The Proteolytic System of Candida dubliniensis

Proteases of Candida dubliniensis have been scarcely studied, these enzymes may play an important role in nitrogen metabolism, post-translational processing, nutritional stress, dimorphism, virulence, etc. In this work, we report the presence of five different intracellular proteases and one extracellular proteolytic activity. The intracellular proteases are: aminopeptidase ycdAPE, carboxypeptidase ycdCP, dipeptidyl aminopeptidase ycdDAP, proteinases ycdPrA and ycdPrB, and extracellular protease Sap activity, measured under several nutritional conditions. C. dubliniensis produced the highest level of intracellular proteolytic enzymes, i.e., ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB in media with peptone during stationary growth phase. Chelating agents affected mainly APE activity; whereas ycdCp, ycdDAP, and ycdPrB were affected by serine protease inhibitors; ycdPrA was affected by pepstatin, an aspartyl protease inhibitor. We found Sap activity in C. dubliniensis in YCB-SBA medium, this activity was inhibited by pepstatin inhibitor. Southern analysis revealed the presence of at least four genes encoding Sap in the C. dublinienisis genome (using as probes SAP1, SAP2, SAP3, and SAP4-6 genes from C. albicans).


INTRODUCTION
Proteolysis, a vital process for cell life, plays an important role in different physiological functions, such as protein digestion, hormone maturation, immune response, inflammation, fertilization, germination, as well as in other morphological processes. Some proteases are important in spores formation and germination, pathogenesis of several microorganisms, and post-translational regulation [1][2][3] .
Candida dubliniensis was first described as a novel species in 1995. This organism is very close to the human yeast pathogen, Candida albicans. However, despite the close phylogenetic relationship between C. albicans and C. dubliniensis, phenotypic and molecular data suggest differences in virulence between these two species. The apparent low virulence of C. dubliniensis could be due to differential gene expression rather than to simple absence or divergence of particular genes [4] .
The study of Saccharomyces cerevisiae has been important in the elucidation of proteinase multiplicity and proteinase function in eukaryotic cells, numerous proteolytic enzymes have been biochemically and genetically characterized [5] . There are several proteases associated with yeast proteasomes that participate in stress-dependent and ubiquitin-mediated proteolysis. They are involved in the degradation of short-lived and regulator proteins that generate small peptides from proteins, these latter are degraded into amino acids by tri-, di-, carboxi-, and amino-peptidases [6][7] .
Extracellular proteolytic activity plays a central role in Candida albicans pathogenicity and is produced by a family of 10 secreted aspartyl proteinases (Sap proteins) [3] . In vitro, animal and human studies have implicated proteinases in C. albicans virulence in one of the following ways: (i) correlation between Sap production in vitro and Candida virulence, (ii) degradation of human proteins and structural analysis in determining Sap substrate specificity, (iii) association of Sap production with other virulence processes of C. albicans, (iv) Sap protein production and Sap immune responses in animal and human infections, (v) SAP gene expression during Candida infections, (vi) modulation of C. albicans virulence by aspartyl proteinase inhibitors, and (vii) the use of SAP-disrupted mutants to analyze C. albicans virulence. Sap proteins fulfill a number of specialized functions during the infective process, which include the simple role of digesting molecules for nutrient acquisition, digesting or distorting host cell membranes to facilitate adhesion and tissue invasion, and digesting cells and molecules of the host immune system to avoid or resist antimicrobial attack by the host [3] .
Detailed analyses of proteases in C. dubliniensis might allow us to determine the proteases produced by this fungus and to examine the production of these enzymes in several nutritional conditions and during the dimorphic transition of yeast to mycelia, which is considered to be an important factor in the pathogenicity of this fungus. Moreover, numerous studies have correlated extracellular proteolytic activity in vitro with the virulence of Candida species and have shown that the most virulent species, such as C. albicans, C. tropicalis, and C. parapsilosis, produce more proteinases in vitro than the less virulent species [3] . Secreted aspartic proteinases have been considered a new target in candidiasis therapy [8] . In this work, we report the presence of five different intracellular proteases and one extracellular proteolytic activity in C. dubliniensis.

