Differential secretion of Sap4–6 proteins in Candida albicans during hyphae formation

Yee-Chun Chen1,2, Chi-Chen Wu2,3, Wei-Lian Chung2,3 and Fang-Jen S. Lee3

Section of Infectious Diseases, Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan1
Department of Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan2
Institute of Molecular Medicine, College of Medicine, National Taiwan University, 7 Chung Shan South Road, Taipei, Taiwan3

Author for correspondence: Fang-Jen S. Lee. Tel: +886 2 2312 3456 ext. 5730. Fax: +886 2 2395 7801. e-mail: fangjen{at}ha.mc.ntu.edu.tw


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multi-gene family and are considered to be putative virulence factors for candidiasis. SAP4–6 mRNAs were first detected during hyphae formation and were assumed to play roles in the development of disseminated candidiasis. Recombinant Sap proteins (Sap2–6) were prepared and specific antibodies were generated against Sap2–6. The presence of Sap4, Sap5 and Sap6, but not Sap2 or Sap3, was demonstrated in culture supernatants of C. albicans after induction of hyphae formation. In parallel to hyphae formation, Sap5 (~40 kDa) was detected as early as ~6 h after induction at neutral pH, and Sap4/6 (~43 kDa) were detected after ~24 h when the culture medium became acidic. The differential secretion of Sap5 and Sap4/6 was affected when the culture medium pH was buffered at pH 6·5 or pH 4·5. In addition, intracellular pools of Sap4–6 seem to exist, and protein is not necessary for Sap4–6 induction. This study provides the first evidence that Sap4–6 proteins in C. albicans are differentially produced and secreted during hyphae formation.

Keywords: Candida albicans, secreted aspartyl proteinase, virulence factor, candidiasis

Abbreviations: Sap, secreted aspartyl proteinase


   INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Candida albicans is an important opportunistic human pathogen, and the number of infections caused by it increased dramatically in the last decade (Chen et al., 1997 ; Edmond et al., 1999 ; Edwards, 2000 ). C. albicans can colonize virtually all body sites. It causes superficial or invasive infection and carries high mortality and morbidity, especially in immunocompromised patients (Edwards, 2000 ; Hung et al., 1996 ; Chen et al., 2001 ). Because the modes of management of such patients are limited, with the increased incidence of C. albicans infections, the investigation of virulence factors and the search for a new target for development of anti-fungal agents and immunotherapy has become increasingly important.

C. albicans possesses a gene family encoding secreted aspartyl proteinases (Saps) with unusually broad substrate specificities (Cutler, 1991 ; Hube, 1996 ; Hube & Naglik, 2001 ). These enzymes have been linked to the virulence of the fungus since their discovery (Staib, 1965 ; Kwon-Chung et al., 1985 ; Cassone et al., 1987 ; De Bernardis et al., 1999 ). The proposed functions of these proteinases during infection include the digestion of host proteins for nutrient supply, evasion of host defences by degrading immunoglobulins and complement proteins, adherence, and degradation of host barriers during invasion (Hube, 1996 ). Although initially C. albicans was believed to express a single SAP gene, further studies have identified the existence of at least 9 closely related SAP genes (White et al., 1993 ; Hube et al., 1994 ; Monod et al., 1994 , 1998 ).

Differences in expression of various SAP mRNAs have been investigated in vitro and in experimental animal models (Morrow et al., 1992 ; Hube et al., 1994 ; White & Agabian, 1995 ; Borg-von Zepelin et al., 1998 ; Monod et al., 1998 ). Experimental infections with various mutants ({Delta}sap1 to {Delta}sap6) generated by targeted mutagenesis suggested the importance of different Saps in the virulence of C. albicans (Hube et al., 1997 ; Sanglard et al., 1997 ; De Bernardis et al., 1999 ; Kretschmar et al., 1999 ). These data suggest that the temporal and specific regulation of SAP expression might be important for the survival of C. albicans in its natural environment and thus in the pathogenesis of this fungus. Yet, because studies on the protein levels are limited, the special role of the individual Sap proteins in infection remains unclear (Cutfield et al., 1995 ; Smolenski et al., 1997 ; Hube & Naglik, 2001 ).

Among the various SAP gene products, deduced amino acid sequences for Sap4–6, which have 75–89% similarity to each other, form a group distinct from Sap1–3 (Hube et al., 1994 ; Miyasaki et al., 1994 ). This subfamily, Sap4–6, was a potential target for intervention because SAP4–6 mRNAs were first identified during hyphae formation at neutral pH (Hube et al., 1994 ). Blood and salivary pH are approximately neutral, and hyphae formation is important during tissue invasion (Edwards, 2000 ); therefore, Sap4–6 proteins were presumed to be important in disseminated candidiasis.

