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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Medically important yeasts of the genus Candida secrete aspartyl proteinases (Sap), which are of particular interest as virulence factors. Six closely related gene sequences, SAP1 to SAP6, for secreted proteinases are present in Candida albicans. The methylotrophic yeast Pichia pastoris was chosen as an expression system for preparing substantial amounts of each Sap isoenzyme. Interestingly, Sap4, Sap5 and Sap6, which have not yet been detected in C. albicans cultures in vitro, were produced as active recombinant enzymes. Different Sap polyclonal antibodies were raised in rabbits and tested before further application by enzyme-linked immunosorbent assay (ELISA) against each recombinant Sap. Two antisera recognized only Sap4 to Sap6. Using these antisera, together with sap null mutants obtained by targeted mutagenesis, we could demonstrate a high production of Sap4, Sap5 and Sap6 by C. albicans cells after phagocytosis by murine peritoneal macrophages. Furthermore, a Δsap4,5,6 null mutant was killed 53% more effectively after contact with macrophages than the wild-type strain. These results support a role for Sap4 to Sap6 in pathogenicity.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Candida albicans is a major source of different types of fungal infections in immunocompromised patients. Chronic superficial Candida infections of the skin or the mucosae are commonly seen in patients with cell-mediated immunological disorders, e.g. AIDS patients. Disseminated infections resulting from C. albicans and related Candida species occur predominantly in patients with severe defects in their phagocytic system, e.g. neutropenic patients (for review, see Lortholary and Dupont, 1997).

Infection with C. albicans generally involves adherence and colonization of superficial tissues. During this process, budding yeasts are able to transform to hyphae (dimorphism) and penetrate into deep host tissues. Among various potential virulence factors proposed, the secreted aspartyl proteinases (Sap) have been intensively investigated, and the presence of Sap antigen has been demonstrated on the surface of fungal elements colonizing mucosa, penetrating tissues during disseminated infection and evading macrophages after phagocytosis of Candida cells (Borg and Rüchel, 1988; 1990; Rüchel et al., 1991). Evidence for the role of Sap in virulence has also been derived from experiments with the specific inhibitor pepstatin A, which is able to block adherence and invasion of C. albicans (Ray and Payne, 1988; Rüchel et al., 1990; Ollert et al., 1993; Fallon et al., 1997).

Under in vitro conditions with bovine serum albumin (BSA) as the sole nitrogen source, C. albicans secretes a dominant aspartyl proteinase, Sap2. In addition, two other aspartyl proteinases, Sap1 and Sap3, have been recovered from a particular C. albicans strain, WO1, that switches from a white to an opaque form at a frequency of 10−2 to 10−4 per cell cycle. Opaque cells produce Sap1, Sap2 and Sap3, while white cells of the same strain secrete Sap2 only (Morrow et al., 1992; White et al., 1993).

A few years ago, three closely related genes, SAP4, SAP5 and SAP6, encoding putative Saps were isolated and sequenced (Miyasaki et al., 1994; Monod et al., 1994). The deduced amino acid sequences for Sap4, Sap5 and Sap6, showing 75–89% similarity to each other, form a group distinct from Sap1, Sap2 and Sap3. The newly found SAP genes (SAP4, SAP5 and SAP6 ) seemed to be of greater interest, as their transcripts were found in several strains during serum-induced germ tube formation at neutral pH (Hube et al., 1994). However, the translation products have never been isolated or characterized. It has been shown recently that a triple deletion of SAP4, SAP5 and SAP6 resulted in attenuated mortality of guinea pigs and mice compared with the wild-type strain when the animals were injected intravenously (Sanglard et al., 1997). These results suggest that Sap4, Sap5 and Sap6 isoenzymes could be important for the development of systemic infections. Using sap null mutants and specific antibodies raised against different Sap recombinant proteins, we show here that Sap4, Sap5 and Sap6 are produced in macrophages after phagocytosis of C. albicans and protect yeast cells from being destroyed.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Expression of Candida SAPs in Pichia pastoris