MATERIALS AND METHODS
Strains and growth conditions: Strains C. dubliniensis CD36 and C. dubliniensis CD92 were provided by Dr.
Derek J. Sullivan of the University of Dublin [9] , C. albicans ATTC 10231 was used as reference strain in this study.
The C. dubliniensis strains were confirmed to the species level using molecular methods described by Bautista et al. [10] . They were routinely maintained on YEPD plates (1% yeast extract, 2% peptone, 2% glucose, and 2% agar) at 28ºC. The ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB activities in soluble and membrane fractions of C. dubliniensis were measured by growing the yeast in YEPD and minimal medium broths containing 0.17% yeast nitrogen base without amino acids or ammonium sulfate, but adding 2% glucose with 0.5% nitrogen source (peptone, proline, or ammonium sulfate) according to manufacturer's instruction. Production of Sap was performed in YCB-SBA medium (1.2% yeast carbon base, 0.2% SBA, pH 4.0 adjusted with 1 M NaOH). To evaluate cell growth, absorbance (A 600 ) of culture samples was measured in a Perkin-Elmer Lambda IA spectrophotometer.
Preparation of crude extracts and differential centrifugation: Biomass from the culture medium was recovered by centrifugation at 5,000 g at 4ºC for 10 min. Cells were fragmented in Braun´s mill using glass beads (0.5 mm diameter). The mixture contained 7.5 g washed acid beads, 12 mL 0.1M Tris-HCl, pH 7.5, and 5 g cells. Total disintegration time was 10 min (10 series, 1 min each). The extent of disintegration was monitored under light microscopy. The crude extract was carefully removed from the glass beads and centrifuged at 10,000 g at 4ºC for 10 min. The supernatant fluid was removed and centrifuged again at 100,000 g at 4ºC for 1.5 h using a Beckman ultracentrifuge. The 100,000 g supernatant (S 100,000 g) was considered as soluble fraction and the membranal fraction was precipitated at 100,000 g (M 100,000 g).

Enzyme assays and protein determination:
Intracellular enzymatic activities were determined as described by Hirsch et al. [5] . The following substrates were used for the different proteases: L-lysyl-4nitroanilide (Lys-4-NA) for aminopeptidase activity (ycdAPE); N-benzoyl-tyrosine-4-nitroanilide (N-BZ-Tyr-4-NA) for carboxypeptidase activity (ycdCP), and L-alanyl-prolyl-4-nitroanilide (Ala-pro-4-NA) for dipeptidyl aminopeptidase activity (ycdDAP), acid denatured hemoglobin for proteinase A activity (ycdPrA), and Hide Powder Azure (HPA) for proteinase B activity (ycdPrB) [11] . Assay for Sap activity was based on the MacDonald and Odds [12] assay, enzyme activity was measured spectrophotometrically following digestion of BSA as substrate. A typical reaction mix containing 500 l 2.0% (w/v) BSA in 50 mM sodium citrate (pH 3.2), 100 l 50 mM sodium citrate (pH 3.2), and 200 l of culture supernatant was incubated for 30 min at 37 o C. The reaction was stopped by adding 200 l 10% TCA, precipitated protein was removed by centrifugation at 22,000 g for 5 min, 75 l of the supernatant was removed and added to 75 l 0.5M NaOH. Tyrosinecontaining peptides in the neutralized sample were determined with the Folin´s reagent according to the method of Lowry. Protein determinations were performed according to the Bradford method [13] .

Effect of protease inhibitors on proteolityc activities:
The effect of protease inhibitors (leupeptin, pefabloc, E-64, EDTA, pepstatin, 1-10, phenanthroline, and PMSF) on the proteolytic activities was measured in 100 mM Tris-HCl buffer, pH 7.5. The soluble fraction (100,000 g) or the culture medium's supernatant, after harvesting cells (for Sap activity), was preincubated with the respective compound for 30 min at 37ºC, followed by the standard enzyme assay.

