Cyclophilin A is the target of the immunosuppressant cyclosporin A (CsA) and is encoded by a single unique gene conserved from yeast to humans. In the pathogenic fungus Cryptococcus neoformans, two homologous linked genes, CPA1 and CPA2, were found to encode two conserved cyclophilin A proteins. In contrast to Saccharomyces cerevisiae, in which cyclophilin A mutations confer CsA resistance but few other phenotypes, cyclophilin A mutations conferred dramatic phenotypes in C. neoformans. The Cpa1 and Cpa2 cyclophilin A proteins play a shared role in cell growth, mating, virulence and CsA toxicity. The Cpa1 and Cpa2 proteins also have divergent functions. cpa1 mutants are inviable at 39°C and attenuated for virulence, whereas cpa2 mutants are viable at 39°C and fully virulent. cpa1 cpa2 double mutants exhibited synthetic defects in growth and virulence. Cyclophilin A active site mutants restored growth of cpa1 cpa2 mutants at ambient but not at higher temperatures, suggesting that the prolyl isomerase activity of cyclophilin A has an in vivo function.
Cyclosporin A (CsA) is a cyclic peptide produced by Tolypocladium inflatum that has potent immunosuppressive activity and is widely used to prevent graft rejection in organ transplant recipients. CsA targets a conserved family of proteins, the cyclophilins, which are widely distributed in both uni‐ and multicellular organisms (Dolinski and Heitman, 1997). Cyclophilins also have peptidyl‐prolyl isomerase activity and catalyze protein folding (Fischer and Schmid, 1990). The ubiquitous and conserved nature of cyclophilins suggests that they have fundamental cellular roles, yet mutants lacking single or multiple cyclophilins have little phenotype in the yeast Saccharomyces cerevisiae (Dolinski et al., 1997).
CsA suppresses the immune system by inhibiting signaling cascades required for T‐cell activation (Schreiber and Crabtree, 1992; Cardenas et al., 1998). CsA binds to the cyclophilin A protein, and the cyclophilin A–CsA complex targets the Ca2+‐regulated phosphatase calcineurin in both S. cerevisiae and human T cells (Liu et al., 1991; Foor et al., 1992; Breuder et al., 1994). In humans, calcineurin regulates nuclear localization of the NFAT transcription factor, which increases gene expression in response to T‐cell activation by antigen presentation to the T‐cell receptor (Crabtree, 2001). In yeast, calcineurin controls cell wall biosynthesis and cation homeostasis via Crz1p, a transcription factor regulating the FKS2, PMR2, PMC1 and PMR1 genes (Matheos et al., 1997; Stathopoulos and Cyert, 1997; Stathopoulos‐Gerontides et al., 1999).
In addition to immunosuppression, CsA has potent antifungal activity and is toxic to the human pathogen Cryptococcus neoformans (Odom et al., 1997). A conserved calcineurin homolog that is required for virulence is the target of CsA action in this fungus (Odom et al., 1997; Cruz et al., 2000b; Fox et al., 2001). CsA analogs with reduced immunosuppressive activity that retain antifungal activity have been identified (Cruz et al., 2000a).
We have cloned, characterized and disrupted the genes encoding two highly related cyclophilin A homologs, Cpa1 and Cpa2, in C. neoformans. We show that CsA toxicity is mediated by CsA in complex with either cyclophilin Cpa1 or Cpa2. Most importantly, we show that Cpa1 has specialized functions in promoting growth at high temperature and virulence, whereas Cpa2 is dispensable under these conditions. This is the first known example in which two cyclophilin A genes have been identified from a single organism and found to encode proteins with both shared and distinct functions. Our studies also reveal that cyclophilin A plays prominent physiological roles in C. neoformans, providing a model genetic system to analyze the functions of this ubiquitous but enigmatic family of enzymes.
