1 Department of Pathology, University of Virginia Health System, PO Box 800904, Charlottesville, VA 22908-0904, USA
2 Department of Microbiology, University of Virginia Health System, PO Box 800904, Charlottesville, VA 22908-0904, USA
Correspondence
Kevin C. Hazen
khazen{at}virginia.edu
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ABSTRACT |
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INTRODUCTION |
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The wall of the commensal yeast Candida albicans is composed of a tight matrix of carbohydrate and protein (Chauhan et al., 2002; Kapteyn et al., 2000
; Klis et al., 2001
). Carbohydrate classes in the cell wall can be broadly separated as glucans, mannans and chitin, which are composed of high-molecular-mass polymers of glucose, mannose and N-acetylglucosamine, respectively. Proteins within the cell wall matrix are typically highly glycosylated mannoproteins, which are cross-linked to other carbohydrate moieties within the wall (Klis et al., 2002
). Biosynthesis of wall components occurs by the addition and maturation of core carbohydrate residues to glycoproteins during transit through the secretory pathway, and by de novo synthesis of glycan polymers within the wall matrix.
Cell-surface hydrophobicity (CSH) has been implicated as a virulence contributor for C. albicans (Hazen & Hazen, 1989; Hazen, 1989
). Cells in candidal lesions are a mixture of yeast and hyphae, and are highly hydrophobic. Additionally, hydrophobic cells attach to cultured endothelial and epithelial cells more readily in static and flow adhesion assays, and are more resistant to killing by macrophages than hydrophilic cells. The hydrophobic state of the cell wall is conveniently regulated in the laboratory by growth temperature and analysis of cell wall extracts from hydrophobic cells has resulted in the identification of a number of protein candidates with hydrophobic character. Further analysis has indicated that hydrophobic and hydrophilic cells are biochemically similar, but the cell walls are ultrastructurally unique (Hazen & Hazen, 1993
).
The first gene product to be cloned that affected the hydrophobic status of the cell was recognized on the basis of reactivity with the 6C5-H4CA (6C5) mAb (Glee et al., 1995, 1996
). The antibody was raised against hydrophobic cell wall extracts and recognizes a single 38 kDa protein which we have referred to as Csh1p (Singleton et al., 2001
). Csh1p (C. albicans unannotated genome build 19, ORF 10206; http://www-sequence.stanford.edu/group/candida; this gene product is identical in sequence to the IFD4 predicted gene product at http://genolist.pasteur.fr/CandidaDB) is one of a family of closely related proteins in C. albicans of unknown function, which are highly homologous to a family of Saccharomyces cerevisiae aryl-alcohol dehydrogenases. Ablation of the family of seven gene products in S. cerevisiae had no discernible phenotype (Delneri et al., 1999
), although knockout of the single gene in C. albicans resulted in a statistically significant reduction in CSH and a concurrent reduction in adhesion in a static assay (Singleton et al., 2001
). Comparison of the csh1 knockout derivative with a wild-type strain has indicated that the knockout is still virulent, but shows a lag in the time-course of disease progression in a mouse infection model (unpublished data).
Inspection of the Csh1p sequence suggested that the protein should demonstrate cytoplasmic localization as opposed to being in the cell wall, as no N-terminal signal sequence directing targeting to the secretory pathway is present. Accordingly total cell extracts released significantly more antigen than limited glucanase digestion of the cell wall (Singleton et al., 2001). However, reproducible amounts of Csh1p are released under conditions maintaining cell integrity (Glee et al., 1996
; Masuoka et al., 1999
; Singleton et al., 2001
) and the mAb is effective in blocking fungal adhesion in static and flow adhesion assays (Glee et al., 2001
), suggesting that the protein is present to some degree in the cell wall. Supporting the cell wall localization of Csh1p, a number of presumptively cytoplasmic proteins have been described in the C. albicans literature to be present within the cell wall and playing a role in Candida pathogenesis (for review, see Chaffin et al., 1998
). The presence of these proteins within the cell wall has been explained as the result of pre-existing or possibly novel transport mechanisms to the cell surface, or by heterologous cell lysis and reassociation of cytoplasmic proteins with neighbouring cells. The latter explanation does not adequately account for the amount of protein recoverable from intact cells.
