Link between Heat Shock Protein 90 and the Mitochondrial Respiratory Chain in the Caspofungin Stress Response of Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an opportunistic mold responsible for invasive aspergillosis. Triazoles (e.g., voriconazole) represent the first-line treatment, but emerging resistance is of concern. The echinocandin drug caspofungin is used as second-line treatment but has limited efficacy. The heat shock protein 90 (Hsp90) orchestrates the caspofungin stress response and is the trigger of an adaptive phenomenon called the paradoxical effect (growth recovery at increasing caspofungin concentrations). The aim of this study was to elucidate the Hsp90-dependent mechanisms of the caspofungin stress response. Transcriptomic profiles of the wild-type A. fumigatus strain (KU80) were compared to those of a mutant strain with substitution of the native hsp90 promoter by the thiA promoter (pthiA-hsp90), which lacks the caspofungin paradoxical effect. Caspofungin induced expression of the genes of the mitochondrial respiratory chain (MRC), in particular, NADH-ubiquinone oxidoreductases (complex I), in KU80 but not in the pthiA-hsp90 mutant. The caspofungin paradoxical effect could be abolished by rotenone (MRC complex I inhibitor) in KU80, supporting the role of MRC in the caspofungin stress response. Fluorescent staining of active mitochondria and measurement of oxygen consumption and ATP production confirmed the activation of the MRC in KU80 in response to caspofungin, but this activity was impaired in the pthiA-hsp90 mutant. Using a bioluminescent reporter for the measurement of intracellular calcium, we demonstrated that inhibition of Hsp90 by geldanamycin or MRC complex I by rotenone prevented the increase in intracellular calcium shown to be essential for the caspofungin paradoxical effect. In conclusion, our data support a role of the MRC in the caspofungin stress response which is dependent on Hsp90.

Keywords: NADH-ubiquinone oxidoreductases; echinocandins; invasive aspergillosis; mitochondria; paradoxical effect; rotenone; transcriptomics.

FIG 1

Transcriptomic analyses of MRC genes in A. fumigatus KU80 (parental strain) and the pthiA-hsp90 mutant under basal conditions (untreated) and after 2 h of caspofungin (CAS) exposure. (A) Gene expression in fold change compared to the reference condition (KU80, untreated). Black, KU80 untreated (KU80); dark gray, KU80 with caspofungin (KU80 CAS); gray, pthiA-hsp90 mutant untreated (phtiA-hsp90); light gray, pthiA-hsp90 mutant with caspofungin (phtiA-hsp90 CAS). The P values are expressed as *, ≤0.01; **, ≤0.001; ****, ≤0.00001; *****, ≤0.000001. Numbers I to V correspond to the MRC complex to which the genes were assigned according to nBlast. ND, not determined. (B) Dried mycelial mass (in milligrams) of the different strains (KU80 and pthiA-hsp90 mutant) under the experimental conditions of the transcriptomic analyses (24 h untreated and 22 h untreated with an additional 2 h of caspofungin exposure). Error bars represent standard deviation of the results from experiments in triplicate. ns, not significant.

FIG 2

Effect of hsp90 repression (pthiA-hsp90 mutant strain) and MRC complex I inhibition (rotenone) on the caspofungin paradoxical effect of A. fumigatus. (A) Pictures were taken after 5 days of growth at 37°C on glucose minimum medium (GMM) agar plates supplemented with caspofungin (CAS) at increasing gradient concentration. Rotenone (ROT) was added at a fixed concentration of 158 μg/ml. (B) Graphs represent the mean diameters of the colonies. Error bars represent standard deviations of the results from experiments in triplicate. P values are represented for comparisons of the diameters of the colonies exposed to caspofungin 1 μg/ml versus 2 and 4 μg/ml in order to demonstrate the paradoxical effect (significant recovery of the growth at concentrations above 1 μg/ml). ****, P ≤ 0.0001; ns, not significant.

FIG 3

Visualization of active mitochondria in KU80 and pthiA-hsp90 mutant in the absence or presence of caspofungin (CAS). (A) Cultures performed on coverslips in GMM broth at 37°C for 24 h in the absence or presence of caspofungin (2 μg/ml after 22 h). Left, light microscopy; right, fluorescence microscopy stained with MitoTracker Red CM-H₂XRos. (B) Graphs represent fluorescence quantification for each condition, measured using the ImageJ software. Fluorescence is expressed in relative fluorescent units (RFU).

FIG 4

Measurement of ATP production by CellTiter-Glo in KU80 and pthiA-hsp90 mutant cell lysates after 24 h growth in GMM broth at 37°C in the absence or presence of caspofungin (CAS; 2 μg/ml added after 22 h). Bars represent means with standard deviations of the results from three biological replicates, with results expressed as fold change in luminescence compared to the untreated KU80 strain. ns, not significant.

FIG 5

Oxygen measurements in KU80 and the pthiA-hsp90 mutant in the absence or presence of caspofungin and rotenone. Graphs represent percentage of oxygen (y axis) in the chambers over time (x axis). (A) KU80 in the absence or presence of 2 μg/ml caspofungin (CAS) added 1 h before start of measurement. (B) pthiA-hsp90 mutant in the absence or presence of 2 μg/ml CAS added 1 h before start of measurement. (C) KU80 and pthiA-hsp90 mutant with addition of 2 μg/ml CAS after 30 min (dashed line). (D) KU80 in the absence or presence of 158 μg/ml rotenone (ROT) added 1 h before the start of measurement.

FIG 6

Intracellular calcium (Ca²⁺) measurement using the bioluminescent reporter aequorin expressed in KU80. (A) The AEQ^ΔakuB strain was preincubated in the absence or in the presence of 4 μg/ml geldanamycin (GDA) or 158 μg/ml rotenone (ROT), added 1 h before start of measurement at room temperature. Caspofungin (CAS; 2 μg/ml) was injected 6 min after start of the measurement. (B) Same experiment as in panel A with the addition of BAPTA (1 mM) for 1 h at room temperature before the start of measurement. Results represent mean curves of triplicates and are expressed in relative luminescence units (RLU) over time.