Scanning Quadrupole Data-Independent Acquisition, Part B: Application to the Analysis of the Calcineurin-Interacting Proteins during Treatment of Aspergillus fumigatus with Azole and Echinocandin Antifungal Drugs

Abstract

Calcineurin is a critical cell-signaling protein that orchestrates growth, stress response, virulence, and antifungal drug resistance in several fungal pathogens. Blocking calcineurin signaling increases the efficacy of several currently available antifungals and suppresses drug resistance. We demonstrate the application of a novel scanning quadrupole DIA method for the analysis of changes in the proteins coimmunoprecipitated with calcineurin during therapeutic antifungal drug treatments of the deadly human fungal pathogen Aspergillus fumigatus. Our experimental design afforded an assessment of the precision of the method as demonstrated by peptide- and protein-centric analysis from eight replicates of the study pool QC samples. Two distinct classes of clinically relevant antifungal drugs that are guideline recommended for the treatment of invasive “aspergillosis” caused by Aspergillus fumigatus, the azoles (voriconazole) and the echinocandins (caspofungin and micafungin), which specifically target the fungal plasma membrane and the fungal cell wall, respectively, were chosen to distinguish variations occurring in the proteins coimmunoprecipitated with calcineurin. Novel potential interactors were identified in response to the different drug treatments that are indicative of the possible role for calcineurin in regulating these effectors. Notably, treatment with voriconazole showed increased immunoprecipitation of key proteins involved in membrane ergosterol biosynthesis with calcineurin. In contrast, echinocandin (caspofungin or micafungin) treatments caused increased immunoprecipitation of proteins involved in cell-wall biosynthesis and septation. Furthermore, abundant coimmunoprecipitation of ribosomal proteins with calcineurin occurred exclusively in echinocandins treatment, indicating reprogramming of cellular growth mechanisms during different antifungal drug treatments. While variations in the observed calcineurin immunoprecipitated proteins may also be due to changes in their expression levels under different drug treatments, this study suggests an important role for calcineurin-dependent cellular mechanisms in response to antifungal treatment of A. fumigatus that warrants future studies.

Graphical abstract

INTRODUCTION

In this study, the utility of the scanning quadrupole DIA method, as described in Part A,¹ is demonstrated by the quantitative analysis of the immunoprecipitated calcineurin protein complexes resulting from three different antifungal drug treatments for Aspergillus fumigatus. Invasive aspergillosis (IA), caused by the fungus A. fumigatus, is a leading infectious cause of death in immunocompromised patients.^2–4 Voriconazole (VOR) is the consensus guideline-recommended first-line antifungal therapy against IA.⁵ However, the emergence of azole antifungal resistance over the past decade has prompted the use of echinocandin antifungals (e.g., caspofungin (CSP), micafungin (MFG)) as much-needed second-line therapeutic options.^2,6–8 Whereas the azoles inhibit ergosterol biosynthesis and cause membrane stress by compromising the integrity of the fungal cell membrane, the echinocandins target fungal cell-wall β-glucan synthesis.^9–11 The calcineurin pathway has an established role in cell-wall integrity in different fungi,¹² and calcineurin inhibitors, cyclosporine (CsA) and tacrolimus (FK506), have shown an in vitro synergistic effect against A. fumigatus in combination with CSP.¹³ Resistance to antifungals has also been linked to the calcineurin signaling pathway,¹⁴ and the combination of calcineurin inhibitors with antifungals has been shown to inhibit the growth of various drug resistant fungi.^15–19 More recently, calcineurin inhibitors were active against multi-drug-resistant strains of A. fumigatus²⁰ and also dimorphic pathogenic fungi,²¹ implicating the importance of calcineurin control over drug-response machinery.

Hyphal growth and extension are necessary to cause invasive fungal disease, and the importance of calcineurin for hyphal growth and cell-wall biosynthesis is well established in A. fumigatus.¹² Although the calcineurin pathway has been shown to impact both the cell-membrane and cell-wall integrity through the regulation of effectors that influence the biosynthesis of ergosterol, chitin, and β-glucan, the exact mechanism of how calcineurin controls these processes is not clearly understood.^12,22 Combinatorial strategies involving the use of the major antifungal classes (azoles and echinocandins), in combination with calcineurin-specific inhibitors (FK506 and CsA) to counteract the emergence of resistance in fungal pathogens, warrant further investigations to aid our understanding and potential exploitation of the calcineurin signaling network in these pathogens.^13,14,23 Calcineurin has, in summary, clearly been established as an important regulator of hyphal growth under stress conditions and in conferring drug resistance in A. fumigatus.

Protein-level changes in the fungus in direct response to antifungal treatments can provide valuable insights into drug actions and also potential biomarkers of drug efficacy. While previous studies were focused on the genomic approaches to decipher alterations in protein profiles as a response to antifungal drug treatment,^24–26 only a few recent studies have examined the response of A. fumigatus to antifungal agents such as amphotericin B,²⁷ CSP,²⁸ and VOR.²⁴ However, no study to date has examined comparative changes in the proteome in response to different drug exposures in A. fumigatus. The alterations in the interactors of calcineurin following treatment of A. fumigatus with the leading antifungals targeting the cell wall (CSP and MFG) and the cell membrane (VOR) were therefore determined, and the relationship between changes in the immunoprecipitated calcineurin protein complements following different drug exposures was examined. To accomplish this, the A. fumigatus strain expressing the calcineurin catalytic subunit (CnaA) tagged to EGFP at its native locus was utilized. The three guideline-recommended antifungals used in clinical management, CSP, MFG, and VOR, were used at slightly subminimal inhibitory concentrations to observe the full effect of their mechanism of action.

EXPERIMENTAL CONDITIONS

Organism and Construction of CnaA-GFP Expression Strain

A. fumigatus wild-type strain akuB^KU80 was used for the construction of CnaA-GFP expression strain from its native locus and Escherichia coli DH5α competent cells were used for subcloning.

