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Characterization of the FKBP12-Encoding Genes in Aspergillus fumigatus

  • Katie Falloon ,

    Contributed equally to this work with: Katie Falloon, Praveen R. Juvvadi

    ‡ These authors are co-first authors on this work.

    Affiliation Duke University School of Medicine, Durham, NC, United States of America

  • Praveen R. Juvvadi ,

    Contributed equally to this work with: Katie Falloon, Praveen R. Juvvadi

    ‡ These authors are co-first authors on this work.

    Affiliation Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham, NC, United States of America

  • Amber D. Richards,

    Affiliation Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham, NC, United States of America

  • José M. Vargas-Muñiz,

    Affiliation Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, United States of America

  • Hilary Renshaw,

    Affiliation Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, United States of America

  • William J. Steinbach

    bill.steinbach@duke.edu

    Affiliations Department of Pediatrics, Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham, NC, United States of America, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, United States of America

Abstract

Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the Δfkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn’t localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don’t play an important role in virulence. While the Δfkbp12-2 and Δfkbp12-3 strains didn’t show any discernable phenotype, the Δfkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated “paradoxical growth effect” at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Introduction

Medical advancement, especially in the fields of transplantation and oncology, has led to a growing population of immunosuppressed patients. Unfortunately, as this population has expanded, the incidence of infections in these patients has also increased and invasive fungal infections are a leading cause of infection-related mortality in the immunosuppressed [15]. Chief among these infections is invasive aspergillosis, largely caused by Aspergillus fumigatus, which kills 40–60% of those it infects [2, 3, 68]. Given the relative ineffectiveness of current antifungal treatment options, an improved understanding of invasive aspergillosis, coupled with novel therapeutic agents, is needed [9].

Paradoxically, the immunosuppressants that accommodate organ transplantation, yet render patients susceptible to opportunistic infections, also possess the ability to halt invasive aspergillosis [911]. Epidemiologically, patients on agents that inhibit the Ca2+/calmodulin (CaM) dependent protein phosphatase calcineurin, such as cyclosporine A and FK506 (tacrolimus), have been shown to be less likely to suffer invasive fungal infections than those receiving other forms of immunosuppression [12, 13]. Additionally, in vitro testing shows cyclosporine A and FK506 both interfere with fungal growth and virulence [14].

Calcineurin inhibitors function by first forming complexes with immunophilins, highly conserved peptidyl-prolyl cis-trans isomerases that serve as chaperones in protein folding in organisms from fungi to humans [1520]. The immunophilins can be further classified into cyclophilins, which bind to cyclosporine A, and FK506-binding proteins (FKBPs), which bind to FK506 or rapamycin [2125]. Immunosupressant-immunophilin complexes then bind to calcineurin between its catalytic (CnaA) and regulatory (CnaB) subunits to exert their inhibitory effects [15, 16, 26, 27]. In humans, this binding prevents activation of the immune system [21, 28, 29]. In A. fumigatus, binding prevents a number of functions important for fungal pathogenesis, including regulation of stress response, cation homeostasis, cell wall integrity, and virulence [3035]. Given this unique mechanism of antifungal activity, as well as the synergism of calcineurin inhibitors with standard antifungals and antifungal activity against drug resistant strains, the calcineurin pathway is an optimal target for drug development [9, 11, 14, 36, 37]. With appropriate chemical modifications, it is possible that a calcineurin inhibitor could be designed for fungal-specific targeting, leaving human calcineurin, and by extension the human immune system, untouched [9]. Therefore, it is important to gain a better understanding of one of the key binding partners of calcineurin, FKBP.

Work on FKBPs in mammals has been extensive, and mammalian FKBPs have been shown to interact with TGF-β as well as with calcium release channels (ryanodine receptors and inositol 1,4,5 triphosphate receptors) via calcineurin and mTOR [3844]. On the contrary, exploration of FKBPs in fungi has been limited. Work in the model organisms Saccharomyces cerevisiae and Neurospora crassa shows no essential role for the FKBPs [45, 46], and orthologs of FKBP12 in both fungi mediated resistance to FK506 and rapamycin [4548]. Studies in the plant pathogens Botrytis cinerea and Fusarium fujikuroi also demonstrated a role for fungal orthologs of FKBPs in FK506 and rapamycin resistance [4951]. In human pathogenic fungi, deletions of the FKBP12 ortholog frr1 in Cryptococcus neoformans and disruptions in the FKBP12 ortholog fkbA in Mucor circinelloides have also led to FK506 and rapamycin resistance [5255]. However, no studies have focused on FKBPs in one of the most common invasive fungal pathogens, A. fumigatus.

In the present study, we identified four orthologs of human FKBP12 in A. fumigatus and characterized their roles in hyphal growth, FK506 sensitivity and virulence. Of the four FKBP12s, FKBP12-1 is critical to target in future drug development, and exploitation of the difference between it and human FKBP12 could prove important in the generation of fungal-specific FK506 analogs.

Materials and Methods

Strains, media, and growth conditions

The A. fumigatus akuBKU80 pyrG- uracil/uridine auxotroph strain was used as the recipient strain in the construction of the Δfkbp12-1, Δfkbp12-2, Δfkbp12-3, and Δfkbp12-4 deletion strains [56, 57]. It was also used in the construction of the fkbp12-1-egfp expression strain in the pyrG- background. The fkbp12-1-egfp pyrG- strain was then used as the recipient strain in the generation of the fkbp12-1-egfpΔcnaA strain. The A. fumigatus akuBKU80 strain was used as the recipient strain in construction of the fkbp12-1-egfp strain, as well as the wild-type control for all experiments [57]. The A. fumigatus Δfkbp12-1 strain was used as the recipient strain in the generation of the Δfkbp12-1Δfkbp12-2 double deletion strain. All A. fumigatus cultures were grown on glucose minimal media (GMM) at 37°C as previously described, unless otherwise specified [58]. Escherichia coli DH5α competent cells (New England Biolabs, Ipswich, MA) were used for cloning and grown on LB media supplemented with appropriate antibiotics at 37°C.

Construction of FKBP12 single and double deletion strains

With a focus on the role of A. fumigatus FKBPs in mediating antifungal resistance or pathogenesis, we constructed deletion strains of all FKBP12-encoding genes (Δfkbp12-1, Δfkbp12-2, Δfkbp12-3, and Δfkbp12-4). Primers used for the construction of the various deletion cassettes are listed in the S1 Table. The Δfkbp12-1 strain was constructed via replacement of the 637 bp fkbp12-1 gene (fkbp1/Afu6g12170, www.aspergillusgenome.org) with the 3.0 kb A. parasiticus pyrG gene to serve as a selectable marker to complement the uracil/uridine auxotrophy of akuBKU80 [31]. Approximately 1 kb of flanking upstream sequence of fkbp12-1 was PCR amplified from A. fumigatus strain AF293 genomic DNA and cloned into the pCDF-Duet-1 vector (Novagen EMD Millipore, Billerica, MA), using the BamHI and EcoRI sites. Fusion PCR was used to generate the ~4.0 kb sequence containing the A. parasiticus pyrG gene and ~1 kb of flanking downstream sequence of fkbp12-1, which was also cloned in the pCDF-Duet-1 vector using the EcoRI and SacI sites. The resulting replacement construct plasmid was used as a template to create the ~4.7 kb PCR amplicon for use in transformation into the akuBKU80 pyrG- strain.

The Δfkbp12-2 strain was constructed via replacement of the 709 bp fkbp12-2 gene (fkbp2/Afu4g04020, www.aspergillusgenome.org) with the 3.0 kb A. parasiticus pyrG gene. Approximately 1 kb of flanking upstream and downstream sequences were PCR amplified from AF293 genomic DNA and cloned into the pJW24 plasmid, using the SalI and EcoRI sites for the upstream sequence and the BamHI and NotI sites for the downstream sequence. The resulting replacement construct plasmid was then linearized via NotI digestion to yield the final construct for transformation into the akuBKU80 pyrG- strain.

The Δfkbp12-3 strain was constructed via replacement of the 485 bp fkbp12-3 gene (fkbp3/Afu2g03870, www.aspergillusgenome.org) with the 3.0 kb A. parasiticus pyrG gene. Approximately 1 kb of flanking upstream and 608 bp of flanking downstream sequences were PCR amplified from AF293 genomic DNA and cloned into the pJW24 plasmid, using the NotI and XbaI sites for the upstream sequence and the EcoRI and SalI sites for the downstream sequence. The resulting replacement construct plasmid was used as a template to create the ~4.7 kb PCR amplicon for use in transformation into the akuBKU80 pyrG- strain.

