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.
INTRODUCTION
Aspergillus fumigatus is a ubiquitous mold which can cause a broad spectrum of diseases, including the devastating invasive aspergillosis (IA), in patients with impaired immunity, such as transplant recipients or cancer patients (1, 2). The treatment of IA remains a challenge, as only three drug classes are available (azoles, polyenes, and echinocandins), and emergence of resistance to azoles, the first-line treatment, is increasingly reported (3). Echinocandins, such as caspofungin, micafungin, and anidulafungin, can be used as second-line therapy for IA or in combination with voriconazole for refractory cases or when azole resistance is suspected (4–6). Echinocandins inhibit the synthesis of (1-3)-β-d-glucan, a major cell wall component. However, their in vitro activity against A. fumigatus is limited and only fungistatic with persistent growth above the MIC threshold. Moreover, a paradoxical effect, defined as a return to growth at increasing concentrations, can be observed with caspofungin, which may have some clinical relevance (7). This phenomenon of tolerance indicates the existence of compensatory mechanisms of the cell wall which are mediated by the heat shock protein 90 (Hsp90) and the calcium-calcineurin pathway (7, 8). Hsp90 is a molecular chaperone playing a key role in the mechanisms of stress adaptation, including the development of antifungal drug resistance or tolerance in A. fumigatus and other pathogenic fungi (9, 10). The essential role of Hsp90 in the caspofungin stress response of A. fumigatus has been previously highlighted (8, 11). However, Hsp90-dependent pathways in this response remain partly unknown. We identified a yet-unrevealed role of the mitochondrial respiratory chain (MRC) in the caspofungin stress response, which was dependent on Hsp90.
RESULTS
Caspofungin stress results in overexpression of genes of the MRC, which is dependent on Hsp90.Our first objective was to determine which genes are involved in the caspofungin stress response in the wild-type A. fumigatus isolate KU80. In order to identify which of them are dependent on Hsp90, we used the pthiA-hsp90 mutant with substitution of the hsp90 promoter by the thiA promoter (8). Exposure to thiamine results in hsp90 repression and complete growth inhibition. However, in the absence of thiamine, this strain has sufficient Hsp90 levels to maintain normal basal growth, but the lack of the native hsp90 promoter does not allow the achievement of appropriate Hsp90 levels for stress adaptation when exposed to caspofungin (8). As a result, the pthiA-hsp90 mutant strain has no basal growth defect but cannot generate tolerance and a paradoxical effect to caspofungin (Fig. 2).
Transcriptomic analyses (RNA sequencing [RNA-seq]) were performed in three biological replicates of whole-RNA extracts of KU80 and the pthiA-hsp90 mutant (without the addition of thiamine) under basal conditions and after 2 h of exposure to caspofungin at 2 μg/ml (i.e., the concentration required to induce the paradoxical effect of caspofungin). As previously demonstrated (8), the expression of hsp90 was significantly decreased (3.1-fold, P = 0.04) in the pthiA-hsp90 mutant compared to KU80 in the presence of caspofungin.
Genes for which a significant increase (fold change ≥2 and P ≤ 0.05) was observed upon caspofungin exposure in KU80 were selected. The transcriptional response of the pthiA-hsp90 mutant in the absence and presence of caspofungin was analyzed for these genes (see Table S1 in the supplemental material). We found that the mitochondrion-carried genes of the mitochondrial respiratory chain (MRC) were strongly induced by caspofungin in KU80 but not in the pthiA-hsp90 mutant (Fig. 1A and Table S1). Although the basal expression of some of these genes was somewhat higher in the pthiA-hsp90 mutant than in KU80, no increase was observed upon caspofungin exposure. The MRC genes of A. fumigatus were identified and classified in their respective complexes (I to IV) by nBlast with other fungi (Aspergillus oryzae and Neurospora crassa). A majority of the genes exhibiting the highest induction of expression in the wild type belonged to complex I (NADH-ubiquinone oxidoreductases) (Fig. 1A).
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.
Because MRC gene expression can be influenced by fungal growth, we have looked for possible variations in the mycelial mass between KU80 and the pthiA-hsp90 mutant in the presence or absence of caspofungin under the same experimental conditions (22 h growth with an additional 2 h with or without caspofungin). Our data show that there was no statistically significant difference in fungal growth between KU80 and the pthiA-hsp90 mutant in the presence or absence of caspofungin at the time of the analysis (Fig. 1B).
