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Eukaryotic Cell, July 2008, p. 1085-1097, Vol. 7, No. 7
1535-9778/08/$08.00+0 doi:10.1128/EC.00086-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
,
B. Zachary Perfect,2
Nadthanan Pinchai,2
Steven Park,3
David S. Perlin,3
Yohannes G. Asfaw,4
Joseph Heitman,1,5,6
John R. Perfect,1,6 and
William J. Steinbach1,2*
Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina,1 Department of Pediatrics, Duke University Medical Center, Durham, North Carolina,2 Public Health Research Institute, International Center for Public Health, UMDNJ-New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey,3 Department of Laboratory Animal Resources, Duke University Medical Center, Durham, North Carolina,4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina,5 Department of Medicine, Duke University Medical Center, Durham, North Carolina6
Received 9 March 2008/ Accepted 17 April 2008
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cnaA (calcineurin A) strain. Like the cnaA strain, the crzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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One potential antifungal drug target in Aspergillus species is the calcineurin pathway (55). The importance of the calcineurin signaling pathway in fungal physiology is vividly demonstrated by gene expression analyses in Saccharomyces cerevisiae that identified 163 genes regulated by the calcineurin/Crz1 signaling pathway. These regulated genes were involved in ion/small-molecule transport, signal transduction, cell wall maintenance, and vesicular transport, indicating the important role that this pathway plays in fungal physiology (66).
Unlike the case for many new candidate antifungal drug targets, drugs that target the calcineurin pathway already exist. The calcineurin inhibitors FK506 and cyclosporine, which are used routinely to inhibit human calcineurin and have revolutionized modern organ transplantation, exhibit antifungal activity in vitro, indicating their potential as therapeutic agents for IPA (3, 20, 54, 56, 57). In addition, our group and another independent research group have shown that deletion of the gene encoding the calcineurin A catalytic subunit in Aspergillus fumigatus resulted in a strain with significant defects in hyphal growth. Furthermore, these cnaA strains are no longer capable of causing IPA in distinct experimental murine models (14, 53).
The importance of the calcineurin pathway in fungal physiology and the dramatic phenotypes associated with pharmacologic inhibition and genetic deletions of this pathway make it an attractive antifungal drug target. However, the critical role of calcineurin in mammalian immune system responses brings into question the feasibility of directly targeting fungal calcineurin pathways in vivo with existing calcineurin inhibitors (8, 27, 34, 36). The impact of calcineurin inhibition on the host, via immunosuppression, may be stronger than its antifungal activity, with the net result being exacerbation of the disease. Thus, further studies are needed to identify fungus-specific components of the calcineurin signaling pathway that are not present in mammals. In this study, we characterize a calcineurin effector protein, CrzA, which is not present in mammals but is integrally linked with calcineurin-mediated signal transduction in many fungi (12, 21, 23, 43, 50).
In S. cerevisiae, calcineurin-dependent transcription is regulated in part by the zinc finger transcription factor Crz1 (30, 50). Calcineurin regulates Crz1 activity via dephosporylation of Crz1, which results in nuclear localization of cytoplasmic Crz1 and requires the karyopherin Nmd5 (39, 51). S. cerevisiae crz1 mutants display many phenotypes, including growth and viability defects, that are similar to those of S. cerevisiae calcineurin mutants (30, 50). Additional downstream effectors of calcineurin have also been identified. In S. cerevisiae, Hph1 and Hph2 have been shown to be novel calcineurin-regulated response components and Crz1-independent downstream effectors of calcineurin (18).
Additional downstream effectors of calcineurin are also likely to exist in the opportunistic human fungal pathogen Candida albicans. C. albicans crz1 mutant strains display a pathobiologic phenotype different from that of C. albicans calcineurin mutants (23, 37, 43). For example, C. albicans crz1/crz1 mutant strains, unlike C. albicans calcineurin mutant strains, are not sensitive to serum and do not display a decrease in virulence in murine models of candidiasis. These results suggest the presence of additional calcineurin downstream effectors in this pathogenic yeast.
However, in the pathogenic mold A. fumigatus downstream calcineurin effectors have not been identified or functionally characterized. In this study, we identified the A. fumigatus Crz1 homolog, CrzA, and tested the hypothesis that CrzA is a primary downstream effector of the calcineurin pathway in A. fumigatus. Our results suggest that CrzA is an important downstream effector for calcineurin in A. fumigatus but that additional calcineurin downstream effectors also exist in this mold. We observed that CrzA is critical for in vivo fungal growth, development, and pathogenicity and thus is an excellent antifungal drug target for treatment of invasive aspergillosis due to its role in pathogenesis and lack of a conserved mammalian homolog.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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crzA) strain (crzA::Aspergillus parasiticus pyrG). AF293.1 is a uracil/uridine-auxotrophic (pyrG–) mutant of A. fumigatus strain AF293 (38), and AF293 was used as the wild-type strain for all experiments. All A. fumigatus cultures were grown on glucose minimal medium as previously described (9, 47, 53) at 37°C. The Escherichia coli One-Shot TOP10 strain (Invitrogen, Carlsbad, CA) was used for routine cloning and grown in Luria broth (Fisher Chemicals, Fair Lawn, NJ) supplemented with appropriate antibiotics at 37°C.
