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Antimicrobial Agents and Chemotherapy, August 2007, p. 2979-2981, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01394-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Calcineurin Inhibition or Mutation Enhances Cell Wall Inhibitors against Aspergillus fumigatus{triangledown}

William J. Steinbach,1,2* Robert A. Cramer Jr.,2 B. Zachary Perfect,1 Christina Henn,1 Kirsten Nielsen,2 Joseph Heitman,2,3,4 and John R. Perfect2,3

Division of Pediatric Infectious Diseases, Department of Pediatrics,1 Department of Molecular Genetics and Microbiology,2 Division of Infectious Diseases and International Health, Department of Medicine,3 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 277104

Received 7 November 2006/ Returned for modification 6 January 2007/ Accepted 5 May 2007


   ABSTRACT
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Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-ß-D-glucan revealed decreased amounts in the calcineurin A ({Delta}cnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.


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Invasive aspergillosis (IA) is a leading cause of infectious mortality in the growing number of immunocompromised patients (7). Our previous in vitro work demonstrated anti-Aspergillus activity of the calcineurin inhibitors FK506 and cyclosporine (11, 12). We subsequently showed with a calcineurin A (cnaA) mutant that calcineurin is critical for A. fumigatus hyphal growth, tissue invasion, and pathogenicity (10). Here we further examine potential enhanced antifungal involvement of calcineurin and cell wall synthesis inhibitors against A. fumigatus. Our results lend support to our hypothesis that the calcineurin pathway is an innovative target for more efficacious strategies against IA.

Patients generally possess blood levels of FK506 from 5 to 25 ng/ml for the prevention of organ rejection. We utilized radial growth assays (8) and found patient achievable levels of FK506 (20 ng/ml) led to significantly stunted A. fumigatus growth similar to the untreated {Delta}cnaA mutant and more effective than therapeutically relevant and greater (300 ng to 1 µg/ml) concentrations of the echinocandin antifungal caspofungin. The higher dose of caspofungin did lead to a phenotypically identical growth arrest as the {Delta}cnaA mutant, suggesting a possible similar or linked mechanism of action on Aspergillus growth. These findings were confirmed with scanning electron microscopy, which demonstrated absent hyphal growth in the {Delta}cnaA mutant or after FK506 (20 ng/ml) treatment and superior antifungal potency compared to caspofungin (1 µg/ml) treatment.

Impact on early fungal growth was also examined. Wild-type A. fumigatus conidia (106 conidia/ml) were incubated for 14 h in RPMI 1640, and subsequent addition of caspofungin (1 µg/ml) resulted in well-described dysmorphic hyphae (6). FK506 acted in a similar fashion to caspofungin, supporting a potentially similar mechanism or pathway (Fig. 1A). Ungerminated wild-type conidia treated with FK506 showed significant delay in germ-tube extension, including growth retardation at only 20 ng/ml. Germination into hyphae was complete by 12 h in the untreated controls, but germination had only begun in the calcineurin inhibitor-treated strains after 60 h (Fig. 1B). Thus, inhibiting the calcineurin pathway caused a profound initial delay in conidial germination.


Figure 1
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  		FIG. 1. (A) Germination of wild-type A. fumigatus for 14 h in RPMI, followed by only 6 h of FK506 treatment, yields concentration-dependent stunted hyphal tips. (B) FK506 treatment of ungerminated conidia in RPMI shows concentration-dependent delays in germ-tube extension at 60 h growth.

 
Given the link between cell wall morphogenesis and hyphal growth in filamentous fungi, we next used the aniline blue fluorescence assay (8) to quantify 1,3-ß-D-glucan after calcineurin inhibition. Measurement was obtained as relative fluorescence units (RFU) normalized to mycelial mass and analyzed using an unpaired t test with significance reported as a two-tailed P value of <0.05. Comparisons were done in triplicate after growth for 24 h with mycelial mass equalized to account for the documented growth defect of the {Delta}cnaA mutant. According to this assay, the {Delta}cnaA mutant had significantly less 1,3-ß-D-glucan than the wild-type (7.677 ± 0.31 RFU versus 2.634 ± 0.05 RFU, P < 0.0001), suggesting calcineurin inhibition perturbs cell wall synthesis. The 1,3-ß-D-glucan from the wild-type strain also decreased after treatment with caspofungin (1 µg/ml) (6.187 ± 0.04 RFU), and FK506 at lower levels (20 ng/ml) (2.909 ± 0.07 RFU) (P = 0.0092). There was an enhanced decrease with caspofungin treatment of the {Delta}cnaA mutant (1.086 ± 0.01 RFU) compared to the untreated {Delta}cnaA mutant (P < 0.0001), demonstrating the additive effects of 1,3-ß-D-glucan and calcineurin inhibition.