The Δfkbp12-4 strain was constructed via replacement of the 1653 bp fkbp12-4 gene (fkbp4/Afu6g08580, www.aspergillusgenome.org) with the 3.0 kb A. parasiticus pyrG gene. Approximately 1 kb of flanking upstream and downstream sequences were PCR amplified from AF293 genomic DNA and cloned into the pJW24 plasmid, using the SalI and EcoRI sites for the upstream sequence and the NotI and SacI sites for the downstream sequence. The resulting replacement construct plasmid was then linearized via digestion with SalI and SacI to yield the construct for use in transformation into the akuBKU80 pyrG- strain.

For strains Δfkbp12-1 through Δfkbp12-4, transformants were selected for growth in the absence of uracil/uridine supplementation.

The Δfkbp12-1Δfkbp12-2 double deletion strain was constructed via replacement of the 709 base pair fkbp12-2 gene (fkbp2/Afu4g04020, www.aspergillusgenome.org) with the 4.4 kb hygromycin B resistance (hph) cassette. Approximately 1 kb of flanking upstream and downstream sequences were PCR-amplified from AF293 genomic DNA and cloned into the pUCGH plasmid, using the HindIII and SbfI sites for the upstream sequence and the EcoRV and NotI sites for the downstream sequence. The resulting replacement construct plasmid was then linearized via digestion with NotI, yielding the construct for use in transformation into the Δfkbp12-1 strain. Transformants were selected for resistance to hygromycin B. Primers utilized to construct this strain are listed in the S1 Table.

To construct the fkbp12-1-egfp strain, 384 bp of the 637 bp fbkp12-1 gene (fkbp1/Afu6g12170, www.aspergillusgenome.org) and ~1 kb of the fkbp12-1 terminator sequence were PCR amplified from AF293 genomic DNA and cloned into the pUCGH plasmid at the N-terminus of egfp, using the KpnI and BamHI sites for the gene and the SbfI and HindIII sites for the terminator sequence. The plasmid was then sequenced to confirm accuracy of the partial sequence of the fkbp12-1 cloned and finally linearized via single restriction enzyme digestion with KpnI. The construct was transformed into the A. fumigatus akuBKU80 strain. Transformants were selected for resistance to hygromycin B. All primers utilized to construct the GFP strain are listed in the S4 Table.

To construct the fkbp12-1-egfpΔcnaA strain, first 384 bp of the 637 bp fbkp12-1 gene (fkbp1/Afu6g12170, www.aspergillusgenome.org) and ~1 kb of the fkbp12-1 terminator sequence were PCR amplified from AF293 genomic DNA and cloned into the pUCGH plasmid at the N-terminus of egfp, using the KpnI and BamHI sites for the gene and the SbfI and HindIII sites for the terminator sequence. The plasmid was then sequenced to confirm accuracy of the partial sequence of the fkbp12-1 cloned and finally linearized via single restriction enzyme digestion with KpnI. The construct was transformed into the A. fumigatus akuBKU80 pyrG- strain. Next, the 3.0 kb A. parasiticus pyrG gene was used to replace the 1.9 kb cnaA gene (calA/Afu5g09360, www.aspergillusgenome.org) as previously described [31] and the resulting replacement construct was transformed into the akuBKU80 pyrG- fkbp12-1-egfp strain.

For all 6 strains, generation of the fungal protoplasts and polyethylene glycol-mediated transformation was performed as previously described [31]. Transformants were initially screened by PCR with primers designed to amplify the deleted genes and also with primers flanking the deleted gene to verify homologous recombination. All primers used to verify proper integration in the deletion strains are listed in the S2 Table. Confirmation of gene deletion was performed via Southern analysis using a digoxigenin labeling system (Roche Molecular Biochemicals, Mannheim, Germany) for all deletion strains. The primers used to generate the probes used for Southern analysis in each strain are listed in the S3 Table. All primers used to verify proper integration in the egfp strains are listed in the S5 Table. Fluorescence microscopy served as the second confirmatory test for the FKBP12-1-EGFP and FKBP12-1-EGFPΔcnaA strains.

Radial Growth

Radial growth on solid media was quantified as previously described for all deletion strains and the FKBP12-1-EGFP strain [31]. All assays were performed in triplicate. To further validate the slight growth defect observed with the Δfkbp12-4 strain, the assays were performed in triplicate in two independent experiments. Statistical comparison was performed using Graph Pad Prism (San Diego, CA).

Antifungal and immunosuppressant susceptibility testing

FK506, cyclosporine A, and caspofungin were obtained as commercial products. Rapamycin was obtained from the National Cancer Institute. An inoculum of 10 μL of 1x106 conidia/mL (104 conidia) was spotted onto GMM plates supplemented with either FK506 (100 ng/mL) or cyclosporine A (10 μg/mL). Testing for caspofungin sensitivity was performed with GMM plates supplemented with either 1 μg/mL or 4 μg/mL of caspofungin and growth was observed after 5 days [34]. Susceptibility to caspofungin was also analyzed in 96 well plates using RPMI 1640 liquid media (RPMI; Roswell Park Memorial Institute) supplemented with either 1 μg/mL or 4 μg/mL of caspofungin. Hyphal growth was visualized microscopically after incubation at 37°C for 24 and 48 hours. Spotting on GMM supplemented with both FK506 (100 ng/mL) and caspofungin (1 μg/mL) was performed to assess the combined effect on antifungal resistance. Susceptibility to FK506 was also analyzed in 96 well plates using RPMI 1640 media and 100 ng/mL of FK506 [59]. Hyphal growth was visualized microscopically after incubation at 37°C for 24 and 48 hours. Given the high MIC of the drug, resistance to rapamycin was analyzed in 96 well plates using RPMI media and 100 μg/mL of rapamycin [14]. Hyphal growth was visualized microscopically after incubation at 37°C for 24 hours. All drug testing was performed in triplicate.

Virulence Testing

Twenty larvae of the waxmoth Galleria mellonella were injected with 5 μl of 1 x 108 spores/ml (total inoculum of 2 x 105 spores) of the wild-type or the respective FKBP12 deletion strains. Infected larvae were incubated at 37°C with survival scored daily for 5 days [60]. Data from this trial was plotted on a Kaplan-Meier curve with log rank pair-wise comparison and statistical analysis was performed using Graph Pad Prism (San Diego, CA).

Light and Fluorescence Microscopy

Conidia of the fkbp12-1-egfp strain were cultured for 18–20 hours at 37°C in petri dishes containing coverslips immersed in 10 mL of GMM liquid media [32]. To assess localization following exposure to FK506, conidia of the fkbp12-1-egfp strain were cultured for 18–20 hours at 37°C in 60x15 mm petri dishes containing coverslips immersed in 10 mL GMM liquid media supplemented with 100 ng/mL of FK506. Following incubation, spores were adherent to the coverslip and could be visualized via microscopy. To confirm that the septal localization observed was due to interaction with calcineurin, both experiments were repeated with the fkbp12-1-egfpΔcnaA strain. Fluorescence microscopy was performed using an Axioskop 2 plus microscope (Zeiss) equipped with AxioVision 4.6 imaging software with Nomarski optics (Differential Interference Contrast) and fluorescence. For nuclear staining, the fkbp12-1-egfp and wild type strains were cultured in GMM liquid medium on coverslips for 18–20 hours and stained with propidium iodide. Briefly, the cultures were washed in 50 mM PIPES (pH 7.0) for 5 minutes, fixed in 8% formaldehyde with 0.2% Triton X-100 for 45 minutes at 25°C, washed in 50 mM PIPES (pH 7.0) for 10 minutes and treated with RNase (100 μg/ml) for 60 minutes at 37°C. After washing with 50 mM PIPES (pH 7.0) for 20 minutes, the fixed sample was stained with propidium iodide solution (12.5 μg/ml) in 50 mM PIPES (pH 7.0) for 5 minutes, washed again with 50 mM PIPES (pH 7.0) for 20 minutes and observed under the fluorescence microscope.

