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
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ABSTRACT |
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Keywords: Candida albicans, secreted aspartyl proteinase, virulence factor, candidiasis
Abbreviations: Sap, secreted aspartyl proteinase
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INTRODUCTION |
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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 (
sap1 to
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 Sap46, which have 7589% similarity to each other, form a group distinct from Sap13 (Hube et al., 1994 ; Miyasaki et al., 1994
). This subfamily, Sap46, was a potential target for intervention because SAP46 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, Sap46 proteins were presumed to be important in disseminated candidiasis.
Recently, it has been demonstrated that a sap46 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 SAP46 genes contribute to the development of disseminated infections. Sap46 proteins have been detected in Candida within macrophages (Borg-von Zepelin et al., 1998
); however, production of the extracellular form of Sap46 during hyphae formation has never been characterized.
In this study, we generated specific antibodies against each of the Sap26 proteins and determined the secretion of Sap46 by C. albicans during hyphae formation. The effect of culture medium pH on secretion of Sap46 during hyphae formation was also examined. Here, we show that production and secretion of Sap46 proteins in C. albicans are differentially regulated during hyphae formation in a pH dependent manner.
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METHODS |
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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(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 413427), Sap4M (VSVRDQLFANVR, aa 179190), Sap5N (GPVAVTLHNEAIT, aa 7789), Sap6C (YTSESNIVAIN, aa 408418) and Sap6N (GPVAVKLDNEIIT, aa 7789) (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 manufacturers instructions.
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RESULTS |
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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(
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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DISCUSSION |
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We attempted to generate antibodies with higher sensitivity and specificity against Sap46; 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 Sap16 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
efg1
cph1 double mutant, which is an avirulent mutant locked in the yeast form (Lo et al., 1997
), there were no detectable levels of Sap46 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,
sap4,
sap5 and
sap6 mutants, and Sap46 proteins were hardly detected even at 48 h in the
sap5 mutant, suggesting that production of Sap5 as well as Sap4/6 was not required for hyphae formation (Figs 6C
and 7B
). SAP46 mRNAs were first detected during serum-induced hyphal induction (Hube et al., 1994
; White & Agabian, 1995
). Our data also showed that Sap46 proteins could not be detected in the culture supernatants until hyphae formation occurred and Sap46 could not be detected in the
efg1
cph1 double mutant, suggesting that hyphae formation might be essential for Sap46 expression. With regard to the time of maximal Sap production during hyphal induction and the existence of intracellular pools of Sap46, 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 efg1
cph1 double mutant, which is defective for hyphae formation (Lo et al., 1997
), there were no detectable levels of Sap46 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 SAP46 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 Sap46 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 SAP46 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
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,
sap4 and
sap5 mutants is earlier than that in unbuffered culture. Moreover, Sap5 was detectable in the
sap6 mutant at a low level in buffered pH 4·5 medium (Fig. 6
). The culture pH affected not only the expression of Sap46 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 SAP46 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 Lees 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 SAP46 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
sap46 (Fig. 8
), suggesting that other Saps, but not Sap46, might be involved in the degradation of BSA. Another scenario is that Sap46 are the main enzymes involved in BSA degradation during hyphae formation, but that other Sap enzymes are up-regulated in the
sap46 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.
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ACKNOWLEDGEMENTS |
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Received 30 April 2002;
revised 22 July 2002;
accepted 8 August 2002.