Exposure of Aspergillus Fumigatus to Atorvastatin Leads to Altered Membrane Permeability and Induction of an Oxidative Stress Response

Journal of Fungi

Article Exposure of Aspergillus fumigatus to Atorvastatin Leads to Altered Membrane Permeability and Induction of an Oxidative Stress Response

Ahmad Ajdidi, Gerard Sheehan and Kevin Kavanagh *

Department of Biology, Maynooth University, Maynooth, Co. Kildare W23F2K8, Ireland; [email protected] (A.A.); [email protected] (G.S.) * Correspondence: [email protected]

Received: 14 February 2020; Accepted: 23 March 2020; Published: 26 March 2020

Abstract: Aspergillus fumigatus is a serious cause of disease in immune-deficient patients and in those with pulmonary malfunction (e.g., cystic fibrosis (CF), asthma). Atorvastatin is a member of the statin drug family, which are the main therapeutic agents used to decrease high serum cholesterol levels by inhibiting (HMG-CoA) reductase enzyme. The aim of the work presented here was to analyse the antifungal activity of atorvastatin and assess its effect on the virulence of A. fumigatus. Atorvastatin demonstrated strong antifungal activity and reduced the growth and viability of A. fumigatus. Exposure of A. fumigatus to atorvastatin led to a reduction in ergosterol content and increased membrane permeability, as evidenced by the release of protein, amino acids and gliotoxin. Proteomic analysis revealed an increased abundance of proteins associated with an oxidative stress response, such as the glutathione s-transferase family protein (+8.43-fold), heat shock protein Hsp30/Hsp42 (+2.02-fold) and 5-demethoxyubiquinone hydroxylase, mitochondrial (+1.73-fold), as well as secondary metabolites such as isocyanide synthase A icsA (+8.52-fold) and non-ribosomal peptide synthetase fmpE (+3.06-fold). The results presented here indicate that atorvastatin has strong antifungal properties and may have potential application in the treatment of A. fumigatus infections alone or in combination with existing antifungal agents.

Keywords: atorvastatin; Aspergillus; ergosterol; infection; proteomics

1. Introduction Aspergillus fumigatus is a serious cause of disease in immunocompromised patients or in those with pulmonary malfunction. A. fumigatus is widely distributed in the environment and produces large numbers of conidia [1,2], which are inhaled on a daily basis. A rapid immune response eliminates these in the immunocompetent host but in cases of pulmonary disease (e.g., cystic ﬁbrosis (CF), asthma) or immunosuppression conidia can quickly germinate and grow in the lung [1,3]. The fungus can cause three types of disease. Saprophytic infection involves the growth of the fungus in the lung and is seen in asthmatic and CF patients [4]. Allergic bronchopulmonary aspergillosis occurs when the body reacts to toxins and allergens secreted by the fungus as it grows in the thick mucus secretion found in the CF or asthmatic lung. Invasive aspergillosis occurs in immunocompromised patients and involves the fungus growing through viable tissue and disseminating to other sites in the body [5]. Invasive aspergillosis can have a mortality rate of 80%–90% and is diﬃcult to diagnose and treat [6] A. fumigatus displays a number of characteristics that facilitate its growth and persistence in the lung. The fungus is thermotolerant and can survive temperatures up to 45 ◦C[7]. It is capable of producing a wide range of enzymes that may facilitate the degradation of tissue and toxins (e.g., gliotoxin, fumagillin), which may play a role in suppressing the local immune response [8,9].

J. Fungi 2020, 6, 42; doi:10.3390/jof6020042 www.mdpi.com/journal/jof J. Fungi 2020, 6, 42 2 of 10

Treatment of aspergillosis involves the use of a range of antifungal drugs. Azole antifungal agents, such as voriconazole, show efficacy against invasive aspergillosis. The polyene Amphotericin B has been widely used for the treatment of invasive aspergillosis and, due to its inherent toxicity, is now usually administered in a liposomal formulation [10,11]. Echinocandins, such as caspofungin, show good efficacy against aspergillosis and are well tolerated in vivo [12]. Statins are widely used for the control of cholesterol and act by inhibiting the action of 3-hydroxy-3-methylglutaryl-CoA (HMG-Co A) reductase and thus block cholesterol biosynthesis [13,14]. Statins may be produced naturally (e.g., lovastatin), semi-synthetically (e.g., simvastatin) or synthetically (e.g., atorvastatin) and were originally obtained from a variety of fungi (e.g., Aspergillus terreus, Penicillium citrinum)[14]. Mammalian cholesterol and fungal ergosterol are structurally similar and their biosynthetic pathways are closely related [14]. Consequently, fungal cells exposed to statins show reduced ergosterol content [15]. Statins have well-established antifungal properties and have been shown to inhibit the growth of a wide range of filamentous fungi (e.g., A. fumigatus, Aspergillus flavus, Aspergillus terreus, and Aspergillus niger)[16] and yeasts [15]. Exposure of Candida glabrata to simvastatin resulted in inhibition of growth, reduced levels of ergosterol and disruption of mitochondrial DNA [17]. Statins can exhibit fungicidal properties but only at concentrations in excess of those that can be clinically achieved [16]. It has been reported that patients on high doses of statins show fewer fungal infections [18–20], prompting the suggestion that statins may represent a well-tolerated antifungal therapy that could be used alone or in combination with conventional therapies for the treatment of recalcitrant infections [21]. The aim of the work presented here was to characterize the response of A. fumigatus to one of the most widely prescribed statins, atorvastatin, with a view to determining its potential as a novel antifungal therapy.