Results

Identification and phylogenic analysis of putative A. fumigatus FKBP12 orthologs

In order to characterize the role of FKBP12 orthologs in A. fumigatus, BLAST analysis was used to identify the four putative orthologs of human fkbp12 (www.aspergillusgenome.org). Four FKBPs (fkbp12-1, fkbp12-2, fkbp12-3 and fkbp12-4) were identified. Comparison with human FKBP12 revealed that FKBP12-1 had the greatest sequence similarity (55% identity, 74% similarity, http://www.ebi.ac.uk/Tools/psa/emboss_needle), followed by FKBP12-2 (47% identity, 68% similarity, http://www.ebi.ac.uk/Tools/psa/emboss_needle), FKBP12-3 (35% identity, 50% similarity, http://www.ebi.ac.uk/Tools/psa/emboss_needle) and finally FKBP12-4 (10% identity, 14% similarity, http://www.ebi.ac.uk/Tools/psa/emboss_needle). Multiple sequence alignment of the proteins (S1 Fig) and phylogenetic analysis (Fig 1A) showed that the FKBP12 proteins from human and other fungal species were closely related. While FKBP12-1, FKBP12-2 and FKBP12-3 were grouped together, FKBP12-4 may belong to a separate clade and diverged from the other members. Based on the sequence similarity between FKBP12-1 and FKBP12-2, it is possible that FKBP12-2 may have been formed due to gene duplication. Comparison of amino acids at the 14 residues previously identified as key for binding to FK506 [61] revealed that FKBP12-1 differed from human FKBP12 at three of the 14 sites, FKBP12-2 at three of the 14 sites, FKBP12-3 at four of the 14 sites, and FKBP12-4 at two of the 14 sites (Fig 1B). The three substitutions in A. fumigatus FKBP12-1 involved the replacement of an acidic amino acid with a basic one (arginine for glutamate, position 55), the replacement of one small nonpolar amino acid with another (glycine for alanine, position 82), and the replacement of a basic amino acid with a nonpolar and bulky one (phenylalanine for histidine, position 88). As is illustrated in Fig 1B, FKBP12 orthologs from other species are mutated only at two residues.

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Fig 1. Phylogenetic analysis of FKBP12 proteins and multiple sequence alignment comparing residues important for FK506-FKBP12 binding.

(A) Phylogram showing orthologous FKBP12 proteins from Human (HsFKBP12), S. cerevisiae (ScFKBP12), C. neoformans (CnFKBP12), M. circinelloides (McFKBP12) and A. fumigatus (AfFKBP12-1, AfFKBP12-2, AfFKBP12-3 and AfFKBP12-4). Phylogenic analysis was performed using the respective amino acid sequences aligned with MUSCLE (v3.8.31) implemented in the PhyML program (v3.1/3.0 aLRT). Graphical representation of the phylogenetic tree was performed with TreeDyn (v198.3). A. fumigatus FKBP12 proteins are designated as AfFKBP12-1, AfFKBP12-2, AfFKBP12-3 and AfFKBP12-4, respectively. (B) 14 residues are known to be important for binding FKBP12 to FK506. Residues distinct from human FKBP12 are highlighted in a different color for each A. fumigatus FKBP12 and for each species-specific FKBP12 homolog.

https://doi.org/10.1371/journal.pone.0137869.g001

The modifications in FKBP12-2, which shares the next most sequence similarity to human FKBP12, include replacement of the neutral phenylalanine with the nonpolar leucine (position 47), replacement of the acidic glutamate with the acidic aspartate (position 55) and replacement of histidine with phenylalanine (position 88). FKBP12-3 differs by four amino acids at similar locations, including replacement of phenylalanine with leucine (position 47), glutamate with arginine (position 55), alanine with glycine (position 82) and histidine with the nonpolar isoleucine (position 88). FKBP12-4, which shares the least sequence similarity to human FKBP12, differs from human FKBP12 at only two of the 14 residues (replacement of arginine with lysine at position 43 and replacement of histidine with leucine at position 88).

Deletion analysis of the FKBP12 genes in A. fumigatus

Based on the in silico analysis, single deletion strains for all four A. fumigatus FKBP12 orthologs were generated (Fig 2A–2D). In addition, due to the higher homology observed between the FKBP12-1 and FKBP12-2 proteins, a double deletion strain of FKBP12-1 and FKBP12-2 (Δfkbp12-1Δfkbp12-2) was generated to verify any coordinated function between the two proteins (Fig 2E). Successful deletion for each strain was confirmed by PCR (data not shown) and Southern analysis (Fig 2). Various recombinant strains generated in this study are listed in Table 1. Radial growth assays of all the FKBP12 deletion strains revealed them to be non-essential in A. fumigatus (Fig 3A). Among the respective deletion strains only the Δfkbp12-4 strain demonstrated slightly impaired growth under basal conditions compared to the wild-type strain (p = 0.016) (Fig 3A and 3B). Apart from the reduced growth rate, however, there were no other visible growth abnormalities in the strain (Fig 3B). The Δfkbp12-1 strain had a statistically significant difference in growth compared to the wild-type strain (p = 0.0405), but by day 5 had reached a similar full growth. All other deletion strains demonstrated radial growth patterns consistent with that seen with the wild-type strain (Fig 3A). In Δfkbp12-1Δfkbp12-2, Δfkbp12-2, and Δfkbp12-3, statistically significant differences in growth were not observed (p = 0.4318, p = 0.2601, p = 0.3138). Thus, of the four FKBP12s, only FKBP12-4 is required for proper growth under basal conditions.

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Fig 2. Construction of the FKBP12 deletion strains.

(A) Construction of Δfkbp12-1 strain. In the Δfkbp12-1 strain, wild-type A. fumigatus fkbp12-1 (637 bp) was replaced by the 3.0 kb A. parasiticus pyrG gene. Three of the five strains validated by PCR were then selected for Southern analyses. SalI-digested genomic DNA was probed with the 646 bp probe of the downstream flanking sequence to confirm homologous recombination. Two of the three tested strains demonstrated the expected ~3.3 kb length, which is contrasted with the WT length of ~1.1 kb. Gel used for Southern analysis was 1% agarose. (B) Construction of Δfkbp12-2 strain. In the Δfkbp12-2 strain, wild-type A. fumigatus fkbp12-2 (709 bp) was replaced with the 3.0 kb A. parasiticus pyrG gene. The strain validated by PCR was then used for Southern analyses. BamHI-digested genomic DNA was probed with the 733 bp probe of the downstream flanking sequence to confirm homologous recombination. The strain demonstrated the expected ~1.1 kb length in contrast with the ~2 kb length in the wild-type strain. Gel used for Southern analysis was 1.5% agarose. (C) Construction of Δfkbp12-3 strain. In the Δfkbp12-3 strain, wild-type A. fumigatus fkbp12-3 (485 bp) was replaced by the 3.0 kb A. parasiticus pyrG gene. Four of the strains validated by PCR were then selected for Southern analyses. SacI-digested genomic DNA was probed with the 446 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~3.9 kb length as opposed to the wild-type length of ~1.4 kb. Gel used for Southern analysis was 1% agarose. (D) Construction of Δfkbp12-4 strain. In the Δfkbp12-4 mutant, wild-type A. fumigatus fkbp12-4 (1653 bp) was replaced by the 3.0 kb A. parasiticus pyrG gene. Four of the strains validated by PCR were then selected for Southern analyses. BamHI-digested genomic DNA was probed with the 677 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~4.5 kb length as opposed to the wild-type length of ~2.0 kb. Gel used for Southern analysis was 1% agarose. (E) Construction of Δfkbp12-1Δfkbp12-2 strain. In the Δfkbp12-1Δfkbp12-2 strain, wild-type A. fumigatus fkbp12-2 (709 bp) is replaced by the 4.4 kb hygromycin B resistance cassette in the Δfkbp12-1 strain. Four of the strains validated by PCR were then selected for Southern analyses. BamHI-digested genomic DNA was probed with the 550 bp probe of the downstream flanking sequence to confirm homologous recombination. All four tested strains demonstrated the expected ~5.2 kb as opposed to the wild-type length of ~1.9 kb.

https://doi.org/10.1371/journal.pone.0137869.g002

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Fig 3. FKBP12-4 is required for complete hyphal growth.

