Isolation and Partial Characterization of Hsp90 from ... - CiteSeerX

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Annals of Clinical & Laboratory Science, vol. 33, no. 1, 2003

Isolation and Partial Characterization of Hsp90 from Candida albicans Edward T. Burt,1 Rhona Daly,2 Deana Hoganson,3 Yuri Tsirulnikov,4 Michael Essmann,5 and Bryan Larsen5 1 Department of Biochemistry, Des Moines University, Des Moines, Iowa 2 Undergraduate exchange scholar, University of Glasgow, Glasgow, Scotland 3 Department of Biology, Drake University, Des Moines, Iowa 4 Medical student, Des Moines University, Des Moines, Iowa 5 Office of University Research, Des Moines University, Des Moines, Iowa Abstract. Hsp90 is a stress-induced protein involved in many cellular processes including the regulation of signal transduction and steroid hormone response pathways in higher eukaryotic cells. Candida albicans hsp90 has a mass of 82 -Da and has previously been implicated as a virulence factor. A 47-kDa C-terminal fragment of Candida hsp90 is a target for an immune response to C. albicans infections. A C. albicans hsp90 specific polyclonal antibody was developed against a synthetic peptide containing a previously defined epitope of the 47-kDa fragment. This antibody was used to investigate the cellular localization and induction of hsp90 in the fungus. By means of cell surface protein extraction, hsp90 is shown to be localized on the cell surface as well as in the cytoplasm. On the cell surface, it appears only as an 82-kDa protein. In the cytoplasm, anti-hsp90 detected the 82-kDa protein as well as 72-kDa and 47-kDa bands on SDS-PAGE gels. The cytoplasmic protein bands were heat inducible and appeared to be estrogen induced as well, suggesting that C. albicans modulates hsp90 expression in response to environmental changes. Since the 82-kDa protein is also found on the surface of the cells, hsp90 may be directly involved in sensing environmental changes. It may also be important for recognition of its host or elements of the host immune system and antibody responses to the molecule and may therefore be useful for diagnostic or prognostic evaluation. (received 24 July 2002; accepted 19 September 2002) Keywords: Candida albicans, estrogen, heat-shock, hsp90, cell surface protein Introduction Candida albicans is an opportunistic fungal pathogen in humans that commonly causes irritation of the mucosal epithelia. In immunocompromised patients, however, C. albicans often causes serious systemic infections. The organism posseses multiple virulence factors, including the yeast/hyphal transition and secretion of proteases [1,2] that are involved in C. albicans transformation from a commensal member of the flora to a pathogenic microbe. Regulation of the expression of virulence Address correspondence to Bryan Larsen, Ph.D., Des Moines University-Osteopathic Medical Center, 3200 Grand Avenue, Des Moines, IA 50312, USA; tel 515 271 1559; fax 515 271 1644; e-mail [email protected].

factors and environmental influences on them is currently under investigation. Nutrient deprivation, pH [3], and steroid hormones [4] are all involved in regulation of the yeast to hyphal transition. Increased colony size [5] and gliotoxin production [6] are stimulated by estradiol and may contribute to increased Candida virulence. O’Connor and coworkers [7] demonstrated that estrogen enhances C. albicans survival under conditions of heat and oxidative stress. Analysis of cytoplasmic extracts showed induction of a number of C. albicans heatinducible proteins in the presence of estrogen and up-regulations of CDR1 (Candida drug resistance) and HSP90 mRNA were reported by our laboratory [8]. Recently, a genomic regulatory region that is

0091-7370/03/0100/0086; $2.00; © 2003 by the Association of Clinical Scientists, Inc.