2. Materials and Methods

2.1. Aspergillus Isolates Conidia of A. fumigatus ATCC 26,933 were isolated from malt extract agar (MEA) plates using PBS-Tween (PBS +0.5% (v/v) Tween 80) and harvested by centrifugation (2056 g) for 10 min. × The conidial pellet was washed twice with 5 mL PBS and resuspended in SAB liquid medium at a concentration of 5 106/mL. × 2.2. Statin Tablets of atorvastatin (20 mg Film-Coated Rowex Ltd) were crushed to a powder and dissolved in sterile PBS. The solution was ﬁltered sterilised using 0.45 µm and 0.02 µm pore ﬁlters.

2.3. Susceptibility of A. fumigatus to Atorvastatin The conidia suspension (100 µL) was added to each well of a 96-well plate containing serial dilutions of atorvastatin in 100 µL of Sabouraud Dextrose Broth. The plate was incubated at 37 ◦C for 48 h. The optical density at 600nm was determined using a microplate reader (Synergy HT, Bio-Tek) and growth was quantiﬁed as a percentage of control.

2.4. Eﬀect of Atorvastatin on A. fumigatus on Biomass Production

Cultures A. fumigatus were grown on MEA plates for 3–4 days at 37 ◦C and the conidia were harvested using sterile 0.5% (v/v) Tween 80. The concentration of conidia was adjusted to 5 106 × conidia/mL in sterile PBS. This conidial suspension was used to inoculate 100 mL Sabouraud dextrose broth supplemented with atorvastatin (1.5 and 6 µg/mL). The culture was incubated at 37 ◦C in a shaking incubator at 200 rpm for 72 h and the fungal hyphae were weighed after collection by ﬁltering through miracloth (Calbiochem) and exclusion of remaining culture medium. J. Fungi 2020, 6, 42 3 of 10

2.5. Ergosterol Extraction and Quantification Ergosterol was extracted from 1 g A. fumigatus mycelium as described in [22]. Ergosterol quantification was performed using a gas chromatograph (Hewlett Packard 5890 series II) with a flame ionization detector and a chrompack capillary column (Chrompack International BV, Middleburg, The Netherlands). The carrier gas was N2, and the injector and detector temperatures were set at 320 ◦C. Ergosterol standards were used to calibrate the instrument.

2.6. Determination of Amino Acid and Protein Leakage A. fumigatus was grown for 72 h and exposed to atorvastatin (1.5, 3 and 6 µg/mL) for 2, 4 and 6 h at 37 ◦C. The release of amino acids was determined using the ninhydrin colorimetric method and expressed in terms of aspartic acid and glutamic acid [23]. Ninhydrin (Sigma-Aldrich) was dissolved in ethanol to give a ﬁnal concentration of 0.35% (w/v) and 200 µL was added to each sample (1 mL) and heated to 95 ◦C for 4 min followed by cooling on ice. The absorbance at 600 nm was recorded on a spectrophotometer (Beckman DU 640 Spectrophotometer). The quantity of protein released from the hyphae in the supernatant was assayed using the Bradford assay (Bio-Rad), with bovine serum albumin (BSA) (Sigma-Aldrich) as standard.

2.7. Gliotoxin Extraction and Qquantification Seventy-two hours A. fumigatus cultures were harvested by filtration using miracloth. Chloroform (20 mL) were added to an equal volume of each filtrate sample and the solution was mixed continually for 2 h. The chloroform was evaporated to dryness in a rotary evaporator. Dried extracts were dissolved in 500 µL methanol (Fisher chemical) and stored at -20 ◦C until assayed. Gliotoxin was determined by reverse-phase HPLC (Agilent 1200 Series). The mobile phase was acetonitrile (Fisher chemical), 0.1% (v/v) trifluoroacetic acid (Sigma-Aldrich) and deionized-distilled water. Gliotoxin extract (20 µL) was injected onto a C18 Hewlett Packard column. Gliotoxin concentrations (50, 100 and 200 ng/mL) were dissolved in methanol (Sigma-Aldrich) to create a standard curve of peak area versus gliotoxin concentration.