(A) Growth of A. fumigatus (104 conidia) on GMM at 37°C for 5 days, with colony diameter measured every 24 hours, revealed no statistically significant difference in growth among Δfkbp12-1, Δfkbp12-2, Δfkbp12-1Δfkbp12-2, Δfkbp12-3 and wild-type strains. Δfkbp12-4 demonstrated reduced growth rate across all 5 days (p = 0.0161). (B) After the 5 day growth period, Δfkbp12-4 demonstrated reduced growth compared to wild-type strain but no other obvious phenotypic abnormalities were noted.

https://doi.org/10.1371/journal.pone.0137869.g003

FKBP12-1 is the key protein that binds to FK506 and inhibits calcineurin

Next, in order to determine which of these putative FKBP12 proteins is involved in the binding of FK506 and inhibition of calcineurin function in A. fumigatus, the respective deletion strains were cultured in the absence or presence of FK506 (100 ng/mL) (Fig 4A and 4B). As shown in Fig 4B, with the exception of the Δfkbp12-1 strain and the Δfkbp12-1Δfkbp12-2 double deletion strain, all deletion strains showed sensitivity to FK506. Δfkbp12-2 and Δfkbp12-3 showed susceptibility to FK506 comparable to that of the wild-type strain (Fig 4B). These results confirmed that the FK506 resistance observed in the Δfkbp12-1Δfkbp12-2 double deletion strain was due to the deletion of fkbp12-1. Δfkbp12-4 showed minimal tolerance to FK506, with slightly less sensitivity to the drug than was seen in the wild type strain (Fig 4B). Testing with another immunosuppressant, cyclosporine A, demonstrated susceptibility indistinguishable from the wild-type strain (Fig 4C), indicating that FKBP12-1 specifically binds to FK506 and inhibits calcineurin function. This is expected given the different mechanism of action of cyclosporine A, which binds to cyclophilin A and causes the inhibition of calcineurin. The Δfkbp12-1 strain also demonstrated resistance to rapamycin (100 μg/mL) (data not shown).

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Fig 4. Δfkbp12-1 is resistant to FK506 but its response to other antifungal agents is unchanged.

(A) A. fumigatus conidia (104 conidia) incubated on GMM at 37°C for 5 days. (B) A. fumigatus conidia (104 conidia) incubated on GMM + 100 ng/mL FK506 at 37°C for 5 days. (C) A. fumigatus conidia (104 conidia) incubated on GMM + 10 μg/mL cyclosporine A (CsA) at 37°C for 5 days. (D) A. fumigatus conidia (104 conidia) incubated on GMM + 1 μg/mL caspofungin (CSP) at 37°C for 5 days (E) A. fumigatus conidia (104 conidia) incubated on GMM + 1 μg/mL caspofungin at 37°C for 5 days (F) A. fumigatus conidia (104 conidia) incubated on GMM + 100 ng/mL FK506 + 1 μg/mL caspofungin at 37°C for 5 days.

https://doi.org/10.1371/journal.pone.0137869.g004

Because the Δfkbp12-4 strain showed reduced growth in comparison to the wild-type strain, we also examined the effect of caspofungin, an anti-cell wall antifungal agent, on all the FKBP12 deletion strains. At 1 μg/mL caspofungin, Δfkbp12-1, Δfkbp12-2, Δfkbp12-3, and Δfkbp12-1Δfkbp12-2 strains demonstrated similar susceptibility to caspofungin, while Δfkbp12-4 demonstrated increased susceptibility (Fig 4D). As is normally observed in the wild-type strain, paradoxical growth effect was noted at higher caspofungin concentrations in all deletion strains except for the Δfkbp12-4 strain (Fig 4E) [6264]. In the presence of the combination of FK506 and caspofungin, the Δfkbp12-1 and Δfkbp12-1Δfkbp12-2 strains demonstrated slightly increased growth compared to other deletion strains as well as the wild-type strain. The growth of Δfkbp12-1 and Δfkbp12-1Δfkbp12-2 strains in the presence of both drugs (FK506+caspofungin) was indistinguishable from their growth in response to caspofungin alone (Fig 4F). To more clearly visualize the inhibition of paradoxical growth at higher caspofungin concentrations in the Δfkbp12-4 strain, the Δfkbp12-4 strain was cultured in RPMI liquid media supplemented with caspofungin at 1 μg/mL and 4 μg/mL (Fig 5A and 5B). In contrast to the akuBKU80 strain the Δfkbp12-4 did not demonstrate paradoxical growth recovery. This lack of paradoxical growth in Δfkbp12-4 may be due to the fact that the Δfkbp12-4 showed reduced growth rate in comparison to the wild-type strain and the other FKBP12 deletion strains.

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Fig 5. Δfkbp12-4 is more susceptible to caspofungin and lacks paradoxical growth at higher concentrations of caspofungin.

(A) A. fumigatus akuBKU80 and the Δfkbp12-4 conidia (104/mL) were cultured in RPMI for 24 hours either in the absence or presence of 1 μg/ml and 4 μg/ml caspofungin (CSP). (B) A. fumigatus akuBKU80 and the Δfkbp12-4 conidia (104/mL) cultured in RPMI for 48 hours either in the absence and presence of 1 μg/ml and 4 μg/ml caspofungin (CSP).

https://doi.org/10.1371/journal.pone.0137869.g005

To visualize hyphal growth in response to FK506, the single deletion strains were also cultured in liquid media supplemented with FK506 (Fig 6). The Δfkbp12-1 displayed full hyphal growth in the presence of FK506 after 24 hours, while Δfkbp12-4 seemed slightly tolerant in comparison to the other deletion strains (Fig 6A). At 48 hours the respective strains demonstrated improved growth, although the inhibitory effect of FK506 was still evident in all except for the Δfkbp12-1 strain (Fig 6B). The Δfkbp12-1Δfkbp12-2 strain was also resistant to FK506 (data not shown).

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Fig 6. Δfkbp12-1 demonstrates full hyphal growth in response to FK506; Δfkbp12-4 is slightly tolerant to FK506.

(A) A. fumigatus conidia (104/mL) incubated in RPMI for 24 hours. (B) A. fumigatus conidia (104/mL) incubated in RPMI with 100 ng/mL FK506 for 24 hours. (C) A. fumigatus conidia (104/mL) incubated in RPMI for 48 hours. (D) A. fumigatus conidia (104/mL) incubated in RPMI with 100 ng/mL FK506 for 48 hours.

https://doi.org/10.1371/journal.pone.0137869.g006

FKBP12-1 localizes to the cytoplasm and nuclei but also shifts to the hyphal septa following exposure to FK506

Previous studies from our laboratory revealed the localization of calcineurin complex at the hyphal septum in a disc-like manner around the septal pore [32, 33]. We took advantage of this localization to verify the binding of FKBP12-1 to calcineurin in the presence of FK506. In order to examine the localization of FKBP12-1 and confirm its association with calcineurin in the presence of FK506, a strain expressing FKBP12-1 tagged to EGFP (fkbp12-1-egfp) at its native locus was generated. Homologous recombination was confirmed by PCR (data not shown). To confirm functionality of the tagged FKBP12-1 protein, radial growth assays and testing with FK506 were performed. No difference between the wild type and the FKBP12-1-EGFP strains were noted (Fig 7A). Under normal growth conditions, FKBP12-1 was localized evenly throughout the cytoplasm and also in the nuclei at the hyphal tips and in the sub-apical compartments (Fig 7B and 7C). Upon exposure to FK506, FKBP12-1 also localized to the septa in the form of a disc-like pattern as noted earlier with calcineurin (Fig 7D; see inset image), suggesting its binding to calcineurin and inhibition of calcineurin activity at the hyphal septum as previously reported [32]. To confirm this, we next constructed an FKBP12-1-EGFP expression strain and deleted the catalytic subunit of calcineurin encoding gene cnaA in this background (Fig 8A–8C and 8D). Akin to what was seen earlier with the wild type FKBP12-1-EGFP, the localization patterns of FKBP12-1 in a calcineurin null strain (fkbp12-1-egfpΔcnaA) revealed nuclear and cytoplasmic localization under basal conditions (Fig 8E). However, upon exposure to FK506 FKBP12-1-EGFP failed to localize to the septa, confirming that FKBP12-1 localizes at the hyphal septum through binding to calcineurin upon exposure to FK506 (Fig 8F).

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Fig 7. FK506 altered the localization of FKBP12-1 to the hyphal septum.

(A) Functionality of the expressed FKBP12-1-EGFP was assessed by comparing the growth of the FKBP12-1-EGFP expression strain with the akuBKU80 strain either in the absence or presence of FK506 (0.1 μg/mL) (B, C) Under normal growth conditions, FKBP12-1 evenly distributes throughout the cytoplasm and is also found in the nucleus at the hyphal tips (panel B) and sub-apical compartment (panel C and panel D) (marked by a white arrow heads in panel B and by red arrowheads in panel C and panel D). It is not seen at the septum (marked by a white arrow in the Fig 7C inset). (C) In the presence of FK506, FKBP12-1 can be seen localized as a double bar on either side of the septa indicating its binding to calcineurin complex at the hyphal septum (marked by a white arrows in the Fig 7D inset).

https://doi.org/10.1371/journal.pone.0137869.g007

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Fig 8. FKBP12-1 localizes to the hyphal septum through binding to CnaA in the presence of FK506.