As a next step, we wanted to know if the induction of MRC gene expression in KU80 was unique to caspofungin or could result from a nonspecific effect of any fungal growth inhibitory drug using a potent antifungal drug, such as voriconazole. The transcriptomic profile of KU80 upon 2 h of voriconazole exposure was analyzed and did not show any significant increase in expression of the MRC genes compared to the untreated KU80 strain (Fig. S1).
We thus hypothesized that the MRC could play a unique role in the caspofungin stress response, as illustrated by the significant increase in expression of the MRC genes in KU80 upon caspofungin exposure but not upon voriconazole exposure. Furthermore, the lack of induction of MRC genes in the pthiA-hsp90 mutant suggests that MRC induction in the caspofungin stress response may be dependent on Hsp90 and that this effect is not a consequence of a growth defect in the pthiA-hsp90 mutant compared to the parental KU80 strain.
MRC complex I activation is required for the paradoxical effect of caspofungin.In order to further investigate the potential role of the MRC in the caspofungin stress response and paradoxical growth, we assessed the effect of various MRC inhibitors on KU80 growth with increasing concentrations of caspofungin. Exposition to rotenone, a MRC complex I inhibitor, resulted in a loss of paradoxical growth at increased caspofungin concentrations, an effect similar to that observed in the pthiA-hsp90 mutant (Fig. 2). Rotenone was also able to abolish the paradoxical effect of caspofungin in other A. fumigatus strains, such as the wild-type AF293 strain (data not shown). However, paradoxical growth at high caspofungin concentrations was conserved in the presence of other MRC inhibitors, such as antimycin A (complex III inhibitor), oligomycin (ATPase inhibitor), and azide (complex IV inhibitor), or under hypoxic growth conditions (Fig. S2). These results show that MRC complex I is important for the caspofungin stress response and paradoxical effect.
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.
Mitochondrial activity is impaired in the pthiA-hsp90 mutant under caspofungin stress.As a next step, we attempted to demonstrate the functional effect of hsp90 repression and downregulation of MRC genes on the activity of the mitochondria in response to caspofungin stress. Staining of mycelia with MitoTracker Deep Red FM (staining all mitochondria irrespective of their activity) did not show any difference between KU80 and the pthiA-hsp90 mutant (data not shown). We then used MitoTracker Red CM-H2XRos, which fluoresces only upon oxidative activity of mitochondria. Exposure to caspofungin (2 μg/ml) for 2 h induced fluorescence in KU80 but not in the pthiA-hsp90 mutant (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).
Mitochondria use oxygen to produce ATP via the MRC. In order to measure ATP production, we used a luciferase assay (CellTiter-Glo luminescent cell viability assay), which produces a luminescent signal proportional to ATP quantity. A significant increase in ATP production was observed in KU80 upon 2 h of caspofungin exposure. Although the pthiA-hsp90 mutant exhibited a higher basal level of ATP production, no increase was observed in the presence of caspofungin (Fig. 4). Finally, analyses with an oximeter showed that the consumption of oxygen by KU80 increased upon caspofungin exposure compared to the untreated condition. However, the pthiA-hsp90 mutant strain exhibited a basal defect in oxygen utilization and was unable to increase oxygen consumption under caspofungin stress (Fig. 5). This impairment of oxygen consumption was not related to a growth defect in the pthiA-hsp90 mutant strain, as illustrated by the comparisons of mycelial mass with the parental KU80 strain, showing no significant differences (Fig. 1B). We concluded that activation of the mitochondria leading to ATP generation in response to caspofungin was impaired in the pthiA-hsp90 mutant.
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.
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.