To address the intriguing presence of Csh1p within the cell wall, we have characterized the nature of attachment of Csh1p to the wall and have labelled surface-accessible protein specifically. Several techniques to categorize peripherally and covalently attached cell wall materials were used to release Csh1p relative to the total cellular pool. A membrane-impermeable surface-labelling reagent was also used to tag surface-accessible Csh1p to allow the comparison of labelled protein with total Csh1p. The results show that the presence of Csh1p on the cell wall is correlated with the level of expression of the protein.
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METHODS |
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Protein extraction.
Proteins from yeast were extracted with a variety of methods to isolate total proteins and cell wall proteins preferentially. For total cell lysates, cell pellets from 5 ml cultures were suspended in a 1·5 ml microfuge tube in 500 µl ddH2O plus a protease inhibitor cocktail of 1 mM AEBSF (Roche), 1 µM pepstatin A (Roche) and 1 mM EDTA (Baker). A volume of glass beads equal in size to the cell pellet was added and the suspension was vortexed 4 °C for 5 minutes. After vortexing, 250 µl concentrated reducing Laemmli SDS-PAGE sample buffer was added and the sample was boiled for 3 min, followed by centrifugation at 10 000 g for 3 min to pellet insoluble material (Singleton et al., 2001).
For preferential extraction of cell wall materials, two methods were used. In the first method, cell pellets from 5 ml cultures (approx. 5x108 cells) were suspended in a 1·5 ml microfuge tube in 500 µl 0·1 M Tris/HCl, pH 7·6/0·1 M EDTA/0·9 M sorbitol plus AEBSF (Roche) and pepstatin A (Roche) as above. The spheroplasting enzyme Zymolyase 100T (ICN) was added as a 50 µl aliquot of a 0·3 mg ml-1 stock (30 µg ml-1 final concn) and a 50 µl aliquot of diluted 2-mercaptoethanol (30 mM final concn). Cells were treated for 60 min at 37 °C with gentle shaking (modified from Glee et al., 1995). Cells were gently pelleted from the extract by centrifugation at 1000 g for 5 min and the supernatant was boiled with reducing sample buffer as above. In the second method, cell pellets were suspended in 0·5 ml 0·1 M Tris/HCl, pH 9·4, plus the protease inhibitor cocktail, and 1 M DTT was added to a final concentration of 10 mM. Cells were incubated for 15 min at 37 °C with gentle shaking, then pelleted as above (Cleves et al., 1996
). Supernatants were again boiled with reducing sample buffer.
Cell surface labelling.
Surface proteins of yeast were biotinylated with sulfo-LC-NHS-biotin (Pierce, after Masuoka et al., 2002) and isolated by precipitation with immobilized avidin (Pierce). Stationary-phase cultures of LGH1095 were grown as above in YNB2G, pelleted and washed with water followed by 100 mM sodium phosphate buffer. After counting, 5x108 cells were suspended in 2·5 ml cold NaPO4, pH 8, buffer and 250 µl sulfo-LC-NHS-biotin (10 mg ml-1 stock in ddH2O) was added. Biotinylation was carried out for 1 h, then the cells were washed sequentially four times with ice-cold 50 mM NaPO4, pH 6, then once with 10 mM NaPO4, pH 7·4. After the final wash, cells were suspended in 3 ml of the pH 7·4 buffer and split into three tubes for protein extractions as above, with the exception that sample buffer was not added to the samples when finished. Following extraction, supernatants were brought up to 1 ml final volume by the addition of 100 µl 10x Tris-buffered saline (TBS, pH 7·4), 100 µl of a 50 % slurry of TBS-washed streptavidin-agarose and ddH2O to volume. Biotinylated proteins were allowed to bind to the matrix while rotating at room temperature. Beads were pelleted at 1000 g for 30 s and washed four times with TBS. Washed beads were suspended in 100 µl reducing sample buffer and boiled prior to electrophoresis and blotting.
