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

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.

(extrinsic to the methodology) is the fact that we only examined one time point of activation (30 min).The interactome will likely shift over time, and some time-points may contain more of the known TLR2 interacting proteins.A limitation that is intrinsic to the methodology is that membrane proteins, due to their hydrophobic characteristics and lipid-rich environment, require significant optimizations for detection by MS (new reference Schey et al., 2013).In addition to that, the amino acid compositions of some proteins obstruct the discovery of cross-linked residues, since the cross-linking reactions can alter the enzymatic digestion of protein peptides (new reference Dau et al., 2019).Of note, Kamal et al (PMID: 31221720) also utilized a cross-linking and immunoprecipitation followed by LC-MS approach (to study the effect of statins on TLR2 signaling induced by PAM) and did not recover known adapter proteins.
In the revised manuscript we include a full discussion of these limitations (p.14).
2-(Results lines 88-118).In this same section, STRING analysis predicted that vinculin interaction with TLR2 is possible by is indirect via an interaction with paxlilin.
The manuscript does a superb job exploring the TLR-vinculin interaction, but does not clarify experimentally the mechanism of direct vs indirect interaction -in other words is paxillin required for the TLR2-vinculin interaction to occur?
Response: We realize that the presentation of the STRING analysis adds confusion since it suggests a potential indirect association whereas our results all support a direct association of vinculin and TLR2.We had used the STRING analysis to help indicate which of the immunoprecipitated proteins have been shown to bear any relationship to TLR2.In fact, we did not find paxillin or SRC in the immunoprecipitated proteins, and since vinculin peptides were modified by the crosslinker we hypothesized, and experimentally pursued, a direct interaction with TLR2.
In the revised manuscript we removed the referral to the STRING analysis -the papers supporting focal adhesion complex members such as paxillin associating with TLR2 (the basis for the STRING result) are referenced and discussed in the discussion.
3-The team does excellent work with THP-1 cells which are an established model cell used for exploring macrophage function.Is there any evidence that primary human macrophages recapitulate the findings with the THP-1 cells?
Response: We found that it is a significant technical challenge to immunoprecipitate native TLR2 from primary human macrophages (in fact, we are unable to find any references where TLR2 was immunoprecipitated from primary human macrophages).
We are unable to visualize a strong band for immunoprecipitated TLR2 from primary macrophages (differentiated from PBMCs), in contrast to the THP1 cells.After crosslinking primary macrophages with DSP (the cross-linker used in our studies), and immunoprecipitation of TLR2, we do see vinculin, but only if we load the blot with more protein than usual, and although there is a difference between control and P. gingivalis-infected macrophages, the difference is not as pronounced as for the THP1 cells.A new supplementary figure showing this finding is included in the revised manuscript (Supplementary Figure 1B), and a statement of the limitation of the study is included in the revised discussion section (p.14, lines 299-302, p. 19lines 414-21).
Additions are also made to the methods and acknowledgements sections.4-In Figure 5D.Was an irrelevant isotype-matched Ab used as a control?
Response: We had used the isotype control in experiments run in parallel to the presented experiments, and found that it did not have an effect on WT THP-1 macrophages.However, in recognition of the critique, we re-ran the experiments of 5-Statistical analyses.In some of the assessments made by the authors, there appears to be 3-way comparisons; however 2-way t-tests are indicated as the sole way data were analyzed.Unclear why ANOVA analyses were not considered?
Response: We re-analyzed the experiments that had multiple comparisons using ANOVA analysis.These analyses are incorporated to new figures 4D and 5D.In general, the significance of the outcome was strengthened in the new analysis.PAM signal in the split ubiquitin system, which was much lower than the signal induced by

Figure
Figure 5D using anti-TLR2 and its isotype control for the WT, shCntrol and shVCL macrophages, and replaced the figure with the new figure.