Both MRC and Hsp90 inhibition prevent the increase of intracellular calcium in response to caspofungin stress.An increase in intracellular calcium (Ca2+) triggers the calcineurin pathway and was shown to be essential for caspofungin stress response and paradoxical growth in A. fumigatus (12). ATP is required for the uptake of extracellular Ca2+ by ATP channels of the cell membrane and also for the release of Ca2+ stores from the endoplasmic reticulum (13). We hypothesized that Hsp90 and the MRC are essential for caspofungin stress response by generating the ATP required for the increase in cytoplasmic Ca2+. For this purpose, we used a KU80 strain harboring the bioluminescent Ca2+ reporter aequorin (AEQΔakuB) to measure intracellular calcium (14). As previously reported (12), we observed an increase in intracellular Ca2+ upon caspofungin exposure (Fig. 6A). However, this response was abolished in the presence of geldanamycin (Hsp90 inhibitor) or rotenone (MRC complex I inhibitor), since no Ca2+ increase was observed (Fig. 6A). The peak of Ca2+ could also be abolished in the presence of the Ca2+ chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid] (Fig. 6B), which suggests that it results from an external calcium source, as previously reported (12). These results further support the link between Hsp90 and the MRC in the caspofungin stress response, which could be essential for ATP production and extracellular Ca2+ uptake by ATP-dependent Ca2+ channels.
Intracellular calcium (Ca2+) measurement using the bioluminescent reporter aequorin expressed in KU80. (A) The AEQΔakuB 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.
DISCUSSION
Echinocandins are gaining interest as salvage or combination therapy for IA since resistance to azoles is emerging. Their limited in vitro antifungal activity against A. fumigatus is the consequence of a compensatory stress response, as illustrated by persistent growth above the MIC and the so-called paradoxical effect, with a loss of efficacy at increasing concentrations. In this study, we provide further insights into the mechanisms of this tolerance phenomenon, highlighting a previously unknown role of the mitochondrial respiratory chain (MRC) which is dependent on the molecular chaperone Hsp90. Exposure of the wild-type A. fumigatus to caspofungin resulted in activation of the MRC, with increased oxygen consumption and ATP production. This stress response seems to be unique to caspofungin, as we did not observe an increase in the expression of MRC genes in the presence of voriconazole. Compromising Hsp90 function (by loss of the native hsp90 promoter) resulted in a lack of activation of the MRC genes, in particular, the NADH-ubiquinone oxidoreductases (complex I). This coincided with mitochondrial dysfunction, as illustrated by an impaired use of oxygen and ATP production in the presence of caspofungin. Furthermore, inhibition of MRC complex I by rotenone resulted in effects similar to those of hsp90 repression, with abolition of the paradoxical effect of caspofungin and the absence of an increase in intracellular calcium, which is known to be essential for the paradoxical effect (8, 12).
Mitochondria are important for many cellular processes in eukaryotes. In A. fumigatus, genes involved in mitochondrial dynamics and in the endoplasmic reticulum-mitochondria encounter structure (ERMES) were shown to play a role in virulence and antifungal resistance (15, 16). However, little is known about the role of MRC. The MRC is composed of four electron transfer complexes (I to IV) located in the inner membrane of mitochondria. The transfer of electrons generates a proton gradient and allows ATP production by an ATPase (complex V) (17). Complex I (NADH-ubiquinone oxidoreductases) plays an important role in energy conversion, and the loss of its activity results in mitochondrial dysfunction (18). In the model mold Neurospora crassa, complex I consists of 35 subunits, of which 7 are encoded by mitochondrial DNA (ND1, ND2, ND3, ND4, ND4L, ND5, ND6) (18). An alternative pathway involved in respiratory activity exists and consists of a single polypeptide located in the mitochondrial inner membrane that oxidizes NADPH and is not coupled to proton pumping (18). In our transcriptomic data, only the mitochondrion-carried genes of the MRC were overexpressed in the presence of caspofungin, while no significant changes were observed for the chromosomally carried genes of the MRC and the aox gene of the alternative pathway.
Grahl et al. demonstrated a role of mitochondrial respiration in the oxidative stress response and virulence, which was compromised after the deletion of cytochrome c (cycA, complex III) (17). However, we did not observe an increase in cycA expression in the presence of caspofungin in our study. Bromley et al. showed that mitochondrial complex I enzymes are involved in resistance to azoles (19). Deletion or mutation of the chromosomal gene encoding the 29.9-kDa subunit of this complex (Afu2g10600) resulted in azole resistance. The same effect was achieved by pharmacological inhibition by rotenone. While the role of MRC in echinocandin resistance of A. fumigatus has not been previously investigated, Chamilos et al. showed that inhibition of the MRC by antimycin A (complex III inhibitor) and benzohydroxamate (BHAM; an alternative pathway inhibitor) could increase susceptibility to caspofungin in the pathogenic yeast Candida parapsilosis (20). In the present study, enhanced caspofungin activity against A. fumigatus was not observed with the addition of antimycin A or azide, only with rotenone (complex I inhibitor). Taken together, these data suggest that the MRC may have crucial and distinct roles in modulating antifungal drug stress responses.