2.8. Protein Extraction and Preparation for Label-Free Mass Spectrometry Analysis Label-free shotgun semi-quantitative proteomics was conducted on A fumigatus hyphae pre-grown in the presence of atorvastatin (3 µg/mL) for 72 hours. Protein was extracted as described by [24]. Briefly, protein (75 µg) was reduced with dithiothreitol (DTT; 200 mM) (Sigma-Aldrich), alkylated with iodoacetamide (IAA; 1 M) (Sigma-Aldrich) and digested with sequence grade trypsin (Promega, Ireland) at a trypsin:protein ratio of 1:40, overnight, at 37 ◦C. Tryptic peptides were purified for mass spectrometry using C18 spin filters (Medical Supply Company, Ireland) and 1 µg of peptide mix was eluted onto a Q-Exactive (ThermoFisher Scientific, U.S.A) high resolution accurate mass spectrometer connected to a Dionex Ultimate 3000 (RSLCnano) chromatography system. Peptides were separated by an increasing acetonitrile gradient on a Biobasic C18 Picofrit™ column (100 mm length, 75 mm ID), using a 65 min reverse-phase gradient at a flow rate of 250 nL/min. All data were acquired with the mass spectrometer operating in an automatic data-dependent switching mode. A high-resolution MS scan (300–2000 Dalton) was performed using the Orbitrap to select the 15 most intense ions prior to MS/MS.

2.9. Protein Data and Analysis Protein identification from the MS/MS data was performed using the Andromeda search engine in MaxQuant (version 1.2.2.5; http://maxquant.org/) to correlate the data against the proteome of A. fumigatus obtained from Uniport. The following search parameters were used: first search peptide tolerance of 20 ppm and second search peptide tolerance of 4.5 ppm, with carbamidomethylation of the cysteines set as a fixed modification, while oxidation of methionines and acetylation of N-terminals were set as variable modifications and a maximum of 2 missed cleavage sites allowed. False Discovery Rates J. Fungi 2020, 6, 42 4 of 10

(FDR) were set to 1% for both peptides and proteins and the FDR was estimated following searches against a target-decoy database. Peptides with minimum length of seven amino acid length were considered for identification and proteins were only considered identified when more than one unique peptide for each protein was observed. Results processing, statistical analyses and graphics generation J. Fungi 2020, 6, x FOR PEER REVIEW 4 of 10 were conducted using Perseus v. 1.5.5.3. LFQ intensities were log2-transformed and ANOVA and t-tests betweenacid the length proteome were considered of 24-h control for identification and atorvastatin-treated and proteins were (3 onlyµg/ mL)consideredA fumigatus identifiedwas when performed usingmore a p-value than one of 0.05unique and peptide significance for each wasprotein determined was observed. using Results FDR processing, correction statistical (Benjamini–Hochberg). analyses Proteinsand that graphics had non-existentgeneration were values conducted (indicative using of absencePerseus v. or 1.5.5.3. very low LFQ abundance intensities inwere a sample) log2- were also usedtransformed in statistical and ANOVA analysis and of t-tests the total between differentially the proteome expressed of 24-h control group and following atorvastatin-treated imputation of the zero values(3 μg/mL) using A fumigatus a number was close performed to thelowest using a valuep-value of of the 0.05 range and significance of proteins was plus determined or minus theusing standard FDR correction (Benjamini–Hochberg). Proteins that had non-existent values (indicative of absence deviation. After data imputation these proteins were included in subsequent statistical analysis. The or very low abundance in a sample) were also used in statistical analysis of the total differentially Blast2GOexpressed software group was following applied imputation to determine of the zero gene va ontologylues using a terms number (GO close terms) to the relatinglowest value to biological of processesthe range (BP) of and proteins molecular plus or function minus the (MF). standard deviation. After data imputation these proteins were included in subsequent statistical analysis. The Blast2GO software was applied to determine gene 2.10. Statisticalontology terms Analysis (GO terms) relating to biological processes (BP) and molecular function (MF). Results presented in this paper are the mean of at least two independent determinations and 2.10. Statistical Analysis the results are presented as the mean standard error. Experimental data were tested for statistical ± significanceResults using presented a Student’s in this paper t-test. are For the allmean experimentation of at least two independent a p-value determinations of 0.05 was and considered the results are presented as the mean ± standard error. Experimental data were tested≤ for statistical statistically significant All the statistical analyses listed were performed using GraphPad Prism. significance using a Student’s t-test. For all experimentation a p-value of ≤0.05 was considered statistically significant All the statistical analyses listed were performed using GraphPad Prism. 2.11. Data Availability The2.11. MSData proteomicsAvailability data and MaxQuant search output files have been deposited to the ProteomeXchangeThe MS Consortiumproteomics data [25] viaand the MaxQuant PRIDE partner search repositoryoutput files with have the been dataset deposited identifier to PXD015254the . ProteomeXchange Consortium [25] via the PRIDE partner repository with the dataset identifier 3. ResultsPXD015254.

