Argentilactone Molecular Targets in Paracoccidioides brasiliensis Identified by Chemoproteomics

Argentilactone extraction.The leaves of H. ovalifolia were harvested during the summer of 2016 in relevant areas of the savanna from the Goiânia municipality of Goiás, Brazil. Their essential oil was obtained by hydrodistillation, and argentilactone was isolated and identified as previously described by de Oliveira et al. (9).

Microorganisms and culture conditions.P. lutzii Pb01, P. brasiliensis Pb18, P. americana Pb02 and Pb03, and P. restrepiensis (PbEPM83, Pb339, and Pb60855 were incubated in liquid Fava-Netto medium (51) (0.3% protease peptone, 1% peptone, 0.5% [wt/vol] meat extract, 0.5% [wt/vol] yeast extract, 1% brain heart infusion [BHI], 4% glucose, 0.5% NaCl, 5 μg/ml gentamicin), pH 7.2, for 48 h at 37°C under shaking. The cells then were centrifuged and washed with phosphate-buffered saline (0.09% Na₂HPO₄, 0.02% KH₂PO₄, 0.8% NaCl, 0.02% KCl, pH 7.2), transferred to chemically defined medium RPMI 1640 (Sigma-Aldrich), and incubated for 16 h at 37°C under shaking for the adaptation of the fungal cells.

Determination of the MIC.The determination of the MIC was performed by following recommendations from the Clinical and Laboratory Standards Institute and adapted according to Paracoccidioides spp., as described by De Paula e Silva et al. (52). In each well of the microplate, dilutions of argentilactone were added, with addition of the fungal suspension to a final concentration of 1 × 10⁵ cells/ml. To determine the maximum fungal growth (positive control), cells were placed in the presence of only RPMI 1640 medium. The plates were maintained at 36°C under agitation for 48 h. Subsequently, 20 μl of the 0.02% resazurin solution was added and incubated for 24 h under the same assay conditions. The MIC was visually determined at the point at which there was no change in the original blue color of the reagent.

Determination of the MFC.The minimum fungicidal concentration was determined by following Takahagi-Nakaira et al. (53). The Paracoccidioides species cells were incubated with argentilactone, which was serially diluted while maintaining the same concentrations and culture conditions used in the MIC test. For each well, a subculture was performed by transferring 50 μl of the corresponding MIC material to a petri dish containing solid Fava-Netto medium. As a control for fungal growth, an aliquot of the positive control was also plated. The plates were incubated at 37°C for 7 days, with subsequent absorbance readout. The MFC was defined as the lowest concentration at which fungal growth was not visualized.

Cytotoxicity against mammalian cells.The A549 (ATCC CCL-185), BALB/c 3T3 clone A31 (ATCC CCL-163), and AMJ2-C11 (ATCC CRL-2456) cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (Nutricell, São Paulo, Brazil). A total of 1 × 10⁵ cells/ml were incubated with different concentrations of argentilactone for 48 h at 37°C and 5% CO₂. A volume of 20 μl of 0.02% resazurin was added to each microwell and incubated for 24 h. The cytotoxic concentration (CC) was visually determined at the point at which there was no change in the original blue color of the reagent. The following expression determined the selectivity index of the argentilactone: SI = CC/MIC.

Cytoplasmic protein extraction.P. brasiliensis cells of strain Pb18 (ATCC 32069) were grown in liquid Fava-Netto's medium (0.3% protease peptone, 1% peptone, 0.5% meat extract, 0.5% yeast extract, 1% BHI, 4% glucose, 0.5% NaCl, and 5 μg/ml gentamicin, pH 7.2) for 3 days at 37°C with shaking. The cells were subsequently centrifuged and resuspended in ammonium bicarbonate buffer (57 mM, pH 8.8). Protein extraction was performed by mechanical cell lysis using glass beads. The cell lysate was centrifuged at 5,000 × g for 15 min at 4°C to obtain a supernatant containing protein extract. The integrity of the protein extract was verified by SDS-PAGE and quantified with Bradford reagent (54).