The P. pastoris expression system was used to express the different C. albicans Saps, which, like many other microbial secreted proteinases, are synthesized as precursors in the form of the preproproteins. The prepropeptide begins by a signal sequence of 14–21 amino acids with one to four putative signal peptidase cleavage sites (von Heijne, 1986). The isoenzymes Sap1 to Sap6 have a 32- to 60-amino-acid-long propeptide containing one Lys-Arg Kex2 processing sequence (Julius et al., 1984) immediately preceding the amino-terminus of the mature form of the enzyme. DNA fragments encoding each Sap with its propeptide (Table 1) were cloned into the pHIL-S1 expression cassette on pKJ113 (Fig. 1) downstream of the PHO1 signal peptide sequence. The pHIL-S1 constructs insert into the P. pastoris genome via homologous recombination at the AOX1 site and carry, in addition to the cloned coding sequence of interest, the HIS4 gene for selection. Therefore, P. pastoris GS115 was used as a host strain for transformation with SmaI-linearized DNA, and His+ mut transformants were selected and screened for proteinase production. C. albicans Sap1, Sap3, Sap4, Sap5 and Sap6 were secreted as single proteins (Fig. 2). Two bands were observed on SDS–PAGE when Sap2 was expressed. Partial amino acid sequencing of both proteins was accomplished by in situ Edman degradation (Kratzin et al., 1989). The initial amino acid residues of the amino-terminus of the 45 kDa and 40 kDa proteins were determined to be SAGFVALDF and QAVPVTL, corresponding to amino acids 26–34 and 57–63 of the Sap2 translation product respectively. These results indicated that a partial processing of the Sap2 translation product was taking place in P. pastoris.

Table 1. . Materials used for the expression of the different Saps in P. pastoris and yields of recombinant proteins (Hube et al. (1991); Wright et al. (1992); White et al. (1993); Miyasaki et al. (1994); Monod et al. (1994)). a. In parenthesis, amino acids encoded by the XhoI restriction site sequence, and added to the N-terminal extremity of the Sap prosequences.Thumbnail image of
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Figure 1. . Map of plasmid pKJ113. REP, pCL1920 replicon (Lerner and Inouye, 1990); SPC, spectinomycin resistance gene; LACPO, LACZ promoter; 5′AOX, P. pastoris alcohol oxidase gene (AOX ) promoter; SS, signal peptide sequence of the P. pastoris acid phosphatase gene (PHO1 ); 3′AOX(TT), AOX terminator; HIS4, P. pastoris histidinol dehydrogenase gene; 3′AOX, 3′AOX downstream sequence; LACZ, pUC19 LACZ fragment.

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Figure 2. . Protein profile of the culture supernatant of P. pastoris strains producing one specific Sap among Sap1 to Sap6. The proteins in 5 μl of supernatant were loaded without further treatment onto 12% SDS–PAGE. The gel was stained with Coomassie brilliant blue. M, molecular mass markers (phosphorylase B, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; bovine carbonic anhydrase, 31.0 kDa; soybean trypsin inhibitor, 21.5 kDa).

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Yield and properties of recombinant proteinases

Depending on the proteinase produced, a yield of 20–80 μg ml−1 protein of culture supernatant was obtained (Table 1). No appreciable difference in yield was observed between transformants expressing the same proteinase. So far, no clones with integration of multiple copies of the expressed gene and therefore producing more proteinase have been obtained. Interestingly, Sap4 to Sap6, which have never been detected in C. albicans supernatants, were recovered in P. pastoris culture supernatants and were shown to be active. They were inhibited by the classical aspartyl proteinase inhibitor pepstatin. The pH-dependent enzymatic activity of the recombinant Saps was determined in citrate buffer between pH 2.0 and 7.0 (Fig. 3). Sap2 and Sap3 had a pH optimum at 3.5, whereas Sap1 was optimally active at pH 4.5. Sap3 differed from the other Saps in showing substantial activity at pH 2.0, thus confirming previous observations made with Sap3 purified from C. albicans supernatants (Smolenski et al., 1997). The pH optima for the three isoenzymes, Sap4, Sap5 and Sap6, were much less acidic (5.0), and their activity was still important at neutral pH. The specific activity of the six recombinant Saps was similar to that of Sap2 isolated from C. albicans grown in vitro with BSA as sole nitrogen source (data not shown). Supernatants of P. pastoris GS115 did not show any proteolytic activity. Consequently, it can be assumed that the proteolytic activities measured were not affected by the activities of possible contaminating P. pastoris (aspartyl) proteinases.

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Figure 3. . Effect of pH on the activity of C. albicans Sap1 to Sap6 isoenzymes with resorufin-labelled casein in sodium citrate buffer. The activities are depicted as a percentage (%) of the activity at the optimum pH.

