Trifluoroacetic acid (TFA) was obtained from Applied Biosystems (Warrington, WA). High performance liquid chromatography (HPLC)-grade acetonitrile, methanol, acetic acid, HPLC-grade water, ammonium bicarbonate, dithiothreitol (DTT), acrylamide, iodoacetamide, urea, and 3-[3-(Cholamidopropyl) dimethylammonio]-1-proanesulfonate (CHAPS) were obtained from Fisher (Fair Lawn, New Jersey). Carrier ampholytes (pH 3–10) and formic acid were purchased from Fluka Chemicals (Milwaukee, WI). SYPRO Ruby fluorescent dye was obtained from Invitrogen Molecular Probes (Carlsbad, CA). α-Cyano-4-hydroxycinnamic acid, bathophenathrolinedisulfonic acid (BPS), and protease inhibitor cocktail for fungal and yeast cells (P8215) was purchased from Sigma Aldrich (St. Louis, MO). Sequence grade trypsin was obtained from Promega (San Luis Obispo, CA). 4-chloro-1-naphthol HRP development was obtained from BioRad (Hercules, CA). HRP conjugate secondary antibodies were purchased from Pierce (Rockford, IL). HSP70 mouse monoclonal antibody was purchased from Sressgen (Victoria, BC Canada). Cyclophilin mouse monoclonal antibody was a gift from Francisco Gomez (University of Cincinnati, Infectious Disease).
H. capsulatum yeast (strain G217B) were prepared by inoculating 50 ml of medium, previously described , with 3 × 106 cells/ml from 3–5 days old slants to give a concentration of 1.5 × 108 cells/ml after 3 days. H. capsulatum yeasts were grown in medium set at pH 7.5 for 48 hr and switched to medium containing 5 μM apo-transferrin or medium free of apo-transferrin. One ml aliquots (~108 cells) were removed following 24 hr and 48 hr incubation with 5 μM apo-transferrin and medium free of apo-transferrin. Samples were then centrifuged at 1500 × g for 10 min. The supernatant was removed and 250 μl of lysis buffer (9 M urea, 2% CHAPS, 1% DTT, and 10 mM fungal and yeast cell protease inhibitor) was added. Cells were disrupted with a Min-Beadbeater (BioSpec Products, Bartlesville, OK) using an equal volume of glass beads for 30 sec and placed on ice for 30 sec. This was repeated 3 times. Following cell disruption, 250 μl of lysis buffer was added and samples were allowed to cool on ice for 10 min. Samples were then centrifuged at 15,000 × g for 30 min. An equal volume of water was added to each sample and further concentrated to 100 μl using an Amicon Ultra Centrifugal Filter Device. The clear supernatant was removed and protein concentrations were determined using the Bradford method.
We confirmed that apotransferrin did reduce iron levels by measuring the amount of iron bound to transferrin using the iron chelator bathophenathrolinedisulfonic acid (BPS) as described previously [42, 43]. One ml of media containing 5 μM apo-transferrin was concentrated using Amicon Ultra Centrifugal Filter Device. To the concentrated transferrin (100 μl), nitric acid was added to give a 3% concentration followed by the addition of 38 mg/ml sodium ascorbate, 1.7 mg/ml BPS, and ammonium acetate (saturated ammonium acetate dilute 1/3) were added to the concentrated transferrin effectively denaturing the protein and reducing all iron to the ferrous form. After 5 min the absorbance was recorded at 535 nm for the concentrated retintate and flow through. Based on measuring the amount of iron bound to apo-transferrin the quantity of iron in the medium prior to addition of apotransferrin was .0008 g/L and following 24 hr in 5 μM apo-transferrin the concentration was reduced by ~83%. At 5 μM apo-transferrin changes in the amount of iron bound to apo-transferrin in the presence or absence of H. capsulatum was not detected.
