Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a fungal pathogen that causes meningitis in patients immunocompromised by AIDS, chemotherapy, organ transplantation, or high-dose steroids. Current antifungal drug therapies are limited and suffer from toxic side effects and drug resistance. Here, we defined the targets and mechanisms of antifungal action of the immunosuppressant rapamycin in C. neoformans. In the yeast Saccharomyces cerevisiae and in T cells, rapamycin forms complexes with the FKBP12 prolyl isomerase that block cell cycle progression by inhibiting the TOR kinases. We identified the gene encoding a C. neoformans TOR1 homolog. Using a novel two-hybrid screen for rapamycin-dependent TOR-binding proteins, we identified the C. neoformans FKBP12 homolog, encoded by the FRR1 gene. Disruption of the FKBP12 gene conferred rapamycin and FK506 resistance but had no effect on growth, differentiation, or virulence of C. neoformans. Two spontaneous mutations that confer rapamycin resistance alter conserved residues on TOR1 or FKBP12 that are required for FKBP12-rapamycin-TOR1 interactions or FKBP12 stability. Two other spontaneous mutations result from insertion of novel DNA sequences into the FKBP12 gene. Our observations reveal that the antifungal activities of rapamycin and FK506 are mediated via FKBP12 and TOR homologs and that a high proportion of spontaneous mutants in C. neoformans result from insertion of novel DNA sequences, and they suggest that nonimmunosuppressive rapamycin analogs have potential as antifungal agents.

FIG. 1

C. neoformans TOR1 kinase and FRB domains are highly conserved. The predicted C. neoformans TOR1 (CnTOR1) protein sequence from amino acid residues 1842 to 2200 is aligned with analogous regions from S. cerevisiae TOR1 (ScTOR1) (amino acids 1952 to 2309) (15), S. cerevisiae TOR2 (ScTOR2) (amino acids 1955 to 2313) (40), mammalian mTOR (amino acids 2015 to 2372) (54), and S. pombe TOR (SpTOR) (amino acids 1814 to 2171). The positions of kinase conserved motifs are underlined. Asterisks indicate conserved amino acid residues of the FRB domain of TOR.

FIG. 2

Sequences of the C. neoformans FRR1 gene and FKBP12 protein. The DNA sequence of the C. neoformans genomic FRR1 locus, encoding the FKBP12 protein, is depicted. The sequence was determined from cDNA and genomic clones as described in Results and in Materials and Methods. The 5′ and 3′ untranslated regions and intronic sequences are in lowercase, and introns are numbered in order of occurrence from 5′ to 3′. Exons of the FRR1 open reading frame are in uppercase, with the translated protein sequence below the DNA sequence in uppercase single-letter amino acid abbreviations. Consensus splice donor (GTNNGY), branch (CTRAY), and acceptor (YAG) sites are underlined. Start and stop codons are in boldface.

FIG. 3

The C. neoformans FKBP12 protein has marked sequence identity with FKBP12 proteins from other organisms. The alignment of the human (64), Drosophila melanogaster (accession no. U41441), Neurospora crassa (68), C. albicans (25), S. cerevisiae (34), and C. neoformans FKBP12 proteins is depicted. The overall levels of amino acid identity are shown as percentages. Amino acid residues that form the hydrophobic rapamycin- and FK506-binding pocket of FKBP12 are in boldface. The arrow indicates the conserved tryptophan residue in the frr1-3 mutant.

FIG. 4

A rapamycin-resistant TOR1 mutation prevents FKBP12-rapamycin binding. The GAL4(DB)-TOR1 FRB domain wild-type and Ser1862Leu mutant fusion proteins were coexpressed with the GAL4(AD)-FKBP12 fusion protein in the two-hybrid host strain SMY4 (fpr1 TOR1-3), with or without rapamycin (1 μg/ml). β-Galactosidase activity was measured by CPRG assay, and the values were determined in triplicate.