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Cross-reactivity of the antibodies with the different recombinant C. albicans Saps in enzyme immunoassays

The availability of recombinant Sap1 to Sap6 allowed us to test the specificity of different polyclonal antibodies raised against Sap2 purified from C. albicans (α-Sap2) against recombinant Sap6 (α-Sap6) and against the 163-amino-acid C-terminal polypeptidic chain of Sap6 fused to gluthathione S-transferase (α-Sap6F). For comparison, the cross-reactivity of these antibodies against α-2-macroglobulin and cathepsin D, representative lysosomal enzymes of the macrophages, was also examined. Under the same conditions, cross-reactivity with C. albicans mannoproteins was assayed as well.

The cross-reactivity against the different Saps is shown in Fig. 4. In parallel tests, α-Sap6 and α-Sap6F reacted strongly with denatured recombinant Sap4 and Sap6, whereas the reaction of α-Sap6 with Sap5 was weaker. This result can be explained by the lower degree of similarity of Sap5 (75%) than that seen for Sap4 (88%) when compared with Sap6. α-Sap6 showed only a slight cross-reaction with Sap2, while α-Sap6F also reacted distinctly with Sap1. α-Sap2 reacted strongly with Sap2 and Sap3 and clearly with Sap1, but less strongly than with the former Saps. A slight reaction was seen with Sap4 to Sap6. None of the antibodies reacted with C. albicans mannoproteins and cathepsin D. Only α-Sap6F revealed a distinct cross-reactivity with α2-macroglobulin (data not shown).

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Figure 4. . Cross-reactivity of the antibodies α-Sap6 α-Sap6F and α-Sap2 with the different denatured proteinase antigens Sap1 to Sap6 expressed in P. pastoris. The tests were performed in enzyme immunoassays. The microtest plates were coated with 0.5 μg ml−1 proteinase antigen under experimental conditions testing the reactivity with denatured antigen. The tests were evaluated in an automatic microtest plate reader (Dynatech) at 490 nm.

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Infection assay and detection of Sap antigens by immunofluorescence staining

Mouse peritoneal macrophages, which adhered to glass cover slips, were infected with log-phase blastoconidia of the wild-type C. albicans strain SC5314 as well as with several mutants in which at least two genes among SAP4, SAP5 and SAP6 were disrupted (Table 2) The infection was performed at a ratio of five blastoconidia per macrophage. After 2 h of co-incubation, almost all blastospores were ingested by the phagocytes. After 4 h, the phagocytosed blastoconidia started to resist their phagocytes by forming germ tubes. After 8 h of phagocytosis, specimens were fixed with freshly prepared paraformaldehyde and cold methanol and examined for the presence of the different Candida proteinase antigens by immunofluorescence staining.

Table 2. . C. albicans strains used in this work and summary of the results of phagocytosis experiments with respect to their reactivity with α-Sap6 and α-Sap6F antibodies (Sanglard et al. (1997)). The symbols + + +, + and (+) are for strongly positive, positive and weak fluorescence. − is for the absence of fluorescence.Thumbnail image of

The immunoreaction with α-Sap6 and α-Sap6F yielded the same picture demonstrating the presence of Sap4 to Sap6 antigens on intracellular and obviously viable blastoconidia and pseudomycelia of C. albicans SC 5314 (Fig. 5A and C). The presence of Sap4 to Sap6 is a consequence of the yeast–macrophage interaction, as these antibodies did not detect proteinase antigen on the surface of pseudomycelia outside the macrophages.

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Figure 5. . Immunofluorescence micrograph of macrophages 14 h after ingestion of blastoconidia of C. albicans SC5314. The immuno- fluorescence reaction was performed with α-Sap6 (A) and α-Sap6F (C) antibodies. As shown, blastoconidia ingested by the macrophages and evading their host cells carried the Sap4 to Sap6 proteinase antigen on the surface of the cell as well as on the tips of the penetrated pseudomycelia (A and C). Fungal elements outside the macrophages were not detected by the antibodies. B and D. Light micrographs of the same macrophages as in A and C for comparison.