(A) Confirmation of generation of the FKBP12-1-EGFP expression strain by PCR and fluorescence microscopy. (B) Cytosolic localization of FKBP12-1-EGFP. (C) Septal localization of FKBP12-1-EGFP in the presence of FK506 (indicated by white arrows). (D) Confirmation of generation of the cnaA deletion in the FKBP12-1-EGFP expression background strain by PCR and fluorescence microscopy. (E) Cytosolic localization of FKBP12-1-EGFP in the calcineurin null strain. (F) Absence of septal localization of FKBP12-1-EGFP in the calcineurin null strain in the presence of FK506 (indicated by white arrows).

https://doi.org/10.1371/journal.pone.0137869.g008

Nuclear localization of FKBP12-1 was confirmed by propidium iodide staining of nuclei (Fig 9). Although we could not identify any nuclear localization signal consensus sequence in FKBP12-1, we speculate that FKBP12-1 might translocate into the nucleus by binding to other protein/s.

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Fig 9. FKBP12-1 is seen in the cytoplasm and in the nuclei under basal conditions.

(A, B) Propidium iodide staining confirms FKBP12-1 localization to the nucleus in the apical compartment (Fig 9A; marked by white arrow heads) and the sub-apical compartment (Fig 9B; marked by white arrow heads).

https://doi.org/10.1371/journal.pone.0137869.g009

FKBP12-1 proteins do not play a key role in virulence

Earlier reports on the human pathogenic bacterium, Legionella pneumophila, and the human parasitic protozoan, Trypanosoma cruzi, have revealed the association of the FKBP12 proteins with virulence [65, 66]. While in the plant pathogenic fungus Botrytis cinerea disruption of the only ortholog of FKBP12, BcPIC5, caused a reduction in pathogenicity [50], in another plant pathogen Fusarium graminearum the interaction of FKBP12 with a virulence factor FGL1 encoding a secreted lipase was demonstrated [67]. In order to verify if A. fumigatus FKBP12s played a role in virulence, we employed a screening systemic aspergillosis infection model using the heterologous invertebrate host Galleria mellonella. Infection of the larvae with all the FKBP12 deletion strains led to survival comparable to that seen in the wild-type strain (p = 0.64) (Fig 10). No difference in melanization of the Galleria, which serves as an indication of immune response, was noted following infection with the wild type strain or FKBP12 deletion strains.

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Fig 10. Deletion of FKBP12 encoding genes did not alter virulence of A. fumigatus.

Larvae of the wax moth Galleria mellonella were injected with 5 μl of 1 x 108 spores/ml (a total inoculum of 2 x 105 spores) of the wild type, Δfkbp12-1, Δfkbp12-2, Δfkbp12-3, Δfkbp12-4, andΔfkbp12-1Δfkbp12-2 strains. 20 larvae were included in each arm. Infected larvae were incubated at 37°C with survival scored daily for 5 days.

https://doi.org/10.1371/journal.pone.0137869.g010

Discussion

Previous work has demonstrated the potential of drugs currently used as immunosuppressants, such as FK506, as possible therapeutic agents against invasive fungal infections, including A. fumigatus [1113, 68, 69]. While in humans the FK506-FKBP12 complex binding to calcineurin suppresses the immune system, in A. fumigatus binding of the FK506-FKBP12 complex to fungal calcineurin leads to impaired growth and virulence [9, 28]. Thus, drugs like FK506 could be chemically modified or repurposed for targeted inhibition of fungal-specific calcineurin in the treatment of invasive fungal infections. While study of FKBP12, one of the key proteins through which FK506 and rapamycin exert their effects, has been extensive in humans, work in fungi has been limited [50, 51, 67]. Deletion of orthologs of FKBP12 have been found to mediate resistance to FK506 in pathogenic fungi, including C. neoformans and M. circinelloides [52, 53, 55, 70]. However, studies in A. fumigatus, a leading cause of death secondary to invasive fungal infection as well as the pathogen with the largest financial burden of all invasive fungal infections [70], have not yet been undertaken [61]. In this study, we characterized the four putative A. fumigatus FKBP12 orthologs through deletion analysis coupled with phenotypic and virulence studies, and identified FKBP12-1 as responsible for binding to FK506 and inhibiting calcineurin and FKBP12-4 as involved in basal growth.

Given that FKBP12-1 is the ortholog with the most sequence similarity to human FKBP12, it is not surprising that deletion of FKBP12-1 encoding gene led to FK506 resistance, presumably through a lack of binding to FK506. Septal localization pattern of FKBP12-1 only in the presence of FK506 in the wild-type but not in the cnaA deletion background further supports the hypothesis that loss of binding to FK506 is responsible for the resistance. Localization under basal conditions to the cytoplasm and nucleus is consistent with FKBP localization in other organisms [61], while the presence of the protein at the septa in the presence of FK506 in the FKBP12-1-EGFP strain but not in the FKBP12-1-EGFPΔcnaA strain suggests calcineurin-binding and inhibition [33]. However, three of the 14 residues important for binding to FK506 are different from human FKBP12 in the A. fumigatus FKBP12-1 protein. On the other hand, FKBP12-2 and FKBP12-3 are mutated at three and four of the 14 residues, respectively, and deletion of these proteins does not lead to resistance to FK506. Thus, the impact of residue changes in FKBP12-2 and FKBP12-3 on binding to FK506 is unclear–it is possible that binding to FK506 is retained despite these changes. Alternatively, the FKBP12-2 and FKBP12-3 proteins may not be endogenously expressed in the fungus and the susceptibility to FK506 observed may be the result of the actions of FKBP12-1 alone on calcineurin, a hypothesis supported by the resistance to FK506 seen in the Δfkbp12-1Δfkbp12-2 deletion strain. Interestingly, FKBP12-4, which shares the least sequence similarity to human FKBP12 but also differs at only two of the 14 residues noted to be involved in FK506 binding, shows minimal tolerance to FK506. This suggests that perhaps its mutations lessen but do not preclude binding to FK506. Alternatively, it is possible that binding to FK506 remains intact but the long N-terminal region of the protein interferes with binding of the FKBP12-FK506 complex to calcineurin. However, the tolerance is difficult to interpret as far as biologic relevance in the face of the observed growth defect in Δfkbp12-4 strain.

Taken together, these results suggest that homology to human FKBP12 can be predictive in determining resistance to FK506, but they also suggest that the number of residues mutated at the 14 residues previously found to be critical for binding is less predictive. Indeed, while FKBP12-4 has the fewest number of mutations in residues involved in FK506 binding, only the Δfkbp12-1 strain, that which still retains the the most sequence similarity to human FKBP12, is resistant to FK506. Most significantly, the present data suggest that even with differences from human FKBP12 that include alterations in polarity and size, binding of FKBP12-1 to FK506 can still occur. Therefore, designing a FK506 analog that will fit into the altered binding pocket of the fungal FKBP12-1, but not into the binding pocket of the human FKBP12, should be explored.

In addition to determining the A. fumigatus FKBP12 responsible for mediating resistance to FK506, we sought to better understand the role of this important family of proteins in fungal biology and thus characterized all four deletion strains as well as a double deletion strain (Δfkbp12-1Δfkbp12-2). As FKBP12s have previously been found to be dispensable for growth in other fungi and model organisms, except under some stress conditions, it is not surprising that the Δfkbp12-1, Δfkbp12-2, Δfkbp12-3, and Δfkbp12-1Δfkbp12-2 strains all demonstrated radial growth consistent with that seen in the wild-type strain [19, 20, 45, 46]. While the Δfkbp12-1 strain did have a statistically significant difference in growth compared to wild type (p = 0.0405), that the Δfkbp12-1Δfkbp12-2 strain did not, and that the Δfkbp12-1 strain did reach full growth by the end of the 5 day incubation period, suggest a lack of biologic relevance.