Two-dimensional electrophoretic separation.
Samples were prepared from a csh1/csh1 derivative of C. albicans strain CAI4 and an isogenic CSH1 reintegrant by glass bead breakage as described above. Protein concentration was determined by a modified BCA assay (Pierce) of supernatants from broken cells and 100 µg protein was precipitated using a universal protein precipitation reagent (UPPA; Genotech). Precipitated protein was dissolved in equilibration buffer [8 M urea, 2 % CHAPS, 0·5 % pH 310 IPG buffer (Pharmacia), 0·002 % bromophenol blue, 0·28 % DTT] for application to 18 cm pH 310 immobilized gradient strips (Pharmacia) and separated to isoelectric equilibrium using a four-step voltage protocol. For this procedure, strips were rehydrated under load with the sample for 12 h at 30 V, then the sample was electrophoresed for 500 V h (500 V maximum), 1000 V h (1000 V max.), 32 000 V h (8000 V max.) for a total run time of 18 h, and 33 862 V h. Following the run, strips were equilibrated in 50 mM Tris/HCl, pH 8·8/6 M urea/30 % glycerol/2 % SDS/0·002 % bromophenol blue prior to application onto 12 % denaturing PAGE slab gels for electrophoretic separation by mass. Gels were transferred to nitrocellulose for Western blot analysis with mAbs 6C5-H4CA or YOL1/34 (Accurate Chemical). Blots were blocked in 4 % non-fat dry milk in phosphate-buffered saline plus 0·1 % Tween-20 (PBS-Tween), incubated in affinity-purified primary antibody solution overnight at 4 °C and an HRP-conjugated isotype appropriate secondary antibody for 1 h at room temperature. Blots were developed using HRP-dependent chemiluminescence and visualized by exposure to film.
RNA isolation.
Total RNA was prepared from cells grown to stationary phase as above in 10 ml YNB2G culture using a method from Ausubel et al. (1999). Cells were collected by centrifugation at 2000 g for 5 min and suspended in 400 µl AE buffer (50 mM NaOAc, pH 5·3, 10 mM EDTA). Cells were broken by vortexing in 40 µl 10 % SDS and 500 µl phenol (pre-equilibrated with AE buffer). The mixture was incubated at 65 °C for 10 min, vortexed and placed on dry ice for 4 h. Phases were separated by centrifugation at 10 000 g for 10 min and the aqueous phase was extracted with an equal volume of chloroform/isoamyl alcohol (24 : 1). RNA was precipitated overnight at -20 °C from the aqueous phase by the addition of 0·1 vols 3 M NaOAc and 2·5 vols 100 % EtOH. RNA was pelleted by centrifugation at 10 000 g for 15 min at 4 °C, rinsed with cold 70 % EtOH and dissolved in 10 mM Tris/HCl, pH 8, 1 mM EDTA. RNA was quantified by spectroscopy and 10 µg total RNA from each sample was separated by denaturing formaldehyde-agarose electrophoresis (Ausubel et al., 1999
), followed by transfer to charged nylon membranes for Northern blotting.