Recently, it has been demonstrated that a {Delta}sap4–6 triple mutant resulted in attenuated mortality after systemic infection in guinea pigs and mice (Sanglard et al., 1997 ), and that less tissue damage occurred in a murine peritonitis model (Kretschmar et al., 1999 ). These results suggest that the SAP4–6 genes contribute to the development of disseminated infections. Sap4–6 proteins have been detected in Candida within macrophages (Borg-von Zepelin et al., 1998 ); however, production of the extracellular form of Sap4–6 during hyphae formation has never been characterized.

In this study, we generated specific antibodies against each of the Sap2–6 proteins and determined the secretion of Sap4–6 by C. albicans during hyphae formation. The effect of culture medium pH on secretion of Sap4–6 during hyphae formation was also examined. Here, we show that production and secretion of Sap4–6 proteins in C. albicans are differentially regulated during hyphae formation in a pH dependent manner.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strains and growth conditions.
The C. albicans wild-type strain SC5314 (Gillum et al., 1984 ) and the sap null mutant strains {Delta}sap1, {Delta}sap2, {Delta}sap3, {Delta}sap4, {Delta}sap5, {Delta}sap6 and {Delta}sap4–6 (Hube et al., 1997 ; Sanglard et al., 1997 ), and {Delta}efg1 {Delta}cph1 (Lo et al., 1997 ) were used in this study (Table 1). C. albicans SC5314, a blood culture isolate from a patient with disseminated candidiasis, was used as the DNA template for cloning individual SAP genes and expression of His-tagged fusion proteins in E. coli. The following liquid media were used: YPD (1% yeast extract, 2% peptone, 2% glucose) complex medium for general use; YNB-BSA medium for induction of Sap2 protein [0·17%, w/v, yeast nitrogen base (Difco), 2% glucose, 0·2% BSA (Sigma), pH 5·0]; and modified Lee’s medium (pH 4·5 and pH 6·5) for induction of Sap4–6 proteins (Lee et al., 1975 ; Morrow et al., 1992 ). BSA (Sigma) was added as indicated. C. albicans was grown in an orbital incubator at the indicated temperatures.


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Table 1. C. albicans strains used in this study

 
Cloning, expression and purification of Sap proteins.
Plasmids were constructed according to standard protocols (Sambrook et al., 1989 ). The following pairs of primers were used to amplify the ORFs of C. albicans SAP2, SAP3, SAP4, SAP5, SAP6 genes: SAP2.1 (5'-CCA TGG ATC AAA AAC TTA ATG TTA TTG TTG AT-3', nt 356–387) and SAP2.2 (5'-TTC CAC CCC TTC ATC TTA GGT-3', nt 1335–1315); SAP3.1 (5'-ATA ACA TAT GTT TTT AAA AAA TAT CTT T-3', nt 222–249) and SAP3.2 (5'-ATC AGG ATC CAG AGG AGT ACT TCC CCC T-3', nt 1452–1424); SAP4.1 (5'-ACC ACA TAT GTT CTT ACA AAA TAT CTT GAG-3', nt 131–160) and SAP4.2 (5'-ACA AGG ATC CTA AAA CCA ATG TCG TCT AAT-3', nt 1416–1387); SAP5.1 (5'-CAT ATG TTC TTG AAA AAT ATC TTG AG-3', nt 265–290) and SAP5.2 (5'-TTA ATC TAA AGT CAA AGT TCT TTA-3', nt 1545–1522); SAP6.1 (5'-CAT ATG TTC TTG AAA AAT ATC TTG AG-3', nt 220–245) and SAP6.2 (5'-ATC TAA AAC CAA AGT TTT CTA-3', nt 1497–1477). SAP4.1N (5'-CTT CCA TAT GAA TAA CGG TTC CTA TTC TCC-3', nt 533–562) and SAP4.2 primers were used to amplify an N-terminally deleted fragment of the ORF (including the catalytic domain of the aspartyl proteinase) of the SAP4 gene [SAP4({Delta}N)]. The pT7Blue T-Vector Kit (Novagen) was used for cloning of PCR products. All PCR products were purified, subcloned and sequenced by the dideoxy chain termination method to confirm their identity (Sanger et al., 1977 ). For the N-terminal His-Tag fusion proteins, the BamHI–SalI fragment of the pT7Blue T vector containing the SAP2 ORF was ligated to the expression vector pET30a (Novagen) and the NdeI–BamHI fragment of the pT7Blue T vector containing SAP3–6 was ligated to pET15b (Novagen).


