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Originally published In Press as doi:10.1074/jbc.M509297200 on November 3, 2005

J. Biol. Chem., Vol. 281, Issue 2, 688-694, January 13, 2006
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Glycosylphosphatidylinositol-anchored Proteases of Candida albicans Target Proteins Necessary for Both Cellular Processes and Host-Pathogen Interactions*

Antje Albrecht{ddagger}, Angelika Felk{ddagger}§, Iva Pichova¶, Julian R. Naglik||, Martin Schaller**, Piet de Groot{ddagger}{ddagger}, Donna MacCallum§§, Frank C. Odds§§, Wilhelm Schäfer§, Frans Klis{ddagger}{ddagger}, Michel Monod¶¶, and Bernhard Hube{ddagger}1

From the {ddagger}Robert Koch-Institut, D-13353 Berlin, Germany, §Molecular Phytopathology and Genetics, University of Hamburg, D-22609 Hamburg, Germany, Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, 166 10 Prague 6, Czech Republic, ||Department of Oral Medicine and Pathology, Kings College London, SE1 9RT London, United Kingdom, **Department of Dermatology, University of Tuebingen, D-72076 Tuebingen, Germany, {ddagger}{ddagger}University of Amsterdam, 1018 WV Amsterdam, The Netherlands, §§Aberdeen Fungal Group, School of Medical Sciences, University of Aberdeen, AB25 2ZD Aberdeen, Scotland, United Kingdom, and ¶¶Centre Hospitalier Universitaire Vaudois, CH-1011 Lausanne, Switzerland

Received for publication, August 23, 2005 , and in revised form, October 27, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular and secreted proteases fulfill multiple functions in microorganisms. In pathogenic microorganisms extracellular proteases may be adapted to interactions with host cells. Here we describe two cell surface-associated aspartic proteases, Sap9 and Sap10, which have structural similarities to yapsins of Saccharomyces cerevisiae and are produced by the human pathogenic yeast Candida albicans. Sap9 and Sap10 are glycosylphosphatidylinositol-anchored and located in the cell membrane or the cell wall. Both proteases are glycosylated, cleave at dibasic or basic processing sites similar to yapsins and Kex2-like proteases, and have functions in cell surface integrity and cell separation during budding. Overexpression of SAP9 in mutants lacking KEX2 or SAP10, or of SAP10 in mutants lacking KEX2 or SAP9, only partially restored these phenotypes, suggesting distinct target proteins of fungal origin for each of the three proteases. In addition, deletion of SAP9 and SAP10 modified the adhesion properties of C. albicans to epithelial cells and caused attenuated epithelial cell damage during experimental oral infection suggesting a unique role for these proteases in both cellular processes and host-pathogen interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteases possess multiple functions in nature ranging from regulation of subtle cellular processes by activating distinct proproteins to nonspecific degradation of proteins for recycling of biomolecules. Several pathogenic microorganisms have adapted this biochemical property to fulfill a number of specialized functions during the infective process. The substrate specificities of these proteases may be very narrow, as in the case of bacterial toxins responsible for botulism or anthrax. In contrast, facultative pathogens may secrete proteases that have more general and much broader effects and play important roles in both saprophytic growth and infection.

The yeast Saccharomyces cerevisiae has served as a eukaryotic model organism to study functions of regulatory proteases including the Kex2 protein, a proprotein processing subtilisin-like serine protease of the late Golgi compartment (1–5). Soon after the discovery of Kex2, numerous Kex2-like processing systems were identified in other eukaryotic species, including humans (6), plants (7), the fission yeast Schizosacchoromyces pombe (8), and the plant pathogenic fungus Ustilago maydis (9). Characteristic of target proteins of Kex2-like proteases are N-terminal processing sites with dibasic amino acids, in particular Lys-Arg. Although Kex2 was shown to be the major protease for processing at dibasic residues, alternative pathways exist in S. cerevisiae (10). Gene products of YPS1 (yapsin 1) and YPS2 (yapsin 2), two closely related glycosylphosphatidylinositol (GPI)2-anchored aspartic proteases, were able to cleave the {alpha}-factor pheromone precursor (a well known Kex2 substrate) at Lys-Arg sites (10–12), and overexpression of YPS1 and YPS2 partially compensated for the loss of Kex2 (10, 13). However, the biological roles of yapsins are unknown.

