Generation of the recombinant mlDL isoforms in E. coli
DNA fragments that encode the mlDL-Ds1, 2, 3 [25] were de novo synthesised from oligonucleotides. These fragments were optimised for expression in E. coli. A list of the oligonucleotides and a scheme for synthesising the fragments are presented in Additional file 2: Table S1 and Additional file 3, respectively. The commercially available plasmid pET-15b (Novagen, USA) was modified by adding new multiple cloning sites. The resulting plasmid was termed pET15MCS (Additional file 3: Figure S2). The DNA fragments encoding the mlDL isoforms and plasmid pET15MCS were treated with the restriction endonucleases BamHI and SalI. The restriction products were ligated. Prepared plasmids were subsequently used for the standard transformation procedure of E. coli Top10 cells. PCR selection of the colonies was performed using the oligonucleotides T7 and T7t. Next, the plasmids were extracted and sequenced. Because of the identity of the plasmids that encoded the three mlDL isoforms, we termed them pET15/Dest. A map of the pET15/Dest plasmid is shown in Additional file 3: Figure S3. The E. coli strain BL21(DE3)-gold was transformed by the plasmids that encoded the three mlDL isoforms. The transformed cells were plated on a selective solid medium containing ampicillin (150 ng/ml) and then incubated at 37 °C for 16 h. Individual colonies of E. coli were inoculated into super broth (SB) medium containing potassium nitrate (2 g/l) and cultured at 37 °C overnight. The cultures were then added to the fresh SB medium containing potassium nitrate at a 1:20 dilution, and the cells were grown until optical density OD600 = 0.8. Next, lactose was added to a final concentration of 10 mM, and the cultures were incubated again at 37 °C for 6 h. Inclusion bodies were then isolated and solubilised in denaturing buffer containing 8 M urea. The mlDL-Ds1, 2, 3 were isolated using metal chelate affinity chromatography under denaturing conditions (for details, see Additional file 4).
The purified mlDL isoforms were diluted in 8 M urea at a final protein concentration below 1 mg/ml and renatured using stepwise dialysis against the solution containing 20 mM NaH2PO4, 150 mM NaCl with the following decreasing urea concentration: 4, 2, 1, and 0 M. Each step lasted at least 8 h. The precipitate was separated by centrifugation at 10,000 × g for 20 min.
MALDI-TOF analysis
The protein bands after 1D PAGE were subjected to trypsin ingel hydrolysis. Gel pieces (2 mm3) were excised and washed twice with 100 ml of 0.1 M NH4HCO3 and 40 % acetonitrile for 30 min at 37 °C, dehydrated with 100 ml of acetonitrile and air-dried. Then, they were treated with 4 ml of a 12.5 mg/ml solution of modified trypsin (Promega) in 40 mM NH4HCO3 and 10%acetonitrile for 16 h at 37 °C. The peptides were extracted with 8 ml of an aqueous solution of 0.5 % trifluoroacetic acid for 20 min. Aliquots (2 ml) of the sample were mixed on a steel target with 0.3 ml of a 2,5-dihydroxybenzoic acid (Brucker Daltonics, Germany) solution (75 mg/ml in 30 % acetonitrile/0.5 % trifluoroacetic acid). Mass spectra were recorded on an Ultraflex II MALDI-ToFToF mass spectrometer (Brucker Daltonics, Germany) equipped with an Nd laser. The [MH+] molecular ions were measured in reflector mode; the accuracy of the mass peak measurement was 0.005 %. The fragment ion spectra were generated by laser-induced dissociation slightly accelerated by low-energy collision-induced dissociation using helium as the collision gas. The accuracy of the fragment ion mass peak measurement was 5 Da. The MS/MS fragments were identified using the Biotools software (Brucker Daltonics, Germany) and Mascot MS/MS ion search. Protein identification was performed using a peptide fingerprint search with the Mascot software (MatrixScience Inc., USA). One missed cleavage, Met oxidation and Cys-ropionamide were permitted. Protein scores greater then 49 were supposed to be significant (p < 0.05).
