부산대학교 도서관

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. Author summary: Porphyromonas gingivalis is an anaerobic bacterium strongly associated with periodontitis. Remarkably, P. gingivalis flourishes in the presence of inflammation and activated innate immune cells. P. gingivalis is sensed by TLR2, however, sensing leads to escape from macrophage bactericidal activity in a manner dependent on PI3K/Akt signaling. We used chemical cross-linking to identify induced interacting partners of TLR2 that contribute to bacterial immune escape, and found that infection induces TLR2 association with vinculin (VCL). Modeling the TLR2-VCL interaction highlighted critical amino acid residues of the TLR2 intracellular domain that were then experimentally validated by site-directed mutagenesis. We then confirmed that the induced TLR2-VCL interaction contributes to P. gingivalis immune evasion by enabling PI3K/Akt activation. Our results demonstrate the importance of induced TLR2 intracellular interacting partners in orchestrating downstream signaling that influences the outcome of the host-pathogen encounter. [ABSTRACT FROM AUTHOR]