C. albicans ATCC 90028 was obtained from ATCC and grown in Sabouraud dextrose media. The protoplasts were prepared from an overnight culture of fully-grown mycelia using zymolyase (Cat. No. L5263, Sigma, St. Louis, MO, USA) as per the manufacturer’s instructions. Protoplasts were lysed in a chaotrophic salt solution and genomic DNA was isolated according to manufacturer’s instruction (Qiagen, Hilden, Germany). The final eluate was reprecipitated with ammonium acetate and isopropanol and DNA quantified by UV spectrophotometer (Spectramax Gemini XS, Molecular devices, CA. USA).

Oligos were designed with codon changes made for 4 ^th and 271 ^st serine residues. Full length HOS2 gene was amplified by using 4 different primers ( Table 1) using splicing by overlap extension (SOE PCR), so that codon usage could be maintained in any heterologous expression system. The full-length blunt end PCR product was cloned in to pJET1.2 cloning vector (CloneJET PCR Cloning Kit Thermo Scientific) and confirmed by restriction digestion using BamH1 and Not1. DNA sequencing using T7 promoter (forward, 5´-TAATAC GACTCACTATAGGG-3´) and pJET1.2 (reverse, 5´-AAGAACATCGATTTTCCATGGCAG-´3) sequencing primer ascertained the sequence of the recombinant Hos2 gene.

Recombinant baculoviruses were generated using the Bac-to-Bac ^® baculovirus expression system according to the instructions of the manufacturer (Cat. No. 10359-016, Invitrogen, Carlsbad, USA). Sf9 insect cells (Cat. No. B825-01, Invitrogen, Carlsbad, USA) were cultured in complete TMN-FH medium (Cat. No. 554760, BD Bioscience, NJ, USA) served as host cells for virus generation and/or protein production. The HOS2 gene was cloned in frame with N-terminal hexa-histidine tag into the transfer vector pFastBac-HT B (Cat. No. 10584-027, Invitrogen, Carlsbad, USA) and transformed in to Escherichia coli DH10Bac cells (Cat. No. 10361-012, Invitrogen, Carlsbad, USA). Sf9 cells were transfected using cellfectin reagent (Cat. No. 10362-100, Invitrogen, Carlsbad, USA) with the recombinant bacmid, and the resulting viruses were tested for their ability to produce recombinant Hos2 protein using western blot. Production cultures were performed T-150 cell culture flasks (Greiner bio-one GmbH, Germany) at a density of 16×10 ⁶ cells per flask. The cultures were inoculated with recombinant baculovirus stocks at multiplicities of infection of 10. At 4 days after infection, cells were harvested by centrifugation at 1200 g for 15 min at 4°C in a tabletop centrifuge (Model 5804R, Eppendorf AG, Hamburg, Germany). Cell pellets were stored at -80°C until protein purification.

The infected Sf9 cell pellets were resuspended in in-house ice-cold lysis buffer [10 mM Tris pH 7.5, 130 mM NaCl, 1% triton X-100, 10 mM NaF, 10 mM NaPi (sodium phosphate), and 10 mM NaPPi (sodium pyrophosphate)] with 1 X EDTA free protease inhibitor cocktail (Cat. No. 539134, Calbiochem, Merck-Millipore, San Diego, CA). Insoluble material was removed by centrifugation (Model 5804R, Eppendorf AG, Hamburg, Germany) at 16000 g, 4°C for 15 min. The cleared lysate was processed for nickel affinity chromatography. Briefly, the cell supernatant was loaded onto a 3-ml nickel-nitrilotriacetic acid-agarose resin (Ni-NTA agarose, Cat. No. 30210, Qiagen, Hilden, Germany) packed column pre-equilibrated with equilibration buffer (25 mM Tris-Cl pH 8.0, 300 mM NaCl). The column was washed with wash buffer (25 mM Tris-Cl pH 8.0, 300 mM NaCl, 20 mM imidazole). His-tagged Hos2 protein was eluted with buffer containing 300 mM imidazole (Sigma, St. Louis, MO, USA). The Hos2 protein preparation was dialyzed into buffer (25 mM Tris, pH 8.0, 138 mM NaCl, 10% glycerol) and kept in 100 μl aliquots at -70°C. The concentration of Hos2 protein in the final eluate was estimated by Bradford assay (Bio-Rad Laboratories, CA, USA).