Mass spectrometry was also used to measure the reduction of iron levels. The inductively coupled plasma mass spectrometer (ICP-MS) based quantification method has been commonly used to determine accurate concentration of metals in complex matrices. Specific iron detection in our samples was performed with an ICP-MS 7500cx (Agilent Technologies, Tokyo, Japan) equipped with an octopole collision/reaction cell system. Two isotopes of iron, 56Fe+ and 57Fe+, were monitored using He as collision gas at 4.0 ml min-1 to reduce the 40Ar16O+ and 40Ar16O1H+ interferences. The RF power was 1450 W and carrier gas flow rate was 1.02 L min-1. Apo-transferrin medium was filtered through a 10 kD membrane filter before analyzed by ICP-MS. Total iron levels were lowered from 57.15 ppb (± .84), to 5.93 (± 1.27) ppb when 5 μM of apo-transferrin was added to the medium, a reduction of ~90% of the total iron. Standard deviations were calculated from 3 biological replicates.
2D-gel electrophoresis and statistical analysis
Fifty μg of protein were mixed with rehydration buffer (9 M urea, 2% CHAPS, 1% dithiothreitol (DTT), and carrier ampholyte (pH 3–10) solution, 20 μl/1 ml). A total of 350 μl of this rehydration solution was loaded onto 24 cm immobilized protein gradient (IPG) strips pH 3–10 (Amersham Biosciences, GE Healthcare, Piscataway, NJ) and incubated (rehydrated) for 12 hr. Isoelectric focusing (IEF) was performed with an IPGphor system (Amersham Biosciences) using a step gradient starting from 0–500 V for 1 hr, 500–1000 V 1 hr, 1000–8000 V 12.5 hr, and 8000V for 2.5 hr. Following IEF, strips were stored at -70°C.
Strips were then equilibrated for 30 min in 15 ml of 6 M urea, 20% glycerol, .05 M Tris, 2% SDS, 75 mg DTT, and a few crystals of bromophenol blue. Strips were then loaded onto a 10% SDS PAGE gel. The second dimension was run at 200 V for 15 min followed by 300 V for 4–5 hr. Gels were next fixed for 15 minutes in 10% methanol/7% acetic acid. Gels were then stained overnight with SYPRO Ruby fluorescent dye and destained for 20 minutes in fixing solution. Gel images were produced using a GE Healthcare Typhoon scanner. Before image analysis, gels were washed with water for 5–10 min.
2D gel spot detection, matching, and spot volume, calculations were performed using SameSpots (Nonlinear Dynamics, Durham, NC) software on three biological replicates, six gels per 24 and 48 hr apo-transferrin treatments and their respective controls. Spot volumes for all protein spot matches within each analysis were calculated and normalized to the total spot volume yielding a spot volume ratio. This ratio enabled the calculation of average abundance changes across all 3 replicates within each test group and the application of ANOVA statistical test. Individual spots were ranked by p-value from a one way global ANOVA analysis, with maximum fold change based on spot normalized volume. P values of < 0.05 were considered statistically significant and reflect the probability that the observed change did not occur from random noise.
H. capsulatum viability test
To verify that the proteins found altered in our analysis did not result from the death of H. capsulatum, the organism was plated on solid medium following 24 and 48 hr growth with apo-transferrin. One hundred yeast were plated from cultures growing with or without apo-transferrin for 48 h. Eighty four colonies were counted on plates from H. capsulatum cultures containing apo-transferrin while 82 were counted from cultures free of apo-transferrin. Thus any changes we might find would be a direct response from the stress we were applying, and not from H. capsulatum death.
Protein bands were excised and diced into small pieces (~1 mm) and placed into 0.65 mL siliconized tubes (PGC Scientific). Each gel piece was briefly incubated and washed with 100 μl of water. Gel pieces were resuspended in 100 μl of acetonitrile and placed in a speed vacuum until dry. Next, 30 μL of 10 mM DTT in 25 mM ammonium bicarbonate was added to the dried gel pieces. Samples were vortexed and centrifuged briefly. The reaction was allowed to proceed at 56°C for 1 hr. The supernatant was removed and 30 μl of 55 mM iodoacetamide was added to the gel pieces. Samples were vortexed and centrifuged briefly. The reaction was allowed to proceed in the dark for 45 min at room temperature. Supernatant was removed and the gels were washed with 100 μl of ammonium bicarbonate by vortexing for 10 min. Supernatant was removed and gel pieces were dehydrated in 100 μL of 25 mM ammonium bicarbonate/50% ACN, vortexed for 5 min, and centrifuged. This step was repeated. Gel pieces were placed in a vacuum centrifuge until dry. Thirty microliters of a 12.5 ng/ml trypsin solution was added to the gel pieces and rehydrated on ice at 4°C for 10 min. Supernatant was removed, samples were centrifuged, and 25 μL of 25 mM of ammonium bicarbonate was added. Samples were centrifuged briefly and incubated at 37°C overnight. Following overnight digestion the solution was placed into a clean 0.65 mL siliconized tube. To the gel pieces, 50 μL of 50% acetonitrile/5% trifluoroacetic acid acid (TFA) was added. Samples were vortexed for 30 min, sonicated for 5 min, and the solution was combined with the first aqueous extraction. This was repeated and samples were then dried in a vacuum centrifuge.