Generation of crzA and crzA + crzA reconstituted strains of A. fumigatus.
The 2.3-kb A. fumigatus crzA gene (Afu1g06900 [www.cadre.man.ac.uk]) was replaced with the 3.1-kb A. parasiticus pyrG gene to generate crzA (Fig. 1A). Approximately 1 kb of upstream flanking and 1.2 kb of downstream flanking sequence of crzA was cloned to flank the 3.1-kb A. parasiticus pyrG in plasmid pJW24 (a gift from Nancy Keller, University of Wisconsin, Madison) to generate the replacement construct. This resulting replacement construct plasmid was used as a template to generate the approximately 4.5-kb PCR amplicon for use in transformation.
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crzA strains. Transformants were additionally screened by PCR for the junctions of the crzA::pyrG allele to indicate replacement and homologous recombination. Strains with both absence of crzA and evidence of homologous recombination, as determined by PCR, then underwent single-spore isolation. Confirmation of gene replacement was performed via Southern analysis using the digoxigenin labeling system (Roche Molecular Biochemicals, Mannheim, Germany) and three separate probes, including a 500-bp probe for crzA, a 1.2-kb probe for the 3' crzA flanking sequence, and a 600-bp probe for the A. parasiticus pyrG gene.
crzA was complemented to generate the crzA + crzA complemented strain by transformation with native crzA, 550 bp of flanking upstream sequence, and 1.7 kb of flanking downstream sequence. The native crzA and flanking sequences were cloned into the TOPO TA pCR 2.1 system (Invitrogen, Carlsbad, CA), followed by EcoRV blunt cloning of a 1.4-kb segment of the E. coli hygromycin B phosphotransferase gene (hph), conferring hygromycin B resistance, from plasmid pCB1636 (59). The approximately 4.5-kb complementation cassette was amplified from this new TOPO vector (named pZP5) and the PCR product transformed as detailed above into the recipient crzA strain. Putative complemented transformants, with ectopic integrations of the complementation construct, were selected on medium supplemented with hygromycin B (150 µg/ml). PCR analyses were performed to screen for crzA as described above, and strains with amplicons underwent single-spore isolation followed by Southern analyses to confirm that isolates contained both crzA and hphB.
Radial growth, conidiation, and germination.
Radial growth on solid medium was obtained by measuring colony diameters of the wild-type, crzA, and crzA + crzA complemented strains once every 24 h over a period of 5 days as previously described (53). Experiments were performed in triplicate, and the mean and standard error of the colony diameter at each 24-hour period are reported.
Conidiation was quantified by harvesting conidia from the wild-type, crzA, and crzA + crzA complemented strains as previously described (53). Harvests from each strain were performed in triplicate, and the mean and standard error of the total number of conidia per ml collected from each strain are reported.
The germination percentage and rate were determined with the wild-type, crzA, crzA + crzA complemented, and cnaA strains. Conidia (1 x 106 conidia/ml) of the respective strains were inoculated into RPMI 1640 broth supplemented with 0.15% (wt/vol) Junlon to promote dispersal as previously described (7) and were incubated over a time course at 37°C. Sampling was performed at 4, 6, 8, 10, 12, 16, 24, and 48 h, and samples were then fixed in 10% neutral buffered formalin and analyzed by light microscopy. Germination rates were calculated in triplicate by scoring 100 conidia for germination to calculate a germination percentage. Hyphal morphology was also assessed at each time point. Conidia (1 x 105/ml) were incubated in RPMI for 48 h as described above, and hyphae and ungerminated conidia were examined for viability using the fluorescent dye 5,(6)-carboxyfluorescein diacetate, which stains live cells. (7, 54).
Quantification of cell wall β-1,3-glucan. The aniline blue fluorescence assay is highly specific for quantifying β-1,3-glucan (46). Using a previously reported (54) slight modification of an earlier study (22), flasks containing 25 ml of RPMI were inoculated with 1 x 107 conidia/ml of the A. fumigatus strains with and without antifungals and grown for 48 h with shaking at 200 rpm and 37°C. The mycelia were vortexed, harvested, washed three times in 0.1 M NaOH, and lyophilized. Mycelial tissue was weighed and normalized to place 5 mg of tissue from each sample in a new tube containing 0.25 ml of 1 M NaOH. This tube was then sonicated with a microprobe. Tubes were incubated at 52°C for 30 min, and aliquots of 50 µl were treated with aniline blue reagent (22, 46) for 30 min at 52°C followed by 30 min at 25°C. Relative fluorescence units were measured on a SpectraMax M2 fluorimeter (Molecular Devices, Sunnyvale, CA) at 405-nm excitation and 460-nm emission wavelengths. A standard curve was created using curdlan, a β-1,3-glucan analog (Sigma, St. Louis, MO), to normalize values. Values are expressed as relative fluorescence units per mg of mycelial tissue; the values were analyzed using an unpaired t test and statistical significance is reported as a two-tailed P value, with significance defined as a P value of <0.05.