Buffer solutions for activity assays (standard buffers)
We assessed the pH effect on the enzymatic activities using the following standard buffers [29]: citric acid/sodium citrate buffer for the pH range of 1.0–5.0, sodium citrate/sodium phosphate buffer for the pH range of 5.0–7.0, sodium phosphate/sodium hydroxide buffer for the pH range of 7.0–12.0, and potassium chloride/sodium hydroxide buffer for the pH range of 12.0–13.0. The total concentration of the components in each buffer was kept at 10 mM. After determining the optimal pH conditions that corresponded to the activity peaks, we assessed the ionic strength effect on the enzymatic activities at the predetermined optimal pH conditions. In this case, the ionic strength of the solutions varied with the concentration of NaCl.
Muramidase activity assay
Muramidase activity was determined via clarifying the suspension of lyophilised M. lysodeikticus cells (0.5 mg/ml; Sigma, USA) after treatment with the mlDL isoforms [30, 31]. Hen egg white lysozyme (HEWL; Sigma, USA) was used as a reference lysozyme with known muramidase activity under the definite experimental conditions: at 25 °C in 20 mM Na-phosphate buffer, pH 7.4. The concentrations of both mlDL isoforms and HEWL varied from 0 to 20 μg/ml, and the sample volume was 200 μl. Incubation took place in the wells of a 96-well plate at 25 °C for 30 min. The optical densities were measured using a photometer (Multiskan Ascent, ThermoFisher Scientific, USA) at the wavelength of 405 nm. The muramidase activities of the mlDLisoforms were calculated using the following formula:
$$ \mathrm{A}\ \left(\mathrm{activity}\ \mathrm{units}\right) = \left({{\Delta \mathrm{O}\mathrm{D}}_{405}}^{\mathrm{d}}{{/\Delta \mathrm{O}\mathrm{D}}_{405}}^{\mathrm{h}}\right)*{\mathrm{A}}^{\mathrm{h}}, $$
(1)
where ΔOD405
d and ΔOD405
h are the differences in optical densities were measured before and after treatment with the mlDL isoforms at different pH values and HEWL at pH 7.4, respectively, and Ah is the known muramidase activity of HEWL at pH 7.4, which was provided by the producer (in units). Negative values that were calculated using formula (1) were set to zero.
Lytic activity assay
Lytic activity was estimated by measuring the amount of total protein that was released from the E. coli (strain Top10) and B. subtilis (strain 186RT) cells following treatment with the mlDL isoforms. The overnight cultures of E. coli and B. subtilis were reseeded in fresh LB medium at a dilution of 1:20 and subsequently grown until OD600 = 1. Two hundred microlitres of the cell culture was precipitated by centrifugation at 10,000 × g for 2 min. Next, the pellet was resuspended in an equal volume of standard buffer followed by the addition of mlDL isoforms at concentrations of 20 μg/ml. The mixture was incubated at 37 °C for 30 min. Next, the sample was diluted with distilled water to a final volume of 1 ml and then centrifuged at 10,000 × g for 10 min. The supernatant was collected, and the amount of total protein released from the cells was measured using the Bradford method (QuickStart™ Bradford Protein Assay, Bio-Rad, USA) according to the manufacturer’s recommendations. It is well known that many gram-negative bacteria are lysed only in the presence of ethylenediaminetetraacetic acid (EDTA), which contributes to outer membrane disruption. Therefore, we have also determined lytic activity in the presence of EDTA (5 mM) in E. coli.
Isopeptidase activity assay
Isopeptidase activity was determined by cleavage of the chromogenic substrate L-γ-Glu-pNA (Sigma, USA) following treatment with the mlDL isoforms [32]. The mlDL isoforms at concentrations of 300 μg/ml were added to standard buffers containing L-γ-Glu-pNA at a concentration of 1 mg/ml. The solutions (each sample was 200 μl) were incubated in a 96-well plate at 37 °C for 48 h. The standard buffers containing L-γ-Glu-pNA without mlDL isoforms treatment were used as control samples to exclude pH and ionic strength effects on substrate integrity. The optical densities were measured using a photometer (Multiskan Ascent) at a wavelength of 405 nm. The isopeptidase activity was calculated using the following formula:
$$ \mathrm{A}\ \left(\mathrm{activity}\ \mathrm{units}\right) = \left({{\Delta \mathrm{O}\mathrm{D}}_{405}}^{\mathrm{d}}{{\hbox{--} \Delta \mathrm{O}\mathrm{D}}_{405}}^{\mathrm{c}}\right)*1000, $$
(2)
where ΔOD405
d and ΔOD405
c are the differences in the optical densities, which were measured before and after treatment with mlDL isoforms for the experimental and control samples, respectively, at the same pH values. Negative values that were calculated using formula (2) were set to zero.