E. coli BL21(DE3) competent cells were transformed with expression plasmids and grown on LB plates containing ampicillin (100 µg ml-1). For identification of colonies with the highest levels of expression of fusion proteins, transformants were grown for 3 h, followed by induction with IPTG (final concentration 0·5 mM) for 3 h. The total lysates prepared from 1 ml samples of each transformant were first assessed by SDS-PAGE under reducing conditions, followed by Coomassie blue R-250 (Bio-Rad) staining. For large-scale expression, 5 ml overnight culture were used to inoculate 1 l LB broth containing ampicillin and protein expression was induced with 0·5 mM IPTG for 3 h after the OD600 reached about 0·6. Cell pellets were suspended in 30 ml phosphate-buffered saline (pH 7·4) containing lysozyme (0·5 mg ml-1) and disrupted by a nitrogen bomb. The lysate was centrifuged after the addition of 6 M urea and 1% Triton X-100, and His-tagged fusion protein was isolated on Ni2+-nitrilotriacetic acid resin (Qiagen) as described by the manufacturer. The protein concentration was determined with a BCA Protein Assay kit (Pierce). Protein extracts were analysed by SDS-PAGE followed by Coomassie brilliant blue R-250 staining or silver staining (Pharmacia).

Polyclonal antibody preparation and Western blot analysis.
The His-tagged fusion proteins [Sap2, Sap3, Sap4({Delta}N), Sap5 and Sap6], purified from Ni2+-nitrilotriacetic acid resin, were further purified by SDS-PAGE separation because trace amounts of proteins of E. coli origin were contaminating the eluant samples. Denatured purified proteins were sliced out of the SDS-PAGE gel to be used as antigens to generate polyclonal antibodies in rabbits as described (Harlow & Lane, 1988 ; Huang et al., 1999 ). In an attempt to generate Sap4-, Sap5- and Sap6-specific antibodies, the following peptides were designed and synthesized by Genemed Synthesis: Sap4C (AQVKYTSQSNIVGIN, aa 413–427), Sap4M (VSVRDQLFANVR, aa 179–190), Sap5N (GPVAVTLHNEAIT, aa 77–89), Sap6C (YTSESNIVAIN, aa 408–418) and Sap6N (GPVAVKLDNEIIT, aa 77–89) (Fig. 1). Peptides for Saps 4, 5 and 6 were conjugated to keyhole limpet haemocyanin (Pierce) as described by the manufacturer and used as antigens to generate antibodies. We used an anti-His monoclonal antibody (Clontech; diluted 1:5000) to detect purified His-tagged fusion Sap proteins. For immunoblot analysis, antibodies were diluted in phosphate-buffered saline (pH 7·4) containing 0·1% Tween 20 and 5% dried skimmed milk. Sample proteins were separated by SDS-PAGE, transferred onto Immobilon-P membrane (Millipore) and incubated with the primary specific antibodies at room temperature for 60 min, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG (diluted 1:5000). Bound antibodies were detected with the ECL system (Amersham) according to the manufacturer’s instructions.



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Fig. 1. Diagrams of recombinant Sap2–6 proteins and synthetic peptides used to generate antibodies. The deduced amino acid sequences of Sap2–6 include a signal sequence (black box), a propeptide (grey box) and a mature protein (white box). GenBank accession numbers of the SAP genes are M83663 (SAP2), L22358 (SAP3), L25388 (SAP4), Z30191 (SAP5) and Z30192 (SAP6). The positions of the amino acids of recombinant proteins and synthetic peptides are numbered as indicated.

 
Induction of hyphae formation.
For hyphae growth, C. albicans were incubated in modified Lee’s medium (pH 4·5) (Lee et al., 1975 ; Morrow et al., 1992 ) at 25 °C for 48 h (stationary phase), transferred to pre-warmed modified Lee’s medium (pH 6·5) containing 0·2% BSA (final cell density approximately 5x106 cells ml-1), then incubated at 37 °C for 48–72 h. Growth was measured at OD600 and the pH values of the culture media were determined at the indicated times. Hyphae formation was visualized by phase-contrast microscopy and percentage of hyphae formation was calculated by counting of ~200 cells. For kinetic study, proteins prepared from culture media obtained from wild-type and mutant strains before and after hyphae induction were evaluated. To determine the expression of the intracellular Sap4–6, we prepared Candida total lysates by use of liquid nitrogen, followed by addition of lysis buffer as previously described (Lee et al., 1997 ).


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Specificity and sensitivity of polyclonal antibodies against Sap proteins
His-tagged fusion proteins of full-length Sap2, Sap3, Sap5 and Sap6 were expressed in E. coli and purified as described in Methods. N-terminally deleted Sap4, Sap4({Delta}N), was cloned and expressed because E. coli transformants carrying full-length Sap4 grew very poorly (data not shown). Purified recombinant His-tagged Sap proteins and synthetic peptides for specific Saps (Fig. 1) were used to immunize rabbits and produce specific polyclonal antibodies.