Cell wall inhibitor effects on radial growth. The activities of a selective β-1,3-glucan synthesis inhibitor (caspofungin at 1 µg/ml), a chitin synthesis inhibitor (nikkomycin Z at 1 µg/ml), and a calcineurin inhibitor (FK506 at 20 ng/ml) were measured using minimal medium impregnated with clinically relevant concentrations. The antifungal activity of each was measured by radial growth following inoculation of 1 x 104 conidia (10 µl of a solution of 1 x 106 conidia/ml) of the A. fumigatus strain. Plates were incubated at 37°C, and images were obtained after 4 days of growth. Each experiment was performed in triplicate.
Transcriptional profiling of calcineurin and cell wall-related genes.
One-step Sybr green I quantitative reverse transcriptase PCR (qRT-PCR) was performed for transcriptional profiling of calcineurin and cell wall-related genes. Primers were designed to the calcineurin, cell wall-related genes, and the housekeeping beta-tubulin gene using Beacon Designer software version 5.0 (Premier Biosoft International, Palo Alto, CA). All primers used in this study are listed in Table S1 in the supplemental material. All qRT-PCRs were performed using a Stratagene Mx3005p quantitative PCR system instrument (Stratagene, La Jolla, CA). Each primer set was optimized by using serially diluted total RNA extracted from wild-type A. fumigatus and the Brilliant II Sybr green qRT-PCR Master Mix Kit, 1-Step (Stratagene), in a 25-µl reaction volume according to the manufacturer's assay optimization instructions. The optimized qRT-PCR assay for each RNA sample was carried out in 25-µl reaction volumes that consisted of 1x Sybr green reaction mix, 50 nM of each primer, 
100 ng of total RNA, and 1 µl of the RT-RNase block enzyme mixture. No-template and no-RT controls for each primer set were also assayed to confirm that no primer-dimers and no DNA contamination were present, respectively. The thermal cycling parameters consisted of a 30-min RT step at 48°C and a 10-min Taq polymerase hot start at 95°C, followed by template amplification of 45 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. Fluorescence was measured during the extension step (72°C). Following amplification, a disassociation analysis (melt curve) was performed to confirm that a single amplified product was present. The qRT-PCR assay was performed in duplicate. The threshold cycle (CT) for each reaction was determined using the Stratagene MxPro v.4 software's "adaptive baseline" algorithm enhancements. For each RNA sample, expression levels of all genes of interest were normalized to beta-tubulin. The 2–CT method of analysis was used to determine fold changes of gene expression in the mutants relative to the wild-type AF293 A. fumigatus strain.
Light and scanning electron microscopy. All light microscopy was performed on a Zeiss microscope using Nomarski optics (differential interference contrast). All scanning electron microscopy images were obtained using an environmental scanning electron microscope (Philips XL30 ESEM TMP; FEI Company, Hillsboro, OR) as previously described (53).
Murine inhalational model of invasive aspergillosis.
Six-week-old CD1 male mice (Charles River Laboratories) (mean weight, 22.5 g) were immunosuppressed with both cyclophosphamide (Cytoxan; Bristol-Myers Squibb, Princeton, NJ) at 150 mg/kg intraperitoneally on days –2 and +3 of infection and triamcinolone acetonide (Kenalog-40; Bristol-Myers Squibb, Princeton, NJ) at 40 mg/kg subcutaneously on days –1 and +6 of infection as previously described (53). Mice were housed under sterile conditions and supplied sterile drinking water supplemented with vancomycin (1 mg/ml), gentamicin (0.2 mg/ml), and clindamycin (1 mg/ml). Four groups of 20 immunosuppressed, unanesthetized mice each inhaled 40 ml of an aerosolized suspension of the 1 x 109 conidia/ml of AF293 wild-type, crzA, or crzA + crzA complemented strain or a diluent control (0.05% Tween 80) in a Hinners inhalational chamber for 30 min as previously described (53). Mice were evaluated for morbidity and mortality in a blinded fashion twice daily. Survival was plotted on a Kaplan-Meier curve for each Aspergillus strain, and the log rank test was used for pairwise comparison of survival (GraphPad Prism 5.0; GraphPad, San Diego, CA). Statistical significance was defined as a two-tailed P value of <0.05.
Histopathologic evaluation of fungal burden. To evaluate the in vivo histopathologic progression of disease, four groups of four additional mice were similarly infected in the murine inhalational model with each strain and euthanized at defined time points (days 4 and 7 after infection). Lungs were harvested and prepared for histologic examination as previously described (53).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The predicted calcineurin interaction site in S. cerevisiae Crz1, PIISIQ, was not identified in the amino acid sequence of A. fumigatus CrzA, nor was the known docking site for calcineurin on the mammalian transcription factor NFAT, PxIxIT identified (Fig. 2) (6). However, significant amino acid identity was shared between Crz1 and CrzA in the serine-rich region known to be involved in calcineurin-dependent regulation of Crz1 localization, activity, and phosphorylation (51). Several serine residues are conserved between Crz1 and CrzA in this region (amino acids 186 to 232) (Fig. 2).