The human pathogenic fungus Candida albicans is closely related to S. cerevisiae, sharing over 60% orthologous genes (14). However, the function of these genes may have altered, because C. albicans has been long adapted to the human host, and these alterations may therefore be directly linked to survival and pathogenicity. Furthermore, the C. albicans genes with no counterpart in S. cerevisiae may have evolved because of the specific demands of the human host environment. These include a gene family encoding secretory aspartic proteases (Sap) (15). Saps were shown to contribute to several virulence attributes of C. albicans during the infection process, with Sap1–3 required for mucosal infections and Sap4–6 for systemic infections (16, 17). All SAP genes are translated into preproenzymes with dibasic motifs in the propeptide. For Sap2, a prototypic member of this family that can digest a variety of host proteins in the extracellular space, processing by Kex2 has been demonstrated (18). However, alternative processing pathways appear to exist in C. albicans, as a {Delta}kex2 mutant was able to activate proSap2 in a yapsin-like manner.

In this study we show that Sap9 and Sap10 are crucial for the infection process. In contrast to the other family members, they are GPI-anchored yapsin-like aspartic proteases that target proteins of fungal origin necessary for cell surface integrity, cell separation, and adhesion.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Strains and Growth Conditions—C. albicans strains SC5314 (19) and CAI-4 carrying CIp10 (20) were used as wild type controls. CAI-4 (21) was used to produce mutants either lacking or overexpressing SAP genes. For phenotypic screening, cells were diluted to give concentrations of 2000, 200, 20, and 2 cells/µl. Five µl of these cell suspensions were dropped onto agar supplemented as indicated. To determine differences in cell separation cells were grown in liquid SD medium until mid-log phase.

Real-time Reverse Transcription-PCR—Cells were harvested in mid-log phase in liquid SD medium. Total RNA was extracted and reverse transcribed into cDNA, and SAP9 and SAP10 were amplified using the Qiagen QuantiTectTM reverse transcription-PCR kit as described by the manufacturer. The gene ACT1 was used as an internal standard. Amplification and detection of specific products was performed with the ABI Prism 7000 sequence detection system (PE-Applied Biosystems).

In Vivo SAP Expression in Patient Oral Samples—Whole saliva samples were collected from 40 patients with active C. albicans infection (clinical signs of disease and >2000 C. albicans colony-forming units/ml of saliva) and 29 asymptomatic C. albicans carriers (no clinical signs and harboring 50–800 C. albicans colony-forming units/ml of saliva) attending the Oral Medicine clinic at Guy's Hospital, London. Upon collection, the total RNA was isolated and examined for ACT1 (positive control), SAP9, and SAP10 expression using a modified radioactive 32P-based reverse-transcriptase-PCR system (Access, Promega) as described previously (23, 24).

Gene Disruption and Overexpression—The Ura-blaster protocol (21) was used to disrupt SAP9 and SAP10. An internal fragment (bp +1169 to +1246) of SAP9 was replaced with the hisG-URA3-hisG cassette to give pAF9ura. For deletion of SAP10, the hisG-URA3-hisG cassette was flanked with the SAP10 region from positions +11 to +521 and +804 to +1206 to give pAA10ura. pAF9ura and pAA10ura were linearized and transformed into strain CAI-4 and 5-fluoroorotic acid-treated (Ura–) {Delta}sap9. Two rounds of Ura-blasting were performed to disrupt both alleles of SAP9 and SAP10 to give {Delta}sap9 and {Delta}sap10 and the double mutant {Delta}sap9/{Delta}sap10, respectively. Each step of integration and disruption was confirmed by PCR and Southern blot.

To rescue the wild type phenotype in mutants lacking SAP9 and SAP10, the entire regions from SAP9 (bp –981 to +1769 bp) and SAP10 (bp –807 to +1567) were cloned into CIp10 to give CIp10-SAP9 and CIp10-SAP10. Plasmids were transformed into Ura– {Delta}sap9 or {Delta}sap10, respectively, after linearization. For overexpression of SAP9 (bp +1to +1879) and SAP10 (bp +1to +1567), fragments were cloned under the control of the C. albicans ACT1 promoter (25) to obtain pACT1-SAP9 and pACT1-SAP10. Plasmids were linearization and transformed into CAI-4. In the corresponding controls, linearized CIp10 and pACT1 (22) were transformed into CAI-4, (Ura–) {Delta}sap9, or {Delta}sap10. Single integration of these plasmids at the RPS1 (formerly RPS10) locus was confirmed by Southern blot analysis.

