Bacterial culture conditions
P. gingivalis wild type strains ATCC 33277 (ATCC, Manassas, VA) and W50, and the W50-derived Kgp proteinase and Rgp proteinase mutant strains (K1A and E8, respectively) were a kind gift from Dr. M.A. Curtis (Molecular Pathogenesis Group, Queen Mary, University of London). P. gingivalis strains were grown in anaerobic conditions (80 % N2, 10 % CO2, and 10 % H2) at 37 °C in an anaerobic chamber (Concept 400 Anaerobic Workstation; Ruskinn Technology Ltd., Leeds, United Kingdom). The bacterial concentration was adjusted to correlate with approximately 109 CFU/ml, which was determined by viable count by culturing the bacteria on fastidious anaerobe agar (45.7 g/l, pH 7.2, Acumedia, Neogen, Lansing, USA), supplemented with 5 % defibrinated horse blood for 5 days.
The Lactobacillus strains L. plantarum NC8, L. plantarum 44048 and L. brevis 30670 (Culture Collection, University of Gothenburg, Sweden) were grown on deMan Rogosa Sharp (MRS, BD Science) supplemented with agar (Difco) at 37 °C for 24 h in a jar containing an oxygen-free environment (Anaerobic pouch system EZ, BD Biosciences, CA, USA). Fresh cultures were used to inoculate MRS broth (Difco, BD Biosciences, CA, USA), and grown statically and anaerobically for 24 h at 37 °C. Lactobacillus were grown from a 0.5 % inoculum for 24 h at 37 °C under anaerobic and static growth conditions, and were then used for further experiments. The two-peptide bacteriocin from L. plantarum NC8, PLNC8 α and β, were purchased from GL Biochem (Shanghai) Ltd, China. Amino acid sequences for the peptides are:
Liposomes were prepared according to methods that are well established in the field [17, 18]. Briefly, liposomes were prepared by dry film formation, hydration and finally extrusion through a polycarbonate membrane to form monodisperse large unilamellar vesicles. The lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) (Avanti Polar Lipids, Alabaster, USA) were mixed at molar ratios 1:99, 5:95 and 10:90 while dissolved in chloroform. A dry lipid film was formed by evaporation of the chloroform by nitrogen flow and overnight lyophilization. The film was hydrated with either 10 mM phosphate buffer (PB) pH 7 or 10 mM phosphate buffer saline (PBS) pH 7, and the solution was vortexed for 1 min and put on a shaker for 1 h before extruded 21 times through a 100 nm pore-sized polycarbonate membrane. For fluorescence leakage assay the lipid film was hydrated with buffer (PBS) containing self-quenching concentration (50 mM) of 5 (6)-carboxyfluorescein (CF) (Sigma Aldrich) and liposomes were prepared as described above. Removal of unencapsulated CF was done by gel filtration using a PD-25 column (GE Healthcare, Uppsala, Sweden) and liposomes with encapsulated CF were eluted with PBS.
Dynamic light scattering and Zeta potential
In order to mimic P. gingivalis membrane composition and potential, the hydrodynamic radius and zeta potential was measured on liposomes suspended in 10 mM PB pH 7 and suspensions containing microvesicles from P. gingivalis W50, using a Malvern ZetaSizer Nano S (Malvern Instruments Ltd, UK) and a disposable cuvette.
Carboxyfluorescein (CF) release assay
Leakage of the liposome encapsulated fluorophore CF due to additions of the bacteriocins was recorded using a fluorescence plate reader (Infinite 200, Tecan, Austria) where λex = 485 nm and λem = 520 nm. CF was encapsulated at self-quenching concentration, and CF release results in an increased fluorescence signal. Liposomes were diluted to 25 μM (total lipid concentration) in PBS, followed by additions of 0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1 and 2 μM of the peptides PLNC8 α and β, separately and combined. In order to estimate the maximum release from each sample, a final addition of 0.5 % Triton X-100 was made at the end of all measurements and the total amount of CF (100 % release) was estimated after 15 min incubation. The CF release is presented as percentage release for each time interval (measurements taken every minute). The percentage CF release is calculated as 100 × (F – F0)/(FT – F0) where F0 is the initial fluorescence intensity of CF before peptide addition, F is the fluorescence intensity of CF at time point t and FT is the maximum fluorescence after the addition of Triton X-100.
Circular Dichroism (CD) spectroscopy
Bacteriocins are often unstructured in solution but typically adopt a well-defined secondary structure when bound to the bacterial cell membrane as a result of membrane partitioning . The secondary structure of the bacteriocins was investigated using CD spectroscopy. CD spectra were recorded using a Chirascan spectropolarimeter (Applied Photophysics, UK) and a 1 mm quartz cuvette at 20 °C with a sampling interval of 0.5 nm. All measurements were done in triplicates and averaged before converted to mean residue ellipticity (MRE) and curves smoothened using Savitzky-Golay algorithm.