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Generation of A. fumigatus crzA strains.
Transformation of the uracil/uridine-auxotrophic A. fumigatus AF293.1 with the crzA replacement construct yielded 26 transformants, including 7 which had PCR products with primers designed to amplify the predicted crzA replacement locus after homologous recombination, and no detectable PCR amplicon with primers designed to amplify the wild-type crzA locus (data not shown). These seven strains underwent Southern analysis with SacI/XbaI-digested genomic DNA and a 1.2-kb probe for the 3' segment of crzA flanking sequence, which revealed that two of the seven transformants had successful homologous recombination events at the crzA locus without presence of the wild-type allele (data not shown). An additional Southern analysis using SmaI-digested genomic DNA (Fig. 1B) and a 500-bp probe from a section of the 3' flank of the crzA replacement construct was performed. This hybridization yielded two bands (4.7 kb and 2.7 kb) in the wild-type strain and the two predicted bands for a gene replacement (4.7 kb and 6.2 kb) in the crzA strain due to successful homologous recombination at the crzA locus. A final Southern analysis with XhoI-digested genomic DNA and a 550-bp probe for the A. parasiticus pyrG gene yielded a single 3.9-kb band in both crzA strains and the absence of a band in the wild type, confirming the homologous replacement of crzA with a single insertion of the gene replacement cassette. The two crzA strains were phenotypically identical, and one was selected to continue as the crzA strain for further experimentation.
To complement the crzA defect, a 4.5-kb complementation cassette (from pZP5) was transformed into the recipient crzA strain for ectopic integration. A total of 85 transformants which grew on the hygromycin B selection medium were obtained. PCR screening of transformants with the same 500-bp section of crzA revealed two colonies with the expected crzA product. Southern analysis with SmaI-digested genomic DNA (Fig. 1B) revealed the persistence of the crzA locus (4.7-kb and 6.2-kb bands) and an additional 2.7-kb band corresponding to the wild-type crzA allele integrated in an ectopic location in the genome. Additionally, the complemented strain was verified by probing for the hygromycin B resistance (hph) gene (data not shown). One strain was selected as the crzA + crzA complemented strain for further experimentation.
Confirmation of crzA gene replacement and complementation was also done using real-time RT-PCR, which revealed similar crzA mRNA abundances in both the wild-type and crzA + crzA complemented strains but no expression in the crzA strain (data not shown).
crzA is necessary for germination, radial growth, and asexual development.
Conidia of the crzA strain displayed a significant delay and defect in germination at 37°C in RPMI medium (Fig. 3). At 8 h postinoculation, 48% and 59% of the crzA + crzA complemented and wild-type strains, respectively, had germinated, while only 18% and 6% of the cnaA and the crzA strain conidia, respectively, germinated. Up until 24 h postinoculation, the crzA strain displayed an even greater defect in conidial germination than the cnaA strain. By 48 h postinoculation only 37% of the crzA conidia had germinated, compared with 81% and 95% of conidia from the crzA + crzA complemented and wild-type strains, respectively. Importantly, 5,(6)-carboxyfluorescein diacetate viability staining revealed that the ungerminated crzA conidia after 48 h of incubation were still viable (data not shown). These results demonstrate the importance of calcineurin signaling, as primarily directed through CrzA, for A. fumigatus conidium germination.
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crzA strain demonstrated a severe defect in polarized hyphal growth (Fig. 4A and B). Radial growth assays demonstrated a 68% decrease in hyphal growth compared with the wild-type and crzA + crzA complemented strain (P < 0.0001). Furthermore, the crzA strain was also defective in conidium production (P < 0.0001) (Fig. 4C); when normalized to the decreased colony area, the crzA strain still possessed a 73% decrease in conidia produced per mm2 compared to the wild-type strain. Scanning electron microscopy of the hyphae of the strains also revealed that crzA hyphae were slightly misshapen with blunted hyphal tips compared with the wild-type and crzA + crzA complemented strains (Fig. 5). However, the hyphal morphology defect was not as severe as previously seen in the cnaA strain (53). Similar to the case for the cnaA strain, the conidial surfaces of the crzA strain were relatively smooth compared to those of the wild-type and crzA + crzA complemented strains (Fig. 6) yet not as smooth as those of the cnaA strain. This may be due to a lack of rodlet formation; however, this was not definitively examined. These results indicate that the crzA strain has a phenotype remarkably similar to that of the cnaA strain, with a slightly more severe defect in germination but less of a defect in hyphal growth and conidial morphology. These results also suggest the likely existence of additional calcineurin effectors involved in polarized hyphal growth and morphology.
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cnaA and crzA strains.