Sap-Gfp Protein Fusion—To construct pACT1-SAP9-GFP and pACT1-SAP10-GFP, GFP was fused to 5'- and 3'-fragments of SAP9 and SAP10 containing sequences encoding the putative Kex2 processing sites and the entire C-terminal sequences that prevented secretion into the culture in Pichia pastoris clones (Fig. 1). Plasmids were integrated into the RPS1 locus of CAI-4.

Immunoelectron Microscopy—Sap9 and Sap10 were localized ultrastructurally by immunoelectron microscopy of C. albicans strains containing pACT1-SAP9-GFP, pACT1-SAP10-GFP, pACT1-GFP (control 1), or pGFP (control 2) with anti-Gfp, as described previously (22).

N-Glycosidase F Treatment—To verify putative N-glycosylation of Sap9 and Sap10, heterologously expressed proteins were incubated with and without N-glycosidase F according to the manufacturer's instructions (Roche Applied Science). Proteins were separated on a 12% SDS-polyacrylamide gel and stained.

Adherence Assay—Strains SC5314, CAI-4::CIp10, {Delta}sap9, {Delta}sap9[SAP9], {Delta}sap10, {Delta}sap10[SAP10], and {Delta}sap9/{Delta}sap10 were grown on SD-agar plates. Buccal epithelial cells (BECs) were gently scraped from a healthy donor with cotton swabs. C. albicans, and BEC suspensions were washed in phosphate-buffered saline, pH 7.2, and resuspended at a concentration of 107 cells/ml and 105 cells/ml, respectively. Suspension were mixed in equal volumes (100 µl) and incubated for 45 min at 37 °C. BECs were collected on a polycarbonate filter, and nonadherent C. albicans cells were washed through the filter pores. The percentage of BECs with C. albicans cells attached was monitored for at least 100 epithelial cells per sample.

Infection Models—To model systemic infection, mice were injected intravenously and monitored as described previously (20). All animal work was carried out under the terms and conditions stipulated by the Home Office, United Kingdom. Reconstituted human epithelium (RHE) infected with C. albicans as described previously (22) provided a model for oral infection. RHE medium was sampled to assay the release of lactate dehydrogenase as a measure of epithelial cell damage, and histological sections of the RHE were examined.

Cell Wall Isolation and Determination of Protein and Chitin Contents—To assess any possible changes in cell wall composition resulting from deletion of SAP9 or SAP10, cell walls were isolated as described previously (26). Protein and chitin contents were determined using a modified method according to Yabe et al. (27), which involves acid hydrolysis to break down chitin to glucosamine.

Production of Recombinant Proteases—Expression plasmids were constructed by insertion of PCR products into the P. pastoris expression vector pKJ113 (28). P. pastoris transformation, and production of recombinant enzymes was described previously (29). Culture supernatants were harvested and dialyzed against 20 mM sodium citrate buffer, pH 6.5, containing 150 mM NaCl (buffer A). Proteases were purified on a SuperdexTM 75 column equilibrated in buffer A using Acta explorer FPLC (Amersham Biosciences).

Synthesis of Peptides and Substrate Specificity—Peptides derived from protein sequences of C. albicans Sap2, Sap7, and Sap9 and human vimentin were synthesized by the solid-phase method. Standard conditions for the cleavage of peptides were as follows. 10 mM sodium citrate buffer, pH 6.5, containing 200 mM NaCl, 330 µM peptide, and 0.6 µM purified heterologously expressed Sap9 or Sap10 in a total volume of 120 µl, incubated overnight at 37 °C. Cleavage products were separated by reverse-phase high pressure liquid chromatography on a Vydac C18 column in a methanol/H2O system, and fractions were analyzed by amino acid analysis and mass spectrometry analysis on a matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF, Bruker Daltonics).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
SAP9 and SAP10 Encode Aspartic Proteases with Structural Similarities to Yapsins—SAP9 and SAP10 code for preproenzymes with a signal peptide removed in the endoplasmic reticulum and a propeptide with Lys-Arg residues, known as proteolytic processing sites for Kex2 (Fig. 1). Like all other members of the Sap family, the mature Sap9 and Sap10 proteins possess four conserved cysteine residues and two conserved aspartate residues. Sequence comparisons revealed that C. albicans Sap9 and Sap10 differ from the other Sap1–8 isoenzymes not only by sequence similarity (Fig. 2) but also by multiple N-glycosylation sites and putative GPI anchor attachment sequences at their C termini (28), a recognized structural property of yapsins.