FIG. 5

Disruption of the C. neoformans FRR1 gene encoding FKBP12. (A) Diagram of the frr1::ADE2 gene disruption. The restriction map of the genomic FRR1 gene is shown. The FRR1 gene was disrupted by inserting a blunt-ended ADE2 gene into an RsrII restriction site. R, EcoRI; H, HindIII. (B) Confirmation of the frr1::ADE2 disruption by Southern analysis. Genomic DNA from the isogenic FRR1 wild-type strain M049 (H99 Δade2) and four frr1::ADE2 disruption mutant strains was cleaved with EcoRI (R) and HindIII (H), electrophoresed in a 0.8% agarose gel, and transferred to nitrocellulose. The membrane was hybridized to a random-primed ³²P-labelled 700-bp gel-purified fragment spanning the FRR1 gene. Positions of DNA size markers are shown on the left. Note that there is a HindIII site in the ADE2 marker; hence, two fragments arise from the frr1::ADE2 allele. In addition, integration of tandem copies of the frr1::ADE2 disruption allele results in more intense hybridization with the frr1::ADE2 allele than with the wild-type (WT) locus. (C) An frr1::ADE2 mutant strain is rapamycin and FK506 resistant. Wild-type FRR1 strain M049 and an isogenic frr1 disruption mutant lacking FKBP12 were grown for 72 h on YPD medium containing 1 μg of rapamycin or FK506 per ml at 37°C.

FIG. 6

Spontaneous frr1 mutations confer rapamycin and FK506 resistance in C. neoformans. (A) FRR1 wild-type (H99, JEC20, and JEC21) and isogenic frr1::ADE2, frr1-1, frr1-2, frr1-3, and FKR1-1 mutant strains were grown on YPD medium containing rapamycin (1 μg/ml), FK506 (1 μg/ml), or CsA (100 μg/ml). R and S, drug resistant and sensitive, respectively. (B) Southern analysis of genomic DNA from isogenic wild-type FRR1 and frr1 rapamycin-FK506-resistant mutant strains. Genomic DNA was cleaved with EcoRI (R), HindIII (H), or PstI (P), electrophoresed in a 0.8% agarose gel, transferred to nitrocellulose, and hybridized to a 700-bp FRR1 gene probe. Positions of DNA size markers are shown on the left. (C) PCR amplification of the frr1 mutant locus in strains C20F1, C20F2, and C21F3. Genomic DNA was PCR amplified with a pair of primers directed against the N-terminal region of the FKBP12 gene (primers 1 and 2) or against the C-terminal region of the FKBP12 gene (primers 3 and 4). The longer PCR products in lanes 5 and 6 resulting from strains C20F1 and C20F2 were cloned and sequenced, revealing novel DNA sequences of ∼2,200 and ∼780 bp that have inserted into the FKBP12 locus in strains C20F1 and C20F2 and are flanked by FKBP12 gene sequence in both cases. Lane M, markers. (D) Rapamycin-resistant mutants fail to express FKBP12. Protein extracts from wild-type and isogenic rapamycin-resistant mutants were subjected to SDS-PAGE, transferred to a nitrocellulose membrane, and probed with rabbit polyclonal antiserum against yeast FKBP12. Strains were FRR1 wild-type serotype A strain H99 and the isogenic frr1::ADE2 mutant strain, wild-type FRR1 serotype D strain JEC20 and the isogenic frr1-1 (C20F1) and frr1-2 (C20F2) mutant strains, and wild-type FRR1 serotype D strain JEC21 and the isogenic FKR1-1 (C21F2) and frr1-3 (C21F3) mutant strains. One hundred micrograms of protein from the same extracts was analyzed by Western blotting with antiserum against the C. neoformans cyclophilin A protein (CypA) as a loading control.

FIG. 7

FKBP12 is not required for virulence of C. neoformans. (A) Rabbits were immunosuppressed with steroids and inoculated intrathecally with wild-type FRR1 C. neoformans M049 expressing FKBP12 (○) (wild-type M049 ADE2 reconstituted) and the isogenic frr1::ADE2 mutant strain lacking FKBP12 (●). CSF was removed on days 4, 7, 10, and 14 following inoculation, and the number of surviving C. neoformans cells (expressed as the mean log₁₀ CFU per milliliter of CSF from eight rabbits at each time point) was determined by serial dilution and plating on YPD medium with growth for 3 days at 30°C. Error bars indicate the standard error of the mean. (B) Mice (10 each) were injected in the lateral tail vein with 10⁷ cells of the FRR1 wild-type strain H99 or the isogenic frr1::ADE2 mutant strain lacking FKBP12. Survival was monitored and plotted with respect to time.