Defects in hyphal growth may be due to defects in the synthesis of important cell wall components such as chitin and β-1,3-glucan. We utilized an aniline blue fluorescence assay to measure the β-1,3-glucan content in hyphae of the wild-type, cnaA, and crzA strains. Figure 7 reveals that the cnaA strain had a statistically significantly reduced β-1,3-glucan content in hyphae compared to the wild-type strain (P = 0.03). While a difference in fluorescence was consistently seen between the crzA and wild-type strains, this was not statistically significant (P = 0.09), and it may reflect the greater ability of the crzA strain to form hyphae in vitro due to an uncharacterized crzA-independent pathway in A. fumigatus.
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cnaA strain compared to cnaA strain with caspofungin, P < 0.0001; crzA strain compared to crzA strain with caspofungin, P = 0.01). However, when the strains were treated with the chitin synthesis inhibitor nikkomycin Z, a statistically significant compensatory increase in β-1,3-glucan content was seen in the cnaA strain (P < 0.0001) but not in the crzA (P = 0.68) and wild-type (P = 0.12) strains. These results suggest that the calcineurin pathway is involved in mediating β-1,3-glucan biosynthesis in A. fumigatus but that CrzA plays a limited role in this regulation. In addition, it seems apparent that calcineurin-independent pathways for β-1,3-glucan biosynthesis also exist in A. fumigatus and may be activated in cells confronted with chitin synthesis inhibitors.
We also transcriptionally profiled genes predicted to be involved in the calcineurin pathway and cell wall biosynthesis in A. fumigatus to help clarify the role of CnaA and CrzA in A. fumigatus physiology (Table 1). With the exception of gelA, the mRNA abundance of genes involved in β-1,3-glucan biosynthesis was consistently decreased in the cnaA strain while remaining at virtually wild-type levels in the absence of CrzA. These data correlate with the decrease in the β-1,3-glucan content observed in the cnaA strain and suggest that, unlike in other fungi, CrzA does not appear to play a significant role in the regulation of β-1,3-glucan biosynthesis in A. fumigatus. A gene replacement mutation of gelA (gel1) was shown to have no effect on the cell wall composition of A. fumigatus. Thus, it is not surprising that expression of gelA in the cnaA strain was not able to rescue the defect in β-1,3-glucan biosynthesis (32). It therefore appears that calcineurin A is involved in regulating β-1,3-glucan biosynthesis in A. fumigatus but that this regulation is not mediated solely by CrzA.
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52-fold, in calmodulin mRNA in the cnaA strain highlights the importance of calcineurin in mediating responses to calcium (Table 1). Interestingly, a similar compensatory increase in calmodulin mRNA was not observed in the crzA strain. With the exception of a fourfold increase of calcineurin mRNA in the crzA strain, elimination of CrzA had minor effects on the mRNA abundance of the genes examined in this study (Table 1). These results strongly suggest that CrzA-independent calcineurin effectors controlling cell wall biosynthesis and responses to calcium exist in A. fumigatus.
Cell wall inhibitors inhibit hyphal growth in calcineurin pathway mutants.
Our data suggest that the calcineurin pathway plays an important role in cell wall biosynthesis in A. fumigatus but that this may be CrzA independent. Evidence that additional calcineurin-independent mechanisms of cell wall biosynthesis and hyphal growth exist in A. fumigatus is observed by the enhanced effects of calcineurin and/or CrzA inhibition and specific cell wall inhibitors (Fig. 8). Inhibition of the calcineurin pathway via FK506, of β-1,3-glucan biosynthesis via caspofungin, or of chitin biosynthesis via nikkomycin Z has clear effects on hyphal growth in the wild-type strain (Fig. 8). Additional evidence for the existence of calcineurin-independent regulation of β-1,3-glucan biosynthesis is the increased inhibition of hyphal growth with caspofungin treatment of the cnaA strain. Treatment of the crzA strain with FK506 resulted in a phenotype virtually identical to that of the cnaA strain (Fig. 8). This result may suggest that CrzA is dependent upon the presence of calcineurin in the cell for its activity. Additional support of this conclusion is the 4-fold decrease in crzA mRNA in the fungal strain lacking calcineurin (Table 1).
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crzA strain, we hypothesized that this strain would be unable to establish invasive disease in an immunosuppressed murine model of IPA, similar to our results with the cnaA strain (53). To test the virulence of crzA, a persistently neutropenic inhalational murine model of IPA was utilized (52). Infection with the wild-type strain yielded 85% mortality by 14 days after infection (Fig. 9A), while infection with the crzA strain or inoculation of mice with 0.05% Tween 80 did not lead to any mortality (P = 0.0011). Mice infected with the crzA + crzA complemented strain had 65% mortality by 14 days after infection, which was not statistically different from the mortality caused by the wild-type infection. Animals infected with the crzA strain were symptomatically indistinguishable from uninfected control animals during the entire experimental course. Animals infected with the wild-type and crzA + crzA complemented strains displayed progressive and severe signs of invasive disease that have been previously well described, including ruffled fur, hunched posture, weight loss, and an increased respiratory rate.
