Strains and Growth Conditions
Escherichia coli JM109 (Promega, Madison, WI) was used for cloning, and E. coli BL21(DE3) (Novagen Inc, Madison, WI) was used for the expression of recombinant histatin 3 variants. Bacteria were grown on Luria-Bertani (LB) agar plates or in modified LB liquid medium containing ampicillin at concentrations of 50 µg·l−1 and 100 µg·l−1, respectively. C.albicans ATCC44505 was grown on Sabouraud dextrose agar (Difco, Detroit, MI) at 37 °C for 48 hrs.
PCR Reactions and Amplification of Histatin 3 Variants
The PCR primers used in this study are listed in and all reagents were from Invitrogen (Carlsbad, CA). Standard PCR reactions were performed using a Gene Amp PCR system 2400 (Applied Biosystems, Foster City, CA) with an initial denaturation step at 94°C for 4 min followed by 35 cycles at 94°C (30 s), 55°C (30 s), and 68°C (45 s) and finally a 10 min extension at 68°C. Reaction products were analyzed on a 2% agarose gel and DNA fragments were excised and purified using standard procedures.
PCR primers used in this study.
Plasmid Construction and Transformation
ReHst3 1-mer, reHst3 2-mer, reHst3 3-mer, and reHst3 4-mer constructs were cleaved with BamHI and ligated into the BamHI site of the pGEX-3X, which is a bacterial GST expression vector (GE Healthcare, Piscataway, NJ). Transformation of the resulting plasmids into JM109 cells was performed according to the manufacturer’s instructions.
PCR screening of transformant colonies was performed with pGEX-3X sense and antisense primers and products were analyzed on a 2% agarose gel. Positive colonies were inoculated into 5 ml of LB medium and plasmids were isolated from cultures grown for 16 hr at 37°C and sequenced in the Department of Genetics and Genomics at Boston University Medical Center to verify in frame ligation of histatin inserts.
Expression of Recombinant Histatin 3 Variants
To express the recombinant histatin 3 constructs, E. coli BL21 (DE3) cells were transformed with each of the pGEX-3X expression plasmids. Single colonies were transferred to 100 ml of LB/amp medium and cultures were grown for 18 hr at 37°C with vigorous shaking. A 1.0 ml aliquot of the liquid culture was transferred into 2 l of LB/amp medium and the culture was incubated as described above until the OD600 reached a value of 0.8 to 1.2 absorbance units. Expression of recombinant protein was induced by the addition of IPTG to a final concentration of 0.1 mM. After 3 hr at 37°C, cells were harvested by centrifugation and the pellet was washed twice with ice cold 20 mM Tris-HCl at pH 7.5 and resuspended in lysis buffer containing 20 mM Tris-HCl at pH 7.5, 5 mM MgCl2, 10 µg/ml DNase I and 0.25 mg/ml lysozyme. Subsequently, cells were disrupted on ice using a Branson sonicater with 3 bursts of 30 sec each and the lysate was centrifuged at 18,000×g for 20 min at 4°C.
Membrane Protein Preparation
Preliminary experiments showed that the reHst3 1-mer and reHst3 2-mer were present in the bacterial membrane fraction while the larger constructs were present in inclusion bodies. To isolate the reHst3 1-mer and reHst3 2-mer products, the pellet from the cell lysate was suspended in extraction buffer containing 20 mM Tris-HCl, 1% Triton X-100, 1% deoxycholic acid and 1 mM EDTA, pH 7.5. Membranes were solubilized by sonication to clarity. The solution was dialyzed against deionized water for 16 hr at 4°C and the resulting membrane protein preparation containing the reHst3 1-mer or reHst3 2-mer were lyophilized and stored at –20°C until use. To isolate reHst3 3-mer and reHst3 4-mer from inclusion bodies, the pellet from the cell lysate was suspended in extraction buffer and membranes were dissolved by sonication. Inclusion bodies were recovered by centrifugation at 11,000×g for 20 min at 4°C. The pellet containing the inclusion bodies was suspended in 20 mM Tris-HCl, containing 1 mM DTT and 6 M urea and solubilized by sonication. The solution was dialyzed for 16 hr against deionized water resulting in the precipitation of inclusion bodies which were subsequently harvested by centrifugation at 11,000×g for 20 min at 4°C. Pellets were washed with deionized water, centrifuged again and stored frozen until further use.