HDAC inhibitors, namely SAHA, TSA or MS-275 were dissolved in DMSO as 10 mM stock and subsequently diluted in 1X assay buffer (50 mM Tris Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂, 1 mg/ml BSA). Enzymatic assay was carried out using the fluorogenic Class I HDAC substrate, Boc-Lys (Ac)-AMC (Cat. No. I1875, Bachem AG, Bubendorf, Switzerland). Briefly, 0.5 μg of purified recombinant protein in a volume of 10 μl of assay buffer was incubated with 50 μl of appropriate concentration of HDAC inhibitors and 20 μM of substrate at 37°C for 1 hr. Reactions were terminated by the addition of trichostatin A (TSA)/suberoylanilide hydroxamic acid (SAHA), trypsin (1 mg/mL) (Sigma, St. Louis, MO, USA) and left at 37°C for 15 min, before reading the plates in a fluorimeter (Spectramax Gemini XS, Molecular devices, CA. USA) at wavelengths 360 nm (ext) and 460 nm (emi).

Female BALB/c mice were purchased from The Jackson Laboratory (Bar Harbor, ME) at 4 weeks of age and then housed at the animal facility, Orchid chemicals and Pharmaceuticals for 2 weeks in a specific-pathogen free facility with a 12 h light cycle (6 am–6 pm) and a 12 h dark cycle (6 pm–6 am). Groups of four mice were housed in sterilised polypropylene cages covered with stainless steel grid top, lined with autoclaved clean rice husk bedding. All animal experimentations were approved by the institutional animal ethics committee (Protocol No. 01/IAEC-05/PPK/2009). The native Hos2 protein (expressed in pET-32 bacterial vector system and purified using nickel affinity chromatography under denaturating conditions) was emulsified in complete Freund’s adjuvant and injected subcutaneously into two female BALB/c mice (20 μg/mice). Booster doses of the deacetylase antigen (20 μg/mice) were given on the 14 ^th and 21 ^st day in Freund’s incomplete adjuvant. Mice were bled 7 days after the second booster dose and polyclonal anti sera was separated after clotting the blood ²⁶ .

Jurkat, a human T lymphocyte cell line, and HeLa, a human cervical adenocarcinoma cell line was obtained from ATCC and were cultured in DMEM (Gibco, Life technologies) supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin (Gibco, Life technologies).

Acetylated histones were isolated from HeLa cells treated with the HDAC inhibitor SAHA as per published protocol ²⁷ . The histone pellet was then resuspended in ultra pure water, stored in 50 μl aliquots at -70°C and the protein concentration was determined using a BCA kit (Pierce).

C. albicans ATCC 90028 mycelia (~ 5 gm wet weight) were washed with water, centrifuged at 10,000 rpm for 10 minutes at 4°C and the mycelial pellet was resuspended in 50 ml of 0.1 mM Tris-HCl, pH 9.4, 10 mM DTT. The sample was incubated with shaking at 30°C for 15 min and pelleted. The pellet was washed with 50 ml of sorbitol/HEPES buffer (1.2 M sorbitol, 20 mM Hepes, pH 7.4) and left resuspended in the same buffer containing lyticase (1000 units) overnight at 30°C for spheroplasting ²⁸ . The sample was pelleted and proceeded to histone isolation using acid extraction as described previously ²⁹ . Acetone was added at 3:1 (vol/vol) to precipitate the histones, which were subsequently dissolved in 10 mM Tris, pH-8.0.

Acetylated histones isolated from SAHA treated HeLa cells were used for the deacetylation assay with recombinant Hos2 enzyme. In brief, purified acetylated histones (2 μg) were incubated with different amounts of recombinant Hos2 enzyme. Histone deacetylation with 300 ng of rhHDAC1 or rhHDAC6 (expressed in-house using Baculo-viral expression system) was used as a positive control. Deacetylation assays were carried out for 1 hr at 37°C in reaction buffer (50 mM Tris Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂, 1 mg/ml BSA). At the end of incubation, the reaction was stopped by the addition of 1X Lammeli sample buffer. The protein samples were resolved by SDS-PAGE and immunoblotted with anti-acetylated H3 Histone (Ac-K-9) antibody to study the deacetylation of H3-histone by rHos2.

Whole cell extracts from TSA (Sigma, St. Louis, MO, USA) treated Jurkat cells were used for the α-tubulin deacetylation assay with recombinant Hos2 enzyme. In brief, whole cell extract (10 μg) were incubated with 5 and 8 μg of recombinant Hos2 protein. Deacetylation assays were carried out for 3 hr at 37°C in reaction buffer (50 mM Tris-Cl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 2.5 mM MgCl ₂). At the end of incubation, the reaction was stopped by the addition of 1X Lammeli sample buffer. The protein samples were resolved by SDS-PAGE and immunoblotted with either anti-acetylated α-tubulin or anti-α-tubulin antibodies.