Table 2 shows that most antibodies can detect ~10 ng of their specific recombinant Sap antigens; however, antibodies against synthetic peptides were less sensitive. Fig. 2 shows that antibodies against Sap2 recombinant protein, Sap4M peptide, Sap5N peptide and Sap6N peptide reacted specifically with their own recombinant protein. Antibodies against recombinant Sap3, Sap6 and an N-terminally deleted fragment of Sap4 protein [Sap4({Delta}N)] proteins, as well as Sap4C and Sap6C peptides, had significant cross-reactivity with other members of the Sap subfamily, highlighting the difficulty of generating specific and sensitive antibodies against each Sap.


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Table 2. Sensitivity and specificity of polyclonal antibodies against recombinant fusion proteins and peptides determined by immunoblotting

 


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Fig. 2. Specificity of antibodies against recombinant Sap proteins. (A) ~100 ng of the indicated recombinant Sap were subjected to SDS-PAGE in 12·5% gels and visualized by silver staining. Positions of protein standards (kDa) are shown on the left. Proteins were transferred to Immobilon-P and reacted with anti-Sap2 (B), anti-Sap3 (C), anti-Sap4({Delta}N) (D), anti-Sap4M (E), anti-Sap5N (F), anti-Sap6 (G) or anti-Sap6N antibodies (H), followed by detection with the ECL system. The asterisk in (A) marks contaminant protein of E. coli origin, which was co-purified from the Ni2+-nitrilotriacetic acid resin.

 
Differential secretion of Sap4–6 proteins during hyphae formation
Proteins prepared from culture supernatants of wild-type strain SC5314 and sap mutants, induced for hyphae formation, were used to characterize the specificity of these antibodies and to study the differential expression of Sap4–6 proteins in C. albicans (Fig. 3). A single band (~40 kDa) was detected by anti-Sap5N in the wild-type strain, and in all strains except those lacking the SAP5 gene (Fig. 3B). No immunoreactivity was detected after incubation of anti-Sap5N antibodies, which were pre-depleted with purified recombinant Sap5 (data not shown). We inferred that the ~40 kDa band was Sap5 protein. In addition to the 40 kDa band, a second band (~43 kDa) was identified by anti-Sap4M in {Delta}sap6 strains (Fig. 3C). Furthermore, the upper band was denser in the {Delta}sap6 mutant than that in the {Delta}sap4 mutant or the wild-type strain, where we used anti-Sap4M or anti-Sap6 antibodies (Fig. 3C, D). We inferred that the ~43 kDa band was Sap4 protein (in the {Delta}sap6) or Sap6 protein (in the {Delta}sap4 mutant), or a mixture of both proteins. There was no detectable Sap2 or Sap3 under the same conditions (Fig. 3E, F). Anti-Sap6N and anti-Sap4M were specific for their own recombinant proteins; however, the sensitivity of these antibodies is not good enough for detection of secreted Sap4–6 in culture supernatants (data not shown). Although anti-Sap6 cross-reacted with Sap4 and Sap5 (Fig. 3D, {Delta}sap6 mutant), its high sensitivity was chosen for detecting Sap4–6 in the following studies.



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Fig. 3. Identification of Sap4–6 proteins in C. albicans after 24 h induction of hyphae formation. Wild-type strain SC5314 and sap mutants of C. albicans were grown in modified Lee’s medium (pH 4·5) for 48 h at 25 °C. Cells were then transferred to modified Lee’s medium (pH 6·5) containing 0·2% BSA at 37 °C to induce hyphae formation. Supernatants (30 µl) were collected from individual cell cultures (2 ml) at 24 h, subjected to SDS-PAGE in 12·5% gels, and proteins visualized by Coomassie brilliant blue R-250 staining (A). Proteins were transferred to Immobilon-P and reacted with anti-Sap5N (B), anti-Sap4M (C), anti-Sap6 (D), anti-Sap2 (E), and anti-Sap3 (F) antibodies, followed by detection with the ECL system. Positions of protein standards (kDa) are on the left.