Unexpectedly, we found that the FKBP with the least sequence similarity to human FKBP12, FKBP12-4, plays a role in growth of the pathogen under basal conditions. The Δfkbp12-4 strain displayed universally slow growth throughout the 5 day testing period. The FKBP12-4 protein is significantly larger than the other three FKBP12s (489 amino acids versus 112 to 134 amino acids in FKBP12-1 through FKBP12-3), with an extended N-terminal sequence.

Though more comparable in size to the other A. fumigatus FKBP12s, human FKBP12.6, an isoform of human FKBP12, has an N-terminal sequence important for binding to the ryanodine receptor [71]. Perhaps the long N-terminal sequence of A. fumigatus FKBP12-4 may similarly have residues important for binding to a target, which in this case is related to growth regulation or cell wall stability. It is also possible that the growth defect is a result of changes in amino acids in the regions FKBP12-4 shares with the other A. fumigatus FKBPs. As FKBPs are involved in protein folding, FKBP12-4 may be required for efficient folding of a protein important to growth in A. fumigatus. Regardless, the probable reason for this growth defect may be related to a defect in cell wall integrity.

The role of FKBPs in the pathogenesis was also studied through the use of G. mellonella, which has been shown to be a reliable screening model of infection in A. fumigatus [72, 73]. There was no statistically significant difference in virulence between the FKBP12 deletion and wild-type strains.

In conclusion, we have identified four A. fumigatus FKBP12 orthologs, and through deletion analysis confirmed FKBP12-1 binding to FK506 in A. fumigatus. We have also established that FKBP12-4 leads to a growth defect under basal conditions. Future directions for this work include further study of FKBP12-1, specifically via more in depth identification of fungal-specific residues most important for binding to FK506 through targeted mutagenesis.

Supporting Information

S1 Fig. Multiple sequence alignment comparing A. fumigatus Fkbp12 proteins to those in other organisms.

Multiple sequence alignment was performed comparing orthologous FKBP12 proteins from Human (HsFKBP12), S. cerevisiae (ScFkbp12), C. neoformans (CnFkbp12), and A. fumigatus (FKBP12-1, FKBP12-2, FKBP12-3 and FKBP12-4) using ClustalW (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

https://doi.org/10.1371/journal.pone.0137869.s001

(TIF)

S1 Table. Primers Used in the Generation of Deletion Strains.

https://doi.org/10.1371/journal.pone.0137869.s002

(DOCX)

S2 Table. Primers Used for PCR Verification of Deletion Strains.

https://doi.org/10.1371/journal.pone.0137869.s003

(DOCX)

S3 Table. Primers Used for Generation of Probes for Southern.

https://doi.org/10.1371/journal.pone.0137869.s004

(DOCX)

S4 Table. Primers Used in the Generation of the EGFP Strains.

https://doi.org/10.1371/journal.pone.0137869.s005

(DOCX)

S5 Table. Primers Used for Verification of Fkbp12-1-EGFP and Fkbp12-1ΔCnaA Strains.

https://doi.org/10.1371/journal.pone.0137869.s006

(DOCX)

Author Contributions

Conceived and designed the experiments: KAF PRJ WJS. Performed the experiments: KAF PRJ ADR JMV HR. Analyzed the data: KAF PRJ ADR JMV HR WJS. Wrote the paper: KAF PRJ WJS.