3.1. Eﬀ3.ect Results of Atorvastatin on Growth of A. fumigatus

6 Exposure3.1. Effect of of AtorvastatinA. fumigatus on Growthconidia of A. (initial fumigatus density 1 10 /mL) to atorvastatin for 48 h at 37 ◦C × resulted in reduced growth. An atorvastatin dose of 6.25 µg/mL inhibited growth by 86.4% 2.7% Exposure of A. fumigatus conidia (initial density 1 x 106/mL) to atorvastatin for 48 h at 37 °C ± (p < 0.0001) while doses of 3.12 and 1.56 µg/mL reduced growth by 83% 2% and 47.6% 4.7% resulted in reduced growth. An atorvastatin dose of 6.25 μg/mL inhibited growth± by 86.4% ± 2.7% (p ± (p < 0.002),< 0.0001) respectively, while doses of at 3.12 the and same 1.56 timepoint μg/mL reduced (Figure growth1). Exposure by 83% ± 2% of culturesand 47.6% of ± 4.7%A. fumigatus (p < to atorvastatin0.002), respectively, lead to a signiﬁcant at the same inhibition timepoint in biomass(Figure 1). accumulation Exposure of atcultures 72 h. Culturesof A. fumigatus were pre-grown to for 24atorvastatin h and then lead supplemented to a significant inhibition with atorvastatin in biomass accumulation at the concentrations at 72 h. Cultures stated were for pre-grown a further 48 h. Biomassfor was24 h reducedand then fromsupplemented 3.45 0.13 with g/ 100atorvastatin mL in control at the concentrations cultures to 2.28 stated0.08 for ga/ 100further mL 48 (p h.= 0.0018) Biomass was reduced from 3.45± ± 0.13 g/100 mL in control cultures to 2.28 ± 0.08± g/100 mL (p = 0.0018) at an atorvastatin concentration of 1.5 µg/mL and at a concentration of 6 µg/mL biomass was reduced at an atorvastatin concentration of 1.5 μg/mL and at a concentration of 6 μg/mL biomass was reduced to 0.488 0.04 g/100 mL (p < 0.0001) (Figure S1). Incorporation of atorvastatin into malt extract agar to 0.488± ± 0.04g/100 mL (p < 0.0001) (Figure S1). Incorporation of atorvastatin into malt extract agar also ledalso to led a reductionto a reduction in growthin growth of ofA. A. fumigatus fumigatus coloniescolonies (Figure (Figure S2). S2).

120

100

A. fumigatus ** 40

20 *** *** ***

% Gr owt h *** *** *** *** 0 2 5 0 0 rol 78 56 .1 .25 2. 25 50 0. 1. 3 6 1 10 20 ont C Atorvastatin concentration (µg/ml)

Figure 1. Eﬀect of atorvastatin on growth of A fumigatus. A. fumigatus conidia (initial concentration 1 10Figure6/mL) 1. were Effect exposed of atorvastatin to atorvastatin on growth of in A SABfumigatus culture. A. fumigatus medium. conidia Growth (initial (%) concentration was calculated 1 by × x 106/mL) were exposed to atorvastatin in SAB culture medium. Growth (%) was calculated by comparing atorvastatin-treated A. fumigatus to control cells after 48 h growth (** p < 0.01; *** p < 0.001). comparing atorvastatin-treated A. fumigatus to control cells after 48 h growth (** p < 0.01; *** p < 0.001).

J.J. Fungi Fungi 20202020,, 66,, x 42 FOR PEER REVIEW 5 5of of 10 10 J. Fungi 2020, 6, x FOR PEER REVIEW 5 of 10 3.2. Measurement of Ergosterol from A. fumigatus Mycelium Exposed to Atorvastatin 3.2. Measurement Measurement of Ergosterol from A. fumigatus Mycelium Exposed to Atorvastatin Ergosterol was extracted from mycelial exposed to atorvastatin as described and quantified by GC analysis.Ergosterol Exposure was extracted of A. fumigatus from mycelial cultures exposed to atorvastatin to atorvastatin (3 μg/mL) as describedfor 72 h lead and to quantified quantiﬁed a significant by µ reductionGC analysis. in ergosterolExposure Exposure of production A.A. fumigatus fumigatus (Figure culturescultures 2) (con to atorvastatin trol = 0.088 (3 ± 0.008μg/mL)g/mL) mg/g for and72 h atorvastatin-treatedlead to a significant signiﬁcant reduction in ergosterol production (Figure(Figure2 2)) (control (control == 0.088 ± 0.008 mgmg/g/g and atorvastatin-treated mycelia = 0.006 ± 0.0012 mg/g (p < 0.01). ± mycelia = 0.0060.006 ± 0.00120.0012 mg/g mg/g (p (p << 0.01).0.01). ± 0.10 0.10 Control Control 0.08 Atorvastatin 3 μ∝g/mLg/ml 0.08 Atorvastatin 3 μ∝g/mLg/ml 0.06 0.06 0.04 0.04