Sap9 and Sap10 are N-Glycosylated and GPI-anchored on the Cell Surface—To determine whether Sap9 and Sap10 are in fact GPI proteins, we used a series of constructs to express native and C-terminal truncated versions of Sap9 and Sap10 in the yeast P. pastoris (Fig. 1). Expression of native Sap9 and Sap10 prevented secretion of these proteases. However, secretion was observed when parts of the C-terminal sequences of the GPI anchor consensus sequence were deleted and was highest when the complete consensus sequence was removed. These data suggest that the C termini prevent secretion of Sap9 and Sap10.


Figure 1
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  		FIGURE 1.
Sap9 and Sap10 are GPI proteins located in the cell membrane (Sap9) or distributed in the cell membrane and cell wall (Sap10). Panel A, schematic structure of Sap9 and Sap10. SP, processing site of signal peptidase; KR, Lys-Arg; D, conserved Asp residues of the catalytic center; C, conserved Cys residues; P, processing site in Sap9 for disjunction into {alpha}- and beta-units; {omega}, omega consensus site for GPI anchoring; S/T, serine/threonine-rich region; H, hydrophobic region. Panel B, secretion of C-terminal truncated versions of Sap9 and Sap10 in P. pastoris. Panel C, localization of Sap9- and Sap10-Gfp fusion proteins as shown by immunoelectron microscopy and gold-labeled anti-Gfp antibodies. Strains carrying pGFP or pACT1-GFP (22) did not show labeling of cell membrane or cell wall.

 


Figure 2
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  		FIGURE 2.
Dendrogram displaying the relationship of the C. albicans Sap isoenzyme family and the two yapsins of S. cerevisiae that showed highest similarity to Sap9 and Sap10.

 
For cellular localization of the two proteases, we constructed Sap9- and Sap10-Gfp fusion proteins by inserting the GFP gene into the protease sequences directly after the Kex2 processing site and attaching the C-terminal sequences that prevented secretion in P. pastoris. Immunoelectron microscopy using an anti-Gfp antibody revealed that both fusion proteins are located on the cell surface of C. albicans (Fig. 1). However, the distribution of antigen was different for the two fusion proteins; Sap9-Gfp was predominantly located in cell membranes, whereas Sap10-Gfp was located in both the cell membrane and the cell wall (Fig. 1).

SDS protein gel analysis of purified Sap9 and Sap10 proteins revealed two subunits for Sap9, with sizes of ~68 and 10 kDa and only one polypeptide with 60 kDa for Sap10 (Fig. 3). In contrast to other members of the Sap family, which possess a maximum of one potential N-glycosylation site within the sequence of the mature enzyme, Sap9 and Sap10 contain, respectively, five and eight such sites. Treatment of Sap9 and Sap10 with N-glycosidase F caused a clear band shift for both proteases, suggesting that Sap9 and Sap10 are N-glycosylated (Fig. 3). Thus, Sap9 and Sap10 are highly N-glycosylated proteases, which are GPI-anchored on the cell surface.


Figure 3
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  		FIGURE 3.
Posttranslational modification of Sap9 and Sap10. A, Sap9 consists of two subunits of ~68 and 10 kDa, and Sap10 consists of one polypeptide of 60 kDa. B, treatment of Sap9 and Sap10 with N-glycosidase F (+) caused a band shift for both proteases, whereas treatment without N-glycosidase F (–) did not, suggesting that Sap9 and Sap10 are N-glycosylated.

 
Sap9 and Sap10 Digest Peptides at Distinct Sites—Because Sap9 and Sap10 were exposed to the extracellular space but attached to the cell membrane or cell wall, we concluded that proteolysis by Sap9 and Sap10 must take place on the cell surface and that putative substrates of these proteases may be of either host or fungal origin.