 
To determine the differential expression of Sap4–6 in the induction of early hyphae formation, we took supernatants from wild-type strain and sap mutant cultures at the indicated times, and the immunoreactivity of Sap4–6 was detected by use of anti-Sap6 antibody (Fig. 4). Hyphae formed within 4 h of induction in wild-type strain, {Delta}sap4, {Delta}sap5 and {Delta}sap6, and the representative (wild-type) hyphae formation is shown in Fig. 4C. The percentages of hyphae formation in wild-type and mutants ({Delta}sap4, {Delta}sap5, {Delta}sap6 and {Delta}sap4–6) showed no significant difference, and >95% of cells formed hyphae within 6 h of induction (Fig. 4D). Sap5 (lower band in Fig. 4B) was first detected in supernatant from the wild-type culture after ~8 h of induction, whereas it was detected after ~4 h induction from the {Delta}sap4 and {Delta}sap6 mutants (Fig. 4B). However, Sap4/6 (upper band) was faintly detected after 24 h induction in the {Delta}sap4 and {Delta}sap6 mutants. Figs 3 and 4 together demonstrate that the upper band is Sap4 in the {Delta}sap6 mutant, Sap6 in the {Delta}sap4 mutant or a mixture of both in the wild-type strain. The denser bands in the {Delta}sap6 mutant may represent up-regulation of Sap4 and Sap5. Sap4–6 proteins were also detected during hyphae formation induced by temperature/pH shift in modified Lee’s medium without BSA (Buffo et al., 1984 ) (data not shown).



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Fig. 4. Differential secretion of Sap4–6 proteins during hyphae formation. Wild-type strain SC5314 and sap mutants of C. albicans were grown in modified Lee’s medium (pH 4·5) for 48 h at 25 °C. Cells were then transferred to modified Lee’s medium (pH 6·5) with BSA at 37 °C to induce hyphae formation. Supernatants (30 µl) were collected from individual cell cultures (2 ml) at the indicated times, subjected to SDS-PAGE in 12·5% gels and proteins visualized by Coomassie brilliant blue R-250 staining (A). Proteins were transferred to Immobilon-P and reacted with anti-Sap6 antibody (B), followed by detection with the ECL system. Hyphae formation in wild-type strain was visualized by use of phase-contrast microscopy at the indicated times (C). The percentages of hyphae formation in wild-type ({diamondsuit}), {Delta}sap4 ({blacksquare}), {Delta}sap5 ({blacktriangleup}), {Delta}sap6 ({circ}) and {Delta}sap4–6 ({square}) mutants were determined at the indicated times (D).

 
To determine whether Sap4/6 protein was produced in the intracellular form, but was not secreted into the culture medium after 24 h hyphae induction, we took cell lysates from wild-type strain and sap mutant cultures at 24 and 48 h, and the immunoreactivity of Sap4–6 was detected (Fig. 5). The small GTPase Arf1p was used as internal control in this study (Lee et al., 1997 ). Sap5 was clearly detected in all cell lysates except in {Delta}sap5; however, Sap4/6 was hardly detected in the wild-type and sap mutants after 24 h induction (Fig. 5). Consistent with the result shown in Fig. 4, Sap4 could clearly be detected in both cell lysate and culture supernatant after 48 h induction in the {Delta}sap6 mutant.



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Fig. 5. Production of intracellular Sap4–6 proteins during hyphae formation. Cell lysates (A) and supernatants (B) were prepared from wild-type strain and sap mutant cultures after 24 h or 48 h hyphae induction as described in Methods. Sap4–6 was reacted with specific antibodies as indicated, followed by detection with the ECL system. The small GTPase Arf1p was used as the internal control.

 
Effects of medium pH on the expression of Sap4–6 proteins
Hyphae formation was induced in modified Lee’s medium at neutral pH, and the pH of the medium fell gradually to 3·0–4·0 after 18–24 h (Fig. 6A, wild-type), which was approximately the time that Sap4/6 was detected in the medium (Fig. 6C). Under the same growth conditions, expression of Sap5 was detected at ~4 h ({Delta}sap4 or {Delta}sap6 mutant) or ~8 h (in wild-type) (Fig. 4B), and reached a higher level after ~48 h of hyphae induction (Fig. 6C). The drop in the medium pH was also observed in the {Delta}sap4, {Delta}sap5 and {Delta}sap6 cultures. To investigate whether a pH change in the culture medium could affect the expression of Sap4–6, different C. albicans strains were first grown in modified Lee’s medium (pH 4·5) at 25 °C for 48 h, then transferred to buffered modified Lee’s medium (pH 6·5) containing 0·2% BSA at 37 °C for induction of hyphae formation. The pH of the culture medium was buffered with sodium citrate to pH 6·5 or pH 4·5, at a final concentration of 50 mM. Hyphae formation of all tested strains was visualized by use of phase-contrast microscopy, and representative cells with hyphae formation at the indicated times are shown in Fig. 7. In the unbuffered control and buffered pH 6·5 cultures, hyphae formation was observed at ~2 h induction, approached ~90% after 3–4 h (Fig. 7), and >95% at 6–48 h (data not shown). Compared to the expression in the unbuffered control culture, the expression of Sap5 was the same or only slightly lower in a buffered pH 6·5 medium in the wild-type and in the {Delta}sap4 and {Delta}sap6 mutants. The upper band was detected as early as ~12 h in the wild-type and the {Delta}sap4 and {Delta}sap5 mutants, but was hardly detected even at 48 h in the {Delta}sap6 mutant (Fig. 6C). The results suggested that the upper band detected at pH 6·5 at 12 h was Sap6. In the buffered pH 4·5 culture, most hyphae were formed as pseudohyphae at ~2 h induction and approached ~80% after ~6 h induction (Fig. 7). Interestingly, Sap5 was detected at significant levels only in {Delta}sap6, and Sap4/6 could still be detected at low levels in the wild-type and {Delta}sap4 mutant in buffered pH 4·5 cultures (Fig. 6C).