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crzA + crzA complemented strains (Fig. 9B). In mice infected with the crzA strain, hyphal growth was occasionally visible, consistent with the in vitro growth phenotype of this mutant strain. However, compared to that in mice infected with the wild-type and crzA + crzA complemented strains, hyphal growth was significantly decreased in crzA strain-infected mice. In addition, there was minimal lung inflammation in animals infected with the crzA strain and a greater number of open alveoli. In animals infected with the wild-type and crzA + crzA complemented strains, infiltration of the alveoli with phagocytic cells and debris and alveolar destruction were extensively observed. Uninfected control mice showed no architectural or inflammatory changes in the lungs.
Pulmonary infarct scores at each time point during infection were greater for the wild-type and crzA + crzA complemented strains than for the crzA strain. The mean pulmonary infarct score at 7 days after infection for the crzA strain was 3, compared to mean scores of 5.125 for the wild-type strain and 4.75 for the crzA + crzA complemented strain (P < 0.05). In the immunosuppressed, uninfected control mice there were no findings of necrosis, hemorrhage, or edema at any time point. These results from our animal model strongly suggest that CrzA is required for in vivo fungal growth and disease development.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The role of calcineurin-mediated signaling in fungal physiology has been extensively explored in S. cerevisiae and a host of other important fungi, including the medically important opportunistic human fungal pathogens C. albicans, Cryptococcus neoformans, and, recently, A. fumigatus (55). In S. cerevisiae, calcineurin mediates resistance to various cation stresses, including Na+, Mn2+, and Li+, and regulates Ca2+ homeostasis, Ca2+-mediated G2 arrest, and the onset of mitosis (12, 13, 16, 31, 63-65). In the human pathogenic yeast C. neoformans, calcineurin mediates pH and CO2 homeostasis, temperature-sensitive growth, antifungal drug tolerance, mating/haploid fruiting, resistance to Li+, and fungal pathogenicity (10, 15, 35, 60). Fungal pathogenicity and growth in human serum are also mediated in part by calcineurin in C. albicans, along with resistance to both cation stress and antifungal drugs (1, 2, 4, 11, 42).
While these studies have clearly illustrated the importance of calcineurin in the physiology of fungi, they also demonstrate unique attributes of calcineurin-mediated signal transduction in different fungal species. Recently, we began an exploration of calcineurin-mediated signal transduction in the opportunistic human mold, A. fumigatus (53, 54). Previous studies with A. nidulans have suggested that calcineurin is essential and required for cell cycle progression through G1/S, nuclear division, and polarized hyphal growth in this species (33, 40). Our results and the results of a complementary study, however, suggested that while calcineurin was not essential for survival in A. fumigatus, calcineurin mutants displayed severe defects in conidial germination, polarized hyphal growth, and conidium development. Importantly, A. fumigatus calcineurin mutants were incapable of causing disease in several experimental murine models of IPA (14, 53).
In this study, we sought to identify the homolog of S. cerevisiae Crz1 in A. fumigatus and examined the hypothesis that CrzA was a downstream effector of calcineurin signaling in A. fumigatus as in other fungi. Bioinformatic analyses revealed the presence of a single putative homolog of Crz1 in the A. fumigatus genome sequence, which we named CrzA. Replacement of the crzA gene in A. fumigatus resulted in a strain with phenotypes similar to those of the calcineurin A mutant but differing in their severity. For example, while the calcineurin A mutant was virtually incapable of polarized hyphal growth, the crzA strain was capable of limited polarized hyphal growth, with a greatly decreased growth rate after 3 days. Furthermore, similar to the cnaA strain, the crzA strain possessed significant defects in conidium germination and conidium morphology, with an apparent absence of the bumpy and echinulate surface typically found on A. fumigatus conidia. In addition, the crzA strain did not display as significant a defect in β-1,3-glucan content as the cnaA strain, indicating that in A. fumigatus, β-1,3-glucan biosynthesis may largely be CrzA independent.
Treatment with nikkomycin Z led to a compensatory increase in β-1,3-glucan content in the cnaA strain, indicating that β-1,3-glucan synthesis can occur in the absence of calcineurin-mediated signaling. While we did observe an increase in mRNA abundance of the β-1,3-glucosyltransferase in the cnaA strain in liquid culture, this did not prevent the loss of β-1,3-glucan. However, it may be possible that treatment with a cell wall stress agent induces higher levels of gelA mRNA that account for the compensatory increase in β-1,3-glucan content. Alternatively, nikkomycin Z may induce calcineurin-independent pathways for β-1,3-glucan biosynthesis. A similar compensatory effect on β-1,3-glucan content, however, was not seen in the crzA strain treated with nikkomycin Z, so it is possible that this pathway is activated primarily in the absence of calcineurin in the cell and is dependent on a functional CrzA. Thus, it appears that calcineurin A is involved in regulating β-1,3-glucan biosynthesis in A. fumigatus, as previously postulated (54), but that this regulation is not solely dependent upon CrzA.