Cellular Extracts Preparation and GFP-Trap Purification

For all of the proteomic analyses experiments and protein-level comparisons between the different drug treatments, the A. fumigatus strain expressing CnaA-GFP fusion protein was utilized. The CnaA-GFP expression strain cultured in the absence of the drugs served as the control. To identify proteins binding to calcineurin, the strain was cultured separately in 200 mL of GMM liquid medium (in a 500 mL culture flask) for 24 h, shaking at 250 rpm, 37 °C, by inoculating a defined amount of spore suspension (10⁷/mL or 10⁸/mL) in the absence or presence of the different antifungals. Voriconazole (Pfizer, New York, NY) was used at 0.125 μg/mL; Caspofungin (Merck, Kenilworth, NJ) and Micafungin (Astellas, Tokyo, Japan) were each used at 1 μg/mL concentrations. The 24 h mycelial cultures were collected by vacuum filtration using a miracloth (CalBiochem, Billerica, Massachusetts) over a sintered glass funnel and washing extensively with ice-cold distilled water (with 250–500 mL for each).The mycelia were semidried on filter paper and ∼1 to 2 g (wet weight) mycelia was used for protein extraction. The mycelia were homogenized to a fine powder using liquid nitrogen in a mortar and pestle. Later, 5 mL of lysis buffer, comprising 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.01% Triton X-100, 1 mM DTT, 1 mM PMSF, and 1:100 Protease Inhibitory Cocktail (Sigma-Aldrich, St. Louis, MO) was added and mixed. The homogenized mycelial suspension was centrifuged (5000 rpm for 10 min at 4 °C) to remove mycelial debris, and the crude supernatant was again clarified by centrifugation at 7000 rpm for 15 min at 4 °C. The supernatant (crude extract) was carefully collected into a fresh tube, and the total protein in the crude extract was quantified by Bradford method and normalized to contain ∼10 mg protein/5 mL in the sample before GFP-Trap affinity purification (Chromotek, Planegg-Martinsried, Germany). 50 μL of GFP-Trap resin was equilibrated by washing three times in 500 μL of ice-cold dilution buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 1:100 Protease Inhibitory Cocktail) according to the manufacturer instructions and finally resuspended in 100 μL of ice-cold dilution buffer. The GFP-Trap resin suspension was then mixed with total crude cell lysate containing ∼10 mg total protein and incubated at 4 °C by gentle agitation for 2 h. After incubation for 2 h, the suspension was centrifuged at 2000 rpm for 10 min at 4 °C and the pelleted GFP-Trap resin was washed once in 500 μL of ice-cold dilution buffer and then twice with 500 μL of wash buffer (10 mM Tris-HCl pH 7.5, 350 mM NaCl, 0.5 mM EDTA, 1 mM PMSF, 1:100 Protease Inhibitory Cocktail). Protein-bound GFP-Trap resin was finally washed three times with 100 μL of 50 mM ammonium bicarbonate, pH 8.0, and then resuspended in 50 μL of 50 mM ammonium bicarbonate, pH 8.0.

Protein Digestion

A. fumigatus immunoprecipitated samples were supplemented with RapiGest SF (Waters Corporation, Milford, MA) surfactant to a final concentration of 0.1%, reduced with 10 mM DTT for 30 min at 80 °C, and alkylated with 20 mM IAA for 45 min at room temperature. On resin proteolytic digestions were accomplished by the addition of 500 ng sequencing-grade trypsin (Promega) for 18 h at 37 °C as previously described.²⁸ Following the removal of supernatants, peptides were acidified to pH 2.5 with TFA and incubated at 60 °C for 1 h to hydrolyze the RapiGest SF, which was removed by centrifugation. Next, all samples were lyophilized to dryness and resuspended in 40 μL of 1%TFA/2% acetonitrile.

LC–MS Configuration

LC separations were performed using a nanoACQUITY system (Waters Corporation) equipped with a Symmetry C18 5 μm, 2 cm × 180 μm precolumn and an HSS T3 C18 1.8 μm, 20 cm × 75 μm analytical column. The samples were transferred with aqueous 0.1% (v/v) formic acid to the precolumn at a flow rate of 5 μL/min. Mobile phase A was water containing 0.1% (v/v) formic acid, while mobile phase B was acetonitrile containing 0.1% (v/v) formic acid. The peptides were eluted from the precolumn to the analytical column and separated with a gradient of 5 to 40% mobile phase B over 90 min at a flow rate of 300 nL/min. The analytical column temperature was maintained at 35 °C. The lock mass compound, [Glu¹]-Fibrinopeptide B (200 fmol/μL), was delivered at 600 nL/min to the reference sprayer source of the mass spectrometer.

Mass-spectrometric analysis of tryptic peptides was performed using a Xevo G2-XS QTOF mass spectrometer (Waters Corporation, Wilmslow, United Kingdom). The mass spectrometer was operated with a resolution of 35 000 FWHM, and all analyses were performed in positive ion mode ESI. The ion source block temperature and capillary voltage were set to 100 °C and 3.2 kV, respectively. The time-of-flight (TOF) mass analyzer of the mass spectrometer was externally calibrated with a NaCsI mixture from m/z 50 to 1990. LC–MS data were collected using a novel data-independent mode of acquisition (SONAR). In this acquisition mode the quadrupole is continuously scanned between m/z 400 to 900, with a quadrupole transmission width of ∼24 Da. The oa-TOF records mass spectra as the quadrupole scans and stores these MS data into 200 discrete bins. Two data functions (modes) are acquired in an alternating fashion, differing only in the collision energy applied to the gas cell. In the low-energy MS1 mode, data are collected with a constant gas cell collision energy of 6 eV. In the elevated energy MS2 mode, the gas cell collision energy is ramped from 14 to 40 eV (per unit charge). As such, the resulting data contain both peptide precursor ions and all associated fragment ions. The spectral acquisition time in each mode was 0.5 s with a 0.02 s interscan delay. The reference sprayer was sampled every 60 s and the data postacquisition lock mass corrected.

Data Processing and Database Searching

SONAR quadrupole scanning DIA data were processed using Progenesis QI for proteomics v2 (PQIp) (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom) using optimized threshold and search parameters. The data acquisition methodology and associated analytical validation are presented in extensive detail in the accompanying Part A manuscript. In brief, in PQIp, all data features are aligned between samples based on their accurate mass and retention time values. All quantitative values are calculated from the high-resolution and quadrupole isolated selected ion chromatograms (SICs), expressed as the area under the curve of these SICs from the MS1 analyses. Protein and peptide identifications were obtained by searching an A. fumigatus NCBI (9150 RefSeq entries, April 2016) database. The results have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository²⁹ with data set identifier PXD005848. All abundance values were normalized to calcineurin across all samples. All fold changes refer to protein-based values derived from the fold changes of the peptides to which they map. Relative protein abundances, log₂ and square-root-transformed, assuming Poisson distribution, were considered significant when the 95% confidence interval was exceeded. Significance (p) fold-change values were calculated using a z-test with the median CV value of all replicate protein abundance measurements (n = 2144) used as an estimation of the biological variance.