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Fig. 6. The pH of the culture medium affects expression of Sap4–6 proteins. Wild-type strain SC5314 and the {Delta}sap4, {Delta}sap5 and {Delta}sap6 mutants were grown in modified Lee’s medium (pH 4·5) at 25 °C for 48 h. Cells were then transferred to modified Lee’s medium (pH 6·5) with 0·2% BSA, with or without sodium citrate buffer [pH 6·5 ({circ}) or 4·5 ({bigtriangledown})], at 37 °C to induce hyphae formation. {bullet}, Control. The pH of the culture media was measured at the indicated times (A). Supernatants (30 µl) were collected from individual cell cultures (2 ml) at the indicated times, subjected to SDS-PAGE in 12·5% gels and proteins visualized by Coomassie brilliant blue R-250 staining (representative {Delta}sap6 in B). Proteins were transferred to Immobilon-P and reacted with anti-Sap6 (C), followed by detection with the ECL system.

 


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Fig. 7. The pH of the culture medium affects hyphae formation. Wild-type strain SC5314 and the {Delta}sap4, {Delta}sap5 and {Delta}sap6 mutants were grown and hyphae formation induced as described in Fig. 6. Hyphae formation was visualized by use of phase-contrast microscopy at the indicated times. Hyphae and pseudohyphae formation (%) of wild-type strain SC5314 is shown in (A). {diamondsuit}, Control; {blacksquare}, pH 6·5; {blacktriangleup}, pH 4·5. After 6 h hyphae induction, phase-contrast images of hyphae and pseudohyphae formation of wild-type and {Delta}sap5 strains at different culture pH are shown in panel B as indicated. Parts of (B) are composite images.

 
Effects of Saps on the degradation of BSA during hyphae formation
Comparing the residual amount of BSA in culture medium at different time intervals (Fig. 6B), degradation of BSA was noticed after ~48 h induction in the {Delta}sap6 mutant and was correlated with the detection of higher expression of Sap4 in the culture supernatants. In buffered medium, no degradation of BSA was observed after 48 h induction. Despite the relatively stable level of expression of Sap5 in the {Delta}sap6 mutant during 12–48 h (with or without control of the medium pH), there was no significant degradation of BSA as shown on SDS-PAGE, suggesting that other Saps, but not Sap5, might be involved in the degradation of BSA. To determine further whether specific Saps play a role in the degradation of BSA during hyphae formation, wild-type strain SC5314, {Delta}sap1, {Delta}sap2, {Delta}sap3, {Delta}sap4, {Delta}sap5, {Delta}sap6, {Delta}sap4–6 and {Delta}efg1 {Delta}cph1 (defective for hyphae formation; Lo et al., 1997 ) mutants were grown and hyphae formation was induced. Fig. 8 shows that, compared with the {Delta}sap6 mutant, degradation of BSA was also evident, to a lesser extent, in the wild-type and {Delta}sap4–6 mutants after ~48 h induction. Interestingly, there were no detectable levels of Sap4–6 proteins after induction in the {Delta}efg1 {Delta}cph1 double mutant.



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Fig. 8. Effect of Saps on the degradation of BSA during hyphae formation. Wild-type strain SC5314 and the {Delta}sap1, {Delta}sap2, {Delta}sap3, {Delta}sap4, {Delta}sap5, {Delta}sap6, {Delta}sap4–6 and {Delta}efg1 {Delta}cph1 mutants were grown and hyphae formation was induced as described in Fig. 6. (A) Supernatants (30 µl) were collected from individual cell cultures (2 ml) at the indicated times, subjected to SDS-PAGE in 12·5% gels and proteins visualized by Coomassie brilliant blue R-250 staining. (B) Proteins were transferred to Immobilon-P and reacted with anti-Sap6, followed by detection with the ECL system.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Sap4–6 production and characterization
The present study was the first to demonstrate detectable levels of Sap5 (~40 kDa) and Sap4/6 (~43 kDa) in culture supernatants of C. albicans during hyphae formation in Lee’s medium. Our data agree with the previous finding that the relative molecular mass of Sap5, expressed and secreted in the Pichia pastoris system, is lower than those of Sap4 and Sap6 (Borg-von Zepelin et al., 1998 ). The difference in the relative molecular mass of Sap4–6 might be due to potential glycosylation sites in Sap4 and Sap6, but not Sap5.