Results and Discussion
Cyclophilin A is encoded by two homologous genes in C. neoformans
Cyclophilin A is the conserved target of CsA in S. cerevisiae and humans. To understand CsA antifungal activity, cyclophilin A homolog(s) were identified from C. neoformans. By degenerate PCR, a region of a cyclophilin A homolog was isolated and used as the probe for Southern blots, which revealed two related genes: CPA1 and CPA2 (DDBJ/EMBL/GenBank accession Nos AF180336 and AF180337). The CPA1 and CPA2 genes are highly conserved, and share 78% nucleotide identity in the coding regions. The CPA1 and CPA2 genes encode 162‐amino‐acid proteins that share 93% protein sequence identity (Figure 1), and 65–75% identity with human and yeast cyclophilin A. The CPA1 (AF333996) and CPA2 (AF333997) genes were also present in a divergent serotype D C. neoformans strain (Figure 1).
Interestingly, the CPA1 and CPA2 genes were found to be linked by Southern blotting, hybridization to common BAC genomic clones and long‐range PCR (Figure 2). In chromosome blots, the CPA1 and CPA2 genes hybridized to one of two co‐migrating ∼2.2 Mb chromosomes (Figure 2A). With isolated BAC DNA as template, CPA1 and CPA2 gene‐specific primers amplified an ∼20 kb fragment linking the genes (Figure 2B).
These findings suggest that the two CPA genes arose by gene duplication. Duplication of the cyclophilin A gene occurred prior to the divergence of serotype A (var. grubii) and serotype D (var. neoformans) strains, estimated to have occurred ∼20 million years ago (Xu et al., 2000). The CPA1 and CPA2 genes are more divergent in serotype A (12 amino acid changes) compared with serotype D (five amino acid changes), suggesting that mutagenesis rates or selective pressures may differ between the two. This is the first known example in C. neoformans in which two highly related genes are linked, and could be a common feature in the C. neoformans genome. We recently found that the MFα2 and MFα3 genes are similarly linked and divergently transcribed. A similar example of gene duplication and divergence is the cyclophilin 40 homologs Cpr6 and Cpr7 in S. cerevisiae (Duina et al., 1996a,b; Dolinski et al., 1997). Two pairs of cyclophilins localized to the secretory pathway in S. cerevisiae, Cpr2/Cpr5 and Cpr4/Cpr8, may represent additional examples of cyclophilin gene duplication and divergence (Dolinski et al., 1997).
Disruption of the CPA1 and CPA2 genes
To determine the functions of cyclophilin A, the CPA1 and CPA2 genes were disrupted individually and in combination. The CPA1 gene was disrupted with a cpa1::ADE2 or cpa1::URA5 allele; the CPA2 gene was disrupted with a cpa2::URA5 allele (Figure 3A). The cpa1::ADE2 allele was transformed into the ade2 strain M049, and five cpa1 mutants from 48 Ade+ isolates (10%) were identified by PCR and verified by Southern analysis (Figure 3B). Five cpa1::URA5 and four cpa2::URA5 mutants were obtained from 49 (10%) and 32 (12.5%) Ura+ isolates. Finally, a cpa1::ADE2 mutant was plated on 5‐fluoroorotic acid (5‐FOA) medium and the resulting cpa1::ADE2 ura5 derivative used as a recipient for transformation with the cpa2::URA5 allele. Seven cpa1 cpa2 double mutants were obtained from 98 Ura+ isolates (7%). The cpa1, cpa2 and cpa1 cpa2 mutants were reconstituted with the CPA1, CPA2 and CPA1+CPA2 genes, and verified by PCR and western blotting (Figure 3B and C).
Cpa1 and Cpa2 proteins are both expressed in C. neoformans
Proteins were extracted from the wild‐type and cyclophilin A mutants, and analyzed by western blotting with polyclonal antiserum against the Cpa1 protein. This antiserum recognized an 18 kDa protein in wild‐type cells but not in cpa1 cpa2 mutants (Figure 3C); 18 kDa proteins were also detected in extracts from the cpa1 and the cpa2 single mutants, and represent the Cpa2 and Cpa1 proteins, respectively. Similar 18 kDa protein(s) were detected in the cpa1 cpa2+CPA1 CPA2 reconstituted strain (Figure 3C). Thus, the antibody raised against Cpa1 also recognizes Cpa2, and the CPA1 and CPA2 genes are both expressed and have been disrupted by homologous recombination.
