Candida albicans Constitute a Family with at Least Three Members

INFECrION AND IMMUNrrY, Aug. 1993, p. 3240-3243 0019-9567/93/083240-04$02.00/0 Copyright © 1993, American Society for Microbiology

Vol. 61, No. 8

The Genes Encoding the Secreted Aspartyl Proteinases of Candida albicans Constitute a Family with at Least Three Members BEATRICE B. MAGEE,1 BERNHARD HUBE,2 RACHEL J. WRIGHT,3 PATRICK J. SULLIVAN,3 AND P. T. MAGEE'*

Department of Genetics and Cell Biology, University of Minnesota, St. Paul, Minnesota 551081; Department of Molecular and Cell Biology, Marischal College, University of Aberdeen, Aberdeen AB9 lAS, Scotland2; and Department ofBiochemistry, University of Otago, Dunedin, New Zealand3 Received 1 March 1993/Accepted 11 May 1993

The secreted aspartyl proteinase activity from Candida albicans is thought to be a potential virulence factor. Four laboratories have cloned a gene from C. albicans encoding this enzyme. When two of these genes sharing 77% homology at the DNA level are hybridized under conditions of high stringency to contour-clamped homogeneous electric field chromosome separations of four different strains, they label different chromosomes: chromosome 6 for SAPI and chromosome R for S4P2. The existence of different genes for the two sequences was confirmed by polymerase chain reaction. Genomic Southern blots probed with the genes and washed at low stringency revealed several cross-hybridizing bands. Contour-clamped homogeneous electric field chromosome separations probed at low stringency indicated that there was a cross-hybridizing sequence on chromosome 3 in addition to those on chromosomes R and 6. The genes for the secreted aspartyl proteinase activity in C. albicans thus constitute a gene family which we have called the SAP family.

The opportunistic pathogenic fungus Candida albicans is a major source of infections in immunocompromised patients (15). This organism, almost always isolated in the diploid state, has no sexual cycle and is thus rather difficult to manipulate genetically (5, 20). Among its more interesting biological properties are the ability to grow in both a yeast form and a hyphal form and its ability to undergo phenotypic transitions in colony morphology and cell shape (21, 22). Despite a great deal of work, no virulence factors have been definitively identified, although several cellular properties have been suggested to play a role in pathogenesis (20). The property whose role in virulence has been most carefully studied is the extracellular acid proteinase activity, first identified by Staib (23) and later examined in detail by Ruechel and his collaborators (1, 18). Such an activity could play an important role in facilitating adhesion of the cells or in promoting tissue penetration. In order to investigate the role of this enzyme in virulence, several laboratories have carried out studies with variants lacking enzymatic activity to see whether such variants are less pathogenic than their parents (7, 10). These studies showed that in a live-mouse model, strains lacking the enzyme are significantly less virulent than those with the activity. Although all were carefully controlled, each of these studies was flawed by the possibility that the mutagenesis used to isolate the variant caused secondary genetic damage which was responsible for the reduction in virulence. In order to resolve the questions about the role of the enzyme in virulence, genes for the acid proteinase were cloned by Hube et al. (6) from strain ATCC 10231 and by Ganesan et al. (4) from strain SC5314. Each group verified that its clone contained the gene for the secreted proteinase: Hube et al. compared the amino acid sequence of the purified protein with the predicted gene product, and Gane*

san et al. demonstrated that a fusion protein reacted with a monospecific antiserum. Recently, Wright et al. have reported the cloning of the enzyme from yet a third strain, ATCC 10261 (26). The sequence of the gene cloned by Ganesan et al. is not available, but a comparison of the cloned sequence from ATCC 10231 with that obtained by Wright et al. suggests that there is 77% homology at the DNA level and 73% homology at the protein level. This lack of complete homology was initially somewhat surprising. However, it could be due to strain differences, since in an organism without a sexual cycle, the possibility of genetic drift is relatively high. However, the lack of homology was interpreted to mean that there are two genes for the extracellular proteinase, differing in function or genetic location. This interpretation was borne out by Southern analyses indicating that there are two genes for the proteinase in C. albicans (26). Recently, Morrow et al. (13) have shown that a gene differentially regulated in cells undergoing the phenotypic white-opaque transition encodes aspartyl proteinase. This gene is apparently identical to that cloned by Hube et al. (6). Surprisingly, although the mRNA for this gene is abundant in opaque cells grown on minimal medium and is somewhat elevated in the same cells grown with protein as the sole nitrogen source (inducing medium [7, 17]), little or no mRNA is apparent in either white cells or nonswitching cells actively secreting the proteinase (26). These findings further suggest that the proteins encoded by the two homologous