Southern hybridization:
Genomic DNA from C. dubliniensis was digested with EcoRI endonuclease, subsequently separated by electrophoresis in agarose and transferred to a Nylon membrane positively charged (Amersham Pharmacia Biotech, UK), as described by Sambrook et al. [14] . Hybridization was performed at 65ºC with SAP1, SAP2 SAP3, or SAP4-6 specific probes labeled with digoxigenin-dUTP (20deoxyuridine 50-triphosphate) using a random-primed digoxigenin (DIG) DNA labeling detection kit (Roche, Mannheim, Germany). Hybridization and immunological detection were performed as recommended by the supplier. SAP DNA probes to be used in hybridization experiments were generated for PCR from C. albicans SAP genes: SAP1, SAP2, SAP3, or SAP4-6 as described by Bautista-Muñoz et al. [10] . Genomic DNA from C. albicans ATCC 10231 was used as template.

ycdAPE, ycdCP, ycdDAP, ycdPrA, ycdPrB, and Sap proteolytic activities under different nitrogen sources:
C. dubliniensis presented ycdAPE, ycdDAP, ycdCP, ycdPrA and ycdPrB intracellular activities. The highest levels of the three exopeptidases ycdAPE, ycdDAP, ycdCP proteolytic enzymes were reached in the medium enriched with peptone as nitrogen source: YPD and YNB-peptone, Table 1. These intracellular exopeptidases were accumulated during the stationary phase (48 h). All of them were localized in the soluble fraction (S 100,000 g), Table 2. The highest level of intracellular acidic proteinase ycdPrA was reached in the medium with amonium as nitrogen source during the logarithmic phase (6 h), Table 1. This activity was localized in the soluble fraction (S 100,000 g) although considerable ycdPrA activity was also found in the membrane fraction (M 100,000 g), Table 2. The production of proteinase ycdPrB was regulated by the nitrogen source, the highest level of this proteinase was reached in the medium with peptone and proline during early stationary phase, Table 1. This activity was localized in the soluble fraction (S 100,000 g), Table 2. It was not possible to detect Sap activity in YPD and YNBdifferent nitrogen sources media. The medium YCB-SBA has been described as inductor medium for the production of Sap in C. albicans, this medium was also an inductor for the production of C. dubliniensis Sap activity. There was no difference in the level of Sap activity between C. dubliniensis and C. albicans, Fig. 1.