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affected by estradiol was identified by deMicheli and coworkers [9] and appears responsible for induction of synthesis of CDR1and CDR2 gene products. C. albicans contains one essential hsp90 gene [10] that shares 84% homology with Saccharomyces cerevisiae hsp82. Although the physiological role of C. albicans hsp90 has yet to be elucidated, over expression of hsp90 in S. cerevisiae has been reported to increase the virulence of this organism for mice [11], adding support for its designation as a virulence factor. Antibody against a 47-kDa antigen was identified in patients who recovered from systemic candidiasis [12], and the eliciting antigen appears to be part of the C-terminal portion of C. albicans hsp90 [10,13]. Antibodies against the 47-kDa Cterminal portion of hsp90 reportedly cross-reacted with a 92-kDa heat-inducible protein, identified as the native hsp90 [14], while a clone of C. albicans hsp90 [10] and subsequent antibody studies [15,16] suggest that native Candida hsp90 actually has a mass of approximately 80-kDa. Immuno-electronmicroscopy using monospecific antibodies has localized the 47-kDa antigen to the cytoplasm and cell wall of cultured C. albicans [17]. These studies suggest that hsp90 localized to the cell wall was already cleaved, by a yet to be described cellular process producing the 47-kDa antigen. In this report, we use isolated Candida hsp90 and an anti-C. albicans hsp90 to determine the mass of native Candida hsp90, and to show that in addition to the cytoplasmic pool, native hsp90 is located in the cell wall of C. albicans. Materials and Methods Organisms. The organisms used for the following experiments included a clinical isolate of Candida albicans originally designated as strain GT 157 (now ATCC MYA-1023) [7] and the ATCC strain 10231. The clinical isolate was originally cultured on BiGGY agar (Nickerson’s medium, DIFCO Laboratories, Detroit, MI), producing brown colonies with typical microscopic morphology. The cultured yeast cells produced germ tubes when placed in human serum and incubated at 37°C, identifying them as C. albicans. All cultures were maintained at 4°C on Sabouraud’s dextrose agar (1%

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peptone, 2% glucose, 1.5% agar) and subcultured at 3-month intervals. Isolation of C. albicans hsp90. C. albicans hsp90 was purified using a modification of the methods described by Srivastava [18]. Yeast cells were grown in yeast peptone dextrose (YPD) medium to earlyor mid-log phase at 25°C. The cells were pelleted by centrifugation at 3000 x g for 5 min at room temperature. The cell pellet was resuspended in 1.4 ml of TE buffer (100 mM Tris, 100 mM EDTA, pH 8.0)/g wet cells and osmotically shocked by addition of deionized water to a final volume of 3.5 ml/g wet cells. Mercaptoethanol (17.5 µl/g wet cells) was added and the cells were incubated for 45 min at 30°C with gentle shaking. The cells were pelleted as previously described and washed with 4.0 ml/g wet cells of S buffer (1.0 M sorbitol, 10 mM PIPES, pH 6.5). The washed cells were resuspended in S buffer (4.0 ml/g wet cells) and zymolyase 20T (50 U/g wet cells) and incubated at 30°C for 1 hr with gentle shaking. Spheroplasts were collected by centrifugation for 5 min at 3000 x g and 4°C and washed twice with S buffer (2 ml/g wet cells). Spheroplasts were lysed by suspension in 5 ml of deionized water followed by addition 5 ml of lysis buffer (40 mM Tris, 2 mM EDTA, 100 mM NaCl, 1mM DTT, pH 7.4, 1 Mini-Complete Protease Inhibitor Tablet, Boehringer Mannheim Gmbh, Mannheim, Germany). Cell debris was removed by centrifugation (20,000 x g at 4°C) for 1 hr. The lysate was applied to a 15 ml DE-52 anionexchange column equilibrated with 20 mM Tris, 1 mM EDTA, 50 mM NaCl, pH7.4. Hsp90 was eluted with the same buffer by a linear gradient from 50 mM to 500 mM NaCl. Fractions containing hsp90 were identified by SDS-PAGE gel electrophoresis and dialyzed against 10 mM potassium phosphate pH 6.8. The dialyzed fractions were loaded on a 10 ml hydroxyapatite column equilibrated with the same buffer. Hsp90 was eluted by a linear gradient from 10 mM to 400 mM potassium phosphate pH 6.8. Fractions containing hsp90 were identified by SDS-PAGE gel electrophoresis. Electrophoresis and Western blotting. Proteins were separated on 7.5% discontinuous polyacrylamide