Phenotypic analysis of cpa1, cpa2 and cpa1 cpa2 mutants
The effects of cpa1 and cpa2 mutations on growth, CsA sensitivity and mating of C. neoformans were tested. The cpa1 and cpa2 single and double mutants exhibited different phenotypes. cpa1 mutants grew normally at 30 and 37°C, but exhibited a growth defect at 39°C, similar to but less severe than calcineurin mutants (Figure 4A). In contrast, cpa2 mutants exhibited normal growth at all temperatures (Figure 4A). cpa1 cpa2 double mutants exhibited a more severe growth defect, and grew poorly at 24, 30 and 37°C and were inviable at 39°C (Figure 4A). We conclude that Cpa1 plays a unique role in growth at 39°C, and that Cpa1 and Cpa2 have partially overlapping functions required for normal cell growth. We note that Cpa1/2 function is specific, as this organism expresses another prolyl isomerase (FKBP12) that is insufficient to protect from loss of cyclophilin A.
Growth of the wild‐type and both the cpa1 and cpa2 single mutants was sensitive to CsA at 37°C. In contrast, growth of the cpa1 cpa2 double mutant was completely CsA resistant (Figure 4B). Because mutations in calcineurin (V344K, V344R) known to block cyclophilin A–CsA binding also confer CsA resistance in C. neoformans (Cruz et al., 2000b), we conclude that CsA toxicity is mediated by inhibition of calcineurin by cyclosporin in complex with either Cpa1 or Cpa2.
In mating assays, cpa1 or cpa2 single mutants were fully fertile and formed abundant filaments, basidia and basidiospores (not shown). In contrast, the cpa1 cpa2 mutant was sterile and formed no heterokaryotic filaments, basidia or basidiospores, indicating that Cpa1 and Cpa2 are required for mating (Figure 5A).
Reconstitution of the cpa1 mutant with the wild‐type CPA1 gene restored growth at 39°C. Reconstitution of the cpa1 cpa2 mutant with the CPA1 and CPA2 genes fully restored growth, CsA sensitivity and mating (Figures 4 and 5). Thus, the cpa1 and cpa2 mutations confer the observed phenotypes.
The CPA1 and CPA2 genes encode proteins that share marked amino acid sequence identity and yet confer distinct phenotypes when mutated. By western blotting, the Cpa1 and Cpa2 proteins are both expressed. The antiserum raised against Cpa1 should cross‐react equally or slightly less well with Cpa2. Based on this analysis, the Cpa1 protein is not >2‐fold more abundant than Cpa2, and this seems unlikely to explain their differences in function. The promoter regions of the two genes are completely dissimilar, and thus the CPA1 and CPA2 genes could be differentially regulated under some conditions. Alternatively, the few subtle differences in amino acid residues between Cpa1 and Cpa2 could endow each with the ability to interact preferentially with different targets.
The phenotypes exhibited by cpa1 cpa2 mutants are similar to but more severe than calcineurin mutants. Both mutants are inviable at 39°C and avirulent. However, cpa1 cpa2 cyclophilin mutants are unilaterally sterile and growth impaired at 30°C, whereas calcineurin mutants are bilaterally sterile and grow normally at 30°C. Given that Cpa1 and Cpa2 bind CsA and inhibit calcineurin, Cpa1 and Cpa2 could regulate calcineurin normally. By 125I‐labeled calmodulin overlay blotting (Cruz et al., 2000b), similar levels of the calcineurin A Cna1 protein were detected in wild‐type, cpa1, cpa2 and cpa1 cpa2 mutants, and reconstituted strains (Figure 3D). No marked difference in calcineurin activity was apparent in crude extracts of wild‐type and cpa1 cpa2 mutant strains; however, this assay is neither highly sensitive nor highly specific (not shown). Calcineurin mutations that block cyclophilin A–CsA binding by impairing protein–protein interactions (Cna1 V344K, V344R) were fully functional in vivo (not shown). Further studies will be required to address whether cyclophilin regulates calcineurin assembly or localization; previous studies have suggested that cyclophilin A may regulate calcineurin in S. cerevisiae (Cardenas et al., 1994). Based on phenotypic differences between cyclophilin A and calcineurin mutant strains, cyclophilin A is likely to have targets in addition to calcineurin.