Ergosterol mg/gm 0.02 **

Ergosterol mg/gm 0.02 ** 0.00 0.00 Condition Condition

FigureFigure 2. AtorvastatinAtorvastatin (3 (3 μµg/mL)g/mL) exposure reduced the ergosterol content of A. fumigatus . Ergosterol Ergosterol quantificationquantificationFigure 2. Atorvastatin was was performed (3 μg/mL) using using exposure a a gas reducedchroma chromatograph tographthe ergosterol with with a acontent flame flame ofionization A. fumigatus detector . Ergosterol and and a chrompackquantificationchrompack capillary capillary was performed column. Ergosterol Ergosterol using a gas standards standards chroma we weretographre used with to calibrate a flame the ionization instrument. detector Ergosterol Ergosterol and a contentchrompackcontent was was capillaryexpressed column. in terms Ergosterol of mg/mL mg/mL standards (** p < 0.01).0.01). we re used to calibrate the instrument. Ergosterol content was expressed in terms of mg/mL (** p < 0.01). 3.3. Atorvastatin Induces Leakage from A. fumigatus 3.3. Atorvastatin Induces Leakage from A. fumigatus 3.3. AtorvastatinThe effect of Induces atorvastatin Leakage on from membrane A. fumigatus permeability was assessed by quantifying amino acid, The effect of atorvastatin on membrane permeability was assessed by quantifying amino acid, protein and gliotoxin release from cells. Exposure of A. fumigatus hyphae to atorvastatin (3 µg/mL) for proteinThe and effect gliotoxin of atorvastatin release from on membrane cells. Exposure permeability of A. fumigatus was assessed hyphae by to quantifying atorvastatin amino (3 μg/mL) acid, 4 or 6 h led to an increase in amino acid release from cells (Figure3A). At 4 h amino acid release from forprotein 4 or 6and h led gliotoxin to an increase release infrom amino cells. acid Exposure release offrom A. fumigatuscells (Figure hyphae 3A). Atto atorvastatin4 h amino acid (3 μreleaseg/mL) forcontrol 4 or cells6 h led was to 0.14an increase0.0035 inµ aminog/mL butacid in releas the 3eµ fromg/mL cells atorvastatin (Figure 3A). treatment At 4 h amino it was 0.5acid release0.0037 from control cells was± 0.14 ± 0.0035 μg/mL but in the 3 μg/mL atorvastatin treatment it was± 0.5 ± fromµg/mL control (p < 0.0001). cells was At 60.14 h exposure ± 0.0035 the μg/mL amino but acid in release the 3 fromμg/mL control atorvastatin cells was treatment 0.4 0.004 itµ wasg/mL 0.5 and ± 0.0037 μg/mL (p < 0.0001). At 6 h exposure the amino acid release from control cells± was 0.4 ± 0.004 1.080.0037 0.04μg/mLµg/ mL(p < (p 0.0001).< 0.0001) At from6 h exposure the cells supplementedthe amino acid with release the 3fromµg/mL control atorvastatin. cells was Exposure 0.4 ± 0.004 of μg/mL± and 1.08 ± 0.04 μg/mL (p < 0.0001) from the cells supplemented with the 3 μg/mL atorvastatin. A. fumigatus to atorvastatin also led to increased protein leakage (Figure3B). Exposure to atorvastatin Exposureμg/mL and of 1.08 A. fumigatus ± 0.04 μg/mL to atorvastatin (p < 0.0001) also from led the to cells increased supplemented protein leakagewith the (Figure 3 μg/mL 3B). atorvastatin. Exposure Exposurefor 4 h lead of toA. afumigatus protein concentrationto atorvastatin of also 107.6 led to5.2 increasedµg/mL in protein the 3 µleakageg/mL treatment (Figure 3B). compared Exposure to to atorvastatin for 4 h lead to a protein concentration± of 107.6 ± 5.2 μg/mL in the 3 μg/mL treatment to85.6 atorvastatin6 µg/mL for in 4 the h lead control, to a protein and after concentration 6 h in control of 107.6 the protein ± 5.2 μ releaseg/mL in was the 3 106.8 μg/mL3.1 treatmentµg/mL compared± to 85.6 ± 6 μg/mL in the control, and after 6 h in control the protein release was± 106.8 ± 3.1 compared to 150.585.6 ± 6 3.6μg/mLµg/mL in the in the control, treatment and after (3 µg 6/mL) h in (pcontrol< 0.02). the protein release was 106.8 ± 3.1 μg/mL compared to± 150.5 ± 3.6 μg/mL in the treatment (3 μg/mL) (p < 0.02). μg/mL compared to 150.5 ± 3.6 μg/mL in the treatment (3 μg/mL) (p < 0.02).

FigureFigure 3. 3. EffectEﬀect of of atorvastatin atorvastatin on on the the release release of amino of amino acids acids (a) and (a) and protein protein (b) from (b) from A. fumigatusA. fumigatus. The . effectTheFigure e ﬀofect 3. atorvastatin Effect of atorvastatin of atorvastatin (3 μ (3g/mL)µg/ mL)on on the onA. releaseA.fumigatus fumigatus of aminoaminoamino acidsacid acid and(a) and and protein protein protein release release (b) from was was A.determined determined fumigatus .by byThe a a ninhydrineffectninhydrin of atorvastatin colorimetriccolorimetric (3 assay μassayg/mL) and andon Bradford A. Bradford fumigatus protein protei amino assay,n acidassay, respectively. and respectively. protein Atorvastatin release Atorvastatin was treatment determined treatment resulted by a resultedninhydrinin increased in increasedcolorimetric amino acid amino andassay proteinacid and and release Bradfordprotein relative release protei to relativen control assay, cellsto respectively.control (* p < cells0.05; (* **Atorvastatin p p << 0.05;0.01; ** *** p ptreatment< <0.01;0.001). *** presulted < 0.001). in increased amino acid and protein release relative to control cells (* p < 0.05; ** p < 0.01; *** p < 0.001).