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The specificity of the Sap4 to Sap6 antigens present on fungal surfaces could be shown in assays in which the Δsap4,5,6 triple mutant strain DSY459 had been phagocytosed by the mouse peritoneal macrophages. Immunoreactions with both α-Sap6 and α-Sap6F antisera performed in parallel with the wild-type C. albicans strain SC5314 clearly demonstrated the absence of the proteinase antigens Sap4 to Sap6 on the fungal surfaces of DSY459 (Fig. 6A and C). The absence of antigen was not attributable to a smaller number of ingested yeast cells, as could be shown by light micrographs of this specimen after phagocytosis (Fig. 6B and D).

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Figure 6. . Absence of the proteinase antigens Sap4 to Sap6 on the surface of the Δsap4,5,6 mutant DSY459. The infection assay and the immunofluorescence reaction were performed in parallel with C. albicans SC5314 using α-Sap6 (A) and α-Sap6F (C) antibodies. B and D. Light micrograph of the same macrophages as in A and C for comparison. By comparing with Fig. 5, it is clear that the macrophages had ingested at least a similar number of blastoconidia, which are intracellular. However, the immunoreaction with these antibodies remains negative. There are also some pseudomycelia evading the phagocytic cells.

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The immunofluorescence results of the phagocytosis experiments performed with the mutant strains lacking at least two SAP genes among SAP4 to SAP6 compared with the wild-type C. albicans strain SC5314 were dependent on the antibody used. A clear reduction in immunofluorescence with antibody α-Sap6 was seen in the phagocytosis experiments using the Δsap4,5Δsap5,6 and Δsap4,6 mutants. In contrast, the immunoreaction with antibody α-Sap6F showed no significant signal differences when wild type and the doubly deleted sap mutants were phagocytosed (Table 2). These results suggest that each of the Sap4, Sap5 and Sap6 are produced in the phagocytosed Candida blastoconidia.

Yeast cells were also stained after phagocytosis with the polyclonal α-Sap2 antiserum, which recognizes Sap1 to Sap3. A positive reaction was observed inside and outside the macrophages on blastoconidia and pseudomycelia of C. albicans SC5314 as well as on the surfaces of the mutant strains (Fig. 7). This means that the detection of Sap1 to Sap3 is not dependent on the contact of fungal cells with macrophages. No fluorescence reaction was seen with preimmune sera of the animals used for the production of the polyclonal antibodies (data not shown).

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Figure 7. . Immunofluorescence reaction of C. albicans SC5314 (A) and Δsap4,5,6 mutant DSY459 (C) with α-Sap2 antibody 14 h after ingestion by macrophages. Fungal elements inside and outside the macrophages were detected by the antibody. B and D. Light micrograph of the same macrophages as in A and C for comparison.

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Viability of C. albicans in macrophages

The cytotoxic effect of adherent peritoneal macrophages on ingested and adherent Candida cells was assayed with the wild-type C. albicans strain SC5314 and the Δsap4,5,6 mutant DSY459. The viability of these two strains engulfed by macrophages was compared. At the end of the phagocytosis period, test samples were plated onto Sabouraud glucose agar. Control samples of yeasts without peritoneal macrophages were handled in the same way. The fungal colonies were counted after at least 24 h of growth. A difference in viability between wild-type and Δsap4,5,6 mutant strains engulfed by macrophages was found to be highly significant (< 0.001). As shown in Fig. 8, an average of 43% of the wild-type cells survived at this time point of the assay compared with only 18.6% for the Δsap4,5,6 mutant DSY459. These results revealed that there was a twofold decrease in the survival of the mutant DSY459 after contact with macrophages, indicating a lower pathogenicity potential. The experiment was repeated three times, and similar results were obtained.

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Figure 8. . Comparison of the results of the viability assay of C. albicans SC5314 and of C. albicans DSY459 after ingestion by peritoneal macrophages. The fungal colonies were counted after at least 24 h of growth. Controls for each Candida strain in the absence of macrophages were set to 100%. The percentage of the viable cells of each strain was measured in a cfu assay (for details, see Experimental procedures). The error bars indicate the standard deviations obtained with four counts of fungal colonies.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The P. pastoris protein expression system provides a useful tool for the separate production of each isoenzyme of the same organism, such as Sap1 to Sap6 of C. albicans. When their sequences were expressed behind that of their propeptide, all proteinases tested could be recovered as active enzymes in the P. pastoris culture supernatants with a yield of 20–80 μg ml−1. In contrast, the yield of C. albicans Sap1 and C. tropicalis Sapt1 produced in Saccharomyces cerevisiae with their propeptide and using multicopy yeast expression vectors was about 100 times lower (Togni et al., 1996; Smolenski et al., 1997). Direct expression of a proteinase gene without its propeptide does not lead to secretion of recombinant protein into the culture supernatant of P. pastoris transformants (data not shown).