RESULTS AND DISCUSSION

Study Pool QC Sampling and Data Analyses to Define Proteins Specifically Immunoprecipitated with Calcineurin

To define how the echinocandin (caspofungin, micafungin) and azole (voriconazole) antifungals influence the interaction of calcineurin with different proteins within the fungal cell, candidate interactors were qualitatively and quantitatively characterized by quadrupole scanning DIA LC–MS following GFP-Trap affinity purification. To identify proteins coimmunoprecipitated with calcineurin we utilized an A. fumigatus strain expressing the catalytic subunit of calcineurin (CnaA) tagged to GFP at its native locus. The experimental strategy, as outlined in Figure 1, included culturing of the CnaA-GFP expression strain separately in the absence (control) or presence of caspofungin (CSP), micafungin (MFG), and voriconazole (VOR) for a period of 24 h. Proteins extracted from the respective cultures were normalized across all of the samples prior to GFP-Trap affinity purification to characterize the enrichment of proteins immunoprecipitating with calcineurin under different antifungal treatments. To provide clear metrics on the qualitative and quantitative reproducibility of the overall analyses, a study pool QC (SPQC) sample, composed of equal aliquots of all samples in this study, was run eight times while collecting control and test data. The corresponding eight results files, along with all control and drug treatment sample data analysis results, are included in Supplementary Table 1, sheet 1. An identification summary of proteins qualitatively and quantitatively characterized, as both a total number (538 proteins) and those with two or more peptides (430 proteins), as well as a histogram of the SPQC coefficient of variation for all proteins with a protein and peptide FDR < 1%, is shown in Figure 2A and 2B. The complete data set, including the qualitative and quantitative results in mzIdentML and mzQuantML format, respectively, is available in the PRIDE proteomics data repository (PXD005848). For the proteins quantitated by two or more peptides, the median CV was 8.3%, and for those identified with one or more peptide, the CV was 8.9%. These values are similar in magnitude to the quantitative results described in Part A of this manuscript, where the quantitative analysis of a four protein mixture was examined in detail, reinforcing the precision that can be achieved using a SONAR label-free DIA LC–MS approach for the quantitative analysis of complex biological samples. In this study, the majority (79.9%) of the proteins quantified had at least two sequence unique peptides. Moreover, ∼92% of all detected features, that is, charge state groups, with a total close of about 27 000 features, were codetected in all eight SPQC samples, demonstrating very consistent sampling of the multidimensional retention time, m/z, and quadrupole position space. In all quantitative analyses of the control and antifungal drug-treated samples, all data were normalized to the level of calcineurin, shown in Supplementary Table 1, sheet 1, and those with a fold-change greater than or less than 2-sigma (2σ) and p < 0.05 were considered to be of interest for hypothesis generation.

Figure 1.

Scheme of the experimental strategy to identify proteins binding to calcineurin under different antifungal drug treatments in Aspergillus fumigatus.

Figure 2.

Numbers of quantifiable proteins (A) and %CV histogram for the eight SPQC analyses (B).

A graphical summary of the qualitative analysis is shown by the unsupervised PCA and hierarchical clustering/heat map analysis of the data in Figure 3, illustrating that both of the echinocandins, caspofungin (CSP) and micafungin (MFG), induced the largest changes in the immunoprecipitated calcineurin protein complements of the samples. The PCA results also indicate that voriconazole (VOR) treatment had little effect at the protein level on the calcineurin interactors of A. fumigatus. Following the SPQC analysis, and normalization of the values with respect to calcineurin, the proteins identified as coimmunoprecipitating with calcineurin were compared between untreated (control) and the different drug treatment conditions, as shown in Supplementary Table 1, sheets 2–4. The complete list of proteins identified and quantified using AUC is also shown in Supplementary Table 1, sheet 1. The biological variance, determined as outlined in the Experimental Conditions section, was used to assign significance (p) values to the measured protein abundance fold-change values for the various drug treatment versus control comparisons. The results are graphically summarized in the volcano-style distributions presented in Figure 4, showing significance as a function of fold change. Proteins exclusively detected and quantified that coimmunoprecipited with calcineurin are highlighted in black. The insets of Figure 4A–C illustrate the application of a t test to determine fold-change significance. Considering a ≥2σ change, equaling a ±1.7 fold change on an absolute scale, and p < 0.05 to be significant, treatment with echinocandins showed an overlap of 164 proteins that were common to both CSP and MFG (Supplementary Table 1, sheets 5 and 6), with 63 proteins exclusively coimmunoprecipitating with calcineurin in CSP treatment (Table 1) versus 87 proteins exclusively coimmunoprecipitating with calcineurin in MFG treatment (Table 2). Treatment with VOR resulted in a total of 52 proteins (with ≥2σ change; Supplementary Table 1, sheet 7) coimmunoprecipitating with calcineurin, of which 9 proteins were exclusive to VOR treatment (Table 3). While 26 proteins with ≥2σ change that coimmunoprecipitated with calcineurin were common to all three drug treatments (Supplementary Table 2), 14 proteins were bound to calcineurin exclusively under CSP and VOR treatments and three proteins exclusively bound to calcineurin under MFG and VOR treatments. The results of these binary comparisons are summarized in Figure 5, supporting the results shown in Figure 3 in that echinocandin-induced changes shared the greatest similarity in their altered quantitative protein complement.

Figure 3.

Unsupervised PCA scores distribution (control (CTRL, purple), voriconazole (VOR, green), caspofungin (CSP, red), micafungin (MFG, blue), and study pool QC (SPQC, gray)) (A) and hierarchical clustering (B) of the identified and quantified calcineurin proteins affected by drug treated A. fumigatus.

Figure 4.

Fold-change significance (p, z-test) versus log₂ protein fold-change distributions for caspofungin (A), micafungin (B), and voriconazole (C) antifungal drug-treated versus nontreated (control) A. fumigatus. Black, proteins exclusively coimmunoprecipitated with Calcineurin overviewed in Tables 1–3, respectively; gray, not significantly regulated or not exclusively coimmunoprecipitated with Calcineurin. Shown inset, as references, are volcano, single-degree t-test-based distributions for the same drug treatment versus control comparisons, respectively. Size = fold change.

Table 1.