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gels as described by Laemmli [19,20]. Gels were stained with coomassie blue stain (Gelcode Blue, Pierce Chemical Co., Rockford, IL). For Western blots, the protein bands on SDS-Page gels were transferred to PVDF membranes for peptide sequencing or nitrocelluslose membranes for Western blotting as described by Towbin et al [21] and Burnette [22]. The hsp90 band was detected by incubating the membranes for 1 hr at room temperature with the anti-Candida hsp90 polyclonal antibody (described below), followed by a 1 hr incubation with horseradish peroxidase-conjugated secondary antibodies (Southern Biotechnology Assoc, Inc., Birmingham, AL). Antibody binding was detected with horseradish peroxidase chemiluminescent substrate (Supersignal West Dura Extended Duration Substrate, Pierce), using a Fluor-S MultiImager (BioRad Laboratories, Hercules, CA) and MultiAnalyst software (BioRad). N-terminal sequence analysis. Samples were separated by SDS-PAGE gel electrophoresis and blotted to PVDF membranes as described above. The membrane was stained with Ponceau S. The 82-kDa band was excised from the membrane and washed 6 times with de-ionized water. The Nterminal sequence was determined by sequential Edman degradation [23,24] using a 492 Procise Protein Sequencer/140C Analyzer (Applied Biosystems, Inc., Forest City, CA) Polyclonal antibody production. Two rabbits were injected with a synthetic peptide corresponding to the C-terminal 15 amino acids of Candida hsp90 [25] coupled to keyhole limpet hemocyanin (Zymed Labs, South San Francisco, CA). This peptide contained the previously described epitope STDEPAGESA found in the 47-kDa fragment of Candida hsp90 [14,26]. Dot blots with anti-Candida hsp90. Dot blots employed a modification of the method described by Burt et al [27] to measure estradiol induction of hsp90 in C. albicans. Cultures of C. albicans were grown to mid-log phase at 25°C under similar conditions as described by O’Connor and coworkers [7]. The cultures were treated with either 10-6 M

1,3,5[10]-estratriene-3,17β-diol (17-β-estradiol) (Sigma Chemical Co., St. Louis, Mo.), 10-9 M 17β-estradiol, methanol (volume equal to that used in estradiol cultures), or heat shock at 42°C for 30 min. The cells were pelleted by centrifugation at 3000 x g for 5 min at room temperature. The pellets were resuspended in 100 µl of breaking buffer (100 mM NaCl, 1 mM EDTA, 65 mM Tris pH 8.0) with 10 µl of protease inhibitor cocktail (Sigma). One hundred µl of acid washed, 425-600 mm glass beads were added, and the tubes were shaken in a reciprocating cell disrupter at 3,000 strokes/min for five 1-min cycles, with 1 min incubations on ice between each cycle. After disruption, the samples were centrifuged (12,000 x g) for 5 min and the supernatant was collected. The protein concentration of the supernatant was determined using the BCA protein assay (Pierce). Thirty µl of each supernatant was loaded onto nitrocellulose strips using a dot-blot apparatus (BioRad). The nitrocellulose was placed in blocking buffer (PBS-Tween, 5% dry milk) for 1 hr. The dot-blots were probed with the same antibodies used for Western blotting. Chemiluminescent HRP substrate (the same as used for Western blots) was used for detection of antibody complexes. Dot-blots were scanned with the Fluor-S MultiImager and densitometry was performed with the MultiAnalyst software. Cell surface protein extraction. Proteins were extracted from the cell wall using the method described by Kanbe et al [28]. Yeast cells were grown to mid-log phase at 25°C in yeast nitrogen base. The cells were washed with deionized water and 0.1 M EDTA, pH 7.5. The protein was extracted from the surface of the cells by suspending the cell in 0.3 M 2-mercaptoethanol, 0.1 M EDTA, pH 9.0, for 30 min with gentle agitation. Cells were removed by centrifugation and the supernatant either dialyzed against deionized water and lyophilized or concentrated and dialyzed in a single step using Centricon filters. Protein samples were subjected to western blotting as described above. Flow cytometry. A log-phase culture of C. albicans was divided into two treatment groups; one group

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Table 1. N-terminal sequence analysis of the isolated 82-kDa protein from C. albicans. C. albicans 82-kDa protein: C. albicans Hsp90 [10]:

ADAKVETHEFTAEIS MADAKVETHEFTAEIS

The N-terminal sequence of the isolated C. albicans 82-kDa protein was determined. The C. albicans 82-kDa protein was homologous to the predicted C. albicans Hsp90, minus the N-terminal methionine [10].

Fig. 1. Dot-blot analysis of hsp90 induction in C. albicans (strain ATTC 10231). Hsp90 was detected in cytoplasmic extracts by anti-hsp90 polyclonal antibody. The chemiluminescent signals were normalized to the weight (µg) of protein (A) spotted on the filter (B).

Fig. 2. Detection of hsp90 in extracts of C. albicans (strain GT-157) control cultures (lane 1), heat shock cultures (lane 2), treated with 10-6 M beta-estradiol (lane 3), and treated with 10-9 beta-estradiol (lane 4). Cytoplasmic extracts ( panel a) and cell surface extracts (panel b) on western blots probed with the anti-hsp90 polyclonal antibody.