Effect of inhibitors on enzymes activity:
The effect of seven inhibitors was tested in the S 100,000 g fraction (YPD medium) with ycdAPE, ycdDAP, ycdCP, ycdPrA and ycdPrB, as well as in the supernatant of the culture medium (YCB-SBA) after harvesting cells with Sap activity. Chelating agents, such as EDTA and 1-10 phenanthroline, exerted a clear inhibitory effect on ycdAPE activity, suggesting that this protease is a metalloenzyme. PMSF, pefabloc (serine protease inhibitors) caused inhibition of the three enzymes (ycdAPE, ycdDAP, and ycdCP). E-64 (cysteine proteases inhibitor) had the same effect on ycdAPE activity; however, leupeptin (cysteine and serine proteases inhibitors) only inhibited ycdPrB, Table 3. Acidic proteinases ycdPrA and Sap were inhibited by pepstatin (aspartyl protease inhibitor). Results suggest that ycdAPE is a metallo-serin protease; ycdDAP, ycdCP and ycdPrB are serine proteases; and ycdPrA and Sap are aspartyl proteases.
Presence of SAP genes in C. dubliniensis genome: C. dubliniensis presented extracellular aspartyl proteinase activity with the inductor medium YCB-SBA. We Genomic DNA from C. dubliniensis CD36 and CD92 isolates was digested with EcoRI endonuclease and Southern transfered to nylon membrane after gel electrophoresis. C. dubliniensis CD36 and CD92 gave identical hybridizing fragments for SAP1, SAP2, SAP3, SAP4-6 probes, Fig. 2. The restriction fragment sizes (band hybridizing) to probe SAP1 were: 4.8 and 4.0 kb; to probe SAP2: 4.8, 4.0, and 1.4 kb; to probe SAP3: 4.8 kb; and to probes SAP4-6: 3.3 kb. These results confirm the similitude that exists between nucleotidic sequences of SAP1, SAP2, and SAP3 genes. However, only one band hybridizing to probe SAP4-6 was revealed in the C. dubliniensis genome. Cellular fractions were prepared from cells grown in the corresponding medium. The 100,000 g soluble fraction was used to determine ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB enzymatic activities against Lys-4-NA, Ala-Pro-4-NA, N-Bz-Tyr-4-NA, acid-denatured hemoglobin and HPA. 100 100 0 a Cellular fractions were prepared from cells growing in the optimal YEPD media. Biomass from the culture media was recovered by centrifugation at 5 o C (5,000 g), and fragmented in Braun´s mill using glass beads (0.5 mm diameter).The extent of disintegration was monitored under light microscopy. The crude extract was carefully removed from the glass beads and centrifuged at 10,000 g at 5ºC for 10 min. The supernatant fluid was removed and centrifuged again at 100,000 g at 4ºC for 1.5 h using a Beckman ultracentrifuge. The 100,000 g supernatant (S 100,000 g) was considered as soluble fraction and the membranal fraction was precipitated at 100,000 g (M 100,000 g). Table 3: Effect of specific inhibitors of proteases on the activity of the intracellular proteases ycdAPE, ycdCP, ycdDAP, ycdPrA and ycdPrB and extracellular Sap activity from C. dubliniensis a Expressed as a percentage of the activity obtained in the absence of any added inhibitor, which was assigned a value of 100 %. b nd: non determined.