Proteins Exclusively Co-Immunoprecipitated with Calcineurin in CSP Treatment Exceeding the 2σ (95% probability) Regulation Confidence Interval and p < 0.05 (z-test)

accession number	protein locus and description	CSP vs control fold change	CSP vs control log2 fold change	p
70994944	XP_752248.1 glycerol-3-phosphate dehydrogenase, mitochondrial	11.7	3.5	4.2 × 10−12
70990422	XP_750060.1 ATP synthase delta chain, mitochondrial precursor	9.4	3.2	2.8 × 10−11
71000343	XP_754866.1 peptidyl-prolyl cis–trans isomerase/cyclophilin	5.9	2.6	5.3 × 10−9
70992617	XP_751157.1 NADH-ubiquinone oxidoreductase 39 kDa subunit	4.6	2.2	1.1 × 10−7
70984376	XP_747702.1 amidophosphoribosyltransferase	4.2	2.1	3.9 × 10−7
70990780	XP_750239.1 proliferating cell nuclear antigen (PCNA)	4.1	2.0	5.6 × 10−7
70999105	XP_754274.1 translation initiation factor 2 alpha subunit	4.0	2.0	8.1 × 10−7
146323681	XP_001481556.1 NADH-ubiquinone oxidoreductase 21 kDa subunit	3.8	1.9	1.3 × 10−6
70989077	XP_749388.1 casein kinase I	3.6	1.9	2.9 × 10−6
71000100	XP_754767.1 glucose-6-phosphate 1-dehydrogenase	3.5	1.8	4.2 × 10−6
70990924	XP_750311.1 phenylalanyl-tRNA synthetase, beta subunit	3.5	1.8	4.9 × 10−6
70996594	XP_753052.1 TCTP family protein	3.5	1.8	5.3 × 10−6
70990790	XP_750244.1 serine/threonine protein phosphatase PP1	3.4	1.8	6.3 × 10−6
70999706	XP_754570.1 DNA damage response protein (Dap1)	3.4	1.8	6.5 × 10−6
70986482	XP_748734.1 pyruvate dehydrogenase E1 beta subunit PdbA	3.4	1.7	8.6 × 10−6
70984092	XP_747566.1 mitochondrial import receptor subunit (tom40)	3.3	1.7	1.1 × 10−5
70991753	XP_750725.1 thiazole biosynthesis enzyme	3.2	1.7	1.4 × 10−5
70990216	XP_749957.1 cytochrome C1/Cyt1	3.1	1.6	2.9 × 10−5
70998348	XP_753896.1 NADH-ubiquinone oxidoreductase B12 subunit	3.0	1.6	3.2 × 10−5
70998192	XP_753823.1 protein transport protein Sec61 alpha subunit	3.0	1.6	3.3 × 10−5
70989089	XP_749394.1 aspartyl-tRNA synthetase Dps1	3.0	1.6	3.5 × 10−5
70992211	XP_750954.1 ATP citrate lyase subunit (Acl), putative	3.0	1.6	4.5 × 10−5
70990662	XP_750180.1 40S ribosomal protein S8e	3.0	1.6	4.5 × 10−5
70983670	XP_747362.1 cell wall glucanase (Scw11)	2.9	1.5	6.4 × 10−5
70989229	XP_749464.1 14–3–3 family protein ArtA	2.9	1.5	7.1 × 10−5
70996256	XP_752883.1 mitochondrial phosphate carrier protein (Mir1)	2.8	1.5	8.6 × 10−5
70993698	XP_751696.1 Aha1 domain family	2.8	1.5	1.1 × 10−4
71002366	XP_755864.1 DUF636 domain protein	2.8	1.5	1.2 × 10−4
70985282	XP_748147.1 histone H2A	2.8	1.5	1.2 × 10−4
70984832	XP_747922.1 ubiquinol-cytochrome C reductase complex core protein 2	2.7	1.5	1.3 × 10−4
70995926	XP_752718.1 ubiquinol-cytochrome C reductase complex subunit UcrQ	2.7	1.4	1.7 × 10−4
71000068	XP_754751.1 eukaryotic translation initiation factor 3 subunit 2i	2.7	1.4	1.8 × 10−4
70991661	XP_750679.1 DUF500 and SH3 domain protein	2.7	1.4	1.9 × 10−4
70995090	XP_752311.1 ribosomal protein S23 (S12)	2.6	1.4	2.9 × 10−4
70981416	XP_731490.1 FAD binding domain protein	2.5	1.3	3.7 × 10−4
70991843	XP_750770.1 NADH-ubiquinone oxidoreductase 304 kDa subunit precursor	2.5	1.3	4.1 × 10−4
70999023	XP_754233.1 NADH-ubiquinone oxidoreductase B18 subunit	2.5	1.3	4.5 × 10−4
70995028	XP_752280.1 conserved hypothetical protein	2.4	1.3	6.1 × 10−4
70995343	XP_752429.1 S-adenosylmethionine synthetase	2.4	1.3	7.7 × 10−4
70987264	XP_749110.1 PCI domain protein	2.3	1.2	9.4 × 10−4
70991681	XP_750689.1 cell division control protein 2 kinase	2.3	1.2	1.1 × 10−3
146323719	XP_752147.2 ubiquinol-cytochrome c reductase complex 14 kDa protein	2.3	1.2	1.1 × 10−3
70999514	XP_754476.1 arginase	2.3	1.2	1.3 × 10−3
71002728	XP_756045.1 cell division control protein Cdc48	2.3	1.2	1.3 × 10−3
70993746	XP_751720.1 acetyl-coenzyme A synthetase FacA	2.3	1.2	1.4 × 10−3
71002010	XP_755686.1 translation elongation factor EF-2 subunit	2.2	1.2	1.7 × 10−3
70993400	XP_751547.1 calnexin	2.2	1.1	1.9 × 10−3
70997740	XP_753605.1 iron–sulfur protein subunit of succinate dehydrogenase Sdh2	2.2	1.1	1.9 × 10−3
70993696	XP_751695.1 saccharopine dehydrogenase Lys9	2.2	1.1	2.1 × 10−3
70998594	XP_754019.1 succinyl-CoA synthetase alpha subunit	2.2	1.1	2.1 × 10−3
70984362	XP_747695.1 nascent polypeptide-associated complex (NAC) subunit	2.2	1.1	2.4 × 10−3
70987006	XP_748988.1 CTP synthase	2.2	1.1	2.5 × 10−3
70989661	XP_749680.1 mitochondrial F1F0 ATP synthase subunit F (Atp17)	2.1	1.1	2.8 × 10−3
70986989	XP_748980.1 40S ribosomal protein S13	2.1	1.1	3.5 × 10−3
146322523	XP_750491.2 mitochondrial GTP/GDP transporter Ggc1	2.1	1.1	3.6 × 10−3
70985058	XP_748035.1 septin AspC	2.1	1.0	4.0 × 10−3
70993656	XP_751675.1 conserved hypothetical protein	2.1	1.0	4.0 × 10−3
70983638	XP_747346.1 mitochondrial ATPase subunit ATP4	2.0	1.0	5.1 × 10−3
146324195	XP_001481515.1 ssDNA binding protein Ssb3	2.0	1.0	6.7 × 10−3
70989001	XP_749350.1 40S ribosomal protein S10a	0.5	−1.0	7.0 × 10−3
70992789	XP_751243.1 conserved hypothetical protein	0.4	−1.2	1.5 × 10−3
70993290	XP_751492.1 actin-related protein ArpA	0.4	−1.2	1.4 × 10−3
71000586	XP_754976.1 Rho GTPase Rac	0.4	−1.4	2.5 × 10−4

Table 2.