Prolyl isomerase activity is important for Cpa1 function in vivo
Two active site mutant alleles of the CPA1 gene were constructed and expressed to examine the in vivo function of cyclophilin A prolyl isomerase activity. Previous studies with mammalian cyclophilin A indicate that replacing the conserved Arg55 or Trp121 residues with alanine results in cyclophilin A proteins with reduced activity (0.1% for R55A and 8.7% for W121A) (Zydowsky et al., 1992). The CPA1R55A and the CPA1W121A mutants both restored normal growth to cpa1 cpa2 mutants at 25°C (not shown). At 30°C, the CPA1R55A and CPA1W121A alleles partially and fully restored growth, respectively (Figure 4C), consistent with their level of activity in vitro. Neither mutant restored wild‐type growth at 37 or 39°C, whereas the CPA1 wild‐type gene did (Figure 4C). By western blotting, the cyclophilin A mutants were both expressed under these conditions (not shown). The cyclophilin A active site mutants did not restore CsA sensitivity at 37°C, indicating that these mutants fail to bind CsA or calcineurin (Figure 4C), in accordance with previous studies. Interestingly, either active site mutant restored mating of the cpa1 cpa2 mutant strain at 24°C (not shown), indicating either that activity is not required for mating or that the residual activity of the mutant enzymes suffices. In summary, the Cpa1 protein and its activity are required for function under certain growth conditions.
Cyclophilin A is required for C. neoformans virulence
Because the C. neoformans Cpa1 cyclophilin A protein is required for growth at 39°C, and the Cpa1 and Cpa2 proteins are both required for normal growth, Cpa1 and Cpa2 were tested for roles in virulence. In a rabbit model, the wild‐type and cpa2 mutant persisted in the cerebrospinal fluid (CSF) at similar concentrations (∼106 c.f.u./ml) for the entire 10 days (day 7, p = 0.13) (Figure 6A). In contrast, the cpa1 mutant was reduced by ∼100‐fold (∼104 c.f.u./ml) compared with the wild type and cpa2 mutants (day 10, p = 0.02) (Figure 6A). In comparison, the cpa1 cpa2 double strain was reduced ∼10 000‐fold (∼102 c.f.u./ml) in survival (p = 0.004 on day 10) (Figure 6A). Virulence was partially restored in the cpa1 cpa2+CPA1 CPA2 strain compared with the cpa1 cpa2 mutant (day 10, p = 0.03) (Figure 6A). Similarly to many virulence studies in Candida albicans and C. neoformans, the reconstituted strain did not reach the full wild‐type level, possibly due to other events during transformation or differences in expression levels between the wild‐type and ectopic re‐integrated genes. Taken together, these findings indicate that Cpa1 is important for virulence of C. neoformans and that Cpa2 plays an ancillary role.
Results in a murine model were concordant. The average survival of mice infected with the wild‐type and cpa2 mutant was 17.8 and 20 days, respectively (p = 0.113; Figure 6B). Virulence was partially attenuated in the cpa1 mutant and the average survival was prolonged to 31.4 days (H99 versus cpa1, p <0.001). Complete (100%) lethality occurred by day 37 with the cpa1 mutant compared with day 20 with wild‐type and day 27 with the cpa1+CPA1 reconstituted strain (cpa1 versus cpa1+CPA1, p <0.001) (Figure 6B). The cpa1 cpa2 mutant was even more significantly attenuated for virulence (p <0.0001), and all 10 mice were still alive on day 50 (Figure 6B). Again, virulence was partially but not completely restored in the cpa1 cpa2+CPA1 CPA2 reconstituted strain, and the average survival of infected mice was 30.7 days (cpa1 cpa2 versus cpa1 cpa2+CPA1 CPA2, p <0.001) (Figure 6B).
We conclude that Cpa1 is important for virulence, whereas Cpa2 is not required for virulence in a CPA1 wild‐type strain, but does contribute in a cpa1 mutant background. The cyclophilin A Cpa1 and Cpa2 proteins are likely to contribute to virulence by promoting normal growth and survival under stress conditions. The cpa1 cpa2 double mutant exhibited defects in both melanin production in response to glucose limitation (Figure 5B) and capsule production in Eagle's medium (not shown). These defects are also likely to contribute to the virulence defect, and reflect the inability of the mutant strain to respond to stress conditions. This is the first report of a role for cyclophilin A in virulence.