Furthermore, treatment of the {Delta}cnaA mutant with the chitin synthase inhibitor nikkomycin Z resulted in larger balloon-like hyphal apical tips than treatment of the wild-type strain (Fig. 2). Treatment of the {Delta}cnaA mutant with both caspofungin and nikkomycin Z resulted in thin-walled cells that were unrecognizable as hyphae and supports added benefit with the triple combination of calcineurin, 1,3-ß-D-glucan, and chitin inhibition.


Figure 2
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  		FIG. 2. Caspofungin and nikkomycin Z treatment of both wild-type and {Delta}cnaA mutant strains in RPMI media at 48 h.

 
Next, we used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to measure antifungal activity against A. fumigatus viability and not just growth (1, 15). Cultures germinated at 37°C for 14 h, antifungals or calcineurin inhibitors were added, and the cultures were coincubated for 10 more hours. Fluorescent staining revealed the distal hyphal tips of the {Delta}cnaA mutant or the wild-type strain treated with FK506 were blunted but also viable (Fig. 3), unlike the tips after caspofungin treatment, supporting the hypothesis that cell death is not the cause for the stunted growth and reduction in pathogenicity. After caspofungin treatment against the {Delta}cnaA mutant, DiBAC staining in the distal hyphal tip areas demonstrated that while the majority of {Delta}cnaA mutant cells revealed no stain, ca. 15% of the cells did stain brightly (>500 cells examined) and demonstrated inviable fungal structures not only at the hyphal tip (data not shown). This suggests an additive effect of caspofungin on the viability of the {Delta}cnaA mutant. However, it is a heterogeneous added effect of caspofungin that might possibly be created by a decreased 1,3-ß-D-glucan in the cell wall of the {Delta}cnaA mutant.


Figure 3
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  		FIG. 3. Cell viability staining in RPMI at 24 h growth shows the viable blunted hyphal tips of the {Delta}cnaA mutant strain. Caspofungin (1 µg/ml), FK506 (20 ng/ml). CFDA, 5,(6)-carboxyfluorescein diacetate; DiBAC, bis-(1,3-dibutylbarbituric acid) trimethine oxonol; WT, Wild-type.

 
Overall, we noted that calcineurin inhibition, via pharmacologic inhibitors or the {Delta}cnaA mutant, led to morphological hyphal defects similar or superior to those found with echinocandin treatment of A. fumigatus (6). The radial growth assay (8), demonstrated FK506 at concentrations 50-fold lower than caspofungin limited filamentation and growth. This hyphal dysmorphic effect is even more significant with the {Delta}cnaA mutant than FK506 treatment and suggests that current calcineurin inhibitors are not completely effective at blocking the calcineurin pathway's impact on hyphal growth and therefore allows possibilities that improved drugs for hyphal growth inhibition by blocking calcineurin could be designed. We also observed that 1,3-ß-D-glucan measurements utilized for quantifying caspofungin antifungal activity (8) led to decreases after calcineurin inhibition, supporting the hypothesis calcineurin inhibition operates through cell wall biosynthesis.

Reports have shown an in vitro synergistic effect of 1,3-ß-D-glucan inhibition combined with chitin inhibition against Aspergillus species (2, 3, 9, 13). However, a compensatory mechanism exists for the fungal cell wall, whereby inhibition of glucan synthesis increases chitin content (5, 14) or disruption of the fks1 gene can lead to increased chitin synthesis (4), and shows dynamic mechanisms for survival of a fungal cell wall under attack. Our work with calcineurin inhibition further demonstrates this important principle and suggests that a third agent and pathway targeting the cell wall can lead to complementary fungal destruction. Further discovery of targets along this calcineurin pathway, where the pharmacologic or genetic inhibition of calcineurin A has clear antifungal benefits, could yield promising therapeutic results.


   ACKNOWLEDGMENTS
 
W.J.S. was supported by NIH grant 1 K08 A1061149, R.A.C. and K.N. were supported by NIH/NIAID Molecular Mycology and Pathogenesis Training Program contract no. 5 T32 AI052080, J.H. is supported by NIH grant R01-AI50438 and NIH/NIAID grant R01-AI42159, and J.R.P. is supported by NIH grant AI28388.


   FOOTNOTES
 
* Corresponding author. Mailing address: Division of Pediatric Infectious Diseases, Box 3499, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-3734. Fax: (919) 684-8902. E-mail: stein022{at}mc.duke.edu Back

{triangledown} Published ahead of print on 14 May 2007. Back


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Antimicrobial Agents and Chemotherapy, August 2007, p. 2979-2981, Vol. 51, No. 8
0066-4804/07/$08.00+0     doi:10.1128/AAC.01394-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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