Affinity chromatography.Initially, a wavelength scanning of the argentilactone solution (1 mg/ml) was performed using wavelengths of 205 to 565 nm to verify the absorption range of argentilactone. For the assay, the wavelength of 265 nm was selected to monitor the absorbance of argentilactone. Argentilactone was immobilized on a chromatographic column containing an Octyl-Sepharose resin (GE Healthcare Life Sciences, Chicago, IL). Two milliliters of resin was added to a chromatographic column and then washed with 10 ml of ultrapure water. The resin was subsequently incubated with 1.5 ml of argentilactone (1 mg/ml) for 30 min, performed 5 times for complete immobilization. After each incubation, the absorbance of the solution exiting the system was measured. The saturation of the resin with argentilactone was verified by comparing the absorbance of argentilactone (wavelength, 265 nm) in the initial solution (1 mg/ml) and that after incubation with the resin.

The Octyl-Sepharose column containing immobilized argentilactone and the column without immobilized argentilactone (the control chromatographic column) were incubated with 6 mg of protein from Pb18 for 1 h on ice. After incubation, the column was washed with 10 ml of 50 mM ammonium bicarbonate buffer solution (pH 8.5). The presence of proteins was monitored during the washing process using the Bradford quantification method and polyacrylamide gel electrophoresis to ensure that all noninteracting proteins were removed. The argentilactone-interacting proteins and control column-interacting proteins were eluted by adding 4 ml of 75% acetonitrile. The samples were lyophilized and conditioned at −20°C until their identification. The sample was acquired from three biological replicates.

Tryptic digestion and data acquisition by nanoUPLC-MS^E.The proteins (argentilactone-interacting proteins and column-interacting proteins) were enzymatically digested and processed as described previously (55). To 50 μg of protein extract, 25 μl of RapiGEST 0.2% (vol/vol) (Waters Corporation, Milford, MA) was added, and the sample was vortexed and incubated at 80°C for 15 min. The reduction of disulfide bonds was subsequently performed by adding 2.5 μl of 100 mM dithiothreitol (DTT) (GE Healthcare), and the sample was incubated at 60°C for 30 min. Thereafter, 2.5 μl of a 300 mM iodoacetamide (GE Healthcare) solution was added for cysteine alkylation, and the sample was incubated in the dark at room temperature for 30 min. Ten μl of trypsin (Promega, Madison, WI, USA), prepared in 50 mM ammonium bicarbonate, next was added at 50 ng/μl. The sample was then digested at 37°C for 16 h. A RapiGEST precipitation was performed by adding 10 μl of 5% (vol/vol) trifluoroacetic acid (TFA) (Sigma-Aldrich, St. Louis, MO), followed by incubation at 37°C for 90 min. The sample was centrifuged at 13,000 × g at 6°C for 30 min. The supernatant was dried in a vacuum concentrator. All of the resulting peptides were suspended in 45 μl of a solution containing 20 mM ammonium formate and 150 fmol/μl PHB (rabbit phosphorylase B) (Waters Corporation). The MassPREP protein was used as an internal standard.

The nanoscale liquid chromatography separation of tryptic peptides was performed using a nanoACQUITY system (Waters Corporation) equipped with a nanoEase 5-μm Bridge BEH130 C₁₈ 300-μm by 50-mm precolumn, trap column (5 μm, 180 μm by 20 mm), and BEH130 C₁₈ 1.7-μm, 100-μm by 100-mm analytical reversed-phase column (Waters Corporation). The peptides were separated into five fractions, 10.8%, 14%, 16.7%, 20.4%, and 65%, in acetonitrile–0.1% (vol/vol) formic acid, at a flow rate of 2,000 μl/min. The source was operated in the positive ionization mode of nano-ESI(+). To perform external calibration, the masses were corrected based on Glu-fibrinopeptide B (GFP; Sigma-Aldrich) with a molecular mass of 785.8486. A GFP solution in 50% (vol/vol) methanol and 0.1% (vol/vol) formic acid was used at a final concentration of 200 fmol/μl, as delivered by the reference sprayer of the NanoLockSpray source for mass spectrometer. Mass spectrometry analysis was performed using a Synapt G1 mass spectrometer (Waters) equipped with a NanoElectronSpray source and two mass analyzers, a quadrupole, and a time-of-flight (TOF) sensor operating in the V-mode. Data were obtained using the instrument in MS^E mode. The sample was analyzed from three technical replicates.

