p cdk2  (Cell Signaling Technology Inc)

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: Our findings demonstrate that Akt activation during P. gingivalis –macrophage interactions is not solely triggered by TLR2 engagement, nor is it the only factor responsible for the increased IL-6 secretion caused by the ATCC 33277 strain.

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: Our findings demonstrate that Akt activation during P. gingivalis –macrophage interactions is not solely triggered by TLR2 engagement, nor is it the only factor responsible for the increased IL-6 secretion caused by the ATCC 33277 strain.

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: Our findings demonstrate that Akt activation during P. gingivalis –macrophage interactions is not solely triggered by TLR2 engagement, nor is it the only factor responsible for the increased IL-6 secretion caused by the ATCC 33277 strain.

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: Our findings demonstrate that Akt activation during P. gingivalis –macrophage interactions is not solely triggered by TLR2 engagement, nor is it the only factor responsible for the increased IL-6 secretion caused by the ATCC 33277 strain.

Techniques: Phospho-proteomics, Infection, Mutagenesis, Derivative Assay, Western Blot, Control, Enzyme-linked Immunosorbent 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: 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: Since purified fimbriae may be contaminated with LPS, residual LPS was removed from fimbriae preparations using an endotoxin removal resin, and the depletion of LPS was confirmed with an endotoxin detection kit. hMDMs were then treated with fimbriae isolated from the WT ATCC 33277 strain and its isogenic ∆PPAD mutant, which were either subjected to LPS removal or not.

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: Since purified fimbriae may be contaminated with LPS, residual LPS was removed from fimbriae preparations using an endotoxin removal resin, and the depletion of LPS was confirmed with an endotoxin detection kit. hMDMs were then treated with fimbriae isolated from the WT ATCC 33277 strain and its isogenic ∆PPAD mutant, which were either subjected to LPS removal or not.

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: Since purified fimbriae may be contaminated with LPS, residual LPS was removed from fimbriae preparations using an endotoxin removal resin, and the depletion of LPS was confirmed with an endotoxin detection kit. hMDMs were then treated with fimbriae isolated from the WT ATCC 33277 strain and its isogenic ∆PPAD mutant, which were either subjected to LPS removal or not.

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: Since purified fimbriae may be contaminated with LPS, residual LPS was removed from fimbriae preparations using an endotoxin removal resin, and the depletion of LPS was confirmed with an endotoxin detection kit. hMDMs were then treated with fimbriae isolated from the WT ATCC 33277 strain and its isogenic ∆PPAD mutant, which were either subjected to LPS removal or not.

Techniques: Phospho-proteomics, Infection, Mutagenesis, Derivative Assay, Western Blot, Control, Enzyme-linked Immunosorbent 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: 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: Analysis of the citrullinomes of the P. gingivalis ATCC 33277 WT and W83 strains, along with three clinical isolates, identified several citrullinated or tentatively citrullinated proteins (i.e. proteins in which arginine residues are post-translationally converted to citrulline by peptidyl arginine deiminases), including key virulence factors such as gingipains, RagA, Mfa1, and FimA [ ].

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: Analysis of the citrullinomes of the P. gingivalis ATCC 33277 WT and W83 strains, along with three clinical isolates, identified several citrullinated or tentatively citrullinated proteins (i.e. proteins in which arginine residues are post-translationally converted to citrulline by peptidyl arginine deiminases), including key virulence factors such as gingipains, RagA, Mfa1, and FimA [ ].

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: Analysis of the citrullinomes of the P. gingivalis ATCC 33277 WT and W83 strains, along with three clinical isolates, identified several citrullinated or tentatively citrullinated proteins (i.e. proteins in which arginine residues are post-translationally converted to citrulline by peptidyl arginine deiminases), including key virulence factors such as gingipains, RagA, Mfa1, and FimA [ ].

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: Analysis of the citrullinomes of the P. gingivalis ATCC 33277 WT and W83 strains, along with three clinical isolates, identified several citrullinated or tentatively citrullinated proteins (i.e. proteins in which arginine residues are post-translationally converted to citrulline by peptidyl arginine deiminases), including key virulence factors such as gingipains, RagA, Mfa1, and FimA [ ].

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