References

  1. 1. Latgé J-P. Aspergillus fumigatus and Aspergillosis. Clinical Microbiology Reviews. 1999;12(2):310–50. pmid:PMC88920.
  2. 2. Kontoyiannis DP, Marr KA, Park BJ, Alexander BD, Anaissie EJ, Walsh TJ, et al. Prospective surveillance for invasive fungal infections in hematopoietic stem cell transplant recipients, 2001–2006: overview of the Transplant-Associated Infection Surveillance Network (TRANSNET) Database. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2010 Apr 15;50(8):1091–100. pmid:20218877. Epub 2010/03/12. eng.
  3. 3. Crabol Y, Lortholary O. Invasive Mold Infections in Solid Organ Transplant Recipients. Scientifica. 2014;2014:17.
  4. 4. Marr KA, Carter RA, Crippa F, Wald A, Corey L. Epidemiology and outcome of mould infections in hematopoietic stem cell transplant recipients. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2002 Apr 1;34(7):909–17. pmid:11880955. Epub 2002/03/07. eng.
  5. 5. Wilson LS, Reyes CM, Stolpman M, Speckman J, Allen K, Beney J. The direct cost and incidence of systemic fungal infections. Value in health: the journal of the International Society for Pharmacoeconomics and Outcomes Research. 2002 Jan-Feb;5(1):26–34. pmid:11873380. Epub 2002/03/05. eng.
  6. 6. Steinbach WJ, Marr KA, Anaissie EJ, Azie N, Quan SP, Meier-Kriesche HU, et al. Clinical epidemiology of 960 patients with invasive aspergillosis from the PATH Alliance registry. The Journal of infection. 2012 Nov;65(5):453–64. pmid:22898389. Epub 2012/08/18. eng.
  7. 7. Denning DW. Therapeutic outcome in invasive aspergillosis. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 1996 Sep;23(3):608–15. pmid:8879787. Epub 1996/09/01. eng.
  8. 8. Brakhage AA. Systemic fungal infections caused by Aspergillus species: epidemiology, infection process and virulence determinants. Current drug targets. 2005 Dec;6(8):875–86. pmid:16375671. Epub 2005/12/27. eng.
  9. 9. Steinbach WJ, Reedy JL, Cramer RA Jr, Perfect JR, Heitman J. Harnessing calcineurin as a novel anti-infective agent against invasive fungal infections. Nature reviews Microbiology. 2007 Jun;5(6):418–30. pmid:17505522. Epub 2007/05/17. eng.
  10. 10. Fox DS, Heitman J. Good fungi gone bad: the corruption of calcineurin. BioEssays: news and reviews in molecular, cellular and developmental biology. 2002 Oct;24(10):894–903. pmid:12325122. Epub 2002/09/27. eng.
  11. 11. Blankenship JR, Steinbach WJ, Perfect JR, Heitman J. Teaching old drugs new tricks: reincarnating immunosuppressants as antifungal drugs. Current opinion in investigational drugs (London, England: 2000). 2003 Feb;4(2):192–9. pmid:12669381. Epub 2003/04/03. eng.
  12. 12. Hofflin JM, Potasman I, Baldwin JC, Oyer PE, Stinson EB, Remington JS. Infectious complications in heart transplant recipients receiving cyclosporine and corticosteroids. Annals of internal medicine. 1987 Feb;106(2):209–16. pmid:3541723. Epub 1987/02/01. eng.
  13. 13. Singh N, Avery RK, Munoz P, Pruett TL, Alexander B, Jacobs R, et al. Trends in risk profiles for and mortality associated with invasive aspergillosis among liver transplant recipients. Clinical infectious diseases: an official publication of the Infectious Diseases Society of America. 2003 Jan 1;36(1):46–52. pmid:12491201. Epub 2002/12/20. eng.
  14. 14. Steinbach WJ, Singh N, Miller JL, Benjamin DK, Schell WA, Heitman J, et al. In Vitro Interactions between Antifungals and Immunosuppressants against Aspergillus fumigatus Isolates from Transplant and Nontransplant Patients. Antimicrobial agents and chemotherapy. 2004;48(12):4922–5. pmid:PMC529228.
  15. 15. Hemenway CS, Heitman J. Calcineurin. Structure, function, and inhibition. Cell Biochem Biophys. 1999;30(1):115–51. pmid:10099825. Epub 1999/04/01. eng.
  16. 16. Rusnak F, Mertz P. Calcineurin: Form and Function2000 2000-01-10 00:00:00. 1483–521 p.
  17. 17. Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, Schreiber SL. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell. 1991 Aug 23;66(4):807–15. pmid:1715244. Epub 1991/09/02. eng.
  18. 18. Arevalo-Rodriguez M, Wu X, Hanes SD, Heitman J. Prolyl isomerases in yeast. Frontiers in bioscience: a journal and virtual library. 2004 Sep 1;9:2420–46. pmid:15353296. Epub 2004/09/09. eng.
  19. 19. Galat A. Peptidylprolyl cis/trans isomerases (immunophilins): biological diversity—targets—functions. Current topics in medicinal chemistry. 2003;3(12):1315–47. pmid:12871165. Epub 2003/07/23. eng.
  20. 20. Pemberton TJ. Identification and comparative analysis of sixteen fungal peptidyl-prolyl cis/trans isomerase repertoires. BMC genomics. 2006;7:244. pmid:16995943. Pubmed Central PMCID: PMC1618848. Epub 2006/09/26. eng.
  21. 21. Ho S, Clipstone N, Timmermann L, Northrop J, Graef I, Fiorentino D, et al. The mechanism of action of cyclosporin A and FK506. Clinical immunology and immunopathology. 1996 Sep;80(3 Pt 2):S40–5. pmid:8811062. Epub 1996/09/01. eng.
  22. 22. Handschumacher RE, Harding MW, Rice J, Drugge RJ, Speicher DW. Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science (New York, NY). 1984 Nov 2;226(4674):544–7. pmid:6238408. Epub 1984/11/02. eng.
  23. 23. Jin L, Harrison SC. Crystal structure of human calcineurin complexed with cyclosporin A and human cyclophilin. Proceedings of the National Academy of Sciences of the United States of America. 2002 Oct 15;99(21):13522–6. pmid:12357034. Pubmed Central PMCID: PMC129706. Epub 2002/10/03. eng.
  24. 24. Griffith JP, Kim JL, Kim EE, Sintchak MD, Thomson JA, Fitzgibbon MJ, et al. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell. 1995 8/11/;82(3):507–22. pmid:7543369
  25. 25. Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, et al. Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature. 1995 Dec 7;378(6557):641–4. pmid:8524402. Epub 1995/12/07. eng.
  26. 26. Hemenway C, Heitman J. Calcineurin. Cell Biochem Biophys. 1999 1999/02/01;30(1):115–51. English. pmid:10099825
  27. 27. Huai Q, Kim HY, Liu Y, Zhao Y, Mondragon A, Liu JO, et al. Crystal structure of calcineurin-cyclophilin-cyclosporin shows common but distinct recognition of immunophilin-drug complexes. Proceedings of the National Academy of Sciences of the United States of America. 2002 Sep 17;99(19):12037–42. pmid:12218175. Pubmed Central PMCID: PMC129394. Epub 2002/09/10. eng.
  28. 28. Clipstone NA, Crabtree GR. Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation. Nature. 1992 Jun 25;357(6380):695–7. pmid:1377362. Epub 1992/06/25. eng.
  29. 29. Feske S, Okamura H, Hogan PG, Rao A. Ca2+/calcineurin signalling in cells of the immune system. Biochemical and biophysical research communications. 2003 Nov 28;311(4):1117–32. pmid:14623298. Epub 2003/11/19. eng.
  30. 30. Juvvadi PR, Lamoth F, Steinbach WJ. Calcineurin-Mediated Regulation of Hyphal Growth, Septation, and Virulence in Aspergillus fumigatus. Mycopathologia. 2014 Aug 15. pmid:25118871. Epub 2014/08/15. Eng.
  31. 31. Steinbach WJ, Cramer RA Jr, Perfect BZ, Asfaw YG, Sauer TC, Najvar LK, et al. Calcineurin controls growth, morphology, and pathogenicity in Aspergillus fumigatus. Eukaryotic cell. 2006 Jul;5(7):1091–103. pmid:16835453. Pubmed Central PMCID: PMC1489296. Epub 2006/07/13. eng.
  32. 32. Juvvadi PR, Fortwendel JR, Rogg LE, Burns KA, Randell SH, Steinbach WJ. Localization and activity of the calcineurin catalytic and regulatory subunit complex at the septum is essential for hyphal elongation and proper septation in Aspergillus fumigatus. Molecular microbiology. 2011 Dec;82(5):1235–59. pmid:22066998. Pubmed Central PMCID: PMC3225650. Epub 2011/11/10. eng.
  33. 33. Juvvadi PR, Fortwendel JR, Pinchai N, Perfect BZ, Heitman J, Steinbach WJ. Calcineurin Localizes to the Hyphal Septum in Aspergillus fumigatus: Implications for Septum Formation and Conidiophore Development. Eukaryotic cell. 2008;7(9):1606–10. pmid:PMC2547068.
  34. 34. Juvvadi PR, Gehrke C, Fortwendel JR, Lamoth F, Soderblom EJ, Cook EC, et al. Phosphorylation of Calcineurin at a Novel Serine-Proline Rich Region Orchestrates Hyphal Growth and Virulence in Aspergillus fumigatus. PLoS pathogens. 2013;9(8):e1003564. pmid:PMC3749960.
  35. 35. Kraus PR, Heitman J. Coping with stress: calmodulin and calcineurin in model and pathogenic fungi. Biochemical and biophysical research communications. 2003 Nov 28;311(4):1151–7. pmid:14623301. Epub 2003/11/19. eng.
  36. 36. Onyewu C, Blankenship JR, Del Poeta M, Heitman J. Ergosterol biosynthesis inhibitors become fungicidal when combined with calcineurin inhibitors against Candida albicans, Candida glabrata, and Candida krusei. Antimicrobial agents and chemotherapy. 2003 Mar;47(3):956–64. pmid:12604527. Pubmed Central PMCID: PMC149324. Epub 2003/02/27. eng.
  37. 37. Marr KA, Schlamm HT, Herbrecht R, Rottinghaus ST, Bow EJ, Cornely OA, et al. Combination antifungal therapy for invasive aspergillosis: a randomized trial. Annals of internal medicine. 2015 Jan 20;162(2):81–9. pmid:25599346. Epub 2015/01/20. eng.
  38. 38. MacMillan D. FK506 binding proteins: cellular regulators of intracellular Ca2+ signalling. European journal of pharmacology. 2013 Jan 30;700(1–3):181–93. pmid:23305836. Epub 2013/01/12. eng.
