Heat shock protein 90 is required for conidiation and cell wall integrity in Aspergillus fumigatus

Abstract

Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of Candida albicans to azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogen Aspergillus fumigatus. We investigated the role of Hsp90 in A. fumigatus by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of hsp90 suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors brlA, wetA, and abaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.

Fig 1

Genetic construction of the pniiA-Hsp90 strain and phenotypes under Hsp90 repression. (A) Schematic representation of the genomic locus of hsp90 in the wild-type (WT) and pniiA-Hsp90 strains. Correct integration of the left arm (LA, consisting of a 1-kb sequence located downstream of the putative hsp90 encoding gene) and the right arm (1-kb 5′-end sequence of hsp90) was confirmed by Southern analysis using the left arm sequence as the probe (P) and two different enzyme digestions (BamHI and SacII). Hph, hygromycin B resistance cassette; pniiA, nitrogen-dependent inducible promoter. (B) Southern analysis of the pniiA-Hsp90 strain. Digestion with BamHI revealed 2-kb and a 4-kb fragments in the wild-type and pniiA-Hsp90 strains, respectively. Digestion with SacII shows a 5.6-kb fragment (wild type) and a 2.7-kb fragment (pniiA-Hsp90). (C and D) Morphological changes of the pniiA-Hsp90 strain grown under different levels of ammonium for Hsp90 genetic repression compared to the akuB^KU80 control strain. (C) AMM (where ammonium is the unique source of nitrogen); (D) GMM+Am (ammonium with a concomitant and constant source of nitrate), which is comparable to the pharmacologic inhibition of Hsp90 with geldanamycin (concentrations ranging from 0 to 8 μg/ml) in the akuB^KU80. Pictures were taken after 5 days of growth at 37°C.

Fig 2

Hyphal growth, germination, and conidiation defects of the pniiA-Hsp90 strain under Hsp90 repression. (A) Culture morphology of the control akuB^KU80 and the pniiA-Hsp90 strains on AMM (20 mM ammonium as the sole nitrogen source) showing both radial growth (top) and aerial growth (bottom). Pictures were taken after 5 days of growth at 37°C. (B) Hyphal growth (colony diameter, in mm) of the control akuB^KU80 strain (black bars) and the pniiA-Hsp90 strain (gray bars) over 5 days on AMM (20 mM ammonium). Results are means ± SD for triplicates. (C) Quantification of the transcription of the conidiation-specific genes brlA (black bar), abaA (dark gray bar), and wetA (light gray bar) via real-time reverse transcription-PCR in the pniiA-Hsp90 strain under repression conditions (20 mM ammonium). Results are mean fold change (2^−ΔΔCT) ± SD compared to the wild-type (dotted line). (D) Germination rate (expressed as the mean percentage of germlings observed in triplicates ± SD) of the pniiA-Hsp90 strain (black bars) over time on AMM (20 mM ammonium). Emergence of germ tubes (black arrows) was achieved in only 50% of conidia after 4 days, while hyphal elongation was absent. The picture was taken after 4 days of growth at 37°C.

Fig 3

Effect of Hsp90 repression in cell wall stress response. (A) Transcriptional profile of hsp90 in the A. fumigatus wild-type strain (AF293) under various stress conditions: heat stress (black bar, 23 h growth at 37°C followed by heat shock 55°C for 1 h), caspofungin treatment (dark gray bar, 24 h growth in the presence of 1 μg/ml caspofungin) and FK506 treatment (light gray bar, 24 h growth in the presence of 20 ng/ml FK506). Results are presented as the mean fold change (2^−ΔΔCT) for triplicates ± SD compared to the wild-type values under basal growth conditions (dotted line; 37°C, no treatment). (B) Genetic and pharmacologic inhibition of Hsp90 increases the effect of caspofungin and abolishes the paradoxical effect of caspofungin. Growth of akuB^KU80 (control), pniiA-Hsp90 under genetic Hsp90 repression conditions (GMM+Am with 20 mM ammonium) and AF293 (wild type) under pharmacologic Hsp90 inhibition (geldanamycin [GdA], 8 μg/ml) with increasing caspofungin concentrations (0.125 to 4 μg/ml). Pictures were taken after 5 days of growth at 37°C. (C) Comparative growth of the akuB^KU80 and pniiA-Hsp90 strains under various stress conditions on GMM+Am (20 mM ammonium). The pniiA-Hsp90 strain is unable to grow under heat stress (55°C) and in the presence of Congo red (binding to the cell wall). The effect of FK506 (calcineurin inhibitor) is increased by concomitant Hsp90 repression. Pictures were taken after 5 days of growth. (D) The cell wall stabilizer sorbitol (at a concentration of 1 M) remediates the conidiation defect of the pniiA-Hsp90 strain under repression conditions (GMM+Am with 20 mM ammonium), as shown by the greenish appearance of the colonies. This effect was also observed in the presence of caspofungin or FK506. Pictures were taken after 5 days of growth at 37°C.

Fig 4

Localization of Hsp90-EGFP under standard growth conditions (untreated) and under caspofungin exposure. (A) Schematic representation of the genomic locus of hsp90 in the wild-type (WT) and the Hsp90-EGFP strains. The enhanced green fluorescent protein gene (egfp) and the hygromycin B resistance cassette (Hph) were linked to the 3′-end of the hsp90 gene by homologous recombination of the left arm (LA, consisting of the 1-kb terminal sequence of hsp90) and the right arm (RA, a 1.5-kb sequence downstream of hsp90). Correct integration of the genetic construct was confirmed by PCR analysis showing a 2.3-kb band in the Hsp90-EGFP strain and no amplification in the control strain. (B and C) Localization of Hsp90-EGFP under standard growth conditions (untreated). Hsp90 is widely and homogenously distributed within the cytosol with the presence of some dotted (or punctate) structures (white arrows). (D and E) In the presence of low caspofungin concentration (0.125 μg/ml), Hsp90 moves from the cytosol to the cell wall and the tips of hyphae or to the sites of septum formation (white arrows). (F and G) At a higher caspofungin concentration (0.25 μg/ml), while more hyphal lysis is observed (dotted black arrows), Hsp90 further accumulates at the sites of hyphal regeneration, such as sites of septum formation (white arrows) (F) and tips of intrahyphal hyphae (arrowheads) (G). (H to O) Costaining with calcofluor white (blue) confirms the localization of EGFP-Hsp90 (green) in the cell wall and septa (white arrows). Panels I, K, M, and O show overlay pictures. Pictures were taken by fluorescence microscopy after 17 h growth on coverslips in liquid GMM at 37°C. Bar, 10 μm.

Fig 5

Localization of Hsp90-EGFP under various stress conditions. The Hsp90-EGFP strain was grown for 19 h on coverslips in liquid GMM at 37°C and then exposed to the stress condition (heat shock [55°C] or glucose or nitrogen starvation) for 1 h, before observation by fluorescence microscopy. Bar, 10 μm. (A) Glucose starvation. Hsp90 remains widely distributed in the cytosol, while more dotted structures are observed (compared to the standard growth conditions). (B) Nitrogen starvation. Many dotted structures are observed, similarly to glucose starvation. (C) Heat shock at 55°C. Hsp90 moves from the cytosol to the nuclei (white arrows). (D) Costaining with propidium iodide confirms the nuclear localization of EGFP-Hsp90 on the overlay picture.