While our data indicate a key role of the MRC in caspofungin stress adaptation, the link between the MRC and Hsp90 is more complex to elucidate. Because both Hsp90 and the MRC are important for fungal growth and morphogenesis, an alternative hypothesis could be that the impact of Hsp90 impairment on MRC function is an epiphenomenon resulting from fungal growth defect. To minimize this effect, we used our pthiA-hsp90 mutant, which has sufficient Hsp90 levels to maintain basal growth but cannot achieve appropriate Hsp90 levels under caspofungin stress. Indeed, our measurements of mycelial mass under the experimental conditions of this study confirmed that there were no statistically significant differences in fungal growth between KU80 and the pthiA-hsp90 mutant in the absence or presence of caspofungin. However, we observed some basal alterations in oxygen consumption, ATP production, and MRC gene expression in the pthiA-hsp90 mutant, suggesting some basal impairment of MRC function with potential impact on growth and morphogenesis, albeit not phenotypically apparent. While caspofungin has a more pronounced inhibitory effect on the rate of fungal growth over time in the pthiA-hsp90 mutant, this effect is manifest only after a few days (loss of paradoxical growth in the pthiA-hsp90 mutant) but is not apparent at the early time point of this analysis (e.g., 2 h of caspofungin exposure). This cannot explain the drastically opposite response of MRC gene expression observed immediately after caspofungin exposure with strong MRC induction in the wild-type KU80 and complete lack of MRC activation in the pthiA-hsp90 mutant. Moreover, we did not observe any increase in MRC gene expression in KU80 with another antifungal drug, such as voriconazole, which suggests that MRC activation does not simply reflect the nonspecific effect of growth inhibition but is rather a specific response to caspofungin stress.
The mechanism by which Hsp90 can influence mitochondrial activity remains unclear. We know from our previous work that Hsp90 does not move to the mitochondria upon caspofungin exposure (21). We thus hypothesize that the impact of Hsp90 on MRC function is probably indirect. As an essential molecular chaperone, Hsp90 controls the activation of multiple client proteins, including transcription factors, which may induce MRC gene expression. However, the mechanisms of regulation of mitochondrial genes remain largely unknown. The role of calcium and calcineurin pathway in caspofungin tolerance and paradoxical effect has been previously established (12, 22). Here, we demonstrate that both pharmacologic inhibition of Hsp90 and MRC resulted in a lack of increase in cytosolic Ca2+ in response to caspofungin. Indeed, ATP produced by the MRC may be required for the activity of the Ca2+ channels and Ca2+ homeostasis in stress responses.
We conclude that mitochondria play an important role in the mechanisms of stress response and tolerance to caspofungin in A. fumigatus and that appropriate Hsp90 levels are required for activation of the mitochondrial respiratory chain in this response. These results may open perspectives for identifying novel antifungal targets in this pathway, in particular, the MRC, which remains largely unexplored in A. fumigatus.
MATERIALS AND METHODS
Strains and growth conditions.Three A. fumigatus strains were used in this study, as follows: the akuBKU80 strain (here referred to as KU80), used as the reference strain (23); the pthiA-hsp90 mutant, in which the native hsp90 promoter was replaced by the thiA promoter in the KU80 background (8); and the AEQΔakuB strain (a gift from Nick Read, Manchester, UK), with the aequorin luminescent reporter expressed in the KU80 background, which was used for measurement of intracellular calcium (14).
Cultures were performed on glucose minimal medium (GMM) with supplementation of 1.5% agar for solid plates (24).
The antifungal drugs used in this study were obtained as powder suspensions (Sigma-Aldrich, St. Louis, MO, USA) and dissolved in sterile water (caspofungin) or dimethyl sulfoxide (DMSO) (voriconazole), for a stock concentration of 5 mg/ml.
