Structural analysis of TIFA: Insight into TIFA-dependent signal transduction in innate immunity
Authors: Nakamura, Teruya; Hashikawa, Chie; Okabe, Kohtaro; Yokote, Yuya; Chirifu, Mami; Toma-Fukai, Sachiko; Nakamura, Narushi; Matsuo, Mihoko; Kamikariya, Miho; Okamoto, Yoshinari; Gohda, Jin; Akiyama, Taishin; Semba, Kentaro; Ikemizu, Shinji; Otsuka, Masami; Inoue, Jun-ichiro; Yamagata, Yuriko
TRAF-interacting protein with a forkhead-associated (FHA) domain (TIFA), originally identified as an adaptor protein of TRAF6, has recently been shown to be involved in innate immunity, induced by a pathogen-associated molecular pattern (PAMP). ADP-beta-D-manno-heptose, a newly identified PAMP, binds to alpha-kinase 1 (ALPK1) and activates its kinase activity to phosphorylate TIFA. Phosphorylation triggers TIFA oligomerisation and formation of a subsequent TIFA-TRAF6 oligomeric complex for ubiquitination of TRAF6, eventually leading to NF-kappa B activation. However, the structural basis of TIFA-dependent TRAF6 signalling, especially oligomer formation of the TIFA-TRAF6 complex remains unknown. In the present study, we determined the crystal structures of mouse TIFA and two TIFA mutants-Thr9 mutated to either Asp or Glu to mimic the phosphorylation state-to obtain the structural information for oligomer formation of the TIFA-TRAF6 complex. Crystal structures show the dimer formation of mouse TIFA to be similar to that of human TIFA, which was previously reported. This dimeric structure is consistent with the solution structure obtained from small angle X-ray scattering analysis. In addition to the structural analysis, we examined the molecular assembly of TIFA and the TIFA-TRAF6 complex by size-exclusion chromatography, and suggested a model for the TIFA-TRAF6 signalling complex.
Commensal and Pathogenic Biofilms Alter Toll-Like Receptor Signaling in Reconstructed Human Gingiva
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
Authors: Shang, Lin; Deng, Dongmei; Buskermolen, Jeroen Kees; Roffel, Sanne; Janus, Marleen Marga; Krom, Bastiaan Philip; Crielaard, Wim; Gibbs, Susan
The balance between the host and microbe is pivotal for oral health. A dysbiotic oral microbiome and the subsequent host inflammatory response are causes for the most common dental problems, such as periodontitis and caries. Classically, toll-like receptors (TLRs) are known to play important roles in host-microbe interactions by recognizing pathogens and activating innate immunity. However, emerging evidence suggests that commensals may also exploit TLRs to induce tolerance to the benefit of the host, especially in oral mucosa which is heavily colonized by abundant microbes. How TLRs and downstream signaling events are affected by different oral microbial communities to regulate host responses is still unknown. To compare such human host-microbe interactions in vitro, we exposed a reconstructed human gingiva (RHG) to commensal or pathogenic (gingivitis, cariogenic) multi-species oral biofilms cultured from human saliva. These biofilms contain in vivo like phylogenic numbers and typical bacterial genera. After 24 h biofilm exposure, TLR protein and gene expression of 84 TLR pathway related genes were investigated. Commensal and pathogenic biofilms differentially regulated TLR protein expression. Commensal biofilm up-regulated the transcription of a large group of key genes, which are involved in TLR signaling, including TLR7, the MyD88-dependent pathway (CD14, MyD88, TIRAP, TRAF6, IRAKs), MyD88-independent pathway (TAB1, TBK1, IRF3), and their downstream signaling pathways (NF-kappa B and MAPK pathways). In comparison, gingivitis biofilm activated fewer genes (e.g., TLR4) and cariogenic biofilm suppressed CD14, IRAK4, and IRF3 transcription. Fluorescence in situ hybridization staining showed the rRNA of the topically applied and invaded bacteria, and histology showed that the biofilms had no obvious detrimental effect on RHG morphology. These results show an important role of TLR signaling pathways in regulating host-microbe interactions: when a sterile gingival tissue is exposed to commensals, a strong immune activation occurs which may prime the host against potential challenges in order to maintain oral host-microbe homeostasis. In contrast, pathogenic biofilms stimulate a weaker immune response which might facilitate immune evasion thus enabling pathogens to penetrate undetected into the tissues.