The availability of pure Sap1 to Sap6 helped to determine the specificity of polyclonal antibody, and we have demonstrated that Sap4, Sap5 and Sap6 are expressed in macrophages after phagocytosis of C. albicans. However, these three closely related isoenzymes could never be recovered up to now from C. albicans in vitro cultures. SAP4, SAP5 and SAP6 mRNAs were detected during hyphal cell growth stimulated by serum at neutral pH (Hube et al., 1994; White and Agabian, 1995), but Western blotting analysis of the C. albicans culture supernatant did not detect any Sap protein (data not shown; White and Agabian, 1995).

After the ingestion of yeast cells by phagocytic cells, Sap antigens have been shown to be expressed by C. albicans and C. tropicalis but not by C. parapsilosis (Rüchel et al., 1986; Borg and Rüchel, 1990). However, under in vitro conditions when serum albumin is the major nitrogen source in the growth medium, C. parapsilosis produces large amounts of one dominant Sap, e.g. Sapp1, like C. albicans and C. tropicalis, which produce Sap2 and Sapt1 respectively (MacDonald and Odds, 1980; Rüchel et al., 1983; 1986; Togni et al., 1991; Monod et al., 1994). These three enzymes are similarly regulated, as their genes could compensate for a sapt1 mutation in C. tropicalis (Sanglard et al., 1992; Monod et al., 1994). Therefore, it is likely that the Sap(s) expressed by C. albicans inside and outside the macrophages are different from Sap2. In our phagocytosis model, the Sap(s) expressed by C. albicans outside the macrophages on blastoconidia and pseudomycelia could be Sap1 or (and) Sap3.

Macrophages and granulocytes constitute major obstacles to the establishment of systemic Candida infections. The uptake of invading microorganisms by phagocytes is followed by the fusion of cellular lysosomes containing hydrolytic enzymes with the phagosome-containing microorganisms to form the microbicidal phagolysosomes. The pH within phagolysosomes, of the order of pH 4.7–4.8 (Ohkuma and Poole, 1978), favours the activity of the host's acid lysosomal hydrolases. However, it is also optimal for the enzymic activity of Sap4 to Sap6, as shown in this study.

Pathogenic microbes are able to persist intracellularly in phagocytes (Russel, 1995). As shown previously, engulfed Candida are not digested completely (Marquis et al., 1991). Obviously, one part of the ingested C. albicans cells has the ability to resist the phagocytes, as described for L. monocytogenes (De Chastellier and Berche, 1994). Another possibility is that Sap isoenzymes could act as cytolysins, as described for Trypanosoma cruzi, Listeria and Shigella (Andrews and Portnoy, 1994). Microbial intracellular persistence could be favoured by the fact that the phagosome–lysosome fusion enhances the activity of potential pathogenic factors, as observed previously for T. cruzi or M. tuberculosis (Tardieux et al., 1992). Therefore, it is possible that the synthesis of Sap4 to Sap6 in C. albicans is activated by one or several as yet unknown macrophage intracellular factors. Furthermore, the activity of these enzymes would be enhanced by the change in pH that occurs during the phagosome–lysosome fusion.

If the enzyme activities of Sap4, Sap5 and Sap6 are indeed involved in the direct destruction of intracellular components of the macrophages as reported previously (Borg and Rüchel, 1990), they could also affect some key enzymes of the macrophage oxidative metabolism, which is important for optimal microbicidal activity (for review, see Miller and Britigan, 1997). Sasada and Johnston (1980) described a correlation between the oxidative metabolic response and the microbicidal capacity of macrophages. After interaction with macrophages, C. albicans elicited a weaker respiratory burst and was killed much less effectively than C. parapsilosis. These results could be explained by the existence of fungal factors that are able to influence the oxidative metabolism of the phagocytic defence system negatively. Such factors are not well defined (for review, see Murphy, 1991; Calderone et al., 1994). However, the different Saps can be considered as candidates. The results of the viabilities of wild-type strain C. albicans SC5314 and of the Δsap4,5,6 mutant DSY459 in macrophages indeed support such a conclusion. The role of Sap4 to Sap6 as potential pathogenicity factors has been demonstrated in animal experiments. Mice and guinea pigs infected with DSY459 survived significantly longer than animals infected with SC5314 (Sanglard et al., 1997).