MALDI-TOF mass spectrometric analysis
Dried peptide extracts were dissolved in 5 μL of water. Next, 2.5 μL of sample was mixed with 2.5 μL of alpha-cyano-4-hydroxycinammic acid in 50% acetonitrile/5% TFA, 10 mg/mL, and 1 μL of sample was delivered to a gold plated matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer (MS) sample plate. Mass spectra were collected on a Voyager-DE PRO (Applied Biosystems, Foster City, CA) MALDI-TOF MS. Each spectrum was an average of 200 laser shots. Trypsin autodigestion peaks (2211.11 and 842.51) were used to calibrate each spectrum.
Raw MALDI-TOF data were processed using manufacturer-supplied Data Explorer software, with the following settings: Baseline correction parameters; peak width 32, flexibility .5, and degree .1 Noise filter/smooth was selected with a correlation factor of .7. Each MALDI spectrum was manually inspected to create a peak list. To control for peaks generated from keratin and trypsin, a spot was cut from an area of the gel containing no protein, and prepared with each sample. Peaks which were in both keratin/trypsin control and sample spectra were excluded from peak lists.
For nanoflow liquid chromatography (LC)-MS/MS analysis, dried peptide extracts were dissolved in 5 μL of 2% acetonitrile, 0.1% formic acid in water. Resuspended samples were loaded onto a Symmetry 5 μm particle, 180 μm × 20 mm C18 precolumn (Waters, Milford, MA), then washed 5 min with 1% acetonitrile in 0.1% formic acid at a flow rate of 20 μL/min. After washing, peptides were eluted and passed through an Atlantis 3 μm particle, 75 μm × 100 mm C18 analytical column (Waters, Milford, MA) with a gradient of 1–80% Acetonitrile in 0.1% formic acid. The gradient was delivered over 120 min by a nanoACQUITY UPLC (Waters, Milford, MA) at a flow rate of 250 nl/min, to a fused silica distal end-coated tip nano-electrospray needle (New Objective, Woburn, MA). Data were collected on a Q-TOF Premiere mass spectrometer (Waters, Milford, MA) set for MS survey scans and automatic data-dependent MS/MS acquisitions, which were invoked after selected ions met preset parameters of minimum signal intensity of 10 counts per second, ion charge state 2+, 3+, or 4+, and appropriate retention time. Survey scans of 1 s were followed by collision induced dissociation (CID) of the three most intense ions for up to 6 s each, or until 10,000 total MS/MS ion counts per precursor peptide were attained.
Raw LC-MS/MS data were processed using manufacturer-supplied ProteinLynxGlobalServer 2.2 software, with the following settings: Adaptive background subtraction of polynomial order 5 below a 35% curve, two smooths with a window of three channels in Savitzky Golay mode, followed by fast deisotoping and centroid calculation of the top 80% of peaks based on a minimum peak width of 4 channels at half-height. On the basis of these parameters, pkl files incorporating parent ion mass and peak lists for each corresponding MS/MS spectrum were generated.