Cyanogen Bromide Cleavage
Fusion proteins were cleaved with CNBr at a methionine residue introduced between the GST fusion partner and the first amino acid of the recombinant histatin variants. Inclusion body preparations or lyophilized membrane protein preparations were dissolved in a minimal amount of 70% formic acid in a glass vial. The reaction mixture was agitated on a magnetic stirrer, solid CNBr was added to a final concentration of approximately 10 mg ml−1, and the vial was purged with nitrogen. After 21 hr of incubation, reactions were diluted 3-fold with water and the HCN formed was purged with nitrogen. The solutions containing the CNBr cleavage products were frozen and lyophilized.
CNBr digests were resuspended in a solution containing 4 M urea and 0.1% trifluoroacetic acid. Samples were centrifuged for 20 min at 25,000×g, dialyzed, passed through a filter with a pore size of 0.45 µM and subjected to reversed-phase HPLC using a preparative C-18 column (2.15×30 cm; ODS 120T, Tosohaas, Montgomeryville, PA). Proteins were eluted at a flow rate of 5 ml min−1 with a gradient of eluent A (H2O, trifluoroacetic acid 0.1%), and eluent B (80% acetonitrile, 20% H2O trifluoroacetic acid 0.1%). The proportion of eluent B was linearly increased from 10% to 40% over a time period of 80 min. For each purification, fractions containing the recombinant histatin 3-mer of interest were combined, concentrated to approximately 30% of the original volume by flash evaporation (Rotavapor M, Büchi Labortechnik AG, Flawil, Switzerland) and lyophilized. All fractions were dissolved in 5 ml of water containing TFA (0.1%) and further purified on the same column by isocratic elution using the appropriate acetonitrile concentration containing 0.1% TFA. Fractions containing purified histatin 3 variants were pooled, concentrated by flash evaporation and lyophilized.
Protein Concentration Determination
The protein concentrations in stock solutions of recombinant histatins in water were determined by measuring the absorbance at 280 nm (1). Molar extinction coefficients (ε) of 5120, 6400, 7680 and 8960 (M−1.cm−1) were used for the reHst3 1-mer, 2-mer, -3-mer and 4-mer, respectively.
For blastoconidia killing assays C. albicans
cells were grown at 30°C in ¼ strength Sabouraud dextrose broth (Difco). Partial strength (¼ diluted) broth was used since fungal growth in this medium shows the typical lag-, log- and stationary- phases, whereas in undiluted broth the point at which the stationary phase was reached was less discernible (unpublished observations). A sample of exponentially growing cells was diluted in 10 mM potassium phosphate buffer (PPB), pH 7.4, to give a cell density of approximately 105
. Aliquots of 50 µl of this suspension were added to the wells of a 96-well microtiter plate and cells were allowed to attach for 15 min. Subsequently, 50 µl of the histatin solutions (in 10 mM PPB) were added to the wells and the microtiter plate was incubated for 60 min at 37°C. After incubation, wells were washed with 10 mM PPB and 100 µl of molten Sabouraud's dextrose broth containing 2% agarose (45°C) was added to each well. After 5–6 hr incubation at 30°C, surviving cells had divided and formed colonies whereas dead cells remained as single cells. Microcolonies, defined as a cluster of more than five contiguous cells, and single cells were counted to determine cell viability as described previously 
For germ tube killing assays, C. albicans cells were grown for 36 hr on Sabouraud dextrose agar, suspended in 10 ml RPMI-1640 medium buffered with 10 mM HEPES to pH 7.4, and diluted in the same medium to a concentration of 105 cells ml−1. Aliquots of 50 µl of this suspension were added to the wells of a microtiter plate and incubated for 3 hr at 37°C. These conditions typically converted over 95% of blastoconidia into germinated cells. Subsequently, plates containing the attached germinated cells were washed twice with 10 mM PPB, and 50 µl of the recombinant protein solutions (in 10 mM PPB) were added. Plates were incubated at 37°C for 10, 30, 60, 90 or 120 min and overlayed with molten Sabouraud's dextrose broth (45°C) containing 2% agarose. After 6–8 hr of incubation at 30°C surviving germinated cells growing in micro colonies and single dead cells were counted to determine the percentage of cell survival.
Statistical differences in LC50 values between blastoconidia and germ tubes treated with reHst3 1-mer, 2-mer, 3-mer, or 4-mer were investigated with the Friedman test (1-sided), with a level of significance set at p<0.05, using SPSS v20.0 software.