Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Activation of TLR2 is dependent on PPAD expression and activity. U251 MG cells overexpressing the TLR2 receptor were infected (MOI=100) or treated for 4 h with (A) ATCC 33277 strain or Pam3CSK4 (1 μg/ml), n=5-7; (B) various P. gingivalis strains and ATCC-derived isogenic mutants of catalytically inactive PPAD (C351A PPAD), n=3-5; (C) ATCC 33277 wild-type and PPAD mutant strains and sonicates (cells were lysed by sonication (sonic.)), n=4; or (D) outer membrane vesicles (OMVs) isolated from ATCC 33277 strain and its isogenic PPAD mutants (2 µg/ml), n=4. Results in (A) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Results in (B–D) are presented as a ratio of Firefly luciferase activity to β-galactosidase activity. Data are normalized to those from cells stimulated/infected with the same factor and transfected with an empty vector. Mean + SD; ****p < 0.0001; ***p < 0.001; ns, no statistical significance; 1-way ANOVA both followed by Tukey’s multiple comparisons test. ATCC WT- ATCC 33277.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Expressing, Activity Assay, Infection, Derivative Assay, Mutagenesis, Sonication, Membrane, Isolation, Luciferase, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Fimbriae purified from the wild-type ATCC 33277 strain activate the TLR2 receptor. U251 MG cells overexpressing TLR2 were: (A) infected for 4 h with WT ATCC 33277 and its isogenic major fimbriae (delFimA) mutant strain (MOI=100), n=4-5 (B) infected for 4 h with various ATCC 33277-derived PPAD and FimA mutants as well as the WT W83 strain at different MOI (all strains MOI=20-100 with an additional MOI=5 for WT ATCC 33277), n=3 or (C) infected for 2, 4 or 6 h with WT ATCC 33277 or the PPAD mutant strains (MOI=100), n=3; and (D) treated for 4 h with purified fimbriae (10 µg/ml) from WT ATCC 332771 (FimA WT) or the PPAD mutant (FimA delPPAD) strains; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (E) HEK Blue cells overexpressing TLR2 were treated with purified fimbriae (10 µg/ml) isolated from the WT ATCC 33277 strain (FimA WT) or the PPAD mutant (FimA delPPAD), n=3. Results are presented as the mean ± SD fold induction compared to control (unstimulated) cells. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. In (B) each condition was compared to control uninfected cells and in (C) comparisons were performed for each timepoint separately and significant differences compared to WT ATCC 33277 are depicted in the graph.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Purification, Infection, Mutagenesis, Derivative Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Isolation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: Both active PPAD and fimbriae are crucial for activation of TLR2. U251 MG cells overexpressing the TLR2 receptor were infected for 4 h (MOI=100) with various ATCC 33277-derived isogenic mutants expressing different forms of PPAD (the T1 form, the T2-hyperactive form, and the catalytically inactive C351A mutant form) or FimA; n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. ****p < 0.0001; ns, no statistical significance; 1-way ANOVA followed by Tukey’s multiple comparisons test.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Activation Assay, Infection, Derivative Assay, Expressing, Mutagenesis, Luciferase, Activity Assay, Transfection, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: TLR2 Activation by Porphyromonas gingivalis Requires Both PPAD Activity and Fimbriae

doi: 10.3389/fimmu.2022.823685

Figure Lengend Snippet: The ability of clinical strains to activate TLR2 is much weaker than that of ATCC 33277. (A) Cells were infected for 4 h (MOI=100) with various clinical strains (k1-k10), ATCC 33277 (ATCC WT), or W83, n=4. Results are presented as the mean ± SD ratio of Firefly luciferase activity to β-galactosidase activity and are normalized to cells stimulated/infected with the same factor and transfected with an empty vector. (B) Western blot analysis of laboratory and clinical strain cultures (adjusted to OD 600 ) to detect FimA. (C) PPAD activity in whole laboratory and clinical strain cultures (adjusted to OD 600 ), n=6. Results were compared with those from the ATCC 332777 strain. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no statistical significance. 1-way ANOVA followed by Tukey’s multiple comparisons test. (D) The FimA type determined by sequencing of the fimA gene in clinical strains.

Article Snippet: The next day cells were transiently transfected with: (i) a vector (pGL2-NFκB, 247.5 ng per well) coding the Firefly luciferase gene under control of 5 tandem repeats of the NF-ĸB response element, (ii) a reference pEF vector (5 ng per well) coding β-galactosidase under control of EF-1α elongation factor, and (iii) a vector (247.5 ng per well) encoding the human flag-tagged TLR2 receptor or TLR2-TLR1/TLR2-TLR6 heterodimer (pDUO-hTLR1-TLR2/pDUO-hTLR1-TLR2, Invivogen or an empty vector pcDNA3.1, in total 500 ng per well.

Techniques: Infection, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Western Blot, Sequencing

Journal: Microbial Cell Factories

Article Title: Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

doi: 10.1186/1475-2859-11-161

Figure Lengend Snippet: TLR2 mediated signalling using HEK293T cells. HEK293T cells were transfected with pUNO-hTLR2 (Invivogen) alone (TLR2 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the corresponding plasmids as described in Material & Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml) and LTA (10 μg/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. * p-value < 0.05 compared to the negative control (TLR2(−)).

Article Snippet: In contrast, transfecting the HEK293T cells with the vector pDUO-hTLR6/TLR2 (Invivogen), resulting in expression of both TLR2 and TLR6, did significantly result in activation of the NF-κB reporter plasmid by 10 μg/ml LTA of LGG (Figure ).

Techniques: Transfection, Plasmid Preparation, Incubation, Activity Assay, Negative Control

Journal: Microbial Cell Factories

Article Title: Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

doi: 10.1186/1475-2859-11-161

Figure Lengend Snippet: TLR2/6 mediated signalling using HEK293T cells. HEK293T cells were transfected with pDUO-hTLR6/TLR2 (Invivogen) alone (TLR2/6 (−)) or together with the reporter plasmid pNiFty2-SEAP (Invivogen). Cells were transiently transfected with the respective plasmids as described in Methods. 24h after transfection, cells were incubated with Pam2CSK4 (50 ng/ml), LTA at 0.1, 1 and 10 μg/ml, mLTA1 (10 μg/ml), mLTA2 (10 μg/ml), LGG wild-type or dltD mutant cells (10 8 CFU/ml) for 24 hours. After incubation, NF-κB-induced SEAP activity was assessed using pNPP (p-nitrophynyl phosphate) and measuring the OD at 405nm. Data represent mean values ± SD. * p-value < 0.05 compared to the negative control (TLR2/6(−)).

Article Snippet: In contrast, transfecting the HEK293T cells with the vector pDUO-hTLR6/TLR2 (Invivogen), resulting in expression of both TLR2 and TLR6, did significantly result in activation of the NF-κB reporter plasmid by 10 μg/ml LTA of LGG (Figure ).

Techniques: Transfection, Plasmid Preparation, Incubation, Mutagenesis, Activity Assay, Negative Control