J. Fungi 2020, 6, 42 6 of 10 J. Fungi 2020, 6, x FOR PEER REVIEW 6 of 10

Atorvastatin exposure also induced an increase in the release of gliotoxin from A. fumigatusfumigatus cultures (Figure4). After 72 h growth at 37 C gliotoxin was 14.04 1.01 ng/mL at an atorvastatin cultures (Figure 4). After 72 h growth at 37 ◦°C gliotoxin was 14.04 ±± 1.01 ng/mL at an atorvastatin concentration of 1.5 µg/mL compared to the control, which was 4.2 0.6 ng/mL (p = 0.005). At an concentration of 1.5 μg/mL compared to the control, which was 4.2 ±± 0.6 ng/mL (p = 0.005). At an atorvastatin concentration of 6 µg/mL the gliotoxin concentration was 47.22 5.06 ng/mL (p = 0.01). atorvastatin concentration of 6 μg/mL the gliotoxin concentration was 47.22 ±± 5.06 ng/mL (p = 0.01).

60 * Control 50 Atorvastatin 1.51.5 μ∝g/mLg/ml 40 Atorvastatin 66∝ μg/mlg/mL

20 **

Gliotoxin ng/ml Gliotoxin 10

Condition

Figure 4. Atorvastatin exposure increased the gliotoxin release from A. fumigatus. Gliotoxin Figure 4. Atorvastatin exposure increased the gliotoxin release from A. fumigatus. Gliotoxin quantification was performed using an HPLC. The gliotoxin standards were used to calibrate the quantification was performed using an HPLC. The gliotoxin standards were used to calibrate the instrument. Gliotoxin content was expressed in terms of ng/mL (* p < 0.01; ** p < 0.01). instrument. Gliotoxin content was expressed in terms of ng/mL (* p < 0.01; ** p < 0.01). 3.4. Proteomic Analysis of Response of A. fumigatus to Atorvastatin 3.4. Proteomic Analysis of Response of A. fumigatus to Atorvastatin Proteomic analysis of A. fumigatus cultures exposed to atorvastatin revealed 25,915 peptides representingProteomic 1360 analysis proteins, of A. and fumigatus 128 proteins cultures were exposed determined to atorvastatin to be diff erentlyrevealed abundant 25,915 peptides with a foldrepresenting change of1360>1.5. proteins, In total and 83 128 proteins proteins were were increased determined in abundance to be differently and 45 abundant were decreased with a fold in abundance.change of >1.5. These In total proteins 83 proteins were were subsequently increased usedin abundance to statistically and 45 analyse were decreased the total in di abundance.fferentially expressedThese proteins group were after subsequently imputation ofused the to zero statistica valueslly as analyse described the and total were differentially then included expressed in statistical group analysisafter imputation after data of imputation. the zero values A principal as described component and were analysis then (PCA)included was in performed statistical onanalysis all filtered after proteinsdata imputation. and clearly A distinguishedprincipal component the control analysis and atorvastatin (PCA) was treatedperformedA. fumigatus on all filteredsamples proteins (Figure and S3). Theclearly volcano distinguished plot shows the the control relative and distribution atorvastatin of these treated proteins A. fumigatus (Figure5 ),samples and of the(Figure proteins S3). thatThe increasedvolcano plot in abundance shows the following relative distribution exposure to atorvastatin,of these proteins the most (Figure notable 6), wereand of isocyanide the proteins synthase that A(increased+8.52-fold), in abundance glutathione following S-transferase exposure family proteinto atorvastatin, (+8.43-fold), the kynureninasemost notable 2 were (+4.48-fold), isocyanide and non-ribosomalsynthase A (+8.52-fold), peptide synthetase glutathione fmpE S-transferase (+3.06-fold). family The mainprotein peptides (+8.43-fold), decreased kynureninase in abundance 2 (+4.48- were O-methyltransferasefold), and non-ribosomal (-3.68-fold), peptide allergen synthetase Asp fmpE f 15 (-2.45-fold) (+3.06-fold). and The GNAT main family peptides acetyltransferase decreased in (-2.25-fold).abundance were O-methyltransferase (-3.68-fold), allergen Asp f 15 (-2.45-fold) and GNAT family acetyltransferaseThe main enzyme (-2.25-fold). classes that were altered in abundance in A. fumigatus treated with atorvastatin wereThe oxidoreductases, main enzyme transferases classes that and hydrolaseswere altered (Figure in S4).abundance Oxidoreductase in A. fumigatus enzymes thattreated increased with inatorvastatin abundance were in oxidoreductases, atorvastatin treatment transferases of A fumigatusand hydrolaseswere (Figure pyoverdine S4). Oxidoreductase/dityrosine biosynthesis enzymes familythat increased protein (in+8.52-fold), abundance 5-demethoxyubiquinone in atorvastatin treatment hydroxylase, of A fumigatus mitochondrial were pyoverdine/dityrosine (+1.73-fold), alcohol dehydrogenase,biosynthesis family putative protein ((+8.52-fold),+1.6-fold) and5-demeth Psi-producingoxyubiquinone oxygenase hydroxylase, A (+1.55-fold). mitochondrial Thirteen (+1.73- oxidoreductasefold), alcohol dehydrogenase, enzymes showed putative a decrease (+1.6-fold) in abundance and Psi-producing and included oxygenase aflatoxin A B1-aldehyde(+1.55-fold). reductaseThirteen oxidoreductase GliO-like, putative enzymes ( 2.86-fold),showed a aryl-alcoholdecrease in abundance dehydrogenase, and included putative aflatoxin ( 1.52-fold) B1- − − andaldehyde NADH-dependent reductase GliO-like, flavin putative oxidoreductase, (-2.86-fold), putative aryl-alcohol ( 1.50-fold). dehydrogenase, Enzymes putative with transferase(-1.52-fold) − activityand NADH-dependent increased in abundance flavin oxidoreductase, included glutathione putative S-transferase (-1.50-fold). familyEnzymes protein with (+transferase8.43-fold), phosphatidylinositolactivity increased in transporter,abundance included putative (glutathione+2.00-fold) andS-transferase allergen Aspfamily f 4protein (+1.42-fold), (+8.43-fold), while O-methyltransferasephosphatidylinositol (transporter,3.68-fold), malateputative synthase (+2.00-fold) ( 2.40-fold) and allergen and GNAT Asp f family4 (+1.42-fold), acetyltransferase, while O- − − putativemethyltransferase ( 2.25-fold) (-3.68-fold), were decreased malate insynthase abundance. (-2.40-fold) Nine hydrolytic and GNAT enzymes family wereacetyltransferase, increased in − abundanceputative (-2.25-fold) and these includedwere decreased kynureninase in abundanc (4.48-fold),e. Nine amidase hydrolytic putative enzymes (+2.77-fold), were lipase increased/esterase in putativeabundance (+ 2.12-fold)and these and methionineincluded kynureninase aminopeptidase (4.48-fold), 2-2 (+1.83-fold). amidase In contrastputative two (+2.77-fold), hydrolytic enzymeslipase/esterase were putative reduced (+2.12-fol in abundanced) and (haloalkanoic methionine aminopeptidase acid dehalogenase, 2-2 (+1.83-fold). putative ( In2.99-fold) contrast andtwo − phytase,hydrolytic putative enzymes ( 1.42-fold). were reduced in abundance (haloalkanoic acid dehalogenase, putative (-2.99- fold) and phytase, −putative (-1.42-fold).