To identify possible target proteins of Sap9 and Sap10, several host proteins known to be substrates of other Saps (15) were exposed to heterologously expressed enzymes. None of the tested proteins, including serum albumin, collagen, hemoglobin, keratin, mucin, or immunoglobulins, were hydrolyzed in a Sap2-like manner (data not shown). In contrast, several synthetic peptides containing basic or dibasic amino acid motifs were digested by Sap9 or Sap10 or by both proteases (Table 1). Digests were similar to the activity of S. cerevisiae yapsins or Kex2-regulatory proteases with hydrolysis at KR, KK, or single Lys (K) sites (Table 1). Sap9 and Sap10 preferred cleavage after dibasic (KR, KK) or monobasic (Lys, Arg) residues, similar to yapsin 1 and 2, whereas the Kex2 protease cleaves only C-terminally to clusters of dibasic residues (13, 29, 30). Addition of uncharged Asn into the P1' side in the peptide PISKRNERS abolished cleavage by Sap9 but maintained the processing with Sap10 (Table 1). In addition, Sap10 digested at sites previously unknown for yapsin-like aspartic proteases (between Phe-Ser and His-Asn). Interestingly, the peptide KIHNKLFGF, which is identical to an internal sequence of Sap9 (Lys149 to Phe157 from the N terminus), was processed by Sap9 between Lys and Leu154. This suggests self-processing activity of Sap9 at this site, which possibly accounts for the two subunits observed in this study and by others (11). The N-terminal sequence of the larger beta-subunit was previously shown to be Leu154-Phe-Gly-Phe (11), identical to the N-terminal sequence of one fragment of the digested peptide. These data suggest that Sap9 and Sap10 are proteases that hydrolyze polypeptides at distinct processing sites.


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  		TABLE 1
Digestion by Sap9 or Sap10

Peptides are digested by Sap9 and/or Sap10 between amino acids (in bold). Asterisk, digestion; underline, digestion by Sap9; overline, digestion by Sap10; double underline, digestion by both Sap9 and Sap10.

 
Sap9 and Sap10 Are Necessary for Cell Surface Integrity and Cell Separation during Budding—To determine the possible functions of the two proteases, we constructed {Delta}sap9 and {Delta}sap10 single mutants and a {Delta}sap9/{Delta}sap10 double mutant. All mutant strains grew normally on media containing different carbon sources, at different pH values, on hyphal inducing media or when incubated at different temperatures (data not shown). However, components that directly or indirectly target the fungal cell surface such as hygromycin B (Fig. 4A), amorolfine, calcofluor, Congo red, and itraconazole (not shown) caused significant growth defects for all mutants. In addition, nikkomycin inhibited growth of the {Delta}sap9 mutant (not shown). Observed phenotypes were at least partially restored by mutants carrying plasmid-borne Sap9 or Sap10 (Fig. 4A). Together, these data suggest a role of Sap9 and Sap10 in cell surface integrity. Furthermore, all three mutants exhibited an abnormal budding phenotype, as daughter cells did not separate from mother cells and remained associated in cell chains (Fig. 4B). Because it is known that blockage of protein activities involved in cell membrane or cell wall maintenance can be compensated for by the up-regulation of proteins involved in the production of alternative structural elements, we quantified the protein and chitin levels in the cell walls of the mutants. Chitin levels of the {Delta}sap9 mutant were significantly increased (4.0 ± 0.9% S.D.; t test, p = 0.05), whereas the {Delta}sap10 mutant showed increased levels of both protein (7.5 ± 0.6%; t test, p = 0.05) and chitin (3.4 ± 0.7%; t test, p = 0.05) as compared with the parental strain (protein 5.8 ± 0.7%; chitin 1.9 ± 0.5%).


Figure 4
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  		FIGURE 4.
Hypersensitivity of mutants lacking SAP9 and/or SAP10 ({Delta}sap9, {Delta}sap10, and two isogenic {Delta}sap9/{Delta}sap10 mutants) during growth in the presence of hygromycin B(A) and dysfunction of these mutants during budding in liquid medium (B) as compared with the wild type (WT). Phenotypes were reversed by plasmid (CIp10)-borne Sap9 and Sap10 expression ({Delta}sap9[SAP9], {Delta}sap10[SAP10]). Hygromycin B, 800 µg/ml ({Delta}sap9; {Delta}sap9/{Delta}sap10) and 1100 µg/ml ({Delta}sap10).