One area of clinical and biochemical exploration is the concept of the "paradoxical effect" seen in fungi following treatment with a β-1,3-glucan synthesis inhibitor (62), where in C. albicans there is a reported compensatory increase in chitin content, therefore linking the two cell wall components in a stress response fashion (58). Our study is the first to demonstrate a compensatory increase in β-1,3-glucan following chitin synthesis inhibition in A. fumigatus. It appears that this glucan-chitin interaction is controlled or affected by calcineurin signaling. If we eliminate calcineurin, there does appear to be an increase in β-1,3-glucan biosynthesis during chitin biosynthesis inhibition. This apparent cell wall stress response seems ideal for combination therapy in which calcineurin, β-1,3-glucan, and chitin targets can all be blocked by drugs and certainly supports a multiple-pronged attack on cell wall targets and hyphal growth in A. fumigatus.
While the molecular mechanisms of calcineurin signaling in A. fumigatus are just beginning to be explored, recent experiments with other fungi have started to further elucidate the complex calcineurin signaling pathway and may provide clues to the interplay between calcineurin and other signaling pathways in A. fumigatus. A calcineurin-independent Crz1 homolog, Crz2, was identified in C. albicans and observed to work in concert with the Rim101/PacC pathway to mediate adaptation to alkaline pH environments (25). A putative homolog for Crz2 in A. fumigatus has not been identified, and it may be that CrzA in A. fumigatus performs the roles of both Crz1 and Crz2. In the filamentous mold Botrytis cinerea, a subset of genes were found to be coregulated by the G alpha subunit gene (bcg1), the phospholipase C gene (bcplc1), and the calcineurin pathway, suggesting an interconnection of these important signaling pathways in filamentous molds (45). Future studies will uncover the mechanism of calcineurin-dependent regulation of CrzA and the likely identity of other signaling pathways that may intersect with calcineurin-mediated signal transduction to control hyphal growth and cell wall homeostasis in A. fumigatus.
Similar to the cnaA strain, the crzA strain was incapable of establishing extensive in vivo growth and disease in a persistently neutropenic murine model of IPA. Several explanations may account for the crzA strain's inability to cause disease. First, as in vitro germination assays demonstrated, fewer than 50% of the crzA strain conidia were able to germinate at 37°C in a relatively nutrient-rich medium by 48 h. Thus, during infection, when initial nutrient availability is likely low, the significant delay in germination may allow those innate immune cells that are functional in neutropenic animals to clear the majority of the crzA strain conidia.
On the other hand, histopathological analyses did demonstrate the presence of sparse fungal hyphae in mice infected with the crzA strain, in contrast to our previous study of cnaA strain-infected mice, which contained no histological evidence of hyphal growth. Given the low growth rate observed in vitro with the crzA strain and the abnormal hyphal morphology observed in electron micrographs, it is possible that the crzA strain hyphae are not capable of substantial invasive growth in vivo and production of disease. However, it may also be possible that the low growth rate allows those immune effector cells that remain in the mice to clear the infection and prevent disease development. Furthermore, the crzA strain may be more susceptible to killing by immune effector cells due to its altered cell wall morphology.
Given the close similarity in phenotypes observed in the cnaA and the crzA strains, two conclusions about the role of CrzA in calcineurin signaling in A. fumigatus are possible from our studies. First, like in other fungi, CrzA is an important downstream effector of calcineurin-mediated signal transduction in A. fumigatus for morphology. The significant defects observed in hyphal growth and asexual development in the crzA strain strongly suggest the importance of CrzA-mediated signaling in fungal morphology. Importantly, it appears that CrzA is largely dependent on calcineurin for its activity in A. fumigatus. Treatment of the crzA strain with the calcineurin inhibitor FK506 resulted in a phenotype virtually identical to that of the cnaA strain, with almost complete cessation of hyphal growth (Fig. 8). This result suggests that CrzA has little or no function in a cell lacking calcineurin activity and suggests that CrzA is an important downstream effector of calcineurin in A. fumigatus. However, the more severe conidium germination defect observed in the crzA strain may suggest that additional factors regulate CrzA in A. fumigatus. Interestingly, no obvious calcineurin docking site, similar to the one found in Crz1, was found in CrzA. However, Crz1 and CrzA do share a serine-rich region known to be involved in calcineurin-dependent regulation of Crz1 localization, activity, and phosphorylation in S. cerevisiae (Fig. 2) (51). Future studies will seek to discover the mechanism of calcineurin-dependent regulation of CrzA in A. fumigatus.
Second, our data suggest that additional CrzA-independent downstream effectors of calcineurin signaling exist in A. fumigatus. Given the intermediate phenotypes in growth and morphology observed with the crzA and cnaA strains, we hypothesize that additional calcineurin-dependent downstream effectors exist that partially maintain, albeit in a limited fashion, hyphal growth in the crzA strain. Data to support this hypothesis are observed in the β-1,3-glucan contents of the cnaA and crzA strains and in the mRNA abundance assays, which demonstrated little or no change in the abundance of mRNAs from genes involved in cell wall biosynthesis in the crzA strain (Fig. 7 and Table 1). Importantly, our results are concordant with those reported for crz1/crz1 mutants of C. albicans, which also display a growth and morphology phenotype intermediate to that of the cna1/cna1 mutants, suggesting the presence of additional downstream calcineurin effectors (23, 37, 43). In addition, our crzA strain displays a phenotype similar to that of a crz1 mutant, bccrz1, of the plant pathogenic mold Botrytis cinerea (44). The B. cinerea mutant displayed significant in vitro growth defects and had impaired hyphal morphology, conidiation, and sclerotium formation. Unlike the A. fumigatus crzA strain, the bccrz1 strain displayed significant defects in cell wall and membrane integrity. Thus, like with calcineurin mutants, fungi lacking a Crz1 homolog display similar phenotypes with subtle differences among genera.