  39. 39. MacMillan D, McCarron JG. Regulation by FK506 and rapamycin of Ca2+ release from the sarcoplasmic reticulum in vascular smooth muscle: the role of FK506 binding proteins and mTOR. British journal of pharmacology. 2009 Oct;158(4):1112–20. pmid:19785652. Pubmed Central PMCID: PMC2785532. Epub 2009/09/30. eng.
  40. 40. MacMillan D, Currie S, McCarron JG. FK506-binding protein (FKBP12) regulates ryanodine receptor-evoked Ca2+ release in colonic but not aortic smooth muscle. Cell calcium. 2008 Jun;43(6):539–49. pmid:17950843. Epub 2007/10/24. eng.
  41. 41. MacMillan D, Currie S, Bradley KN, Muir TC, McCarron JG. In smooth muscle, FK506-binding protein modulates IP3 receptor-evoked Ca2+ release by mTOR and calcineurin. Journal of cell science. 2005 Dec 1;118(Pt 23):5443–51. pmid:16278292. Epub 2005/11/10. eng.
  42. 42. Huse M, Chen YG, Massague J, Kuriyan J. Crystal structure of the cytoplasmic domain of the type I TGF beta receptor in complex with FKBP12. Cell. 1999 Feb 5;96(3):425–36. pmid:10025408. Epub 1999/02/20. eng.
  43. 43. Yao D, Dore JJ Jr, Leof EB. FKBP12 is a negative regulator of transforming growth factor-beta receptor internalization. The Journal of biological chemistry. 2000 Apr 28;275(17):13149–54. pmid:10777621. Epub 2000/04/25. eng.
  44. 44. Girgenrath T, Mahalingam M, Svensson B, Nitu FR, Cornea RL, Fessenden JD. N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins. The Journal of biological chemistry. 2013 May 31;288(22):16073–84. pmid:23585572. Pubmed Central PMCID: PMC3668763. Epub 2013/04/16. eng.
  45. 45. Dolinski K, Muir S, Cardenas M, Heitman J. All cyclophilins and FK506 binding proteins are, individually and collectively, dispensable for viability in Saccharomyces cerevisiae. Proceedings of the National Academy of Sciences of the United States of America. 1997 Nov 25;94(24):13093–8. pmid:9371805. Pubmed Central PMCID: PMC24268. Epub 1997/12/16. eng.
  46. 46. Pinto D, Duarte M, Soares S, Tropschug M, Videira A. Identification of all FK506-binding proteins from Neurospora crassa. Fungal genetics and biology: FG & B. 2008 Dec;45(12):1600–7. pmid:18948221. Epub 2008/10/25. eng.
  47. 47. Heitman J, Movva NR, Hall MN. Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast. Science (New York, NY). 1991 Aug 23;253(5022):905–9. pmid:1715094. Epub 1991/08/23. eng.
  48. 48. Hemenway C, Heitman J. Proline isomerases in microorganisms and small eukaryotes. Annals of the New York Academy of Sciences. 1993 Nov 30;696:38–46. pmid:8109845. Epub 1993/11/30. eng.
  49. 49. Teichert S, Wottawa M, Schonig B, Tudzynski B. Role of the Fusarium fujikuroi TOR kinase in nitrogen regulation and secondary metabolism. Eukaryotic cell. 2006 Oct;5(10):1807–19. pmid:17031002. Pubmed Central PMCID: PMC1595341. Epub 2006/10/13. eng.
  50. 50. Gioti A, Simon A, Le Pecheur P, Giraud C, Pradier JM, Viaud M, et al. Expression profiling of Botrytis cinerea genes identifies three patterns of up-regulation in planta and an FKBP12 protein affecting pathogenicity. J Mol Biol. 2006 Apr 28;358(2):372–86. pmid:16497329. Epub 2006/02/25. eng.
  51. 51. Meléndez HG, Billon-Grand G, Fèvre M, Mey G. Role of the Botrytis cinerea FKBP12 ortholog in pathogenic development and in sulfur regulation. Fungal Genetics and Biology. 2009 4//;46(4):308–20. pmid:19116175
  52. 52. Cruz MC, Goldstein AL, Blankenship J, Del Poeta M, Perfect JR, McCusker JH, et al. Rapamycin and less immunosuppressive analogs are toxic to Candida albicans and Cryptococcus neoformans via FKBP12-dependent inhibition of TOR. Antimicrobial agents and chemotherapy. 2001 Nov;45(11):3162–70. pmid:11600372. Pubmed Central PMCID: PMC90798. Epub 2001/10/16. eng.
  53. 53. Cruz MC, Cavallo LM, Gorlach JM, Cox G, Perfect JR, Cardenas ME, et al. Rapamycin antifungal action is mediated via conserved complexes with FKBP12 and TOR kinase homologs in Cryptococcus neoformans. Molecular and cellular biology. 1999 Jun;19(6):4101–12. pmid:10330150. Pubmed Central PMCID: PMC104369. Epub 1999/05/18. eng.
  54. 54. Calo S, Shertz-Wall C, Lee SC, Bastidas RJ, Nicolas FE, Granek JA, et al. Antifungal drug resistance evoked via RNAi-dependent epimutations. Nature. 2014 Jul 27. pmid:25079329. Epub 2014/08/01. Eng.
  55. 55. Bastidas RJ, Shertz CA, Lee SC, Heitman J, Cardenas ME. Rapamycin exerts antifungal activity in vitro and in vivo against Mucor circinelloides via FKBP12-dependent inhibition of Tor. Eukaryotic cell. 2012 Mar;11(3):270–81. pmid:22210828. Pubmed Central PMCID: PMC3294450. Epub 2012/01/03. eng.
  56. 56. d'Enfert C. Selection of multiple disruption events in Aspergillus fumigatus using the orotidine-5'-decarboxylase gene, pyrG, as a unique transformation marker. Current genetics. 1996 Jun;30(1):76–82. pmid:8662213. Epub 1996/06/01. eng.
  57. 57. da Silva Ferreira ME, Kress MR, Savoldi M, Goldman MH, Hartl A, Heinekamp T, et al. The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryotic cell. 2006 Jan;5(1):207–11. pmid:16400184. Pubmed Central PMCID: PMC1360264. Epub 2006/01/10. eng.
  58. 58. Shimizu K, Keller NP. Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans. Genetics. 2001 Feb;157(2):591–600. pmid:11156981. Pubmed Central PMCID: PMC1461531. Epub 2001/02/07. eng.
  59. 59. Fortwendel JR, Juvvadi PR, Pinchai N, Perfect BZ, Alspaugh JA, Perfect JR, et al. Differential effects of inhibiting chitin and 1,3-{beta}-D-glucan synthesis in ras and calcineurin mutants of Aspergillus fumigatus. Antimicrobial agents and chemotherapy. 2009 Feb;53(2):476–82. pmid:19015336. Pubmed Central PMCID: PMC2630655. Epub 2008/11/19. eng.
  60. 60. Reeves EP, Messina CG, Doyle S, Kavanagh K. Correlation between gliotoxin production and virulence of Aspergillus fumigatus in Galleria mellonella. Mycopathologia. 2004 Jul;158(1):73–9. pmid:15487324. Epub 2004/10/19. eng.
  61. 61. Kay JE. Structure-function relationships in the FK506-binding protein (FKBP) family of peptidylprolyl cis-trans isomerases. The Biochemical journal. 1996 Mar 1;314 (Pt 2):361–85. pmid:8670043. Pubmed Central PMCID: PMC1217058. Epub 1996/03/01. eng.
  62. 62. Stevens DA, Espiritu M, Parmar R. Paradoxical effect of caspofungin: reduced activity against Candida albicans at high drug concentrations. Antimicrobial agents and chemotherapy. 2004 Sep;48(9):3407–11. pmid:15328104. Pubmed Central PMCID: PMC514730. Epub 2004/08/26. eng.
  63. 63. Wiederhold NP. Attenuation of echinocandin activity at elevated concentrations: a review of the paradoxical effect. Current Opinion in Infectious Diseases. 2007;20(6):574–8. pmid:00001432-200712000-00004.
  64. 64. Lewis RE, Albert ND, Kontoyiannis DP. Comparison of the dose-dependent activity and paradoxical effect of caspofungin and micafungin in a neutropenic murine model of invasive pulmonary aspergillosis. The Journal of antimicrobial chemotherapy. 2008 May;61(5):1140–4. pmid:18305201. Epub 2008/02/29. eng.
  65. 65. Helbig JH, Konig B, Knospe H, Bubert B, Yu C, Luck CP, et al. The PPIase active site of Legionella pneumophila Mip protein is involved in the infection of eukaryotic host cells. Biological chemistry. 2003 Jan;384(1):125–37. pmid:12674506. Epub 2003/04/04. eng.
  66. 66. Pereira PJ, Vega MC, Gonzalez-Rey E, Fernandez-Carazo R, Macedo-Ribeiro S, Gomis-Ruth FX, et al. Trypanosoma cruzi macrophage infectivity potentiator has a rotamase core and a highly exposed alpha-helix. EMBO reports. 2002 Jan;3(1):88–94. pmid:11751578. Pubmed Central PMCID: PMC1083928. Epub 2001/12/26. eng.
  67. 67. Niu XW, Zheng ZY, Feng YG, Guo WZ, Wang XY. The Fusarium Graminearum virulence factor FGL targets an FKBP12 immunophilin of wheat. Gene. 2013 Aug 1;525(1):77–83. pmid:23648486. Epub 2013/05/08. eng.
  68. 68. Alarcon CM, Heitman J. FKBP12 physically and functionally interacts with aspartokinase in Saccharomyces cerevisiae. Molecular and cellular biology. 1997 Oct;17(10):5968–75. pmid:9315655. Pubmed Central PMCID: PMC232445. Epub 1997/10/07. eng.
  69. 69. Steinbach WJ, Juvvadi PR, Fortwendel JR, Rogg LE. Newer combination antifungal therapies for invasive aspergillosis. Medical mycology. 2011 Apr;49 Suppl 1:S77–81. pmid:20608784. Epub 2010/07/09. eng.
  70. 70. Wenzel R, Del Favero A, Kibbler C, Rogers T, Rotstein C, Mauskopf J, et al. Economic evaluation of voriconazole compared with conventional amphotericin B for the primary treatment of aspergillosis in immunocompromised patients. The Journal of antimicrobial chemotherapy. 2005 Mar;55(3):352–61. pmid:15728146. Epub 2005/02/25. eng.
  71. 71. Lee EH, Rho SH, Kwon SJ, Eom SH, Allen PD, Kim do H. N-terminal region of FKBP12 is essential for binding to the skeletal ryanodine receptor. The Journal of biological chemistry. 2004 Jun 18;279(25):26481–8. pmid:15033987. Epub 2004/03/23. eng.
  72. 72. Fuchs BB, O'Brien E, Khoury JB, Mylonakis E. Methods for using Galleria mellonella as a model host to study fungal pathogenesis. Virulence. 2010 Nov-Dec;1(6):475–82. pmid:21178491. Epub 2010/12/24. eng.
  73. 73. Fallon JP, Troy N, Kavanagh K. Pre-exposure of Galleria mellonella larvae to different doses of Aspergillus fumigatus conidia causes differential activation of cellular and humoral immune responses. Virulence. 2011 Sep-Oct;2(5):413–21. pmid:21921688. Epub 2011/09/17. eng.