Porphyromonas gingivalis induction of TLR2 association with Vinculin enables PI3K activation and immune evasion

Abstract

Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that thrives in the inflamed environment of the gingival crevice, and is strongly associated with periodontal disease. The host response to P. gingivalis requires TLR2, however P. gingivalis benefits from TLR2-driven signaling via activation of PI3K. We studied TLR2 protein-protein interactions induced in response to P. gingivalis, and identified an interaction between TLR2 and the cytoskeletal protein vinculin (VCL), confirmed using a split-ubiquitin system. Computational modeling predicted critical TLR2 residues governing the physical association with VCL, and mutagenesis of interface residues W684 and F719, abrogated the TLR2-VCL interaction. In macrophages, VCL knock-down led to increased cytokine production, and enhanced PI3K signaling in response to P. gingivalis infection, effects that correlated with increased intracellular bacterial survival. Mechanistically, VCL suppressed TLR2 activation of PI3K by associating with its substrate PIP2. P. gingivalis induction of TLR2-VCL led to PIP2 release from VCL, enabling PI3K activation via TLR2. These results highlight the complexity of TLR signaling, and the importance of discovering protein-protein interactions that contribute to the outcome of infection.

Fig 1. TLR2 physically associates with VCL in vitro.

(A) Human macrophage THP1 cells were infected with P. gingivalis (MOI 10) for 30 min followed by cross-linking with DSP (M, marker). TLR2 was immunoprecipitated (IP) and eluates were analyzed for VCL by immunoblot (IB). Whole cell lysates (WCL) were analyzed to control for protein input and loading. (B) THP1 cells were differentiated for three days and left untreated or infected with P. gingivalis at MOI 10 for 30 min. Fixed cells were stained for endogenous TLR2 (green) and VCL (red), and nuclei were counterstained with Hoechst (blue). Data are representative of three independent experiments. (C) HeLa cells were transfected with TLR2-YFP (green). Control or treated cells (PAM vs. P. gingivalis at MOI 10 for 30 min) were stained for endogenous VCL (red), and nuclei were counterstained with Hoechst. Images were captured using a NIKON confocal microscope at 60X magnification. Yellow color indicates the co-localization of green and red channels. Co-localization was quantified using JACoP/ImageJ analysis software. Data are representative of three independent experiments. (ns: non-significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 2. Computational predictions of protein-protein interface residues.

(A) Five core residues of the TLR2 TIR domain are colored blue and labeled (“Core” column). The top row shows the structure for the native (W684, F719), and the second row for the alanine mutated (W684A, F719A), sequence. ODA and NIPs residue values colored on the TLR2 TIR domain crystal structure and core residue list. ODA values are colored in grey-white-red scale (decreasing values), where red indicates lowest energy values (residues with highest contribution to a protein-protein interface stability). NIPs values are colored with a grey-white-orange scale (increasing values), where higher NIP values represent a higher chance of a residue to participate in the specific TLR2-VCL interface. (B) TLR2-TIR domain sequence showing the two residues mutated to alanine by site-directed mutagenesis (arrows), and their proximity to TIR regions implicated in homo- and hetero-oligomerization (the BB loop and αC’ helix). Fig 2B was created with BioRender.com.

Fig 3. Site directed mutagenesis validates TLR2 residues involved in TLR2-VCL interaction.

(A) NF-κB reporter-HEK293 cells were transfected with native TLR2 (W684/F719), or each of the TLR2 mutants W684A, F719A, or W684A/F719A together with TLR1 and MyD88, and then cells were stimulated with PAM (100ng/mL) for 4 h. Cells were lysed and NF-kB activation was measured by luciferase activity. Percent NF-kB induction is shown relative to luciferase values of control cells transfected in an identical manner but not activated with PAM. Data represent mean values of three independent experiments. (B) HeLa cells were transfected with YFP-fused native (WF) TLR2, or W684A, F719A, or AA TLR2 mutants (green). Cells were fixed and stained for VCL (red) and nuclei were stained with Hoechst (blue). Images were captured using a NIKON confocal microscope at 60X magnification. TLR2-VCL co-localization was analyzed using JACoP/ImageJ analysis software. Data are representative of five independent experiments. (ns: non-significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig 4. MaMTH system for validating TLR2-VCL interaction.