Preparation of tryptic peptides from the mlDL isoforms
Trypsin (0.1 μg/ml; Gibco®, USA) was added to the solutions containing mlDL isoforms at the concentration of 200 μg/ml. The solutions were incubated at 37 °C for 1 h. Next, trypsin was inhibited by adding phenylmethanesulfonylfluoride (PMSF; Sigma, USA) at a concentration of 1 mM, and the solutions were subsequently incubated at room temperature for additional 3 h. We used a refolding buffer (20 mM NaH2PO4, 150 mM NaCl) that had undergone the same preparation procedures as a control solution.
Antibacterial activity assay
The antibacterial activity of the mlDL isoforms and their tryptic peptides was independently determined using the following two methods:
Method 1. Determination of the growth curves of bacteria treated with the mlDL isoforms and their tryptic peptides. The overnight cultures of E. coli and B. subtilis cells were inoculated into LB medium at a dilution of 1:20 and then the mlDL isoforms or their tryptic peptides were added at final concentrations varying from 0 to 20 μg/ml along with the twofold dilutions. The cells were subsequently grown in a 96-well plate at 37 °C for 7 h. The sample volume was 200 μl. The optical density (OD600) was measured every 30 min for each sample at a wavelength of 600 nm using a photometer. The total observation time was 7 h for each sample. Next, the growth curves were expressed as the time dependence of OD600.
Method 2. Determination of the minimum inhibitory concentrations (MICs) of the mlDLisoforms and their tryptic peptides [33]. A total of 150 μl of LB medium containing 5*105 CFU/ml E. coli or B. subtilis was inoculated into each well of a 96-well plate. The cells were incubated with mlDL isoforms at 37 °C for 16–18 h, and their tryptic peptides at the concentrations were varied from 0 to 20 μg/ml. Next, the samples were plated onto solid LB medium in Petri dishes. The Petri dishes were incubated at 37 °C for 18 h. The growth colonies were counted, and the MICs were determined.
Fibrinolytic activity assay
Fibrinolytic activity was assayed using the fibrin plate method [34] with a slight modification. Briefly, fibrin plates were prepared by the addition of 6 mg of fibrinogen (Tehnologia-Standart, Russia) and 0.5 U of thrombin (Tehnologia-Standart, Russia) to 15 ml of 0.1 M Na-phosphate buffer (pH 7.4) containing 0.15 M NaCl. The resulting solution was immediately inoculated onto a 90-mm Petri dish. Fibrin gel formation continued at room temperature for 4 h. Droplets (5 μl) of the solutions containing mlDL isoforms at the concentration of 1 mg/ml were carefully placed on the plate and incubated at room temperature for 48 h. We measured the diameters of the lysed zones produced by each droplet. The measurements of each lysed zone were performed in three directions followed by the calculation of the mean value. The experiments were repeated independently in three fibrin plates. Percentage of stabilized fibrin in fibrin clot was determined according to the amount of soluble protein in 2 % acetic acid [35]. Unstabilized fibrin is dissolved in acid, stabilized fibrin is precipitated.
Circular dichroism
The CD spectra were recorded using a Chiroscan CD spectrophotometer (Applied Photophysics, UK). The spectra weremeasured between 180 and 280 nm (1 nm step) at 20 °C. A 2 mm-pathlength cell with a detachable window was used. The protein concentrations were 2 mg/ml in the buffer (5 mM Na2HPO4, pH 2.2, 3.2 and 6.5).