 
Sap9, Sap10, and Kex2 Have Distinct Cellular Functions—Our results, together with previous data (18), suggest that Sap9 and Sap10 and the processing serine protease Kex2 in C. albicans all may be involved in the maintenance of cell surface integrity. To investigate whether Sap9, Sap10, and Kex2 have overlapping functions, we constructed SAP9 and SAP10 overexpression vectors, which were integrated into the single mutants (SAP9 into {Delta}sap10 or {Delta}kex2, respectively; SAP10 into {Delta}sap9 or {Delta}kex2, respectively). Phenotypic screening of these strains showed little or no overlapping function of the three proteases. Real-time reverse transcription-PCR analysis of the mutants revealed a 2-fold over-expression of SAP9 in the {Delta}kex2 mutant, a 2-fold over-expression of SAP9 in the {Delta}sap10 mutant, and a 4-fold over-expression of SAP10 in the {Delta}sap9 mutant, suggesting that the loss of one processing protease gene induces up-regulation of the other genes (data not shown).

Deletion of SAP9 or SAP10 Modifies Adhesion to Epithelial Cells and Causes Attenuated Epithelial Cell Damage during Experimental Oral Infections—To investigate whether dysfunctions in cell surface integrity resulting from SAP9 or SAP10 deletion influenced virulence of C. albicans, we investigated the potential of {Delta}sap9 and {Delta}sap10 mutants to cause infections. Both mutants were only moderately attenuated in a mouse model of systemic infection after intravenous challenge (not shown). However, the same mutants had a significantly reduced ability to invade and damage epithelial cells in a model of oral infection based on RHE (Fig. 5).

One possible explanation for the attenuated virulence phenotype in the RHE model could be a reduced ability of the mutant strains to adhere to epithelial cells, thus resulting in reduced invasion and cell damage. This may indeed be the case for {Delta}sap10, as adherence of this mutant to BECs was reduced (Fig. 6). However, the ability of the {Delta}sap9 mutant to adhere to epithelial cells was dramatically increased as compared with the wild type, suggesting that properties other than reduced adhesion attributes were responsible for the decreased epithelial cell damage of {Delta}sap9. Interestingly, the phenotype of the double mutant {Delta}sap9/{Delta}sap10 showed reduced adhesion resembling the phenotype of the {Delta}sap10 and not the {Delta}sap9 single mutant (Fig. 6). Therefore, activity of Sap9 and Sap10 is necessary for wild type adhesion properties and invasion and cell damage of oral epithelial cells.


Figure 5
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  		FIGURE 5.
Reduced pathogenicity of {Delta}sap9 and {Delta}sap10 as compared with the wild type (WT) in a model of oral candidosis 12 h after infection. Epithelial tissue damage is attenuated by all mutants as indicated by histology and release of the epithelial marker enzyme lactate dehydrogenase (LDH; units/ml) into the medium. Full virulence is restored by reintroducing SAP9 and SAP10 into the mutants ({Delta}sap9[SAP9], {Delta}sap10[SAP10]). Standard deviation is shown in parentheses;*, t test, p ≤ 0.05.

 


Figure 6
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  		FIGURE 6.
Adhesion of {Delta}sap9, {Delta}sap10, the {Delta}sap9/{Delta}sap10 double mutant, and revertant strains to epithelial cells as compared with the wild type. Shown are the percentages of epithelial cells with C. albicans cells attached. Standard deviation is shown in parentheses;*, t test, p ≤ 0.05.

 
In Vivo SAP Expression in Oral Samples from Patients—If SAP9 and SAP10 play a virulence role during oral infections, both of these genes would be predicted to be expressed in vivo by C. albicans strains colonizing and infecting the oral cavity. To demonstrate this possibility, we analyzed SAP9 and SAP10 expression in total RNA samples isolated from the saliva of a large number of patients (40 symptomatic C. albicans infected and 29 asymptomatic C. albicans carriers). SAP9 and SAP10 transcripts were detected in 98 and 93% of C. albicans-infected patients, respectively, and in 83 and 86% of C. albicans carriers, respectively. This indicates that both SAP genes are frequently expressed both in the commensal stage and during infection, suggesting an important role for growth and/or survival in the oral cavity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The model yeast S. cerevisiae and the human pathogenic fungus C. albicans share a large portion of similar genes, which may, however, have different functions because of the different natural environments of these two fungal species. Data presented in this study suggest that C. albicans is equipped with 10 extracellular aspartic proteases, Sap1–10, which are either secreted (Sap1–8) or associated with the cell surface (Sap9 and Sap10). However, the functions of these two subclasses of extracellular aspartic proteases seem to be fundamentally different. Proteases Sap1–6 are known to hydrolyze host proteins and consequently to cause tissue damage (15). In contrast, our data suggest that Sap9 and Sap10 are linked to regulatory processes on the fungal cell surface that are essential for maximal pathogenicity during interaction with oral epithelial tissue.