have different functions. In order to get some insight into this problem and to facilitate further manipulation of these genes, we undertook to assign the clones of the extracellular proteinase genes to the electrophoretic karyotype of C albicans. We also tested for the presence of the two genes by polymerase chain reaction (PCR) and by genomic Southern blots of the two clones. We report here that the two genes map to different chromosomes, that every strain tested so far seems to

genes may

Corresponding author. 3240

contain both genes, and that the nonidentity of the two genes is confirmed both by the Southern blots and by differential PCR fragments. Low-stringency probing of either restriction digests of genomic DNA or chromosomes separated by a contour-clamped homogeneous electric field (CHEF) gives evidence for additional proteinase genes. Thus, the genes encoding the extracellular proteinases constitute a gene family in C albicans. MATERIALS AND METHODS Strains and culture conditions. Strains ATCC 10231 (6) and ATCC 10261 (26) are the sources of the acid proteinase clones. Strain WO-1 was obtained from David Soll (22). Strain C9 has been previously reported (7). Strain SC5314 was obtained from the Squibb Institute for Medical Research. Strains CBS2730 and ATCC 48867 were obtained from Reinhard Ruechel (18). Strain 1001 was obtained from Federico Navarro. The strains were grown on YEPD (3) at 30°C with shaking. Plasmids. pRW2 is a pUC19 derivative into which was cloned a PstI-HindIII fragment of the SAP2 gene (24). CaPra contains a 5.4-kb piece of DNA including 1,173 nucleotides encoding the SAPI gene (6). DNA preparation. DNA was prepared for restriction digests as described by Magee et al. (11). Cells were prepared for pulsed-field gel electrophoresis as described earlier (25). Plasmids were prepared by the method of Lee and Rasheed (8). Pulsed-field gel electrophoresis. CHEF separation of chromosomes was carried out essentially as described by Wickes et al. (25). (For specific conditions, see the legend for Fig. 1.) Hybridization conditions. Gels were transferred to a MagnaGraph (Micron Separations Inc.) and probed with oligoprimed radioactively labeled sequences from the appropriate plasmids. After probing, the blots were washed according to the manufacturer's directions for stringency. PCR. The method described by Saiki et al. (19) was used for PCR. Four primers were synthesized: 1, 5'-ATGT'l'IT TAAAGAATATITTCAT-3', complementary to the minus strand of the first 23 bp of SAPI and SAP2; 2, 5'-GGAC GAGGTITATCACAAGTAAC-3', complementary to the plus strand of bp 282 to 304 of SAP1; 3, 5'-AGATAAAGT GAATAAGCATTCTT-3', complementary to the plus strand of bp 603 to 625 of SAPJ; 4, 5'-CCATCATCACCTTG TAAAGAAGC-3', complementary to the plus strand of bp 1009 to 1031 of SAP2. Approximately 10 ng each of the genomic DNAs of ATCC 10231, ATCC 10261, ATCC 48867 (serotype B), and CBS2730 and of the plasmid DNAs from pRW10 (26) and CaPra (6) were heated with primer 1 and one of the other primers for 3 min at 96°C and then cooled to 57°C for 5 min before Taq polymerase (Amersham) was added. After 5 min at 72°C, the cycle was repeated. Conditions for denaturation, primer annealing, and extension in subsequent cycles were 91, 57, and 72°C, respectively, for 1 min each. After 40 cycles, amplified DNA was analyzed by 5% polyacrylamide gel electrophoresis in TBE buffer (12). RESULTS The CHEF separations of four C. albicans strains, ATCC 10231, ATCC 10261, WO-1, and 1001, probed under conditions of high stringency with the aspartyl proteinase clones pRW2 (from ATCC 10261) and CaPra (from ATCC 10231) are shown in Fig. 1A. It is clear that the two clones have

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FIG. 1. Electrophoretic karyotypes of C. albicans WO-1, ATCC 10231, ATCC 10261, and 1001 were probed with the EcoRV-BamHI fragment of SAPI and the PstI-HindIII fragment of SAP2. The same blot was probed at high (A) and low (B) stringencies. Electrophoretic conditions were a 120- to 300-s switching ramp for 24 h followed by a 420- to 900-s ramp for 48 h, both at 80 V. The gels were 0.6% Amresco pulsed-field gel electrophoresis agarose and the temperature was 14°C. CHR, chromosomes which hybridize.