Proteins Exclusively Co-Immunoprecipitated with Calcineurin in MFG Treatment Exceeding the 2σ (95% probability) Regulation Confidence Interval and p < 0.05 (z-test)

accession number	protein locus and description	MFG vs control fold change	MFG vs control log2 fold change	p
70983245	XP_747150.1 methyltransferase SirN-like	4.8	2.3	3.9 × 10−6
70997461	XP_753478.1 26S proteasome regulatory subunit Mts4	3.8	1.9	7.2 × 10−5
71000828	XP_755095.1 SRP receptor beta subunit (Srp102)	3.7	1.9	8.5 × 10−5
146323801	XP_751845.2 hypothetical protein AFUA_4G09810	3.7	1.9	9.6 × 10−5
70999388	XP_754413.1 ADP-ribosylation factor 6	3.5	1.8	1.8 × 10−4
70992657	XP_751177.1 cytosolic large ribosomal subunit protein L7A	3.5	1.8	2.0 × 10−4
70984619	XP_747816.1 conserved hypothetical protein	3.5	1.8	2.0 × 10−4
70991302	XP_750500.1 26S proteasome regulatory particle subunit Rpn8	3.3	1.7	3.4 × 10−4
70991445	XP_750571.1 proteasome regulatory particle subunit Rpt4	3.2	1.7	4.7 × 10−4
70996212	XP_752861.1 peptidase D	3.2	1.7	5.9 × 10−4
71000275	XP_754832.1 succinate dehydrogenase subunit Sdh1	3.1	1.6	6.8 × 10−4
70997834	XP_753649.1 nitroreductase family protein	2.9	1.5	1.3 × 10−3
70995930	XP_752720.1 transketolase TktA	2.9	1.5	1.3 × 10−3
71001294	XP_755328.1 mitochondrial Hsp70 chaperone (Ssc70)	2.9	1.5	1.4 × 10−3
146322483	XP_750349.2 ribosomal protein L16a	2.9	1.5	1.5 × 10−3
70999940	XP_754687.1 serine hydroxymethyltransferase	2.9	1.5	1.5 × 10−3
70994984	XP_752268.1 methionyl-tRNA synthetase	2.9	1.5	1.5 × 10−3
70998911	XP_754177.1 ketol-acid reductoisomerase	2.8	1.5	1.7 × 10−3
70985200	XP_748106.1 vacuolar dynamin-like GTPase VpsA	2.8	1.5	1.8 × 10−3
70992635	XP_751166.1 hypothetical protein AFUA_6G12880	2.8	1.5	2.0 × 10−3
71000066	XP_754750.1 C1 tetrahydrofolate synthase	2.8	1.5	2.0 × 10−3
71001286	XP_755324.1 fatty acid activator Faa4	2.8	1.5	2.1 × 10−3
70982294	XP_746675.1 NIMA-interacting protein TinC	2.8	1.5	2.1 × 10−3
70983436	XP_747245.1 conserved hypothetical protein	2.8	1.5	2.3 × 10−3
70984557	XP_747785.1 acetylglutamate kinase	2.8	1.5	2.4 × 10−3
70992559	XP_751128.1 AhpC/TSA family protein	2.7	1.4	2.5 × 10−3
146323687	XP_752229.2 glutaredoxin Grx5	2.7	1.4	2.8 × 10−3
146322509	XP_750420.2 t-complex protein 1, eta subunit	2.7	1.4	3.1 × 10−3
70991353	XP_750525.1 glucosamine-fructose-6-phosphate aminotransferase	2.7	1.4	3.3 × 10−3
146323147	XP_748443.2 aspartic-type endopeptidase	2.7	1.4	3.3 × 10−3
70992917	XP_751307.1 5-oxo-L-prolinase	2.7	1.4	3.4 × 10−3
70999438	XP_754438.1 phosphoglucomutase PgmA	2.7	1.4	3.4 × 10−3
70994192	XP_751943.1 DUF757 domain protein	2.6	1.4	3.5 × 10−3
70995532	XP_752521.1 ethanolamine kinase	2.6	1.4	3.8 × 10−3
70990724	XP_750211.1 alcohol dehydrogenase, zinc-containing	2.6	1.4	4.0 × 10−3
70984364	XP_747696.1 ATP synthase D chain, mitochondrial	2.6	1.4	4.0 × 10−3
70986952	XP_748962.1 electron transfer flavoprotein alpha subunit	2.6	1.4	4.2 × 10−3
70997049	XP_753279.1 calcium binding protein Caleosin	2.6	1.4	4.8 × 10−3
70993752	XP_751723.1 NADH-ubiquinone oxidoreductase, subunit F	2.5	1.3	5.8 × 10−3
70995446	XP_752478.1 60S ribosomal protein L6	2.5	1.3	6.8 × 10−3
70989367	XP_749533.1 alpha,alpha-trehalose-phosphate synthase subunit	2.5	1.3	6.8 × 10−3
146324747	XP_747279.2 oligopeptidase family protein	2.5	1.3	7.1 × 10−3
146322982	XP_755704.2 NADH-ubiquinone oxidoreductase 49 kDa subunit	2.4	1.3	7.4 × 10−3
70999742	XP_754588.1 acetolactate synthase, large subunit	2.4	1.3	7.5 × 10−3
70999522	XP_754480.1 proteasome regulatory particle subunit Rpt3	2.4	1.3	7.5 × 10−3
70998630	XP_754037.1 BAR adaptor protein RVS161	2.4	1.3	7.6 × 10−3
71001148	XP_755255.1 60S ribosomal protein L10	2.4	1.3	7.7 × 10−3
70998726	XP_754085.1 40S ribosomal protein S3Ae	2.4	1.3	8.3 × 10−3
70996304	XP_752907.1 DUF1014 domain protein	2.4	1.3	8.4 × 10−3
70985998	XP_748502.1 F-box domain protein	2.4	1.3	8.5 × 10−3
70998710	XP_754077.1 nucleosome assembly protein Nap1	2.4	1.3	8.9 × 10−3
70989083	XP_749391.1 Coatomer subunit gamma	2.4	1.3	9.0 × 10−3
70995173	XP_752351.1 mitochondrial inner membrane translocase subunit TIM44	2.4	1.3	9.3 × 10−3
70984804	XP_747908.1 hypothetical protein AFUA_5G04350	2.4	1.2	9.5 × 10−3
146324059	XP_754050.2 homoserine kinase	2.4	1.2	1.1 × 10−2
146323070	XP_755989.2 phospholipase D (PLD)	2.3	1.2	1.1 × 10−2
146322910	XP_755431.2 ADP-ribosylation factor	2.3	1.2	1.2 × 10−2
70991469	XP_750583.1 Rho GTPase Rho1	2.3	1.2	1.2 × 10−2
70993636	XP_751665.1 alpha-ketoglutarate dehydrogenase complex subunit Kgd1	2.3	1.2	1.3 × 10−2
71000467	XP_754922.1 40S ribosomal protein S4	2.3	1.2	1.3 × 10−2
70993160	XP_751428.1 Hsp70 family protein	2.3	1.2	1.4 × 10−2
70983336	XP_747195.1 alanyl-tRNA synthetase	2.3	1.2	1.5 × 10−2
70992323	XP_751010.1 ribosomal protein L26	2.2	1.2	1.6 × 10−2
70997379	XP_753438.1 Rab small monomeric GTPase Rab7	2.2	1.1	1.8 × 10−2
70986492	XP_748739.1 fatty acid synthase beta subunit	2.2	1.1	1.9 × 10−2
70983023	XP_747039.1 bifunctional catalase-peroxidase Cat2	2.2	1.1	2.0 × 10−2
71001952	XP_755657.1 bifunctional tryptophan synthase TRPB	2.2	1.1	2.1 × 10−2
71001738	XP_755550.1 lectin family integral membrane protein	2.2	1.1	2.1 × 10−2
70991907	XP_750802.1 oxidoreductase, short-chain dehydrogenase/reductase family	2.2	1.1	2.2 × 10−2
70999658	XP_754546.1 40S ribosomal protein S7e	2.1	1.1	2.3 × 10−2
146322408	XP_750101.2 ATP synthase gamma chain, mitochondrial precursor	2.1	1.1	2.5 × 10−2
70986856	XP_748915.1 cytokinesis EF-hand protein Cdc4	2.1	1.1	2.6 × 10−2
70990130	XP_749914.1 kinesin family protein	2.1	1.1	2.8 × 10−2
146324723	XP_747206.2 U5 snRNP component Snu114, putative	2.1	1.0	3.1 × 10−2
146322400	XP_750089.2 60S ribosomal protein L12	2.0	1.0	3.3 × 10−2
71001364	XP_755363.1 40S ribosomal protein S17	2.0	1.0	3.8 × 10−2
70995568	XP_752539.1 ADP-ribosylation factor	2.0	1.0	3.8 × 10−2
70987220	XP_749089.1 cyclopropane-fatty-acyl-phospholipid synthase	2.0	1.0	4.0 × 10−2
70985198	XP_748105.1 vacuolar ATP synthase catalytic subunit A	2.0	1.0	4.2 × 10−2
70985024	XP_748018.1 ubiquitin C-terminal hydrolase (HAUSP)	2.0	1.0	4.3 × 10−2
71002512	XP_755937.1 t-complex protein 1, alpha subunit	2.0	1.0	4.4 × 10−2
70994744	XP_752149.1 iron−sulfur cofactor synthesis protein (Isu1)	0.4	−1.4	3.2 × 10−2
70992913	XP_751305.1 5′-nucleotidase	0.4	−1.5	2.5 × 10−2
70995822	XP_752666.1 SCF ubiquitin ligase complex subunit CulA	0.3	−1.6	2.1 × 10−2
71000215	XP_754812.1 PX domain protein	0.3	−1.6	2.0 × 10−2
146323430	XP_754484.2 arsenite translocating ATPase ArsA	0.3	−1.7	1.4 × 10−2
71001378	XP_755370.1 iron–sulfur cluster assembly accessory protein Isa2	0.2	−2.1	5.8 × 10−3