We attempted to generate antibodies with higher sensitivity and specificity against Sap4–6; however, only anti-Sap4M, anti-Sap5N and anti-Sap6N have characteristics that can specifically detect their recombinant forms. Borg-von Zepelin et al. (1998) , using antibodies generated against recombinant Sap proteinases prepared in the P. pastoris system, also failed to distinguish Sap4 and Sap6. Although recombinant Sap1–6 proteins prepared from the P. pastoris (Borg-von Zepelin et al., 1998 ) and the Escherichia coli expression systems (Koelsch et al., 2000 ) have been purified and characterized, only ‘native’ secreted Sap2p from the culture supernatant of C. albicans has been purified and characterized.

A differential expression of individual SAP mRNAs has been shown in vitro and in animal models (Morrow et al., 1992; Hube et al., 1994 ; White & Agabian, 1995 ; Borg-von Zepelin et al., 1998 ; Monod et al., 1998 ). The temporal and specific regulation of SAP gene expression might be important for the survival of C. albicans in its natural environment and thus in the pathogenesis of this fungus. However, it has been shown recently that the correlation between mRNA and protein levels was insufficient to predict protein expression levels from quantitative mRNA data (Gygi et al., 1999 ). For some genes, where the mRNA levels were the same, the protein levels varied by more than 20-fold. However, invariant steady-state levels of certain proteins were observed with respective mRNA transcript levels that varied by as much as 30-fold. Because studies on the secreted form of Sap protein levels are limited, the special role of the individual Sap proteins in infection remains unclear (Cutfield et al., 1995 ; Smolenski et al., 1997 ; Hube & Naglik, 2001 ).

Differential secretion of Sap5 and Sap4/6
Our differential expression studies showed that Sap5 was produced and secreted earlier than Sap4/6 during hyphae formation (Fig. 4). Although it seems to be impossible to compare an in vitro expression study using defined induction medium and a study with cells from infected tissue, our findings agree with a recent study of in vivo expression of SAP genes in animal models, which showed that SAP5 was the first SAP gene induced after intraperitoneal infection or haematogenous dissemination (Staib et al., 2000 ). SAP5 expression at this stage of the infection did not correlate with the presence of germ tubes or hyphae, and SAP6 gene activation was detected only when C. albicans hyphae also were observed in the infected tissue (Staib et al., 2000 ). Furthermore, in the {Delta}efg1 {Delta}cph1 double mutant, which is an avirulent mutant locked in the yeast form (Lo et al., 1997 ), there were no detectable levels of Sap4–6 proteins after induction. These data suggest that the expression of Sap5 may be induced by host signals that also result in hyphae formation. However, our data indicated that hyphae formation can be induced in wild-type, {Delta}sap4, {Delta}sap5 and {Delta}sap6 mutants, and Sap4–6 proteins were hardly detected even at 48 h in the {Delta}sap5 mutant, suggesting that production of Sap5 as well as Sap4/6 was not required for hyphae formation (Figs 6C and 7B). SAP4–6 mRNAs were first detected during serum-induced hyphal induction (Hube et al., 1994 ; White & Agabian, 1995 ). Our data also showed that Sap4–6 proteins could not be detected in the culture supernatants until hyphae formation occurred and Sap4–6 could not be detected in the {Delta}efg1 {Delta}cph1 double mutant, suggesting that hyphae formation might be essential for Sap4–6 expression. With regard to the time of maximal Sap production during hyphal induction and the existence of intracellular pools of Sap4–6, our findings were consistent with previous studies (Hube et al., 1994 ; White & Agabian, 1995) and suggested that maximal Sap protein production can be later than the maximal mRNA level.

In the {Delta}efg1 {Delta}cph1 double mutant, which is defective for hyphae formation (Lo et al., 1997 ), there were no detectable levels of Sap4–6 proteins after induction (Fig. 8). However, a recent study demonstrated that wild-type expression levels of Efg1 are not sufficient for regular hyphae development, expression of SAP4–6 genes and evasion from macrophage in the absence of CaTec1, a TEA/ATTS transcription factor in C. albicans (Schweizer et al., 2000 ). Besides, the catec1 null mutant is almost avirulent in a systemic model of candidiasis, despite the formation of hyphae. It seems that Sap4–6 proteins are the downstream effector of these either coordinately regulated or independent signalling pathways (Schweizer et al., 2000 ; Felk et al., 2002 ).