Recombinant Hos2 protein or the acetylated/deacetylated nuclear histones from deacetylation assays were separated on 10–15% SDS-polyacrylamide gels. The gels were stained with coomassie blue (Phast gel, Aersham) or electro-blotted onto nitrocellulose membrane (HyBond-C, Amersham), which was blocked with 5% nonfat dry milk (Cat No. 70166, Fluka, Sigma-Aldrich) in 0.1% Tween- 20 in TBS for 1 hr, followed by overnight incubation at 4°C with either the polyclonal mouse serum (dilution 1:4000 of the sera), rabbit polyclonal anti-acetylated H3-Histone (dilution 1:4000, Ac-K-9 of H3-Histone, Cat. No. 06-599; Millipore), mouse monoclonal anti-acetylated tubulin (dilution 1:15000, Cat. No. T7451, Sigma) or mouse monoclonal anti-tubulin antibody (dilution 1:150000, Cat. No. T6199, Sigma). After incubation with the appropriate horseradish peroxidase conjugated secondary antibodies (Bovine anti-mouse IgG HRP conjugate-dilution 1:7500, Santa Cruz, SC-2371, Goat anti-rabbit IgG-HRP Upstate (Millipore) 12–348, dilution 1:4000), supersignal west pico substrate (Thermo Scientific Pierce) was used for detection.

HeLa nuclear extract (2 μg) or recombinant Hos2 enzyme (300 ng) was incubated with NAD+ (500 μM), Fluor de Lys ^®−Sirt1 (Enzo lifescience), varying concentrations of resveratrol (Cat. No. 0219605205, MP Biomedicals, Ohio, USA) (30, 100, 250 and 500 μM) in presence or absence of the pan-HDAC inhibitor trichostatin, at 37°C for 30 min. The reaction was carried out following manufacturer’s protocol (Enzo lifescience).

Codon usage in C. albicans is different from standard genetic code. The 4 ^th serine and the 271 ^st serine of HOS2 are translated as leucine in both mammalian and in insect cell expression systems. Hence, these codons were mutated using oligonucleotide primers to express serine in the recombinant Hos2 protein. The PCR product was cloned in the pFastbac-HTB shuttle vector and subsequently into baculo viral DNA using the Bac-to-Bac expression system.

The Hos2 enzyme was expressed in the baculoviral-insect cell expression system as a NH ₂-terminal hexa histidine tagged fusion protein, which is detected on Western blot as a ~ 52 kDa protein using our own polyclonal anti-Hos2 anti-sera raised against Hos2 protein in mice. SDS-PAGE analysis of purified protein revealed a major band at ~ 52 kDa ( Figure 1A).

Recombinant Hos2 enzyme was assayed for deacetylase activity using the synthetic deacetylase substrate, Boc-Lys (ac)-AMC. The total activity with Boc-Lys (ac) AMC showed the enzyme to be active in deacetylating the lysine residue and the activity increased with an increase in Hos2 concentration ( Figure 1B).

The inhibition of deacetylation activity of recombinant Hos2 was studied using classical HDAC inhibitors namely SAHA, TSA and MS-275. TSA was very potent in inhibiting Hos2 with an IC ₅₀ of 2.8 nM, SAHA inhibited Hos2 with an IC ₅₀ of 65 nM ( Figure 2, Table 2). However, MS-275 showed > 50% inhibition of Hos2 activity only at 10 μM ( Table 3).

The ability of purified Hos2 protein to deacetylate acetylated histones was examined in vitro using acetylated nuclear histone preparation made from SAHA treated HeLa cells. The nuclear histones from HeLa cells were isolated using a modified protocol of Shechter et al. ²⁷ and established the deacetylation assay using rhHDAC1/rhHDAC6 as controls along with rHos2. In these assays it was found that 0.3 μg of rhHDAC1 was able to deacetylate the nuclear acetylated histones as detected by an anti-H3-K9 histone antibody. Recombinant hHDAC1 was more potent in deacetylating lysine residues in H3-histones than rhHDAC6. However, no significant deacetylation of H3-Histone was seen with 0.3 μg of Hos2 ( Figure 3A). Similar results were obtained with acetylated histones from Candida in the in vitro histone deacetylation assay with purified Hos2 protein (up to 3 μg, Figure 3B). In contrast, when recombinant Hos2 was incubated with mammalian acetylated α-tubulin, a significant reduction in acetylation of α-tubulin (K40) could be observed ( Figure 4).

Since Hos2 is a putative NAD+ dependent deacetylase, a feature of sirtuin class of deacetylases, it was of interest to check if the Hos2 protein displayed any sirtuin like activity. The sirtuin-like activity in response to the sirtuin activator resveratrol was studied using fluor-de-lys, a synthetic Sirt1 substrate. HeLa nuclear extract was used as a positive control for sirtuin activity. In the presence of TSA where the HDAC activities are inhibited no appreciable NAD+ dependent deacetylase-like activity was seen, following incubation of Hos2 with different concentrations of resveratrol ( Figure 5).