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Fig. 3. Flow cytometry detection of cell surface hsp90. Control (top panel) and heat shock (bottom panel) treated cells stained with fluorescein labeled anti-Candida hsp90 antibody before (lighter curve) and after (heavier curve) stripping to extract cell surface proteins. The stripped cells show a decrease in fluorescence, indicating that hsp90 is extractable from the surface of both the control and the heat shock treated cells.

was used as the control and the second was heatshocked at 42°C for 30 min. Each treatment group was then divided in half. The first half was diluted 1:100 with YNB growth media and probed with 20 µl of FITC labeled antibody per ml of diluted cells. Prior to being diluted and probed with the FITC labeled antibody, the second half was stripped of cell surface proteins, using the method described above. Cell fluorescence (Fl1-H) was determined using a Becton Dickinson FACScan flow cytometer and plotted as mean channel fluorescence. Results Protein isolation and sequences. Approximately 0.3 mg of hsp90 was isolated from 3.5 g (wet weight) of

GT-157 cells. When the hsp90 isolates were run on SDS-PAGE gels, there were several unidentified protein bands and hsp90 fragments that consistently co-purified with the 82-kDa hsp90 band (data not shown). Since the separation methods used to isolate the fungal hsp90 may not have disrupted all protein complexes in the cell lysates, these proteins may be proteins that are normally complexed with hsp90 in the yeast cells. The N-terminal sequence of the 82-kDa protein was determined and compared to the SWISS-PROT database. The N-terminal 20 amino acids of the 82-kDa band were identical to the sequence predicted from the open reading frame of the previously described hsp90 cDNA [10] (Table 1). The only difference between the sequence of the isolated protein and the predicted sequences was the deletion of the N-terminal methionine residue predicted by the open reading frame (common in eukaryotic proteins). The predicted primary structure of C. albicans hsp90 does not contain any identifiable signal sequences or other sequences that predict proteolytic processing of the protein. Therefore, the N-terminal sequence supports the molecular weight predicted by the nucleotide sequence of the open reading frame and the identification of this protein with a mass of approximately 82-kDa as Candida hsp90 [15,16]. Estrogen induction of cytoplasmic hsp90. Cytoplasmic protein samples were prepared as described and directly blotted to nitrocellulose. Fig. 1 presents the results of a typical experiment, showing that C. albicans hsp90 is induced approximately 200% by heat shock and approximately 80% by 10 -9 M 17-β -estradiol. Densitometry (normalized to protein load) confirms the visually observed differences in the dot blot. Since differences seen in the dot-blots cannot be attributed to changes in expression of a specific protein, western blotting was used to confirm that the changes seen upon heat stress or estradiol treatment are attributable to changes in hsp90 expression (Fig. 2). The polyclonal antibody used for the dot-blots detected bands of 82-kDa, 76-kDa, and approximately 47-kDa in size on Western blots of all cytoplasmic extracts.

Heat shock protein-90 from Candida albicans

Surface expression of hsp90. Cell surface extracts prepared as described, were Western blotted against the anti-hsp90 polyclonal antibody. As seen in Fig. 2b, full-length (“native”) hsp90 was detected in the cell surface extracts. These results are consistent with the reports of hsp90 on the surface of various eukaryotic cells [28-30]. In order to verify that the hsp90 was truly located on the cell surface and not the result of cell lysis, cells were probed by flow cytometry with FITC-labeled polyclonal antibody (Fig. 3). Diminution of fluorescence following surface protein extraction indicates that the hsp90 detected on the western blots of cell surface extracts was not the result of cell lysis. Since the surface extraction method is known to remove proteins from the cell wall, it is concluded that hsp90 is part of the cell wall. Discussion Understanding the function of the 47-kDa fragment and the mechanism whereby it is formed may provide valuable insight into factors controlling C. albicans pathogenesis and may have diagnostic or immunoprophylactic implications. While the 47kDa fragment may be formed by multiple proteases, one candidate protease for the formation of the 47kDa fragment is ycaB [31], the C. albicans variant of the S. cerevisiae, cerevisin (protease B). In S. cerevisiae, this vacuolar protease is known to activate aminopeptidase I by cleavage of a Leu-Glu bond. The reported N-terminus of the 47-kDa fragment corresponds to Glu-313 of Candida hsp90 [10] which is preceded by Leu-312. The formation of the 47-kDa fragment would require the hydrolysis of the Leu-Glu linkage at position 312, which could be accomplished by ycaB. In addition, the cleavage of the Leu-Glu linkages at positions 59 and 78 by ycaB would also produce Candida hsp90 fragments with masses consistent with the fragments reported by Panaretou [15] and the immunoreactive 72-kDa peptide seen in our isolation procedure. It is therefore possible that in the vacuole of Candida, ycaB forms the 47-kDa antigen, which is then released during lytic events of the immune response. The report by Matthews et al [17] that the 47-kDa antigen could be detected in vesicles by

