inhibition zone assay p gingivalis strain w83  (ATCC)

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: P. gingivalis strains with fimbriae depleted of all accessory subunits (DAP) poorly activate MDM pro-inflammatory responses. MDMs were treated for A) 4 h or B) 24 h with purified fimbriae (1 μg/ml) from various P. gingivalis strains, including ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD), as well as mutants of accessory fimbrial subunits, ∆ fimE mutant (fimbriae ∆FimE) and ∆ fimC mutant (fimbriae ∆FimC). A) Relative mRNA expression of PGE2 synthesis-related gene COX2 and cytokines IL6 and IL8 ; n = 4–7. B) Secretion of IL-6 and IL-8; n = 5–9. Data are presented as the mean ± SEM compared to WT fimbriae, with significance levels indicated as **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Purification, Mutagenesis, Expressing

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: LPS contamination in fimbriae preparations is negligible and does not influence the pro-inflammatory activation of MDMs. A) MDMs were treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and ∆PPAD mutant (fimbriae ∆PPAD) strains, as well as LPS-free fimbriae from both strains; n = 3. B) hMDMs were preincubated for 30 min with control (IgG1) or TLR4-blocking antibody (anti-TLR4), then treated for 24 h with purified fimbriae (1 μg/ml) from ATCC 33277 WT (fimbriae WT) and the ∆ ppad mutant (fimbriae ∆PPAD) strains; n = 3. Levels of IL-6 and IL-8 secretion were subsequently measured. Data are presented as mean ± SEM. ‘ns’ indicates not statistically significant. Statistically significant differences are shown in A) between LPS-free fimbriae and fimbriae without the LPS removal step; in B) between IgG1 and anti-TLR4 pretreated samples.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Activation Assay, Purification, Mutagenesis, Control, Blocking Assay

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: PPAD and fimbriae mutants show differences in adhesion and safe entry to host cells. Flow cytometry analysis of MDMs infected for 30 min at MOI = 20 or 100 with P. gingivalis ATCC 33277 WT and PPAD mutant strains—including its isogenic total (∆PPAD) and catalytically inactive (C351A PPAD) strains, as well as fimbriae (∆FimA) mutants—labelled with A) CFSE or B) pHRODO-Red (pHRODO). Phagocytosis and adhesion rates were determined as the percentages of CFSE and pHRODO-Red positive cells, relative to the WT strain; n = 4. Results are shown as mean ± SEM. ** p < 0.01; * p < 0.05; ns, not significant. Comparisons are made to the ATCC WT strain. C) Widefield fluorescence microscopy of MDMs infected for 30 min at MOI 100 with CFSE-labelled P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE ∆FimC, and W83 strains. Nuclei were stained with DAPI, and actin was stained with phalloidin conjugated to Alexa Fluor 647. Scale bars: 30 µm. Images are representative of three independent experiments. White arrows indicate bacteria that are not internalised.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Flow Cytometry, Infection, Mutagenesis, Fluorescence, Microscopy, Staining, Bacteria

Journal: Journal of Oral Microbiology

Article Title: Macrophage activation and invasion by P. gingivalis is modulated by PPAD and accessory fimbriae subunits

doi: 10.1080/20002297.2026.2638646

Figure Lengend Snippet: Phosphorylation of Akt is enhanced by PPAD and accessory fimbriae subunits, with the PI3K/Akt pathway being essential for IL-6 but not for IL-8 secretion. MDMs were infected for A) 10 min, 30 min, or 2 h with P. gingivalis ATCC 33277 WT strain (ATCC WT) and its isogenic PPAD mutant (∆PPAD), or B) for 30 min with ATCC 33277-derived PPAD: total (∆PPAD) and catalytically inactive (C351A PPAD), fimbriae: main FimA subunit (∆ fimA ), accessory subunits FimE (∆ fimE ), and FimC (∆ fimC ) mutants, or the W83 WT strain at MOI 20. Akt phosphorylation levels were determined by western blot analysis, with total Akt as a control and β -actin as the loading control. Representative blots are shown, and densitometry results ( n = 3) are presented as mean ± SEM. *** p < 0.001; ** p < 0.01; * p < 0.05; ns, not significant. In B), data are compared with the ATCC WT strain. C) MDMs were pretreated for 30 min with an Akt inhibitor or left untreated, then infected for 24 h at MOI 20 with P. gingivalis ATCC WT, ∆PPAD, C351A PPAD, ∆FimA, ∆FimE, ∆FimC, and W83 strains. Secretion of IL-6 and IL-8 was measured by ELISA; n = 4−7. Results are shown as mean ± SEM; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05 compared to ATCC WT without Akt inhibitor; ns, not significant.

Article Snippet: We thoroughly assessed how PPAD affects inflammatory and antibacterial responses of macrophages to the fimbriated wild-type P. gingivalis strain ATCC 33277.

Techniques: Phospho-proteomics, Infection, Mutagenesis, Derivative Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: SEM ( A , B ) and TEM ( C – F ) micrographs of P. gingivalis with long bundled fimbriae. The strain designations and fimA type are indicated within each panel. Individual and bundled fimbriae are marked with “i” and “b”, respectively. The diameter of the bundles ranges from approximately 10 to 50 nm, depending on the numbers of fimbriae involved. In contrast, individual fimbria had a maximum diameter of 5 nm. Bundled fimbriae protruded from the cell surface, or in ( D ), from the vesicle surface, into the medium. In ( B , C , E ), the fimbriae of the opposing cells have become entangled.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: P. gingivalis FimA type IV strains (AJW5 = OMI 1049 and afimbriated W83 = OMI 629) and P. gulae (OMI 1080, fimbrial type A) in SEM ( A – C ) and TEM ( D – F ) micrographs. W83 and the P. gulae strain formed a strong capsule and many vesicles but showed no (bundled) fimbriae. In slimy strain OMI 1049, bundled fimbriae are masked but can still be assumed, as depicted in image ( A ).