different chromosomal locations. WO-1 has three different chromosomal translocations, one of which involves chromosome 6 (2). CaPra hybridizes to the intact chromosome 6 in both parental strains and in 1001 and to one of the chromosome 5 or 6 translocation products in WO-1. (This product runs at the same place as chromosome 4.) Therefore, CaPra maps to chromosome 6 (precisely, to the SfiI digestion fragment 60) (2). pRW2, however, maps to the largest band in all three strains, the chromosome R-chromosome 1 region. In ATCC 10261, 1001, and WO-1, both chromosome R homologs migrate at about the same rate, while in 10231, the two homologs separate slightly, proving that chromosome R is the correct assignment. This result implies that there are at least two extracellular protease genes in each of these strains. We are naming these genes SAPJ (the gene from 10231) and SAP2 (the gene cloned from 10261). (These names were chosen to demonstrate that the genes are not analogous to any known proteinase genes of Saccharomyces cerevisiae.) Since so far as is known, none of the four strains (1001, WO-1, and the ATCC strains) are related, it seems likely that the existence of these two genes is very common among C albicans strains. In order to confirm the existence of both sequences in both parent strains, PCR was used. Four primers were used: primer 1 spans the first 23 bp of the coding sequence of both SAPI and SAP2, primer 2 spans bp 304 to 282 of SAPI and is not homologous to any sequence in SAP2, primer 3 spans bp 625 to 603 in SAPI and bp 644 to 622 in SAP2, and primer 4 spans bp 1031 to 1009 in SAP2 but lacks homology to SAPJ. These primers should lead to the following PCR products. Primers 1 and 2 should give a 304-bp fragment from SAPI and no product from SAP2. Primers 1 and 3 should yield a 644-bp fragment from SAP1 and a 625-bp fragment from SAP2, while primers 1 and 4 ought to give a 1,031-bp fragment from SAP1 and no product from SAP2. Figure 2 shows the results of this experiment; in the control (plasmid CaPra, which contains the SAPJ gene) the 644- and 304-bp bands are present, while the 625- and 1,031-bp bands are absent. In each of the Candida strains tested, the predicted bands for both genes are present. Stringent hybridization of the two proteinase gene clones to Southern blots of C. albicans genomic DNA digested with

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various enzymes yields hybridlization only to bands corresponding to the probe used (26) I. Figure 3B shows that SAPI hybridizes to a 7-kb band in a B amHI digest, to two bands of 2.2 and 1.2 kb in an EcoRI diges t, to a band of approximately 30 kb after PstI digestion, and to a single 7.4-kb band in a BglII digest. SAP2 hybridize s to single bands of 9 kb (BamHI), 12 kb (EcoRI), 1.6 k;b (PstI), and 2 kb (BglII) in digests from the same strainqs. In order to verify these results, we probed the same blcat under less stringent conditions. This experiment, the re,sults of which are shown in Fig. 3A, indicated that there arle genes other than SAPI and SAP2 which are part of the SAM ' gene family. It can easily be seen that each of the restrictio: In digests contains fragments which do not hybridize with evither probe under stringent conditions but which show hornology to both probes when the stringency is lowered. For e vxample, EcoRI digests yield

two new bands (1.5 and 4 kb) when probed with SAP2 and four (16, 10.5, 7.3, and 4.1 kb) when probed with SAPI. The fact that the same series of fragments hybridizes with both probes suggests that the homology is specific rather than general. Since the same blot was used for the high- and low-stringency washes, the new bands are not partial digestion fragments. We cannot tell the number of members of the SAP gene family from the number of bands, but it is certainly greater than two. One way to get a minimum estimate of the number of genes is to determine the number of chromosomes which contain a SAP-like gene. We therefore probed a CHEF separation with the two probes under the same conditions used to obtain the results shown in Fig. 3A. Figure 1B shows that chromosome 3 hybridizes to either gene under conditions of low stringency. SAP2 hybridizes with chromosomes R, 3, and 6, while SAPI hybridizes with chromosomes R, 3, 4, and 6. Thus, there are at least three SAP-like genes in C. albicans, and there may be more if several are located on the same chromosome.