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RESULTS |
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DISCUSSION |
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Expression of Csh1p is significantly higher in early-stationary-phase cultures that have been grown at 37 °C as opposed to 23 °C, and the surface detection of the protein is strongly correlated with high levels of intracellular Csh1p expression. Cells grown at 23 °C do have readily detectable levels of the protein, however, so the lack of detection of Csh1p on the surface of these cells may therefore be due to the limits of detection of the protein using the antibody. The steady-state RNA levels of the CSH1 gene inversely reflect that of the protein levels in early-stationary-phase cultures. This discrepancy may be the result of differences in relative population maturity between cultures grown at different temperatures, as cultures grown at 37 °C enter stationary phase somewhat more rapidly (approx. 2 h earlier) than those grown at 23 °C. Transcription levels and steady-state RNA levels would then be lower in the 37 °C cultures than in the 23 °C cultures. This is supported by the observation that mid-exponential-phase cultures grown at 37 °C show higher levels of CSH1 RNA than 23 °C cultures. For the purposes of examining protein expression in the context of CSH, these are the culture conditions generating populations with virtually 100 % hydrophobic or hydrophilic cells. C. albicans is unique among common pathogenic yeasts in that hydrophobicity can be significantly varied in standard in vitro culture conditions. However, hydrophilic cell populations rapidly develop a hydrophobic phenotype upon reinoculation into fresh medium at low cell number (Hazen & Hazen, 1988), even at 37 °C. Expression levels of Csh1p in early-stationary-phase hydrophilic cells and in reinoculated cells is comparable both in total cell lysates and in surface-accessible pools. This result suggests that hydrophilic cells in early stationary phase might be in a state that allows the population to have a pre-existing pool of proteins that directly affect hydrophobicity readily available for surface presentation upon a change in the environment and a concurrent change in the growth rate of the organism.
A number of proteins have been described as cell wall components in C. albicans that lack a defined N-terminal signal sequence for insertion into the secretory pathway. Typically these proteins were reported to be potent elicitors of the host immune response (Ponton et al., 2001) and cell wall localization was later demonstrated by microscopy or other methods. These antigens are frequently glycolytic enzymes and include phosphoglycerate kinase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and enolase, all of which have been shown by biochemical methods to be major expressed proteins secreted by regenerating spheroplasts. A number of heat-shock proteins and chaperonins have also been postulated to be significant cell wall components (Pitarch et al., 1999
, 2002
). No enzymic function in the cell wall has been described for these antigens and presumably any other function of them in the wall is fortuitous and unrelated to the well described biochemical function. A report describing the cross-reactivity of the C. albicans alcohol dehydrogase protein with antibodies directed to human fibronectin receptor suggested the possibility that this protein may function as an adhesin to host extracellular matrix proteins (Klotz et al., 2001
). No interaction of ADH1p with fibronectin was demonstrated and recombinant ADH1p was unable to inhibit fungal cell adhesion in vitro.
The presence of glycolytic enzymes in the fungal cell wall is controversial, primarily because of the lack of a proven mechanism of export (Klis et al., 2002). Speculation on the exact mechanism has focused on heterologous cell lysis and reassociation of pre-synthesized antigen on adjacent cells. Such a mechanism is not extant for Csh1p. Analysis of cell viability in 26 h cultures indicates that greater than 99 % of the cells in the population are metabolically active (data not shown), suggesting the absence of a paracrine-like mechanism for Csh1p association with the cell wall. Similarly, a mechanism used by some moulds to deposit hydrophobins on the wall (McCabe & Van Alfen, 1999
) is not likely as culture filtrates lack detectable Csh1p (unpublished results). Alternatively, proteins might be exported via a non-traditional secretory mechanism. Secretion of the S. cerevisiae a mating factor is facilitated by the STE6 transporter that translocates the mature polypeptide across the plasma membrane. However, the STE6p transporter system is responsible for translocating only a single, small peptide. Recently, Delgado et al. (2003)
demonstrated that a chimera between GAPDH and a non-secreted invertase was able to direct secretion of the construct to the cell wall in S. cerevisiae. Truncation of the GAPDH fusion partner led the authors to map the region necessary for chimera secretion to the N-terminal region of GAPDH. In mammalian systems, a non-traditional secretory pathway is proposed to explain the secretion of the cytokines IL-1
, bFGF and TNF-
, which also lack an N-terminal signal sequence (Rubartelli & Sitia, 1997
). The mechanism of export of IL-1
is unclear, although the majority of the protein is cytoplasmic and it appears to bypass the endoplasmic reticulum to Golgi pathway. No class of proteins demonstrating a secretion pathway analogous to that of IL-1
has been described in lower eukaryotes.