It has been shown recently that a C. albicansΔcph1,efg1 double mutant defective in filamentous growth has no destructive action on macrophages (Lo et al., 1997). Consequently, the Sap4, Sap5 and Sap6 isoenzymes constitute a second group of factors that could contribute to the destruction of the macrophages and influence the pathogenesis of invasive candidiasis.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Strains and plasmids

E. coli strain DH5α was used for transformation with competent cells and propagation of the recombinant plasmids. The low-copy-number plasmid pCL1920 (Lerner and Inouye, 1990) was used to generate new E. coliP. pastoris shuttle vectors. The existing multiple cloning site of pCL1920 was modified to include a SmaI and an EcoRI site between the BamHI and Sal I sites. The newly generated plasmid was called pCL1920b.

The E. coliP. pastoris shuttle vector pHILS1 was provided by the P. pastoris expression system from Invitrogen. A NotI linker (5′-AGCGGCCGCT-3′) was inserted in the SmaI site of the multiple cloning site of pHILS1 to generate the plasmid pHILS1-NotI. The expression cassette of pHILS1-NotI was cloned into pCL1920b to generate the plasmid pKJ113 (Fig. 1). In this plasmid, the pHILS1 expression cassette was flanked by two SmaI sites for linearization of the DNA before transformation of P. pastoris.

The His Mut+P. pastoris strain GS115 used as a host for transformation was provided by Invitrogen. The C. albicansΔsap4,6 double mutant DSY437 and Δsap4,5,6 triple mutant DSY459 have been described previously (Sanglard et al., 1997). The C. albicansΔsap4,5 and Δsap5,6 double mutants DSY456 and DSY462 were constructed like the Δsap4,6 double mutant DSY437 using the SAP4, SAP5 and SAP6 disruption cassettes on plasmids pDS237, pDS210 and pDS198 respectively (Sanglard et al., 1997).

Construction of the expression plasmids

Expression plasmids were constructed by cloning a polymer chain reaction (PCR) product of the C. albicans SAP genes to be expressed in the multiple cloning site of the E. coliP. pastoris shuttle vector pKJ113. In detail, PCR was performed using homologous primers derived from DNA sequences of the different SAP genes (Table 1). Custom-made primers were provided by Microsynth. PCR buffers and AmpliTaq polymerase were from Perkin-Elmer. The buffer composition was 10 mM Tris-HCI (pH 8.3), 50 mM KCl with 1.5 mM MgCl2 containing 0.2 mM dNTPs and 2.5 U polymerase per reaction. The PCR was carried out in a Thermal Cycler 480 (Perkin-Elmer) with a first cycle of denaturation of 4 min at 94°C, followed by 25 cycles of annealing at 54°C for 2 min, elongation at 72°C for 2 min and denaturation at 94°C for 30 s. PCR was completed by a final elongation step at 72°C for 10 min. Previously described plasmids containing individual C. albicans SAP (Monod et al., 1994) were used as templates.

The PCR products were purified using a PCR purification kit (Boehringer) and were digested by restriction enzymes for which a site had been designed previously at the 5′ extremity of the primers (Table 1). Subsequently, the digested PCR products were cloned into the appropriate sites of the multiple cloning site of a E. coliP. pastoris shuttle vector according to standard procedures (Sambrook et al., 1989). The cloned fragments were sequenced further (Microsynth DNA sequencing service), and the absence of possible PCR-induced errors was confirmed. Plasmid DNA was prepared from one E. coli clone harbouring a correct construct and digested by SmaI before P. pastoris transformation.

Expression of the Sap isoenzymes in P. pastoris

P. pastoris GS115 was transformed by preparing spheroplasts as described in the Manual Version 2.0 of the Pichia Expression Kit (Invitrogen) with 10 μg of linearized DNA. Transformants selected on histidine-deficient medium [1 M sorbitol, 1% (w/v) dextrose, 1.34% (w/v) yeast nitrogen base (YNB) without amino acids, 4 × 10−5% (w/v) biotin, 5 × 10−3% amino acids (i.e. 5 × 10−3% (w/v) each of L-glutamic acid, L-methionine, L-lysine, L-leucine, L-isoleucine)] were screened for insertion of the construct at the AOX1 site on minimal methanol plates [1.34% (w/v) YNB without amino acids, 4 × 10−5% (w/v) biotin, 0.5% (v/v) methanol)].