Cell surface-associated proteases that are exposed to the extracellular space with such regulatory functions are rare in nature. The most prominent examples are the yapsins, first discovered in S. cerevisiae. Sap9 and Sap10 of C. albicans share a number of structural characteristics with yapsins. Both proteins show high sequence similarities to Yps1, Yps2, Yps3/4, Yps6, and Yps7 (www.yeastgenome.org) and, in contrast to other Saps, are glycosylated. C-terminal sequences of all of these aspartic proteases contain putative GPI anchor sequences. As has been shown for Yps1 and Yps2 (31), only C-terminally truncated versions of Sap9 and Sap10 are secreted to the extracellular space. Furthermore, using a Gfp reporter protein fused to the C-terminal sequences of Sap9 and Sap10, along with immunoelectron microscopy, we showed that these proteases are targeted predominantly to either the cell membrane (Sap9) or to both the cell membrane and the cell wall (Sap10). In addition, our data shed light on a previous study, which showed that native Sap9 consists of two subunits (11) linked by a disulfide bridge. We demonstrate that Sap9 is able to digest a peptide identical to the putative processing site of the Sap9 precursor, indicating an autocatalytic processing ability for Sap9.

The function of yapsins has yet not been elucidated. Several lines of evidence in this study suggest that Sap9 and Sap10 target proteins of fungal origin with roles in cell surface integrity and cell separation, which they probably share with yapsin-like protease in S. cerevisiae and other fungi. In pathogenic fungi these proteases may have gained functions with specific roles during host-pathogen interactions. In addition to C. albicans, yapsin-like proteases exist in other pathogenic fungi; for example, as many as nine genes encoding yapsin-like aspartic proteases exist in the genome of the emerging pathogen Candida glabrata (32).

Although Sap9 and Sap10 are required for cell surface integrity and both cleave at similar or identical processing sites, they also seem to have unique processing sites. This view is supported by the fact that the single mutants showed phenotypes that were only partially compensated for by overexpression of its paralog Sap isoenzyme, suggesting that each protease has distinct target proteins.

What are the target proteins of Sap9 and Sap10? Because both proteases are exposed to the extracellular space, processing must occur on the cell surface, and thus the fungal target proteins must have been transported, presumably via the secretory pathway, to the outer face of the cell membrane. These may be proteins of the cell membrane (facing the extracellular space), the cell wall, or secreted proteins. Other Saps may also be target proteins. However, total proteolytic activity of {Delta}sap9 and {Delta}sap10 mutants was not reduced as compared with wild type strains (not shown), suggesting that processing of Sap2 (the dominant secreted protease expressed in vitro (33)) can occur independently of Sap9 and Sap10. GPI-anchored proteins are an abundant class of cell surface proteins. As many as 104 putative GPI proteins were identified by in silico approaches in C. albicans (34). Some of them were identified by biochemical analysis (26, 35), including proteins such as Phr1, Ecm33, and Crh1, which are also present in S. cerevisiae. In addition, several proteins and protein families with no significant homology to S. cerevisiae proteins were identified, including the cell wall-associated proteins Als1 and Als4, Rbt5, and Hwp1. Several studies showed that yeast cell wall proteins are associated with a number of functions in cell physiology including cell surface integrity, flexibility and remodeling, cell morphology, growth, and budding (36). In pathogenic yeasts like C. albicans, some of these proteins have been shown to be directly or indirectly involved in virulence functions, in particular adhesion and immune evasion (37). Therefore, proteases such as Sap9 and Sap10 that process proteins of the cell wall may be associated with these functions. In fact, C. albicans strains lacking certain cell wall proteins show characteristics resembling the phenotypes of {Delta}sap9 and {Delta}sap10. For example, mutants lacking Ecm33 show sensitivity to compounds that target the cell wall, including calcofluor white, Congo red, and hygromycin B, similar to the phenotypes of {Delta}sap9 and {Delta}sap10 (38). In addition, it has been shown that the chitin content is increased in cells that are exposed to environmental stresses or that have dysfunctions in the biosynthesis or remodeling of structural elements as a compensatory mechanism (36). For example, mutants lacking KRE5, which encodes an endoplasmic reticulum protein involved in glucan synthesis, had strongly reduced levels of glucan but higher chitin content (39), and disruption of GPI7, encoding a GPI anchor-modifying activity, showed increases in chitin deposition (40). Because mutants lacking Sap9 or Sap10 have higher chitin contents, it is possible that some of the target proteins are associated with the integrity of other structural elements of the cell wall such as glucan. Processing of Sap9 and Sap10 target proteins is not essential for normal growth. However, it is a prerequisite for normal separation of cells after budding. Cell separation defects similar to those found for {Delta}sap9 and {Delta}sap10 in this study have also been observed for mutants lacking the chitin synthase gene CHS1 (41) or ACE2, which encodes a key regulator of cell wall metabolism (42).