DISCUSSION The potential role of the extracellular acid proteinase from C albicans as a virulence factor has led to much work on this

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and its

genes.

Although fewer than 30 C.

albicans genes of known function have been cloned (19), the acid proteinase gene has been cloned four times (4, 6, 13, 24) and the cDNA has been cloned once (14). In setting out to assign two of these clones to a particular chromosome on the electrophoretic karyotype, we discovered that these clones map to different locations; that is, they are separate genes. Low-stringency hybridizations of both genomic Southern blots and CHEF chromosome separations indicate the presence of at least one other gene with significant homology to the SAPI and SAP2 genes. We believe that these genes encode enzymes closely related in sequence to the SAPI and -2 gene products rather than distantly related aspartyl proteinases, because chromosome 2, the genetic location of PEP4, a previously cloned aspartyl proteinase from the vacuole (9), does not show hybridization with our probes.

Thus, the stringency conditions we are using require homology greater than that of the conserved

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active site of this class of enzyme. This is the first gene family discovered in this pathogenic yeast, and it is highly interesting that it encompasses genes for one of the most commonly mentioned potential virulence factors. We have chosen to call this gene family SAP (for secreted acid proteinase) in order to avoid confusion with the PEP genes of S. cerevisiae,

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that SAPJ replace the PEPI designation of Morrow (13) and SAP2 replace the PRA2 designation of Wright et al. (26). A practical consequence of the finding that there are multiple SiAP genes in C. albicans is that the possibility of using gene disruption to investigate the role of this enzyme in infection has become very remote, at least until the technology becomes much better. With present methods, disrupting just the two cloned genes would take either four separate transformations or two transformations followed by mitotic crossing over. Such treatment would leave the disruptant propose

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FIG. 3. Southern blots of resttriction digests of C albicans SC5314, C9, and WO-1 washed firs .tat low stringency (A) and then at high stringency (B). The probes' were the same as those used for Fig. 1. E, EcoRI; Ba, BamHI; Bg, JBglll; P, PstI; The lines in the A HindIII lanes (XHind) indicate the positions of its digestion products.

very much altered and would make the results impossible to interpret. If the other genes must be disrupted, the technical difficulties escalate. Other approaches will have to be found to examine the role of this enzyme in virulence. In Candida

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tropicalis, a single gene seems to exist for the secreted aspartyl proteinase, since disruption of both copies of a single cloned gene gave rise to a strain unable to grow on a medium containing bovine serum albumin as the sole nitrogen source (24). Since the genetic map of C. albicans is not yet highly detailed, one cannot generalize about the role of gene families, but at least one member of the SAP family appears to have significance in the phenomenon called the phenotypic transition, in which cells switch from a particular colony morphology (and sometimes cell shape) to others at a frequency too high for mutation (13, 16). The white-opaque switch in strain WO-1 is the most commonly studied of these transitions, and expression of the SAPI gene for the acid proteinase appears to be limited to the opaque form of this strain. Both our results and those of Morrow et al. seem to rule out regulation of expression by a shift of the gene to an expression site, since the chromosomal location of the gene does not vary during the switch from white to opaque and the pattern of hybridization to a Southern blot of a restriction enzyme digest of genomic DNA also does not change (13). These findings are consistent with the recent observation that there is very little SAPI transcript in ATCC 10261 under inducing conditions, while the SAP2 transcript is abundant under these conditions (26). Little is known about the regulation of the extracellular acid proteinase, except that it is repressed by ammonia and induced by a medium containing high protein concentrations (7, 17). The fact that there are several genes encoding similar enzymes may indicate that these proteins have a variety of functions. For example, they could be required at different times in infection or in different tissues. In that case, we would expect the various members of the gene family to be differentially regulated, as has already been shown for the SAPI gene. Since clones of at least two members of the family are available and the other should be easy to obtain, this hypothesis can be tested. ACKNOWLEDGMENTS We thank Brian Wong and Susan Howell for critical readings of the manuscript and Reinhard Ruechel for helpful discussions. This work was supported in part by USPHS grant AI16567 awarded to P. T. Magee. REFERENCES 1. Borg, M., and R. Ruchel. 1988. Expression of extracellular acid proteinase by proteolytic Candida spp. during experimental infection of oral mucosa. Infect. Immun. 56:626-631. 2. Chu, W.-S., and P. T. Magee. The switching Candida albicans strain WO-1 has a highly rearranged genome. Submitted for

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