Data processing and protein identification.MS raw data were processed using ProteinLynx Global Server, version 2.4 (Waters Corporation). The data were subjected to automatic background subtraction, deisotoping, and charge state deconvolution. After processing, each ion comprised an exact mass-retention time that contained the retention time, intensity-weighted average charge, inferred molecular weight based on charge, and m/z. The processed next spectra were searched against Pb18 protein sequences (Broad Institute; http://www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/Multiome.html) together with the reverse sequences.

The mass error tolerance for peptide identification was under 50 ppm. The parameters for protein identification included the following: (i) the detection of at least 2 fragment ions per peptide, (ii) 5 fragments per protein, (iii) the determination of at least 1 peptide per protein, (iv) carbamidomethylation of cysteine as a fixed modification, (v) the phosphorylation of serine, threonine, and tyrosine and the oxidation of methionine (considered variable modifications), (vi) maximum protein mass of 600 kDa, (vii) one missed cleavage site allowed for trypsin, and (viii) maximum false discovery rate (FDR) of 1%. The minimum repeat rate for each protein in all replicates was 2. The tables of peptides and proteins generated by the PLGS were merged, and the data on the dynamic range, peptide detection type, and mass accuracy were calculated for each sample, as previously described using the software MassPivot v1.0.1 (56), FBAT (57), Spotfire v8.0 (TIBCO software), and Microsoft Excel (Microsoft). The argentilactone-interacting proteins were categorized according to the functional category database Munich Information Center for Protein Sequences (MIPS), which is available at geneontology.org. Their biological functions were proposed by the PEDANT genome database (http://pedant.gsf.de/). The NCBI database was employed to annotate the uncharacterized proteins (https://www.ncbi.nlm.nih.gov/).

The mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (58) with the data set identifier PXD007680 for the sample argentilactone-interacting proteins and PXD008858 for the sample column-interacting proteins.

Enzymatic activity.The enzymatic activities were explored using a crude extract of P. brasiliensis Pb18 cells.

Malate dehydrogenase.The malate dehydrogenase activity was measured using a final concentration of 20 mM oxalacetic acid (Sigma-Aldrich), 2 mM NADH (Sigma-Aldrich), 50 mM Tris HCl, pH 7.5, and 36 μg/ml argentilactone. The control sample was prepared without argentilactone. The reaction was initiated by adding 10 μg of protein extract. The decrease in absorbance due to the oxidation of NADH was monitored at 340 nm for 30 min. A unit of malate dehydrogenase was defined as the oxidation of 1 μmol of NADH per minute at 37°C, based on the standard curve of NADH.

Pyruvate dehydrogenase.The pyruvate dehydrogenase activity was measured by following the manufacturer's instructions (Sigma-Aldrich) at 37°C. Argentilactone at a final concentration of 36 μg/ml and 10 μg of protein extract were added to the assay. The NADH formation was monitored at 450 nm and was quantified using the standard curve. One unit of pyruvate dehydrogenase is the amount of enzyme that generated 1 μmol of NADH per min.