Transformants unable to grow on media containing only methanol as a carbon source were assumed to contain the construct at the correct yeast genomic location by integration events in the AOX1 locus displacing the AOX1 coding region. The selected transformants were grown to near saturation (OD of 20 at 600 nm) at 30°C in 10 ml of glycerol-based yeast media [0.1 M potassium phosphate buffer at pH 6.0, containing 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB without amino acids, 1% (v/v) glycerol and 4 × 10−5% (w/v) biotin]. Cells were harvested and resuspended in 2 ml of the same medium with 0.5% (v/v) methanol instead of glycerol and incubated for 2 days. After 2 days of incubation, the supernatant was harvested, and 10 μl was loaded on SDS–PAGE gels to identify clones expressing Sap.

Small-scale purification of recombinant proteinases

P. pastoris culture supernatant (2.5 ml) was first desalted, and low-molecular-weight solutes were removed by passing through Sephadex G25 (PD10 column; Pharmacia) using 10 mM sodium citrate buffer, pH 6.5. The recombinant proteinases Sap1, Sap2 and Sap3 recovered from the first purification step were loaded on a small polypropylene column filled with 0.5 ml of DEAE-Sepharose (Pharmacia), washed with the same buffer and eluted with 100 mM sodium citrate buffer at pH 5.0. The three closely related Sap4, Sap5 and Sap6 proteins recovered from the G25 Sephadex column were loaded on hydroxyapatite (0.1 g in a small polypropylene column). The column was first washed with 50 mM sodium phosphate buffer (pH 7.0), and the Sap isoenzymes were eluted with 150 mM sodium phosphate buffer (pH 7.0).

Gluthathione S-transferase (GST)–Sap6 fusion protein

A fusion protein consisting of GST and the C-terminal 138 amino acids of SAP6 (GST–Sap6F) was prepared using the GST gene fusion system from Pharmacia. A SAP6 fragment encoding the C-terminal Sap6 protein from Met-281 to Asn-418 was obtained by PCR with primers flanked by BamHI and XhoI sites (5′-CGGGGATCCATGGGACGAAATGTTAATGT-3′ and 5′-CGCTCGAGATTAATAGCAACAATGT-3′). The plasmid pCA6 (Monod et al., 1994) was used as DNA template for the PCR reaction. The amplified fragment was cut by BamHI and XhoI and inserted in the vector pGEX-4T (Pharmacia) previously cut with the same enzymes to obtain an in frame fusion between GST and the C-terminal part of Sap6. The fusion protein was produced and purified by affinity chromatography according to the recommendations of the manufacturer.

Protein extract analysis

Protein concentrations were measured by the method of Bradford (1976). Protein extracts were analysed by SDS–PAGE (Laemmli, 1970) with a separation gel of 9% polyacrylamide. Gels were stained with Coomassie brilliant blue R-250 (Bio-Rad).

Proteolytic assays

The proteolytic activity of Sap isoenzymes was measured with 0.02% resorufin-labelled casein as substrate at different pH values in sodium citrate buffer (50 mM; pH 2.0–7. 0) in a total volume of 0.5 ml. After incubation at 37°C, the undigested substrate was precipitated with trichloroacetic acid (4% final concentration) and separated from the supernatant by centrifugation. The absorbance of the supernatant was measured in the alkaline range at 574 nm after adding 30 μl of 4 N NaOH. For practical purposes, one unit of Sap activity was defined as that producing an absorbance of 0.001 min−1 in a proteolytic assay at optimum pH of activity.

Antibodies

Antisera to Sap2 isolated from C. albicans (α-Sap2), recombinant Sap6 (α-Sap6) and GST-Sap6F (α-Sap6F) were raised in New Zealand White rabbits as already described (Monod et al., 1991).

Cross-reactivity of the antibodies with the different recombinant SAP isoenzymes

The reactions of the different polyclonal rabbit anti-Sap antibodies with each recombinant Sap under denatured conditions with an indirect enzyme immunoassay were performed essentially as described by Rüchel and Böning (1983).