Antimicrobial activity of Lactobacillus
The ability of different Lactobacillus strains to inhibit P. gingivalis growth was assessed on fastidious anaerobe agar plates. Briefly, different P. gingivalis strains (108 CFU in 100 μl) were spread onto fastidious anaerobe agar plates and allowed to dry. Lactobacillus were diluted in MRS broth and 10 μl drops (106 CFU) were placed onto the P. gingivalis layer. The plates were incubated for 4 days, after which images were acquired with Olympus SZX9 at 10× magnification and the zone of inhibition was measured using the software ImageJ.
Antimicrobial activity was also assessed using Lactobacillus culture media (MRS broth), in which different Lactobacillus strains were cultured for 24 h. The bacteria were removed by centrifugation at 7000 × g and the supernatants were sterile filtered (0.2 μm). The pH was measured and the supernatants were used to determine antimicrobial activity against P. gingivalis on fastidious anaerobe agar plates (10 μl drops), as mentioned above.
Antimicrobial activity of bacteriocin PLNC8 αβ
The antimicrobial activity of PLNC8 αβ on P. gingivalis was visualized using the fluorescent dye Sytox® Green, which can only cross damaged membranes and fluoresce upon binding to nucleic acids. P. gingivalis were washed and resuspended in Krebs-Ringer Glucose buffer (KRG) (120 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 1.7 mM KH2PO4, 8.3 mM Na2HPO4, and 10 mM glucose, pH 7.3) and incubated in the presence or absence of PLNC8 αβ in 96-well microtiter plates. Images were captured with Olympus BX41 at 40× magnification.
Transmission electron microscopy (TEM) was used to visualize the damage of P. gingivalis, caused by PLNC8 αβ. Briefly, P. gingivalis ATCC 33277 were pelleted and washed with KRG. The bacteria were then treated with 280 nM of PLNC8 αβ in a molar ratio of 1:2 for 2 min and 10 min, followed by fixation in 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH 7.3. Specimens were washed in 0.1 M phosphate buffer, postfixed in 2 % osmium tetroxide in 0.1 M phosphate buffer for 2 h and embedded into LX-112 (Ladd, Burlington, Vermont, USA). Ultrathin sections (approximately 50-60 nm) were cut by a Leica ultracut UCT/ Leica EM UC 6 (Leica, Wien, Austria). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Hitachi HT 7700 (Tokyo, Japan). Digital images were taken by using a Veleta camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).
Surface plasmon resonance analysis was performed by immobilizing PLNC8 αβ peptides in 1:2 molar ratio onto a carboxymethylated dextran (CM-5 sensor chip, GE-Healthcare GmbH, Uppsala, Sweden) using biacore 2000 instrument equipped with four flow cells (GE-Healthcare GmbH, Uppsala, Sweden). Each channel of the chip was immobilized with 3 different concentrations of PLNC8 αβ (2.8, 28, 280 nM) respectively, and the fourth channel was used as a blank for negative reference subtraction between the channels. HBS-EP (0.01 M HEPES, pH7.4, 0.15 M NaCl, 3 mM EDTA, 0.005 % surfactant P20) (GE-Healthcare GmbH) was used as running buffer and the flow cell temperature was set to 25 °C in all experiments. Immobilization was a 3-step process performed using amine coupling kit (GE-Healthcare GmbH, Uppsala, Sweden) where the chip surface was activated using 200 mM N-ethyl-N0-(3 diethylaminopropyl) carbodimide (EDC) and 50 mM N-hydroxysuccinimide (NHS) mixture. PLNC8 αβ peptides diluted in acetate 4.5 buffer (GE-Healthcare GmbH, Uppsala, Sweden) were immobilized to the activated surface. Ethanolamine-HCl (pH 8.5) was used to deactivate the surface to enable an efficient binding of samples to the immobilized ligand. The contact time was 7 min, which resulted in immobilization levels between 1350 and 1750 response units (RU). One thousand RU corresponds to a surface peptide concentration of about 1 ng/mm2.
P. gingivalis were washed and prepared as described above after which the bacteria were pre-incubated with 2.8, 28 or 280 nM of PLNC8 αβ for 5 min at room temperature before analysis. P. gingivalis without PLNC8 αβ was used as positive control. The binding affinity of the bacteria to the peptides immobilized on each channel of the chip was measured in response units (RU). 1RU = 1 pg/mm2 using Bia-evaluation software .
Immobilization of surfaces with P. gingivalis-specific antibodies produced and tested as described in our previous studies , were used to study the binding affinity of P. gingivalis with and without PLNC8 αβ to the immobilized antibodies. Samples were prepared as described above and the responses obtained from Bia-evaluation software were plotted. The immobilization response for anti-P. gingivalis antibodies was 6400 RU.
All data were analyzed using GraphPad Prism 5.0 (GraphPad Software, La Jolla, CA, USA). One-way ANOVA with Tukey’s multiple comparison test was used for the comparisons between the different treatments. P-values are referred to as *,#p < 0.05; **,##p < 0.01; ***,###p < 0.001.