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immuno-electronmicroscopy further supports this hypothesis of the origin of the 47-kDa fragment. Finally, since protease B expression in S. cerevisiae is stimulated by glucose or nitrogen starvation, it can be considered a stress protein. As such, the activation of ycaB during candidal infection may be a stress response related to the estrogen and heat induction of Candida hsp90. Estrogen has been linked to the pathogenesis of C. albicans by the demonstration of estrogen stimulated Candida growth [5] and yeast to hyphal transition [4]. Hodgetts, et al [11] reported that over-expression hsp90 in S. cerevisiae increased the virulence of this fungus in mice and therefore hsp90 appears to be a virulence factor of C. albicans. The estrogen induction of cytoplasmic hsp90, as seen in Fig. 1, suggests that estrogen’s role in pathogenesis is at least partially linked to the cellular functions of Candida hsp90. By stimulating hsp90 production, estrogen may affect the virulence of C. albicans by direct or indirect regulation of hsp90’s putative role in cell cycle control [32-34] via interactions with signal transduction proteins. This may involve the role of hsp90 in the activation or stabilization of key signal transduction and cell cycle regulator proteins. Hsp90 may also be involved in the folding or conformational stability of other estrogen-induced proteins that appear to be necessary for the change in cellular function and pathogenic development [27]. While these putative functions of hsp90 appear to be wide ranging, both in the cellular systems affected and in the types of protein complexes formed, the fact that hsp90 lacks ligand specificity [35] supports the broad and multiple functions of the protein. Currently, a major function of surface hsp90 appears to be antigen presentation [35] to MHC Class I molecules. Related to this function, surface hsp90 may be among cell surface markers used by the immune system to identify cells as “self.” As might be expected with such a cell surface marker, antibodies against hsp90 are frequently associated with autoimmune diseases [34]. Therefore, the high degree of homology between Candida hsp90 and human hsp90 suggests that surface hsp90 may serve to camouflage Candida cells from the immune

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system, allowing the yeast to exist in the normal human flora and become pathogenic. Since patients that recover from systemic candidiasis have high antibody titers against the 47-kDa antigen [12], ie, the C-terminal portion of hsp90 [10], recovery from systemic candidiasis may involve ability of an individual’s immune system to detect the differences between human hsp90 and Candida hsp90. The 47-kDa antigen being the C-terminal portion of Candida hsp90 is consistent with the lower homology seen between the C-terminal portions of human and Candida hsp90 relative to the homology observed between the N-terminal portions of these proteins. Full-length hsp90 on the surface of C. albicans cells also implies the possibility that the immune response to fragments of hsp90 in recovering patients is actually an autoimmune type response to full-length hsp90 found on the surface the fungal cells. If this hypothesis is correct, it could indicate why development of an immune therapy has not been successful. In contrast to cytoplasmic extracts, cell surface extracts do not contain any of the hsp90 fragments seen in Fig. 2a, or described by Panaretou et al [15]. This suggests that the 47-kDa antigen found in the serum of candidiasis patients must result from either the lysis of Candida cells or the degradation of the hsp90 found on the cell surface. The results reported here support the recent suggestion that one or more cytoplasmic proteases cleaves fungal hsp90 to produce the 47-kDa fragment [15]. If recovery from systemic infection depends on an ability to mount and sustain an immune response to these fragments [36,12], an immune therapy directed toward these fragments could improve survival [37], while release of proteolytic fragments of hsp90 from disrupted cells could produce a toxic effect on the host.

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Acknowledgements and In Memoriam The Iowa Osteopathic Educational Foundation funded this work. Dr. Edward Burt (1961-2002) was working on this manuscript before his untimely death. This paper was finished in tribute to the memory of a scientist who loved his research, his students, and above all his family.

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