A notable alternative to the two export pathways discussed above has been described in S. cerevisiae and may provide an explanation for the appearance of Csh1p in C. albicans. Cleves and co-workers proposed an alternative export pathway for the transport of a heterologously expressed galectin (Cleves et al., 1996; Cleves, 1997
). Expression of mammalian galectin-1 in S. cerevisiae resulted in the extracellular appearance of the mature protein in a sec18-independent manner, indicating that it is secreted by a mechanism that bypasses traditional vesicular transport. Release of extracellular galectin was accomplished by treatment of intact cells with DTT in alkaline conditions, presumably by releasing a secreted fraction peripherally associated with the cell wall. The authors went on to describe an endogenous S. cerevisiae protein (NCE3p) that uses the same export mechanism, and two candidate gene products (NCE1p/2p) of unknown function that were responsible for the export of this class of proteins (Cleves et al., 1996
). Ablation of either NCE1 or NCE2 prevented secretion of heterologously expressed galectin, confirming their roles in effecting secretion of galectin in a sec14-independent manner. NCE1 and NCE2 showed no homology with any other described transporters and to date the mechanism by which they effect secretion of galectin is still unclear. Analysis of the unannotated C. albicans genome (http://www-sequence.stanford.edu/group/candida) has identified the presence of homologues of the NCE1, 2 and 3 proteins, indicating the potential conservation of a non-classical export pathway between the two fungal species and suggesting a possible mechanism for Csh1p export in C. albicans. Studies are under way to address this possibility.
Use of Zymolyase extraction and cell-surface biotinylation allowed us to demonstrate the presence of Csh1p in the C. albicans cell wall. Previously, we have demonstrated that ablation of the CSH1 gene product results in a moderate, although significant decrease in CSH (Singleton et al., 2001), and antibodies recognizing Csh1p are effective in blocking fungal cell adhesion (Glee et al., 2001
; Masuoka et al., 1999
); however, the antibody is not reproducibly effective in indirect immunofluorescence. These results taken together present circumstantial evidence that Csh1p has a surface presence. Our present results confirm by several methods that Csh1p can be biochemically detected at the cell surface. Other workers reporting the surface presence of glycolytic enzymes in C. albicans have not described the relative surface presence to total cellular pool, although it must be assumed that any glycolytic enzymes must be functioning within the cytoplasm. The fraction of Csh1p associated with the extracellular compartment is small in comparison with the total cell-associated Csh1p and is reminiscent of the amount of cell-associated IL1
. Release of Csh1p from the cell wall and the ability to surface-label the protein is correlated with the total level of Csh1p, suggesting that surface presentation of this protein is not a default pathway for it.
CSH status in C. albicans is mediated by a combination of proteins and post-translational modifications within the cell wall. Previous work has demonstrated that the proteinaceous makeup of cell walls from hydrophobic and hydrophilic yeasts are very similar (Hazen et al., 1990; Hazen & Hazen, 1993
), and mannans extracted from each are also similar, with the exception that the length of the acid-labile fraction (composed of
-1,2-oligomannose) of N-glycans from hydrophobic yeasts are generally longer than in hydrophilic cells (Masuoka & Hazen, 1997
, 1999
). Characterization of proteins extracted from hydrophobic yeasts has enabled us to identify potential gene products that might contribute to the CSH phenotype on the basis of the behaviour of the proteins during hydrophobic interaction chromatography (Masuoka et al., 1999
; Singleton et al., 2001
). CSH contributes to virulence in mice and can be regulated experimentally by changes in growth conditions of cultures. The regulation of the cellular protein components that affect CSH may allow us to identify factors that regulate the CSH phenotype itself.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 21 July 2003;
revised 31 October 2003;
accepted 5 November 2003.
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