Transcriptomic analyses.Transcriptomic analyses were performed with the parental KU80 strain and the pthiA-hsp90 mutant in the absence of any drug and in the presence of caspofungin or voriconazole. For untreated conditions, a suspension of about 4.105 spores/ml was inoculated in 250 ml GMM broth and incubated for 24 h at 37°C under constant agitation (225 rpm). For the treated conditions, the spore suspension was grown for 22 h in the absence of any drug, and the antifungal drug (caspofungin or voriconazole) was then added at a concentration of 2 μg/ml for an additional 2 h of incubation. The mycelial mass was then washed with cold sterile distilled water, filtrated, immediately frozen with liquid nitrogen, and reduced to a fine powder. Total RNA was extracted with the RNeasy plant kit (Qiagen, Inc., Venlo, The Netherlands) and purified with the Turbo DNA-free kit (Thermo Fisher Scientific, Reinach, Switzerland). RNA concentration was measured with NanoDrop 1000 spectrophotometer (Witec AG, Switzerland) and adjusted to a concentration of 9 ng/μl in RNA-free water. Tubes were kept frozen at −80°C until analysis.
The quality of the total RNA samples was checked with a Fragment Analyzer (Advanced Analytical Technologies) prior to preparation of the RNA libraries with the TruSeq stranded mRNA library prep kit (Illumina), according to the manufacturer’s instructions. Samples were sequenced in an Illumina HiSeq 2000 sequencing platform using the 100-nucleotide (nt) single-end protocol with all the samples on same lane (25).
The analysis was performed with three biological replicates for each condition. RNA-seq data were processed using CLC Genomics Workbench version 10.1.1 (Qiagen). Reads were aligned to the A. fumigatus genome Af293 and read counts normalized by the quantile approach method. All conditions were compared with each other and filtered according to a false-discovery rate (FDR) cutoff of ≤0.05.
Only genes with significantly increased expression levels (P ≤ 0.05) in the caspofungin-exposed strain compared to the untreated condition were considered.
Quantification of mycelial mass.Conidia of KU80 and the pthiA-hsp90 mutant were harvested from fresh GMM agar plates, counted with a hemocytometer, and adjusted for a quantity of 107 spores in flasks containing 25 ml of GMM liquid medium. Both strains were incubated at 37°C for 22 h and an additional 2 h in the absence or presence of 2 μg/ml caspofungin for the untreated and caspofungin-treated conditions, respectively. The mycelial mass was filtered and completely dried overnight at 60°C before weighing. The experiment was performed in triplicate.
Measurement of caspofungin paradoxical effect.A 10-μl aqueous suspension with 104 conidia of the tested A. fumigatus strain (KU80, AF293, or pthiA-hsp90 mutant) was inoculated on GMM agar plates supplemented with caspofungin at a concentration of 1, 2, or 4 μg/ml. The paradoxical effect of caspofungin was defined as a significant increase in fungal growth between caspofungin concentrations of 1 and 2 or 4 μg/ml. Different inhibitors of the MRC were tested at different concentrations for their ability to abolish the paradoxical effect, as follows: rotenone (MRC complex I inhibitor), antimycin A (MRC complex III inhibitor), oligomycin (ATPase inhibitor), and sodium azide (MRC complex IV inhibitor). The effect of hypoxic conditions on paradoxical growth was also tested in the GENbox anaerobic generator (bioMérieux, France). Pictures were taken after 5 days of incubation at 37°C. Experiments were performed in triplicate.
Quantification of intracellular ATP.The KU80 and pthiA-hsp90 mutant strains were cultured at a concentration of 106 conidia/ml in GMM broth at 37°C under constant agitation (225 rpm) for 24 h. Caspofungin (2 μg/ml) was added at 22 h for an additional 2 h for the treated conditions. Cultures were filtered and washed with sterile water using a Büchner funnel. The mycelial mass was immediately frozen with liquid nitrogen, reduced to fine powder with mortar and pestle, and kept at −20°C. Proteins were extracted with lysis buffer (150 mM NaCl, 10 mM Tris-HCl [pH 7.5], 0.5 mM EDTA, 0.1% Triton, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1× protease inhibitory cocktail). Protein concentration was measured by the Bradford method (26) and adjusted to a concentration of 30 μg/ml for each sample. Lysates of 100 μl of each strain were incubated with 100 μl of CellTiter-Glo one solution (Promega, Fitchburg, WI, USA) in a 96-well plate (black/clear flat-bottom) for 10 min before measurement of luminescence using a LUMIstarOmega microplate reader (BMG Labtech, Ortenberg, Germany). A standard curve was made using ATP at a concentration range of 0 to 1 μM. The experience was performed in three biological replicates, and the mean of the final results was expressed as the fold change compared to KU80 under basal conditions.