DISCUSSION
This paper presents, as far as we know, the first report on intracellular proteases of C. dubliniensis and suggests four encoding SAP genes on the genome of this CNCA (Candida no C. albicans). Proteolysis plays an important role in different physiological functions of the cell and the recently discovered secreted aspartic proteinase multi-gene (SAP) family in C. albicans has complicated assessment of proteolytic activity as a factor in the onset and development of Candida infections.
C. dubliniensis presented a soluble ycdAPE activity, this activity seems to be a metalloprotease, according to the results obtained in the inhibition assays. This inhibitory effect has been observed in other aminopeptidases isolated from yeasts, such as AP-Y from S. cerevisiae [23] , aminopeptidase I from S. pombe [24] , lysine aminopeptidase from Kluyveromyces marxianus [25] , lysine aminopeptidase from Y. lipolytica [19] . The presence of blocking agents from serine and cysteine proteases, such as pefabloc and PMSF, inhibited the activity, suggesting that serine residues and thiol groups might be participating in the catalysis of this enzyme. Most of the aminopeptidases described in lactic bacteria are metalloproteases that have a very similar inhibition pattern to that of the aminopeptidase from C. dubliniensis [26][27] . An immunogenic aminopeptidase of C. albicans has been described as a metallopetidase of 52 kDa and has been proposed for the diagnosis of systemic candidiasis [28] . Two carboxypeptidases have been described mainly in S. cerevisiae: yscCPY and yscCPS [29] . We detected one carboxypeptidase activity that was located in the soluble fraction; ycdCP activity in C. dubliniensis is associated with an endogenous inhibitor, since it was necessary to incubate the extracts with 0.5% sodium deoxycolate to detect the activity of this enzyme [16] (data not shown). ycdCP activity seems to be a serine protease. CYP1 C. albicans gene encodes a protein with 74% identity with CYP from S. cerevisiae. Reports on carboxypeptidase actvities in C. albicans are scarce; howewer, an extracellular carboxyl proteinase produced by the yeast C. albicans enhanced vascular permeability when injected into the dorsal skin of guinea pigs [30] .
We detected a ycdDAP activity localized in the soluble fraction. The activity of ycdDAP was inhibited by E-64, PMSF, and pefabloc, indicating that it is probably a serine protease. It is interesting to note that the dipeptidyl aminopeptidase of S. cerevisiae, called yscIV, is a membrane serine exopeptidase involved in the processing of the -sexual factor [29] . A dipeptidylaminopeptidase encoded in the STE13 gene has been proposed in C. albicans that could participate in the processing of a putative -sexual factor or of other peptides [31] . The ycdDAP found in C. dubiniensis might correspond to the activity that participates in controlling post-translational processing of other peptides.
An acidic proteinase intracellular activity was detected in the soluble fraction (S 100,000 g). This ycdPrA activity is an aspartyl proteinase (inhibited by pepstatin). Probably this activity is similar to yscA from S. cerevisiae, a soluble vacuolar endoprotease that participates in protein degradation, nitrogen metabolism, acting like a vacuolar hydrolase, and precursor proccesing [29] . In other yeasts, like C. albicans, S. pombe, K. lactis, and Ustilago maydis, an intracellular acidic aspartyl PrA, inhibited by pepstatin [2,18,11] has also been described.
The ycdPrB activity was detected in the soluble fraction (S 100,000 g) and seems to be a metallo-serinecysteine protease. In S. cerevisiae, the gene that encodes vacuolar proteinase yscB activity is regulated by nitrogen and carbon sources, and depends on the growth phase [32] . In turn, K. lactis produces a yklB similar to that detected in S. cerevisiae [18] . Likewise, it is known that yeasts S. pombe and C. albicans produce proteinase B activities called yspB and ycaB, respectively [2] .
In S. cerevisiae, yscB participates in the proteolytic processing during maturation of other enzymes [29] . The role played by the ycdPrB detected in C. dubliniesis must still be elucidated. In Saccharomyces carlbergensis, proteinase B activity is regulated by an endogenous inhibitor as occurs in S. cerevisiae [33] .
The studied yeast presents Sap activity that is associated to the logarithmic growth phase, and the enzymatic levels are higher when a protein, such as albumin, provides the nitrogen source. Similarly to the Sap described for C. albicans, the C. dubliniensis Sap is synthesized when the initial pH of the medium is acidic (3.0-5.0), suggesting the relation of one or more aspartyl proteinases, considering that in other Candida species more than one Sap activity have been found [34] . In C. albicans, the sequences of each of the SAP1-SAP10 genes described up to now are highly homologous among them [35] . Southern analysis, using heterologous probes of C. albicans for genes SAP1, SAP2, SAP3, SAP4-SAP6, revealed hybridization bands of different molecular size for the SAP genes in the C. dubliniensis genome. Since the sequence of the genome of this yeast is not known yet, we can only assure that C. dubliniensis possesses four homologous sequences to those of the SAP genes described in C. albicans. Most probably the 4.0 kb hybridization band is the homologous of the SAP1 gene of C. albicans; the 1.4 kb hybridization band would correspond to the SAP2 gene; the 4.8 kb band would be homologous to the SAP3 gene. The single 3.3 kb hybridization band would correspond to any of the three SAP genes (SAP4, SAP5, SAP6) of C. albicans. Currently, our group is working on the deduced nucleotidic and amino-acidic sequence of these four genes.
Gilfillan et al. [36] reported the presence of homologue sequences to the SAP genes described in C. albicans, through Southern analysis of chromosomes separated by PFGE and hybridization of heterologous probes of SAP1, SAP2, SAP3, SAP5, and SAP7. Since genes SAP4, SAP5, and SAP6 were 90% identical and could not be distinguished through hybridization with specific probes for each one, the gene SAP5 was used as probe to search for the presence of SAP4-SAP5-SAP6.
C. dubliniensis is an attractive model to study the production of proteases because it is a recently identified species that is implicated in oral candidosis in HIV-infected and AIDS patients. Numerous reports have described a significant increase in the incidence and diagnosis of opportunistic and systemic candidosis during the last decade [37] . The C. dubliniensis species shares many phenotypic characteristics with and is closely related phylogenetically to C. albicans. Many studies with C. albicans have implicated the Sap proteinases in virulence; hence, it will be necessary to study the role played by these C. dubliniensis proteases.