80-1 P65 P50 P48-1 P37-4 P37-5 P48-2
Infection Infection Infection Infection Infection Infection
Expression Expression Expression Expression Expression Expression Increase
388
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II Infection
46.5 238.5 334.5 273 366 156 633
MW
pI
60.6 60.6 60.3 60.3
5.5 5.4 5.6 5.7
80.3 65.6 50 46 37.5 37.6 48
6.6 5.6 5.6 6.6 5.6 6.1 6.2
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Fig. 4. Two-dimensional Western blot of Candida albicans anti-enolase monoclonal antibody revealed by enhanced chemiluminescence.
in their reactivity with antibodies. Interestingly, the variations in antibody reactivity presented by the isoforms of an antigen of 80 kDa seem to be related with the pI of the antigens, the highest reactivity showed by the isoforms of the antigen with acidic pI, while the isoforms with basic pI lost their reactivity with antibodies present in sera from infected mice. These di¡erences in reactivity showed by the isoforms are likely to be masked in 1D gels. Similar events were found by Woëhl et al. [16], with the appearance of more acidic Hyp2p isoforms as a result of HYP2 overexpression in a S. cerevisiae strain or in the change in antigenicity accompanying the transition from white to opaque in C. albicans [31]. As well, Norbeck and Blomberg [32], by using 2D with non-linear IPG in the ¢rst dimension, found, on some previously identi¢ed spots, quantitative variations of protein expression related to metabolic
processes. These changes could be responsible for the variations in the immune response against C. albicans described in this work. In conclusion, the use of 2D SDS-PAGE and Western blotting revealed by ECL allowed us to detect antibodies to antigens of C. albicans showing small di¡erences in MW and to isoforms of the same antigen. The results presented in this study show that the antibody response in humans and mice with candidiasis is not homogeneously directed to all the isoforms of an antigen.
Acknowledgments This work was ¢nanced by grants UPV 093.310EA042/94 and UPV 093.327-EB131/96 from the Universidad del Pa|ès Vasco and by grant EX97/4 from
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the Departamento de Educacioèn, Universidades e Investigacioèn del Gobierno Vasco. References
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