Citrate synthase.The enzymatic activity of the citrate synthase was determined as previously described by Brock et al. (59). The reaction of 5,5′-dithiobis-2-nitrobenzoate (DTNB) with CoA, forming 5-thio-2-nitrobenzoic acid, gives a yellowish coloration to the reaction mixture detected at 412 nm. Final concentrations of 50 mM Tris-HCl, pH 8.0, 0.2 mM acetyl-CoA (Sigma-Aldrich), 1 mM oxaloacetate (Sigma-Aldrich), 1 mM DTNB (Sigma-Aldrich), and 36 μg/ml argentilactone were used, with the final volume being 150 μl. The sample control was set up and performed without argentilactone. The reaction was started by adding 10 μg of protein extract from Pb18, and the reading was completed after 35 min. One unit was defined as the release of 1 μmol CoA per min per ml at 37°C, based on the standard DTT curve, when varying the concentration from 0.5 mM to 25 mM.

Quantification of cell wall polymers.The cell wall polymers were quantified as described by Tomazett et al. (35). A total of 1 × 10⁵ cells were inoculated in the presence of 36 μg/ml argentilactone. Cells grown in the absence of the compound were used as the sample control. After 48 h of incubation, the cells were centrifuged and washed twice with 50 mM Tris-HCl buffer, pH 7.5, and then were resuspended in the same buffer with added glass beads for mechanical cell lysis. The cell wall was separated from the cytoplasmic fraction after centrifugation at 10,000 × g for 10 min. The soluble fraction was separated from the insoluble fraction after adding 1 M NaOH and 500 mM sodium borohydrate.

To determine the total carbohydrates, 1 mg of the dry weight of the cell walls was dissolved in 1 ml of 75% sulfuric acid and the mixture was stirred for 30 min. Subsequently, 30 μl of 5% phenol was added and the sample was incubated at 90°C for 5 min. The reading was performed using a wavelength of 490 nm. Quantification was performed based on the standard glucose curve.

To quantify N-acetylglucosamine, 2 mg of the cell wall sample was dissolved in 1 ml of 8 M HCl, followed by incubation at 100°C for 4 h. Thereafter, the solution was neutralized with 900 μl of 8 M NaOH. To the neutralized extract, 100 μl of 3 M Na₂CO₃ solution was added, followed by incubation for 20 min at 100°C. Subsequently, 700 μl of ethanol and 100 μl of Ehrlich's reagent were added. The reaction was quantified at 520 nm. The quantification of N-acetylglucosamine was performed in comparison with the standard curve of glycosamine.

To quantify the 1,3-β-d-glycan, 1 mg of the insoluble fraction was added to 200 μl of Tris DTT buffer (50 mM Tris HCl, pH 7.5, and 10 mM DTT) and 2 μl of glucanase, followed by incubation for 12 h at 37°C. The mixture was centrifuged, and the supernatant was collected for quantification. In one tube, 50 μl of the digestion was mixed with 1.5 ml of the 4-hydroxybenzoic acid hydrazide solution, and then the sample was incubated for 10 min at 100°C. Two hundred μl of the mixture was dispensed into the microplate, and the reading was performed at a wavelength of 410 nm. The quantification of 1,3-β-d-glycan was based on the results of the standard glucose curve. Laminarin was used as a negative control.

Evaluation of reactive oxygen species production.A total of 1 × 10⁵ cells were treated with 36 μg/ml of argentilactone for 3, 6, and 12 h at 37°C and then centrifuged at 5,000 × g and incubated with 20 μM 2′,7′-dichlorofluorescein diacetate for 30 min at room temperature under shelter from light. The cells next were washed and resuspended in 1 ml of phosphate-buffered saline. The fluorescence intensity was determined by flow cytometry using an Accuri C6, FL1-H channel (BD Biosciences, Ann Arbor, MI). A total of 10,000 events were considered for analysis.

Statistical analysis.The experiments were performed in triplicate and were arranged on the graph as arithmetic means. Statistical analysis was performed using Student's t test, and samples with a P value of ≤0.05 were considered statistically significant.