Strains and media.
Serotype A strain H99 and its ade2 derivative M049 have been described (Toffaletti et al., 1993; Sudarshan et al., 1999). The H99 ura5 mutant was obtained on 5‐FOA medium (Kwon‐Chung et al., 1992b). All strains used in this study are listed in Supplementary Table I. YPD, YNB, V8 and Niger seed agar were as described (Alspaugh et al., 1997; Wang et al., 2000).
Isolation and characterization of C. neoformans cyclophilin A gene homologs.
A partial cyclophilin A gene was amplified by PCR from strain H99 and used to probe a genomic Southern blot, which revealed two related cyclophilin A genes. A 3.0 kb BamHI fragment (pPWC1) cloned from a partial library of H99 contains the first CPA1 gene. A 1.6 kb HindIII fragment (pPWC2) contains the second CPA2 gene. Complete sequences of the CPA1 and CPA2 genes were obtained by sequencing both DNA strands, and all introns were determined by sequencing RT–PCR‐amplified fragments that overlap the entire coding regions. Primers used to map the CPA1 and CPA2 genes to a common 20 kb fragment were 5′‐AACGGTACCAGAAGCGGCA (2949) and 5′‐ACCCAACGAAGCTAGAAGA (2931), and PCR comprised 30 cycles of 94°C for 20 s, 50°C for 20 s and 72°C for 15 min. DNA from a BAC clone containing the CPA1 and CPA2 genes was used as template.
Disruption of the CPA1 and CPA2 genes, reconstitution of mutants, antibody preparation, and detection of proteins.
See Supplementary data.
Active site mutagenesis of the CPA1 gene.
Point mutations were introduced in the CPA1 gene replacing, respectively, triplets CGA encoding Arg55 or TGG encoding Trp121 with GCA encoding alanine by PCR overlap mutagenesis. To construct the CPA1R55A allele, the 0.85 kb PCR fragment amplified with primers 5′‐TATCGTCTGCGTTGTCTGCT (5267) and 5′‐GGATCACTGCGTGGAAACC (6093), and also the 0.85 kb fragment with 5′‐GGTTTCCACGCAGTGATCC (6092) and 5′‐CGGTATGAGAGTCCAAGAGAAG (1335), were pooled and used as the template for a second round of PCR using primers 5267 and 1335. The CPA1W121A mutant allele was created using the same approach with primers 5′‐GTTACCTCTGCGCTCGACG (6094), 5′‐CGTCGAGCGCAGAGGTAAC (6095), 5267 and 1335. A 1.7 kb fragment was cloned, inserted in pGMC200 (pPWC10), verified by sequencing, and transformed into the cpa1 cpa2 mutant strain. The plasmid with the 1.7 kb wild‐type CPA1 gene (pPWC9) was used as a control.
Animal models of cryptococcal meningitis.
New Zealand White rabbits were used, and the maintenance, immunosuppression, sedation, recovery of CSF, and quantitative evaluation of virulence by counting colony‐forming units were as described (Wang et al., 2000). In the murine model, 50 μl (total 5 × 104 cells) of yeast cells per strain were used to infect 10, 4‐ to 6‐week‐old female A/Jcr mice by nasal inhalation. Mice were monitored twice daily and those that were moribund or in pain were killed by CO2 inhalation (Cruz et al., 2000b).
A Student's t‐test was performed to compare mean CSF yeast counts between strains in the rabbit model, and a Kruskal–Wallis analysis was applied to compare survival in mice. Values of p <0.05 were considered statistically significant. Virulence tests in both animal models were repeated twice.
We thank T. Means for 125I‐labeled calmodulin, and M. Arevalo‐Rodriguez, C. Arndt, L. Cavallo, C. Cruz, D. Fox and K. Lengeler for assistance. This work was supported by NIAID R01 grants AI39115 and AI42159, P01 grant AI44975 and K01 award CA 77075 (to M.E.C.). G.M.C. is a Burroughs Wellcome Fund New Investigator. J.H. is an associate investigator of the Howard Hughes Medical Institute and a BWF Scholar.
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