Protein/genomic databases 1, 2 and 3 were constructed beginning with the H. capsulatum G217B strain genome obtained from Washington University's Genome Sequencing Center website http://www.genome.wustl.edu/home.cgi. Database 1 was constructed using only Fgenesh and parameters setup by Softberry http://www.softberry.com/berry.phtml. Database 2 was assembled using publicly available EST libraries, also retrieved from Washington University's Genome Sequencing Center website http://www.genome.wustl.edu/home.cgi. EST's were generated from H. capsulatum cDNA under both mycelial and yeast phases, and assembled into full gene sequences with the aid of the contig assembly program (CAP)3 genome assembly program . Database 3 was assembled using a combination of the gene finding programs Fgenesh, EAnnot, GeneWise, and SNAP [28, 30–32].
MALDI-TOF MS and LC-MS/MS data were used to search in-house databases 1–3 and publicly available fungal databases using MASCOT (version 2.1, Matrix Science, London, United Kingdom). A protein match from either of the 3 databases would count as a hit. Only protein matches deemed statistically significant (p < .05) by the MASCOT algorithm were accepted as protein sequence matches. For MALDI-TOF data, a 50 ppm mass accuracy threshold was set while a minimum precursor and fragment-ion mass accuracy of 1.2 and 0.8 Daltons was required for LC-MS/MS data. Up to 1 missed trypsin cleavage was allowed while fixed and variable carbamidomethyl were selected for separate searches. A decoy database was constructed by scrambling our in-house database sequences, and used to estimate false positive protein ID rates. Searching MS data against the decoy database did not result in any significant MASCOT scores. In order to annotate the protein sequence matches obtained from searching databases 1–3, each sequence was BLAST searched against the NCBInr sequence database.
Western blot analysis
H. capsulatum proteins prepared for 2D gel analysis were also probed using different antibodies specific for either cyclophilin or HSP 70. Ten micrograms of protein was mixed with 1× sample buffer (5×; 200 mM Tris-HCl, pH 6.8, 50% glycerol, 5% SDS, .5% bromphenol blue, and 5% beta-mercaptoethanol) heated at 95°C, and loaded onto a 12% SDS PAGE gel. Gels were run at 150 V for ~2 hrs. Following electrophoresis, gels were incubated in transfer buffer (25 mM Tris, 192 nM glycine, 15% v/v methanol) for 15 min. Proteins were transferred from 1D gels to nitrocellulose membrane overnight at 30V and 60V for 1 hr. Following transfer, each membrane was placed in 20 ml of blocking buffer for 1–2 hr. Blocking buffer consisted of 1× Tris-buffered saline (TBS) 5 g of non-fat dried milk/2.5 ml of 10% Tween. 10× TBS consisted of 24.2 g Tris base, 80 g of NaCl, pH 7.6. Primary antibody was added in a 1/5000 (v/v) ratio of antibody to blocking buffer and the membranes were incubated overnight at 4°C. This was followed by five 5 min washes in blocking buffer. Secondary horseradish peroxidase (HRP) conjugated antibody was added in a 1/1000 ratio of antibody to blocking buffer and the membranes were incubated for 1 hr. Membranes were then washed 5 times in 1× TBS for 5 min each. Immunoreactive bands were visualized using BioRad HRP color development reagent 4-chloro-1-naphthol and the AlphaImager 950 high-performance CCD camera. Spot densities were quantified using AlphaImager software.
2D gel analysis of H. capsulatum isolated from IFN-γactivated macrophages
Bone marrow derived macrophages were harvested 5 days following incubation with 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF). Macrophages were incubated for 24 hr with 5 ng/ml of IFN-γ. Leucine uptakes assays, as previously described7, showed that macrophages were activated to inhibit H. capsulatum growth by 80% (data not shown). Macrophages were infected with H. capsulatum at a multiplicity infection of 5 for 24 hrs. Following 24 hr incubation, macrophages were washed to remove any H. capsulatum that had not been ingested. Macrophages were then lysed in a buffer containing 19%, ethanol, .1% SDS, and 1% phenol. An H. capsulatum control was also prepared under the exact conditions except for the addition of macrophages. Cells were centrifuged at 2000 G's and the supernatant containing macrophage debris was removed. H. capsulatum cells were washed, lysed, and 2D gel analysis was performed as previously described. In order to retrieve an adequate amount of H. capsulatum protein approximately 3.0 × 107 macrophages needed to be infected for one biological replicate.