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Figure 5. Volcano plot showing the distribution of quantified proteins according to p value ( log10 − p-value)Figure and 5. Volcano fold change plot (log2showing mean the LFQ distribution intensity of di ffquantierence).fied Proteins proteins above according the horizontal to p value line (−log10 are p- consideredvalue) and statistically fold change significant (log2 mean (p value LFQ< intensity0.05) and difference). those to the Proteins right and above left ofthe the horizontal vertical linesline are indicateconsidered relative statistically fold changes significant of 1.5. (p value < 0.05) and those to the right and left of the vertical lines indicate relative fold changes± of ±1.5. 4. Discussion 4. Discussion Statins are widely used for the control of cholesterol and function by inhibiting the action of 3-hydroxy-3-methylglutaryl-CoAStatins are widely used for (HMG-CoA) the control of reductase cholesterol [26 and,27]. function Patients by on inhibiting statin therapy the action display of 3- lowerhydroxy-3-methylglutaryl-CoA incidences of fungal infections (HMG-CoA) [19,28], prompting reductase the [26,27]. suggestion Patients that on statins statin may therapy display display a significantlower incidences antifungal of e fffungalect [20 ]infections and could [19,28], have applications prompting inthe treating suggestion recalcitrant that statins fungal may infections. display a Statinssignificant can inhibit antifungal the growth effect [20] of C. and albicans could [have15] and applications growth inhibition in treating may recalcitrant be via afungal reduction infections. in ergosterolStatins can content inhibit [29 ].the High growth dose of of C. statin albicans treatment [15] and has growth been correlated inhibition with may immune-modulatory be via a reduction in effectsergosterol in some content patients [29]. [30 High,31] dose and, of in statin particular, treatment the inductionhas been correlated of modulation with immune-modulatory in the activity of regulatoryeffects in T cellssome [ 32patients]. [30,31] and, in particular, the induction of modulation in the activity of regulatoryExposure T of cellsA. fumigatus [32]. to atorvastatin reduced fungal growth and induced increased membrane permeabilityExposure as evidenced of A. fumigatus by increased to releaseatorvastatin of protein reduced and aminofungal acids. growth Maximum and induced amino acid increased and proteinmembrane leakage permeability occurred in the as 3evidencedµg/mL treatment by increased at 6 h andrelease this of concentration protein and may amino be optimal acids. Maximum for this effect.amino Statins acid inhibitand protein the production leakage occurred of cholesterol in the 3 in μ mammalsg/mL treatment and have at 6 previouslyh and this concentration been shown tomay inhibitbe optimal ergosterol for biosynthesis this effect. inStatins fungi inhibit [29]. Reduced the production ergosterol of in cholesterol atorvastatin-treated in mammalsA. fumigatus and have cellspreviously may alter thebeen integrity shown of to the inhibit membrane ergosterol and thus bios leadynthesis to increased in fungi permeability. [29]. Reduced Cultures ergosterol treated in withatorvastatin-treated atorvastatin also released A. fumigatus elevated cells levels may of gliotoxin,alter the whichintegrity also of has the been membrane observed and in A. thus fumigatus lead to exposedincreased to amphotericin permeability. B Cultures and may treated indicate with an oxidative atorvastatin stress also response released [33 elevated]. levels of gliotoxin, whichProteomic also analysishas been revealed observed an increasedin A. fumigatus abundance exposed of a range to amphotericin of proteins involved B and inmay dealing indicate with an oxidativeoxidative stress, stress including response pyoverdine [33]. /dityrosine biosynthesis (+8.52-fold), 5-demethoxyubiquinone hydroxylase,Proteomic mitochondrial analysis revealed (+1.74-fold) an increased and alcohol abunda dehydrogenasence of a range (+ of1.61-fold). proteins IninvolvedSaccharomyces in dealing cerevisiaewith , dityrosineoxidative is astress, major componentincluding of thepyoverdine/dityrosine spore wall surface and playsbiosynthesis a role in the(+8.52-fold), resistance of 5- ascosporesdemethoxyubiquinone to environmental hydroxylase, stress [34]. mitochondrial Furthermore, the (+1.74-fold) glutathione and S-transferase alcohol dehydrogenase family protein (+1.61- is increasedfold). In in Saccharomyces abundance (+ 8.43-fold),cerevisiae, dityrosine and this protein is a major functions component to detoxify of the xenobiotics. spore wall The surface fumipyrrole and plays biosynthetica role in the (fmpE) resistance protein of wasascospores also increased to enviro innmental abundance stress (+[34].3.06-fold). Furthermore, Deletion the ofglutathione fmpE in S- A. fumigatustransferaseresulted family in protein reduced is growthincreased and in sporulation abundance of (+8.43-fold), the mutant strains and this but protein virulence functions was not to altereddetoxify as compared xenobiotics. to the The WT fumipyrrole strain in a murinebiosynthetic infection (fmpE) model protein [35]. was also increased in abundance (+3.06-fold).A range of heatDeleti shockon of proteins, fmpE in such A. fumigatus as Hsp30 /Hsp42resulted (+ 2.02-fold),in reduced also growth increased and insporulation abundance of in the response to atorvastatin. Heat shock proteins play an important role in adaptation of the fungal cell to environmental and chemical stresses [36]. A. fumigatus heat shock proteins are also immunodominant and elicit strong humoral immune responses [37]. Proteomic analysis also revealed the reduced J. Fungi 2020, 6, 42 8 of 10 abundance of a number of peptides associated with virulence, including aflatoxin B1 ( 2.86-fold) and − allergen Asp f 15 ( 2.45-fold). Asp f 15 is cell-wall associated and its decreased abundance could be − due to an altered cell-wall composition as a result of an altered membrane composition. Statins inhibit the growth of A. fumigatus and supplementation of media with ergosterol or cholesterol blocked the growth-inhibiting effect of atorvastatin, thus indicating the specificity of statins for the mevalonate synthesis pathway [15]. However, another study found that statins were fungistatic against A. fumigatus, but the MICs were greater than the clinically achievable concentrations [16]. Furthermore, a variety of Candida strains that were resistant to fluconazole and nystatin were all sensitive to atorvastatin [38]. Atorvastatin combined with fluconazole improved cryptococcosis caused by Cryptococcus gattii and mice treated with atorvastatin had increased survival and better clinical outcomes; this was mediated via modifications in the capsule and cell membrane in the presence of a statin that induced cell death within phagocytes [39].