(A) Graphical summary of MaMTH split ubiquitin system for detection of bacterially-induced TLR2-VCL interaction (B) Luciferase activity of untreated or PAM-stimulated HEK293T cells co-transfected with TLR2-bait and the indicated positive and negative control prey constructs. Data are representative of five independent experiments. (C) HEK293T cells co-transfected with TLR2-bait and VCL prey plasmids were left untreated, exposed to PAM (100ng/mL), or infected with P. gingivalis (MOI 10) for 8 h. Data are representative of four independent experiments. (D) Luciferase activity in HEK293T cells co-transfected with wild-type TLR2 bait, or mutants TLR2 bait mutants W684A, F719A or WF, together with VCL prey. Transfected cells were untreated, treated with PAM (100ng/mL), or infected with P. gingivalis (MOI 10) for 8 h. Data are representative of two independent experiments. (ns: non-significant; *P≤0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). Fig 4A was created with BioRender.com.

Fig 5. Cytokine production and bacterial survival in VCL knock-down macrophages.

(A) Representative IB of VCL and GAPDH in whole cell lysates from shCtrl and shVCL-THP1 cells, and RT-PCR showing levels of VCL expression (B) TNF production by shCtrl and shVCL-THP1 cells challenged with P. gingivalis (MOI 10), PAM (10ng/mL), and LPS (10ng/mL) for 4 h, was measured by ELISA. Data are representative of five independent experiments. Unstimulated cells were used as controls (BG, background). (C) PMA-differentiated shCtrl and shVCL-THP1 cells were challenged with P. gingivalis (MOI 10), PAM (10ng/mL), and LPS (10ng/mL) for 4 h and mRNA levels of TNF, IL6, and IL1b were quantified by quantitative RT-PCR. mRNA from unstimulated cells collected at the same time point was used as background (BG). Data are representative of three independent experiments. (D) Intracellular P. gingivalis survival was determined in wild-type (WT) THP1, shCtrl-THP1 and shVCL-THP1 cells using the antibiotic protection assay as per methods with and without prior blocking of TLR2 using T2.5 anti-TLR2 mAb (αTLR2) vs. IgG1 isotype control. CFU were enumerated after 7 days of anaerobic growth (ND, none detected). Data are representative of three independent experiments. (E) shCtrl and shVCL-THP1 cell phagocytosis of FITC-labelled P. gingivalis was measured by flow cytometry (percentage of fluorescent cells above background, and geometric mean of fluorescence). A sample histogram is shown on the left. Data representative of three independent experiments are shown on the right. (ns: non-significant; *P≤0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001).

Fig 6. VCL negatively regulates TLR2-dependent PI3K activation.

(A) shVCL and shCtrl THP1 cells were challenged with P. gingivalis (MOI 10) at different time points and lysates were analyzed by IB for phospho-Akt (P-Akt S473) and total Akt. Representative immunoblot of three independent experiments. (B) shCtrl-THP1 and shVCL-THP1 cells were pre-treated with the PI3K inhibitor LY294 or the control inhibitor LY303 prior to infection with P. gingivalis (MOI 10). Intracellular P. gingivalis survival was determined using the antibiotic protection assay. Data are representative of three independent experiments. (C) P. gingivalis-infected and control THP1 cells were fixed and stained for PIP2 (green), and VCL (red), and nuclei were stained with Hoechst (blue). Images were captured using a NIKON confocal microscope at 60X magnification. PIP2-VCL co-localization was analyzed using JACoP/ImageJ analysis software. Data are representative of two independent experiments. (D) Differentiated THP1 cells were infected with P. gingivalis for 30 min and VCL was immunoprecipitated. Eluates were analyzed by IB with an antibody to PI3k-p85α. Data are representative of three independent experiments. (ns: non-significant; *P≤0.05; **P ≤ 0.01; ***P ≤ 0.001).

Fig 7. Schematic illustration.

In un-infected macrophages [1], VCL associates with PIP2 and p85α, preventing activation of downstream Akt phosphorylation and thereby favoring macrophage killing of P. gingivalis when cells are infected. TLR2 senses P. gingivalis and is induced to interact with VCL [2], which causes VCL to dissociate from PIP2 and p85α [3]. This enables PI3K conversion of PIP2 to PIP3, and subsequent phosphorylation of Akt [4], leading to increased intracellular survival. Fig 7 was created with BioRender.com.