Table 3.

Proteins Exclusively Co-Immunoprecipitated with Calcineurin in VOR Treatment Exceeding the 2σ (95% probability) Regulation Confidence Interval and p < 0.05 (z-test)

accession number	protein locus and description	VOR vs control fold change	VOR vs control log2 fold change	p
70994720	XP_752137.1 14-alpha sterol demethylase Cyp51A	3.2	1.7	1.5 × 10−5
70989235	XP_749467.1 conserved hypothetical protein	3.0	1.6	3.2 × 10−5
70998480	XP_753962.1 DUF89 domain protein	2.0	1.0	5.5 × 10−3
70984753	XP_747883.1 dienelactone hydrolase family protein	0.5	−1.0	5.2 × 10−3
70994244	XP_751969.1 mitochondrial peroxiredoxin Prx1	0.5	−1.1	2.5 × 10−3
70991867	XP_750782.1 conserved hypothetical protein	0.3	−1.6	4.7 × 10−5
70985178	XP_748095.1 thiamine biosynthesis protein (Nmt1)	0.3	−1.6	3.9 × 10−5
70993702	XP_751698.1 fructose-1,6-bisphosphatase Fbp1	0.3	−1.6	2.3 × 10−5
70986306	XP_748647.1 FAD binding monooxygenase	0.3	−2.0	1.0 × 10−6

Figure 5.

Venn intersection of proteins coimmunoprecipitating with calcineurin in the three drug treatments compared with the control with a fold change of greater than or less than 2σ and p < 0.05.