As a first step, round-bottomed microtest plates were coated with the different recombinant Saps at a final concentration of 0.5 μg ml−1 in 0.1 M carbonate buffer (pH 9.8). The microtest plates were then incubated for up to 18 h at 8°C. After this time, the microtest plates were rinsed five times with 10 mM imidazole buffer (pH 6.0) containing 0.1% Tween 20 (rinsing buffer). The antibodies were applied in the rinsing buffer additionally containing 1% BSA and 0.1% milk powder at a 500-fold dilution for 2 h at room temperature. After a further rinsing of the microtest plates (five times with the above-mentioned buffer), the anti-rabbit peroxidase conjugate (Dakopatts) was applied at a 1000-fold dilution in the buffer as above and incubated for 1 h at room temperature. After another series of rinses, the H2O2-activated substrate was applied to the plates. After 20 min of incubation in the dark, the reaction was stopped with 2 M H2SO4. The tests were evaluated in an automatic microtest plate reader (Dynatech) at 490 nm.

Preparation of macrophages

Mouse peritoneal macrophages were elicited by intraperitoneal inoculation of NMRI mice (25 g) with 2 ml of thioglycollate broth. The mice were sacrificed after 2 days, and the stimulated macrophages were recovered from the peritoneal cavity by rinsing with 10 ml of phosphate-buffered saline (PBS). The cells were washed twice with PBS and resuspended in cell culture medium at a concentration of 105 ml−1. One millilitre of the macrophage suspension was placed on a round glass cover slip (12 mm diameter) in a compartment of a flat-bottomed polystyrene plate. Macrophages were incubated for at least 24 h (37°C, 5% CO2) in cell culture medium (MEM + 5% fetal calf serum). During the course of this incubation, the cells adhered to the cover slip.

Phagocytosis assay

After removal of the cell culture medium, adherent mouse macrophages were infected with 2 × 105Candida blastoconidia (preopsonized with normal mouse serum for 30 min at 37°C) in 1 ml of infection medium (mixed medium 1640-199 + 10% fetal calf serum according to Borg et al., 1984). The ratio of yeasts to macrophages was 2:1. The culture plates were then centrifuged (1200 g for 2 min) to facilitate contact between yeasts and phagocytes. The plates were incubated for up to 14 h (37°C, 5% CO2) and the cells were washed with PBS. Before the immunoreaction, cells were fixed with freshly prepared paraformaldehyde (2% in PBS) for 1 h at room temperature and with absolute methanol at −20°C.

Immunofluorescence staining

The fixed specimens were rinsed with PBS. They were then incubated with PBS + 1% BSA and 0.1% milk powder to reduce unspecific binding for 1 h at room temperature. The different specific antisera as well as the preimmunization serum were applied (diluted 100-fold in PBS + 1% BSA + 0.1% milk powder) and incubated at 8°C for at least 12 h. After another series of rinses with PBS, a second antibody–fluorescein isothiocyanate (FITC) conjugate from goat was added (diluted 200-fold in the above-mentioned buffer). After 3 h incubation and five subsequent rinses in PBS, the tests were embedded in p-phenylenediamine according to Johnson et al. (1982). The binding of the conjugate was evaluated with a Zeiss fluorescence microscope using the filter combination BP 450–490/FT 510/LP 520.

Viability of yeasts in macrophages

The assay was performed essentially as described by Ferrante (1989) measuring the killing of ingested and adherent Candida cells. The phagocytosis assay was performed over 90 min at 37°C as described above with 4 × 105 macrophages ml−1 and 2 × 106Candida cells ml−1. At the end of the phagocytosis assay, sterile distilled water was added and left at 37°C for 30 min. Duplicate serial dilutions of the lysate in PBS were plated on Sabouraud glucose agar. After incubation of the plates (n = 4) at 37°C for at least 24 h, the number of colony-forming units (cfu) was determined as a measure of the number of viable Candida cells present. Viability (as a percentage) was compared with that of fungal cells incubated without peritoneal macrophages under the same assay conditions. The difference between the viabilities of wild-type and Δsap4,5,6 mutant strains engulfed by macrophages was analysed statistically using the Student's t-test.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank J. Wyniger, C. Zaugg and Dr U. Reichard for technical assistance, C. Maelicke and Dr H. Pooley for critical review of the manuscript and assistance with the English. This work was supported by the Swiss National Foundation for Scientific Research, grant 3100-043193.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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