5. Conclusions The results presented here conﬁrm previous ﬁndings obtained using statins (e.g., a reduction in ergosterol content); however, the alteration in membrane permeability caused by atorvastatin and the altered proteomic response are indicative of oxidative stress in the cells. Fully understanding the antifungal mode of action of statins may highlight novel ways to utilise these widely used drugs for the control of recalcitrant fungal infections.

Supplementary Materials: The following are available online at http://www.mdpi.com/2309-608X/6/2/42/s1, Figure S1. The biomass of A. fumigatus exposed to atorvastatin (1.5 and 6 µg/mL). Figure S2: Growth area of A. fumigatus on MEA plates supplemented with atorvastatin. Figure S3. Principal component analysis of control and atorvastatin treated A. fumigatus at 24 h showing a clear distinction between control and treatment. Figure S4. Bar chart showing changes to number of proteins involved in enzyme classes at Level 3 ontology. Proteins were assigned groups based on involvement in biological processes for control and atorvastatin treated. Author Contributions: Conceptualization, K.K., A.A. and G.S.; methodology, A.A. and G.S.; formal analysis, A.A. and G.S.; writing—original draft preparation, A.A. and G.S.; writing—review and editing, K.K.; supervision, K.K. All authors have read and agreed to the published version of the manuscript. Funding: Ahmad Ajdidi is the recipient of a PhD Scholarship from the Libyan Ministry of Education; Gerard Sheehan is the recipient of a Maynooth University Doctoral studentship. The Q-exactive mass spectrometer was funded under the SFI Research Infrastructure Call 2012; Grant Number: 12/RI/2346 (3). Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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