Echinocandin Treatments Differentially Influenced the Interaction of Calcineurin with Proteins Regulating Fungal Cell-Wall Biosynthesis and Hyphal Septum Organization

In accordance with the known role for echinocandins in targeting cell-wall biosynthesis machinery,³⁰ we noted that treatment with echinocandins enhanced binding of calcineurin to Rho1 (the regulatory subunit of β-1,3-glucan synthase complex), known to regulate β-1,3-glucan synthase activity.³¹ Although not very significant in the case of CSP treatment, MFG treatment resulted in a 2.3-fold increase in Rho1 binding to calcineurin (Supplementary Table 3). In the presence of CSP, Gel1 (a β(1–3) glucanosyltransferase) and Scw11 (a β-1,3-glucan-modifying enzyme) showed a 6.7- and 2.9-fold respective increase in coimmunoprecipitation with calcineurin. Gel1 β(1–3) glucanosyltransferase plays a major role in the elongation of cell-wall β-1,3-glucan chains.³² Intriguingly, although MFG treatment did not show such an increase in binding to calcineurin with respect to Gel1 and Scw11 proteins, it did increase the interaction of calcineurin with glucosamine fructose-6-phosphate aminotransferase (GFAT) by 2.7-fold. GFAT catalyzes the formation of glucosamine 6-phosphate, the first rate-limiting step in the biosynthesis of cell-wall chitin, and its expression was increased under cell-wall stress.³³ Another enzyme that catalyzes the synthesis of GDP-mannose in cell-wall biosynthesis,³⁴ mannose-1-phosphate guanylyltransferase (Srb1), also showed 2.7-fold enhanced interaction under CSP and MFG treatments. In contrast, VOR treatment did not result in any significant increase in the binding of the respective proteins to calcineurin (Supplementary Table 3), indicating the probability of calcineurin-mediated regulation of these effector proteins only under cell-wall stress induced upon echinocandin treatments. The increased interaction observed between calcineurin and these key cell-wall biosynthesis proteins supports previous reports on the potential role of calcineurin in the regulation of the cell-wall integrity pathway.^13,35,36 Increased interaction of calcineurin with septins AspB, AspC, and AspD, the key cytoskeletal GTPase proteins that localize at the hyphal septa³⁷ and are involved in the organization of septation, was also evident in the presence of both the echinocandins but not in the case of VOR treatment. A recent study from our laboratory has also revealed the interaction of AspB septin with calcineurin during caspofungin treatment.³⁸

Opposed to the observed increased interactions, a two-fold decrease in binding of a α-1,2-mannosyltransferase, Ktr4, with calcineurin was noted in the presence of CSP and MFG in comparison with the VOR treatment (Supplementary Table 3). Mannosyltransferases play a crucial role in the modification of cell-wall proteins through the addition of N-linked or O-linked oligosaccharides and contribute to fungal virulence.³⁹ Ktr4 is responsible for glycosylation of cell-wall mannoproteins and cell-wall integrity,⁴⁰ and its deletion resulted in thinner cell walls and hypersensitivity cell-wall stress. Previous studies in our laboratory have revealed the localization of calcineurin at the hyphal tips and septa,⁴¹ where active cell-wall synthesis occurs, lending support to the observed cell-wall and septation-related protein interactions in this study. The mechanism of how calcineurin regulates these important cell-wall- and septation-associated proteins remains the subject of future investigations. We hypothesize that calcineurin being a protein phosphatase is involved in the dephosphorylation of these substrates.

Treatment with the Echinocandins Altered the Immunoprecipitation of Key Proteins Related to Cytoskeleton Organization and Membrane Trafficking with Calcineurin

We have previously demonstrated that calcineurin localized at the active points of fungal hyphal growth, the hyphal tip and septum. Dynamic movement of calcineurin within the hyphal compartments and its colocalization with the major cytoskeletal component, actin, during septation revealed the possibility of calcineurin’s interaction with yet unknown proteins involved in cytoskeleton structure and function.⁴² It was noted that calcineurin interacted with a number of cytoskeletal proteins and others involved in membrane trafficking, such as ArpA (an actin-related protein), tubulin (α-1, β, and TubB subunits), Rac Rho GTPase, Rab GTPase (Vps21/Ypt51), and the monomeric GTPase SarA under normal growth conditions (Supplementary Table 3). From among these proteins, tubulin α-1 and tubulin β showed a three- to four-fold and four- to sixfold decreased calcineurin binding following treatment with CSP and MFG, respectively. These results suggest that the mode of action of the echinocandins may also involve alterations in the interaction of calcineurin with the tubulin cytoskeletal components and proteins responsible for membrane trafficking. A previous study showed the interaction of calcineurin with α-1 tubulin.⁴³ The precise mechanism of how calcineurin interaction with these cytoskeletal proteins regulates their function remains to be investigated.

Calcineurin Immunoprecipitated Protein Complexes Revealed Differential Enrichment of Proteins Involved in Membrane Biosynthesis during Echinocandin and Voriconazole Treatments

Important trends that also emerged from these analyses are the interactions of calcineurin with the key regulatory enzymes such as the 14α-sterol demethylases (Cyp51A and Cyp51B), squalene monoxygenase (Erg1), and hydroxymethylglutaryl-CoA synthase (Erg13) involved in the biosynthesis of membrane ergosterol. While the interaction of Cyp51A and Cyp51B was particularly enhanced in the presence of VOR when compared with the control (Supplementary Table 3), indicative of the important role for calcineurin in membrane stress response, we cannot rule out the possibility that the expression levels of these proteins in the presence of VOR may also be a contributing factor to the observed increase in calcineurin binding to these proteins. It is known that prolonged VOR exposure induces the expression of both cyp51A and cyp51B mRNAs.⁴⁴ The cyp51A and cyp51B genes encode for 14α-sterol demethylases involved in the synthesis of ergosterol required for fungal membrane structure, and mutations in the cyp51A gene are responsible for emergence of azole resistance.^6–8

Following VOR treatment, there was a 3.2-fold increase observed in the amount of Cyp51A immunoprecipitating with calcineurin in comparison to the control (Supplementary Table 3). The interaction of calcineurin with a putative transmembrane protein, UsgS, increased by 10-fold in the VOR treatment sample. Although we did notice a two-fold increase in the interaction of UsgS under CSP treatment, a four-fold decrease in interaction was noted in MFG treatment, again indicating differential interactions under different drug treatments. These results suggest that although CSP and MFG antifungals belong to the same echinocandin class, they may cause differential effects on calcineurin interactions with different proteins. In support of this, it has been previously demonstrated that CSP at higher concentrations triggers a differential calcium response in comparison with MFG and results in differential phosphorylation of calcineurin in vivo.⁴⁵ Furthermore, while these comparative analyses indicate drug-dependent variations in the interactors of calcineurin, it is also unknown if the echinocandin treatments or other factors contribute to the observed decrease in the interaction of Cyp51A and Cyp51B proteins. Previous reports from Candida species,⁴⁶ C. neoformans,⁴⁷ and Mucorales⁴⁸ suggested fungicidal activity of calcineurin inhibitors (cyclosporine A and FK506) in combination with azoles that are usually fungistatic. However, in A. fumigatus, clinical isolates from transplant and nontransplant patients exposed to a combination of VOR with calcineurin inhibitors were not synergistic,^49,50 but azole-resistant strains were susceptible to calcineurin inhibitors,¹³ indicating calcineurin inhibition as a potential effective strategy to overcome azole resistance. In addition, a recent study showed that combination of FK506 with VOR had synergistic inhibitory activity against Aspergillus biofilm formation.⁵¹