During the writing of this paper, a complementary study reporting the creation of a crzA strain from A. fumigatus strain CEA17 was published (49). The phenotype of the crzA strain in the CEA17 background is virtually identical to the results we report here with the crzA strain in the AF293 background. Those authors observed localization of CrzA to the nucleus in response to calcium chloride treatment. Our analysis of the CrzA amino acid sequence compared to the amino acid sequence of Crz1 in S. cerevisiae revealed a putative NLS. However, this site was downstream of the Crz1 NLS site in CrzA (amino acids 460 to 490) (Fig. 2). This region contains the basic amino acids arginine and lysine, which are typically found in NLS regions, and is serine rich, similar to Crz1's NLS region (Fig. 2). Further experiments utilizing site-directed mutagenesis will confirm whether this site is indeed the NLS site in A. fumigatus.
In addition to the nuclear localization study, Soriani et al. (49) observed that the crzA strain in the CEA17 background was more sensitive to calcium and manganese chloride. While we did not directly measure sensitivity to these chemicals, we observed that germination of crzA strain conidia in the AF293 background was severely inhibited in liquid culture in the presence of 200 mM CaCl2 (data not shown). We also observed a significant decrease in conidium germination in the crzA strain in the AF293 background, while Soriani et al. (49) reported an increase in polar germ tube emergence in the crzA strain in the CEA17 background. The explanation for this significant difference is not readily apparent; however, the differences in methodology may be an important factor. While we measured germ tube emergence over a defined time period of 48 h, Soriani et al. (49) treated germlings with hydroxyurea and, following release from cell cycle arrest over 150 min, measured germling polarization. Interestingly, Soriani et al. were unable to identify germinating conidia or hyphae in their histopathological analyses of mice infected with the crzA strain. However, unlike our animal model results, Soriani et al. did observe 30% mortality in mice infected with the crzA strain. Viable crzA colonies were recovered from mice infected with the crzA strain that perished.
Soriani et al. (49) also examined the mRNA abundances of various ion pump homologs in response to a short pulse of 200 mM CaCl2, while we focused our mRNA abundance assays on components of the cell wall biosynthesis machinery (Table 1). However, we did examine mRNA abundances of pmrA and pmcA in the cnaA and crzA strains and found results similar to those of Soriani et al., i.e., that both pmrA and pmcA appear to be regulated by the calcineurin pathway in A. fumigatus as in S. cerevisiae. These results likely explain the calcium sensitivity of the crzA strain reported by Soriani et al. (49) and observed by us in our experiments.
These results continue to confirm the great potential that inhibition of the calcineurin pathway in pathogenic fungi may have for the treatment of invasive mycoses. There is a high amino acid identity between the A. fumigatus CnaA and human calcineurin A proteins (58.4% and 63.1%, respectively) (17). However, the closest possible homolog to A. fumigatus CrzA is human NFAT, and the amino acid identity between A. fumigatus CrzA and human NFAT is only 14.9% (34). This information suggests that inhibition of A. fumigatus CrzA, which blocks A. fumigatus hyphal growth in vitro and in vivo, would have far less cross-reactivity with potential similar human targets and could more selectively damage A. fumigatus with targeted antifungal therapy.
In conclusion, our results strongly suggest that the A. fumigatus Crz1 homolog, CrzA, is a downstream effector of calcineurin signaling in this important opportunistic human fungal pathogen. A. fumigatus CrzA has a role in polarized hyphal growth, asexual development, and fungal pathogenicity much like that of CnaA. Future studies will seek to discover the mechanism of CrzA-mediated signal transduction and to uncover additional pathways that intersect with the calcineurin signaling pathway that may account for some of the differences observed between the crzA and cnaA strains. Finally, given that CrzA seems to be fungus specific and strongly linked to disease development, this particular component of the calcineurin pathway is an ideal antifungal drug target for invasive aspergillosis and possibly other molds.
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ACKNOWLEDGMENTS |
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R.A.C. was supported by the NIH/NIAID Molecular Mycology and Pathogenesis Training Program (5 T32 AI052080) at Duke University Medical Center and is currently supported by NIH/COBRE grant RR020185 and the Montana State Agricultural Experiment Station. W.J.S. is supported by NIH/NIAID award K08 A1061149, a Basic Science Faculty Development grant from the American Society for Transplantation, and a Children's Miracle Network grant.
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FOOTNOTES |
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Published ahead of print on 2 May 2008.
Supplemental material for this article may be found at http://ec.asm.org/.
Present address: Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT.
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