Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin

Abstract

Attenuated activity of echinocandin antifungals at high concentrations, known as the "paradoxical effect," is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca(2+)/calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the DeltacnaA and DeltacrzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.

FIG. 1.

A. fumigatus exhibits paradoxical growth at high concentrations of caspofungin. (A) Comparison of A. fumigatus wild-type strain Af293 grown for 48 h at 37°C in the presence of 8, 4, 2, 1, and 0.5 μg/ml of caspofungin, micafungin, or anidulafungin. NT, no treatment. (B) Quantification of radial growth rates among wild-type cultures treated with caspofungin, micafungin, or anidulafungin. Results and error bars represent the means ± SD (mm/h) for biological triplicates.

FIG. 2.

The calcineurin pathway controls A. fumigatus paradoxical growth in response to caspofungin treatment. Comparison of wild-type (WT), ΔcnaA, and ΔcrzA strains grown for 48 h at 37°C in the presence of 8, 4, 2, 1, and 0.5 μg/ml of caspofungin. NT, no treatment. Growth of the ΔcnaA mutant was examined at two concentrations, 5,000 conidia (ΔcnaA 5,000) and 10,000 conidia (ΔcnaA 10,000) per inoculum, to clarify growth characteristics in response to caspofungin treatment.

FIG. 3.

Chitin content increases in response to ascending doses of caspofungin. Analysis of total chitin content in the WT (○), ΔcnaA (▪), and ΔcrzA (▴) strains in response to treatment with 0.5, 1, 2, 4, and 8 μg/ml caspofungin. All analyses were performed in biological triplicate for statistical analysis. Results and error bars represent the means ± SD for glucosamine equivalents. NT, no treatment.

FIG. 4.

Calcineurin controls transcriptional upregulation of chsA and chsC in response to caspofungin treatment. Transcriptional profile of the A. fumigatus chitin synthase genes in response to treatment with caspofungin (4 μg/ml) alone for the WT (dark gray bars) and ΔcnaA (light gray bars) strains or cotreatment with caspofungin (4 μg/ml) and FK506 (200 ng/ml) for the WT (black bars). Results are presented as the mean fold change (2^−ΔΔCt) ± SD. All experiments were performed in biological triplicate for statistical analysis.

FIG. 5.

Chitin synthase activity increases in response to caspofungin in a calcineurin-dependent manner. (A) The reproducibility of the nonradioactive chitin synthase assay was verified by analysis of total chitin synthase activity in increasing amounts of starting protein (5 and 10 μg). Results were calculated as stated in Materials and Methods and are presented as the mean ± SD for total chitin (μg/ml) produced during the 90-min incubation. Total chitin synthesis in complete reaction mixture (black bars), total chitin remaining after a 5-h treatment with chitinase (1 U/ml) at room temperature (light gray bars), chitin synthesis in reaction mixture lacking UDP-GlcNAc substrate (dark gray bars), and chitin synthesis in reaction mixture lacking trypsin for activation (white bars) are shown. Experiments were performed in biological triplicate for statistical analysis. (B) Comparison of the total chitin synthase activity of the wild-type (WT) and ΔcnaA strains after exposure to either 4 μg/ml caspofungin alone (gray bars) to that of the WT strain after cotreatment with 4 μg/ml caspofungin and 200 nM FK506 (black bar). Results and error bars represent the means ± SD for chitin (μg/ml). (C) Comparison of total chitin synthase activity in WT microsomal membrane extracts pretreated with TFP (100 or 200 μM) or FK506 (200 or 400 μg/ml) for 30 min at room temperature. Results and error bars represent the means ± SD for chitin (μg/ml). All experiments were performed in biological triplicate for statistical analysis.