Significant Alterations in Other Calcineurin Interactors Were Evident during Different Antifungal Treatments

In the CSP and MFG treatments a significant number of ribosomal proteins (41 proteins) with ≥2σ change were identified to interact with calcineurin (Supplementary Table 4). Such an increase with respect to any of these ribosomal proteins was not observed in VOR treatment. The observed increase in ribosomal protein interactions with calcineurin in CSP and MFG treatments but not in VOR treatment, in particular, indicates the possibility of reprogramming of cellular growth mechanisms during different drug treatments. A previous study on profiling the proteome of A. fumigatus in response to CSP treatment also showed an increase in the abundance of ribosomal proteins following exposure for 24 h.²⁸ The Pma1 plasma membrane H⁺-ATPase, which was previously shown to be down-regulated in the presence of CSP²⁸ but up-regulated in the presence of amphotericin B,²⁷ showed increased interaction with calcineurin in the presence of CSP (3.7-fold) and MFG (2.1-fold) but decreased binding to calcineurin in the presence of VOR (3.5-fold). Pma1 has been previously shown to be a target of calcineurin in S. cerevisiae and A. fumigatus.^52,53 The endoplasmic reticulum calcium ATPase was increased by 2.5- and 2.3-fold following CSP and VOR treatment, respectively, but not in treatment with MFG. Another well known interactor of calcineurin, the calcineurin binding protein (CbpA), which belongs to the RCAN (regulator of Calcineurin) family of proteins and has been shown to both positively and negatively impact calcineurin function in fungi,^28,54 was also found to interact with calcineurin in our analyses. However, we did not observe any significant difference in its binding to calcineurin under the different antifungal drug treatments.

Only in the presence of CSP and MFG was a 9.6- and 3.9-fold respective increase noted in the G-protein complex beta subunit CpcB protein binding to calcineurin (Supplementary Table 1). The Gβ-CpcB protein was recently shown to be involved in cell-wall integrity and virulence of A. fumigatus.⁵⁵ Interestingly, the increased fold change in the interaction of calcium/calmodulin-dependent protein kinase with calcineurin only in the presence of CSP (5.6-fold; Supplementary Table 1, sheet 5) and MFG (5.2-fold; Supplementary Table 1, sheet 6) but not following VOR treatment also indicated differential calcineurin interactions between the azole and the echinocandin treatments. The DUF89 protein, whose function remains unknown, was also increased two-fold in VOR treatment in comparison with the control (Table 3), but not in CSP and MFG treatment. There was also a 2.6-fold increase in binding of calcineurin to the COP II vesicle protein Yip3 in the VOR treatment only (Supplementary Table 1, sheet 7). Several hypothetical proteins were also identified as calcineurin interactors in the presence of different antifungals. However, due to insufficient annotation available for these proteins in the Aspergillus genome database, no significance could be assigned to these proteins. A schematic representation of the major quantitative proteomic findings of calcineurin inhibition and antifungal drug-dependent alterations in the calcineurin interactome is shown in Figure 6.

Figure 6.

Schematic representation of calcineurin inhibition and antifungal drug-dependent alterations in the proteins coimmunoprecipitating with calcineurin. The major echinocandin antifungals (caspofungin and micafungin) target β-glucan synthesis in the fungal cell wall (green arrow) and the azoles (voriconazole) target the ergosterol biosynthesis in the fungal cell membrane (orange arrow). Calcineurin inhibition abolishes azole and echinocandin resistance, making it a suitable drug target for overcoming antifungal drug resistance. Calcineurin is a heterodimer composed of CnaA (catalytic) and CnaB (regulatory) subunits, and its activity is inhibited by the immunosuppressants, FK506 and cyclosporine A (CsA). Calcineurin plays a major role through the binding and activation of several yet unknown downstream effector proteins involved in the regulation of cell-wall integrity, hyphal growth, drug resistance, stress response and virulence. How the echinocandins and azoles differentially influence the calcineurin interactome is unknown (shown in dotted arrows). Proteomic profiling of calcineurin interactors in the presence of the antifungals (echinocandins and azoles) revealed alterations in its interactions (shown in bold solid arrows). Bold arrow with a cross mark indicates the reduced interaction of calcineurin with different cytoskeletal proteins in the presence of echinocandins. Only some relevant proteins are shown.

CONCLUSIONS

Recent studies on the identification of new substrates for calcineurin through advanced proteomic technologies both in the model yeast S. cerevisiae⁵⁶ and in human pathogenic yeast C. neoformans⁵⁷ provided novel insights into additional functions of calcineurin in vivo. Here we performed a comparative study to probe the proteome of A. fumigatus for potential calcineurin interactors under treatment with two important classes of clinical guideline-recommended antifungal drugs, the echinocandins and the azoles, which are currently being used to treat patients suffering from invasive aspergillosis. This is the first label-free quantitative proteomics study to utilize a new scanning quadrupole DIA method to distinguish important changes in the calcineurin-dependent proteome of A. fumigatus in response to different drug treatments. Novel and specific interactors of calcineurin in response to the drug treatments indicative of calcineurin’s role in regulating these effectors have been detected. The only well-characterized substrate of calcineurin to date is Crz1p (ortholog of mammalian NFAT), which mediates the transcriptional response triggered by calcineurin activation in response to stress conditions.^58,59 While Rcn1 belonging to the calcipressin family of proteins has been shown to bind to calcineurin and contribute to fine-tuning calcineurin signaling in the yeast,⁶⁰ other known stress-regulated substrates of calcineurin in the yeast are Hph1 and Hph2,⁶¹ Slm1 and Slm2.⁶² Although the genes encoding Crz1 (CrzA) and Rcn1 (CbpA) proteins have been characterized in A. fumigatus,^28,63–65 several calcineurin substrates remain unknown in A. fumigatus. This study contributes to our understanding of the important, but yet unknown, link between calcineurin and antifungal drug mechanisms. How calcineurin regulates these proteins will be an important aspect for future investigation. Overall, the presented results for the three antifungals at clinically relevant doses provide relevant biological context on how these drugs can differentially modulate protein–protein interactions in vivo and lend further insight into both drug efficacy and resistance. While this study identified previously unknown substrates of calcineurin, strengthening the link between calcineurin, cell-membrane and cell-wall biosynthesis, stress, and repair mechanisms, further studies are nevertheless required to confirm the exact mechanism of calcineurin-mediated regulation of these substrates, as the identified interactors may be regulated through a phosphorylation–dephosphorylation mechanism.