White & Agabian (1995) showed that, of the three SAP4–6 genes, expression of SAP6 mRNA is the highest during hyphae induction at neutral pH. However, our data indicated that production of Sap5 was highest during hyphae induction at pH 6·5. Interestingly, up-regulation of Sap4/6 occurred in the {Delta}sap5 mutant at pH 6·5 (Fig. 6C, pH 6·5 panel). In the buffered pH 6·5 culture, secretion of Sap4/6 in wild-type, {Delta}sap4 and {Delta}sap5 mutants is earlier than that in unbuffered culture. Moreover, Sap5 was detectable in the {Delta}sap6 mutant at a low level in buffered pH 4·5 medium (Fig. 6). The culture pH affected not only the expression of Sap4–6 proteins, but also the induction of hyphae and pseudohyphae formation. De Bernardis et al. (1998) suggested that gene expression and virulence of C. albicans are controlled by the pH of the host microniche. Our data also suggest that the low pH values influence hyphae formation and consequently may change the expression pattern of SAP4–6 genes. Therefore, changes in the pH may affect the expression pattern only indirectly.

It was shown that there was no SAP4 mRNA when SAP6 mRNA was first detected in culture medium in in vitro cell cultures, or in animal models (White & Agabian, 1995 ; Schaller et al., 1998 , 1999 ; Staib et al., 2000 ), except after haematogenous dissemination to the kidneys (Staib et al., 2000 ). Although differential expression of Sap4/6 can be detected when the medium pH is unbuffered or buffered at 6·5 in the wild-type strain and in sap mutants (Fig. 5C), it is difficult to determine which is expressed first. Until specific antibodies against Sap4 or Sap6 are generated, all of these findings can provide only indirect evidence that Sap6 and Sap4 might not be expressed under the same conditions, and/or play a different role in C. albicans. A combination of Sap4/6-specific antibodies and specific mRNA detection may also provide evidence as to which Sap is expressed under which conditions.

BSA degradation in Lee’s medium
Fig. 6(B, C) shows that expression of Sap4/6 was associated with significant degradation of BSA at 48 h hyphae induction, when the culture reached pH 4·0. In the same growth culture, Sap2 and Sap3 were not detected. Our result agrees with that of Hube et al. (1994) , who previously reported that the SAP2 gene was not expressed during serum-induced hyphae formation. BSA or serum is an important component in the induction of Sap2 (Homma et al., 1993 ; White & Agabian, 1995 ) and SAP4–6 expression (Hube et al., 1994 ). This may be important for Candida to survive in hosts because it would enable the yeast to use host proteins as a nitrogen source. In addition, Candida expresses virulence factor(s) to overcome the host immune defence after exposure to intravascular component(s) (Hube & Naglik, 2001 ). In this study, during hyphae formation the medium pH fell gradually, and there was no degradation of BSA at 24 h induction, despite the presence of a significant amount of Sap5. Although expression of Sap4 was observed when BSA was degraded in the culture medium at ~48 h of induction (Fig. 6B, C), there was still significant degradation of BSA in {Delta}sap4–6 (Fig. 8), suggesting that other Saps, but not Sap4–6, might be involved in the degradation of BSA. Another scenario is that Sap4–6 are the main enzymes involved in BSA degradation during hyphae formation, but that other Sap enzymes are up-regulated in the {Delta}sap4–6 mutant and have a compensatory function.

Conclusion
Many members of the SAP gene family have been identified. This raises the question whether they exist in redundancy or play a special role under different environmental or physiological conditions (Hube et al., 1997 ; Sanglard et al., 1997 ; De Bernardis et al., 1999 ; Kretschmar et al., 1999 ; Naglik et al., 1999 ; Staib et al., 2000 ; Felk et al., 2002 ). The present study was the first to demonstrate clearly that secretion of Sap5 in C. albicans was induced during hyphae formation, followed by secretion of Sap4/6 under acidic conditions, which occurred in parallel to the degradation of BSA. These results are consistent with the in vivo expression patterns (Staib et al., 2000 ) and suggest that Sap5 might play a key role in the initial phase of Candida infection.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the National Science Council, R. O. C. (NSC 88-2314-B-002-068), and National Taiwan University Hospital (NTUH 89S1534). The authors are grateful to Drs D. Sanglard, Bernhard Hube, W. A. Fonzi and G. R. Fink for providing the strains of C. albicans. We thank Drs H. M. Hassan, S. J. Libby and R. Haun for critical reading of the manuscript.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 30 April 2002; revised 22 July 2002; accepted 8 August 2002.