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: TEM ( A – E , G ) and SEM ( F , H – J ) micrographs of tubular and chain-like OMEs. P. gingivalis formed tubes (indicated with ➡) that protruded from the cell surface and chains of OMVs that appeared to be interconnected (indicated with ⇨). The transitions between the two forms appeared fluid. Probably due to the preparation process, some of these structures were detached from the cell. Vesicles with a larger diameter were frequently observed at the end of these structures (indicated with ▷). Tubes adhered to bundled fimbriae ( J ). In TEM images, a prominent EDSL was visible on the OMVs and on the chain- and tube-like OMEs. Strain designations are indicated within the panels.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: SEM ( A , B , D ) and TEM ( C , E – I ) micrographs showing OMVs associated with P. gingivalis long fimbriae. OMVs attached to both long single and bundled fimbriae and commonly appeared at the distal tips of fimbriae (indicated with ⇨) or at multiple points along their shaft (indicated with ➡). The TEM micrographs displayed a prominent EDSL on the OMVs. The strain designations and fimA type are indicated within each panel.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: P. gingivalis co-cultured with S. oralis (top row) and F. nucleatum (bottom row) documented by SEM micrographs. To facilitate differentiation, in some images, P. gingivalis was highlighted in purple, S. oralis in green and F. nucleatum in yellow. P. gingivalis cells were found individually and in small colonies between the predominant S. oralis cells ( A ). ( B , C ) illustrate specific details from ( A ). In ( B ), two P. gingivalis cells were observed to adhere to S. oralis , subsequently forming a network with the S. oralis cells. This network likely contained (bundled) fimbriae, OMVs and matrix components. In section ( C ) (( D ) = enlarged detail), a P. gingivalis cell appears to have been attached to the S. oralis cell at its lower edge by a short robust bundle of fimbriae. In the F. nucleatum co-culture, slightly larger P. gingivalis cell clusters were identified ( E , F ). A prevalent finding was the presence of an altered polar cell morphology of P. gingivalis cells ( F , G ). ( G ) shows this in detail. One side of the cell exhibits a kind of cap, while the other side appears to attach to F. nucleatum with a bundle of fimbriae. Bacteria were incubated anaerobically in co-culture for 5–7 days. The coloration was performed using image processing software (Affinity Photo, version 1.10.8, Serif). C = chain(s) of OMVs, F = (bundled) fimbriae.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques: Cell Culture, Co-Culture Assay, Bacteria, Incubation, Software

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: HGFs co-incubated with P. gingivalis ATCC 49417 ( A , C – G ) and OMI 1127 ( H , I ) in comparison to the control of pure HGF culture ( B ) documented by SEM micrographs. HGFs in the co-culture with ATCC 49417 rounded up ( A , C ) compared to the pure culture ( B ), indicative of stress due to anaerobic conditions and/or bacterial toxins. ATCC 49417 exhibited extensive colonization of the HGFs ( C ). The bacterial cells adhered more readily to the HGFs than to the glass slides ( C , D ). At high magnification, direct interactions between P. gingivalis and the fibroblasts were visible ( E , G ). Bundled fimbriae were visible between P. gingivalis and HGFs (( E ) = enlarged section of ( D )). Similar to the co-culture with F. nucleatum , stalked adhesion with partially asymmetrical deformation of the bacteria could be observed ( F ). In contrast, OMI 1127 was concentrated in proximity to fibroblast products, most likely collagen (as its substrate), and was hardly identifiable on the remaining surfaces ( H , I ). The SEM images were subsequently colored using image processing software (Affinity Photo, version 1.10.8, Serif) to facilitate differentiation between P. gingivalis (purple) and HGFs (pink). The samples were incubated anaerobically for 24 h in 75% DMEM (with 10% FBS) and 25% Brucella medium.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques: Incubation, Comparison, Control, Co-Culture Assay, Bacteria, Software

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: Quantities ( A , B ) and ratios ( C , D ) of shaft and accessory proteins of major fimbriae in OMV preparations of two P. gingivalis strains. ( A , B ): Graphical representation of the determined MVs (n = 3) listed in . Of the four proteins, FimA is the most abundant, followed by FimE, then FimC and FimD. The quantities are not absolute and cannot be compared between preparations. ( C , D ): Graphical representation of the determined MRs of the shaft to each accessory protein (n = 3). The ratio of FimA to FimE was significantly smaller than those of FimA to FimC and FimD in both strains. In contrast, the ratios of FimA to FimC and FimD were similar. The iBAQ values were determined using DIA proteomics software (Spectronaut). SDs are displayed within the bars. Statistical analysis was performed using unpaired t -tests with p < 0.05 (ns: non-significant, *: p < 0.05, **: p < 0.01).

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques: Software

Journal: International Journal of Molecular Sciences

Article Title: Porphyromonas gingivalis Bundled Fimbriae Interact with Outer Membrane Vesicles, Commensals and Fibroblasts

doi: 10.3390/ijms27010383

Figure Lengend Snippet: Overview of the observed structures. TEM micrographs of P. gingivalis . ( A ) Bundled fimbriae, ( B ) FAVs, ( C ) tube- and chain-like OMVs. T = tube, C = chain.

Article Snippet: To this end, HGFs were co-incubated anaerobically with each of the two different P. gingivalis strains (ATCC 49417 and OMI 1127) for 24 h. The cultures were again examined using SEM.

Techniques: