Fig 1.
Isolation of highly purified P. gingivalis, T. denticola and T. forsythia OMVs.
OptiPrep density gradient centrifugation of a crude T. forsythia OMV preparation (A1) separated non-OMV associated material (bottom of density gradient) from highly purified OMVs isolated at a higher density. A blank OptiPrep density gradient is shown in A2. SDS-PAGE of P. gingivalis (B), T. denticola (E F) and T. forsythia (I J) Optiprep density gradient fractions taken in eight 1.5 mL gradient fractions from top to bottom following centrifugation. Gradient fractions were subjected to SDS-PAGE and protein bands visualized by SimplyBlue or SyproRuby staining. Molecular mass markers (Novex SeeBlue Plus2 Prestained Standard) are indicated in kDa. OMVs were observed using transmission electron microscopy (TEM) (C G K). Vesicle size and range was further confirmed using dynamic light scattering (DLS) on pooled and washed OMV containing gradient fractions (D H L).
Fig 2.
Enumeration of OMVs by high resolution flow cytometry.
P. gingivalis purified OMVs unlabeled (A G) or labelled with either FITC (B H), AF488 (C I), Syto9 (D J), Fm464 (E K) or PHK-26 (F L) were analysed using an Apogee A50-Micro Flow Cytometer. Dye suitability was observed as determined by Forward/Small Angle Light Scatter (F/S-ALS) (A-F) and fluorescence (G-L).
Table 1.
Compositional analysis of OMVs from P. gingivalis, T. denticola and T. forsythia.
Fig 3.
TLR2 and TLR4 Activation by P. gingivalis, T. denticola and T. forsythia OMVs.
HEK-Blue TLR2 (A, B, C) and TLR4 (D, E, F) Cell lines were incubated with 20μL of either OMVs or ligands Pam3CSK4 (10 μg/mL in 5 fold dilutions) and LPS-EB (10 μg/mL in 10 fold dilutions) respectively. Alkaline phosphatase activity was determined after 20 hours incubation at 620 nm on a spectrophotometer. Crude OMV (B, E) and purified OMV preparations standardised by OMV count (A, D) and general protein (C, F) were used to ascertain the effects of purification and standardisation methods on OMV immunogenicity. Lipoproteins were observed by GC-MS fatty acid analysis to determine the % fatty acid chain lengths present (G). OMVs were subjected to SDS-PAGE and lipopolysaccharide and lipooligosaccharide detected by Pro-Q Emerald 300 LPS Gel Stain (H). Glycoproteins were identified using SyproRuby protein stain (I). Purified P. gingivalis LPS was used as a positive control. Molecular mass markers (Novex SeeBlue Plus2 Prestained Standard) are indicated in kDa next to each sample.
Fig 4.
TLR7, TLR8 and TLR9 Activation by P. gingivalis, T. denticola and T. forsythia OMVs.
HEK-Blue TLR7 (A), TLR8 (B) and TLR9 (C) Cell lines were challenged with 20μL of either purified OMVs or ligands R848 (10 μg/mL in 2 fold dilutions) and dsDNA (100 μg/mL in 2 fold dilutions) respectively. Alkaline phosphatase secretion was determined after 20 hours incubation at 620nm on a spectrophotometer. Monoclonal antibody αdsDNA MAB030 was used to detect DNA in purified OMV preparations and Triton X-114 extracted Outer Membrane Protein (OMP) preparations, used at 0.5 mg/mL protein and 1.5 mg/mL protein respectively, for P. gingivalis (F), T. denticola (I) and T. forsythia (K). OMV preparations were spotted on a nitrocellulose Immuno-Blot PVDF Membrane for Protein Blotting in three 10-fold dilutions. E. coli double stranded DNA was used as a positive control at 50 ng/mL in three 10-fold dilutions (D). Bovine Serum Albumin was used as a negative control at 0.5 mg/mL in three 10-fold dilutions (E). P. gingivalis whole OMVs and extracted OMV DNA were treated with DNase and DNA concentrations determined using Qubit Assay Kits and SYBR Safe DNA gel stain (L).
Fig 5.
NOD1 and NOD2 Activation by P. gingivalis, T. denticola and T. forsythia OMVs.
HEK-Blue NOD1 (A) and NOD2 (B) Cell lines were challenged with 20 μL of either purified OMVs or ligands Tri-DAP (150 μg/mL in 2 fold dilutions) and L18-MDP (50 μg/mL in 2 fold dilutions), respectively. Alkaline phosphatase secretion was determined after 20 hours incubation at 620 nm on a spectrophotometer.