If the target proteins of Sap9 and Sap10 are transported to the surface via the secretory pathway and have processing sites similar to Kex2, why does Kex2 (43) not cleave them? There are at least two possible explanations. First, processing sites of the target proteins may not be exposed to processing by Kex2 in the Golgi because of folding of these proteins. Second, recent studies of GPI-anchored protein sorting has led to the surprising finding that, during their delivery to the surface, different types of plasma membrane proteins are sorted from each other early in this pathway in the endoplasmic reticulum. Therefore, it may be possible that these putative target proteins are bypassing Kex2-containing Golgi compartments and thus are not processed by Kex2 (44).

One of the most interesting and surprising observations was the fact that {Delta}sap9 and {Delta}sap10 mutants had oppositely altered adhesion properties; whereas mutants lacking SAP9 were more adherent to BECs, the loss of SAP10 caused reduced adherence, and mutants lacking both genes behaved like the {Delta}sap10 mutant. This intriguing observation led us to speculate that Sap9 and Sap10 may have defined functions during infection similar to surface-associated proteases of malaria and Toxoplasma parasites (45). Here, distinct surface proteins are trimmed to activate or enhance adhesion to host receptors, whereas other proteases shave resident surface proteins or break connections between surface ligands and the host receptor during the penetration process. A similar function of Sap9 may explain why {Delta}sap9 had reduced abilities to invade epithelial tissue and to cause damage despite enhanced adhesion properties.

Although we cannot exclude the theoretical possibility that the Sap10 protein itself may have a direct adhesion function, this seems very unlikely in the case of Sap9 because disruption of SAP9 caused increased adhesion. Removal of an adhesion protein should not increase the adhesion properties but rather should reduce adhesion. Furthermore, adherence of C. albicans to human mucosa (46), epidermal corneocytes (47), and epidermal keratinocytes (48) has been shown to be inhibited by pepstatin A, suggesting that proteolytic activity of aspartic proteinases is necessary for the adhesion properties of C. albicans.

In C. albicans, loss of the surface protein Als3 or the cell wall regulator protein AceII was shown to reduce adherence to epithelial cell or plastic surfaces (42, 49). However, the loss of the putative adhesin Eap1 or the GPI-anchored cell wall protein Ywp1/Pga24 caused increased adherence, suggesting that aberrant, misprocessing of, or removal of certain cell wall proteins may cause either enhanced or attenuated adhesion (50, 51). Interestingly, Granger et al. (50) observed that Ywp1/Pga24 seems to be processed by proteases other than Kex2 and suggested an expansive role for processing of cell surface proteins in regulating cellular adhesion. The data presented in our study suggest that Sap9 and Sap10 of C. albicans are key enzymes for such activities.


   FOOTNOTES
 
* This work was supported by Grants Hu528/8 and Scha 897/1 from the Deutsche Forschungsgemeinschaft (to B. H. and M. S.), 303/04/0432 from the Grant Agency of the Czech Republic (to I. P.), and QLK2-2000-00795 and MRTN-CT-2003-504148 ("Galar Fungail consortium") from the European Commission. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Robert-Koch-Institut, FG16, Nordufer 20, D-13353 Berlin, Germany. Tel.: 49-18887542116; Fax: 49-18887542605; E-mail: HubeB{at}rki.de.

2 The abbreviations used are: GPI, glycosylphosphatidylinositol; SAP, secretory aspartic protease; GFP, green fluorescent protein; BEC, buccal epithelial cell; SD medium, synthetic defined medium; RHE, reconstituted human epithelium. Back


   ACKNOWLEDGMENTS
 
We thank Barbara Léchenne (Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland) for preparing heterologous Sap9 and Sap10 from P. pastoris, Nina Agabian (University of California, San Francisco) for providing the {Delta}kex2 mutant, and Sascha Thewes (Robert-Koch-Institut, Berlin, Germany) for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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