Visualization of active mitochondria.A total of 104 conidia of KU80 and the pthiA-hsp90 mutant were incubated on microscope coverslips in GMM broth at 37°C for 24 h. Caspofungin (2 μg/ml) was added after 22 h for an additional 1-h incubation. Cultures were incubated with 1 μM MitoTracker Deep Red FM or MitoTracker Red CM-H2XRos (Thermo Fisher, Waltham, MA, USA) for 45 min at 37°C and then observed with a fluorescence microscope (Axioplan 2; Zeiss, Oberkochen, Germany). Fluorescence quantification was determined using the ImageJ software.
Oxygen measurement.Oxygen measurement was performed with the Dual Digital Model 20 oximeter (Rank Brothers, Cambridge, England). KU80 and pthiA-hsp90 mutant strains were cultured at a concentration of 107 conidia/ml in GMM broth at 37°C for 7 h under constant agitation (225 rpm) to allow the start of germination and then transferred to the oximeter chambers that were saturated in oxygen, sealed with Parafilm for 4 h, and maintained at 37°C. Caspofungin (2 μg/ml) was added with a syringe 1 h before measurement or 30 min after the beginning of measurement. The effect of rotenone (158 μg/ml) was also tested. Oxygen consumption was measured every 15 s for 3 to 4 h.
Calcium measurement.A total of 106 conidia of KU80-AEQ were incubated in calcium-free medium (GMM supplemented with 50 mM uridine and 25 mM uracil) in a white 96-well plate for 18 h at 37°C, washed with PGM [20 mM piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) (pH 6.7), 50 mM glucose, 1 mM MgCl2], and incubated for 4 h at 4°C in PGM supplemented by 1.06 μg/ml water-soluble coelenterazine (Sigma-Aldrich, St. Louis, MO, USA) for protein reconstitution (27). Cells were then incubated at room temperature for 1 h in the presence of the inhibitors geldanamycin (4 μg/ml; Sigma-Aldrich) and rotenone (158 μg/ml; Sigma-Aldrich). Caspofungin at 2 μg/ml was added 6 min after the start of luminescence measurement using a LUMIstarOmega microplate reader (BMG Labtech, Ortenberg, Germany). The experiment was repeated with the addition of 1 mM Ca2+ chelator BAPTA (Sigma-Aldrich) 1 h before measurement to remove all source of extracellular Ca2+.
Statistical analyses.For transcriptomic data (RNA-sequencing), statistical analyses were performed in R (version 3.1.1) using the edgeR Bioconductor package implemented in the CLC software (28). This software implements the exact test for two-group comparisons accounting for overdispersion caused by biological variability (29). Nonparametric tests were performed using the software GraphPad Prism 7.03. P values were calculated by multiple comparisons using a Kruskal-Wallis test and considered significant at ≤0.05.
Data availability.Raw sequence reads of the current RNA-seq data can be found under BioProject number PRJNA486252.
ACKNOWLEDGMENTS
This work was supported by the Swiss National Science Foundation (SNSF; Ambizione-Score, grant PZ00P3 161140).
We are grateful to the foundation Santos-Suarez for financial support to the F. Lamoth laboratory.
We acknowledge Johann Weber, Sandra Calderon, and Hannes Richter from the Lausanne Genomic Technologies Facility, Center for Integrative Genomics of the University of Lausanne, for performing transcriptomic analyses. We are grateful to Nick Read and Margherita Bertuzzi for providing the AEQΔakuB strain.
FOOTNOTES
- Received 28 January 2019.
- Returned for modification 9 March 2019.
- Accepted 12 April 2019.
- Accepted manuscript posted online 6 May 2019.
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00208-19.
- Copyright © 2019 American Society for Microbiology.
