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치주염과 관련된 구강나선균의 공통 표면단백질의 병독력과 NLRP3 인플라마좀 활성 : Virulence and NLRP3 inflammasome activation of common surface protein in oral spirochetes associated with periodontitis

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치과대학 치의학과
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구강나선균Tp92 유사단백질염증NLRP3 인플라마좀인테그린 alpha5 beta1치주질환
학위논문 (박사)-- 서울대학교 대학원 : 치의학과, 2013. 2. 최봉규.
Oral spirochetes are composed of an enormously heterogeneous group of Treponema species, and some have been implicated in the etiology of periodontitis. Multiple species or phylotypes of Treponema have often been found in a single patient or a single periodontal pocket. Some outer membrane proteins (OMPs) of oral spirochetes are important in immune modulation, which has been demonstrated in molecular pathogenesis studies. Tp92 of T. pallidum, a surface protein, has been reported to have opsonic potential and to induce a protective immune response against syphilis, T. pallidum infection. It has been reported that the Tp92 homologs are a family of highly conserved OMPs in gram-negative bacteria that may have conserved functions. It is therefore important to find common virulence determinants that are localized on the surface of oral spirochetes and to characterize them in order to elucidate their role in periodontal pathogenesis. The pro-inflammatory and bone-resorptive characteristics of IL-1β are associated with the immunopathology of periodontitis, leading to periodontal tissue destruction. IL-1β secretion is required for caspase-1 activation, which is associated with the inflammasome, which contributes to innate immunity. In this study, Tp92 homologs of four representative oral spirochetes (T. denticola, T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii), which are strongly associated with periodontitis, were identified and characterized for their virulence.
Tp92 homologs from oral spirochetes were expressed in Escherichia coli, and the recombinant proteins were purified. To assess the binding ability of the Tp92 homologs to KB cells (ATCC CCL-17), a carcinoma epithelial cell line was incubated with the FITC-labeled Tp92 homologs or carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled bacteria. The cross-reactivity of the Tp92 homologs was detected with antiserum (IgG fraction) against Td92, a Tp92 homolog from T. denticola. The biological activities of the Tp92 homologs were analyzed by assessing the expression of various factors regarding inflammation and tissue destruction in a human monocytic cell line (THP-1) and periodontal ligament cells (PDL cells).
To identify the molecular mechanisms of T. denticola Td92-induced IL-1β secretion, PMA-differentiated THP-1 cells were stimulated with Td92 in the presence or absence of KCl, oxATP, and K+ channel blockers, and then, caspase-1 activation, proIL-1β expression, and IL-1β secretion were determined. The components of the inflammasome involved in Td92-induced caspase-1 activation were examined using siRNA against NLRP3, ASC, and NLRC4. To determine whether Td92 internalization is a prerequisite for NLRP3 activation, Td92 was internalized into the cells using protein transfection reagents or in the presence or absence of cytochalasin D. A cellular receptor that binds to Td92 and results in inflammasome activation was identified using an affinity ligand-binding assay. To determine which signaling pathway contributes to the Td92-induced pro-IL-1β expression following Td92 binding to the cellular receptor, MAPK pathway activation and NF-κB translocation were determined by immunoblotting and confocal microscopy in the presence or absence of various inhibitors or neutralizing Abs.
The Tp92 homologs of the four oral spirochetes had signal peptides (20-31 amino acids) and molecular masses of 88-92-kDa for the mature proteins. The tp92 gene homologs were successfully expressed in E. coli, and the recombinant proteins were capable of binding to KB cells. Furthermore, the binding of the bacteria to the cells was inhibited by the addition of T. denticola Td92. An antiserum (IgG fraction) raised against T. denticola Td92 cross-reacted with the Tp92 homologs from the three other species of treponemes, including T. lecithinolyticum, T. maltophilum, and T. socranskii subsp. socranskii. The Tp92 homologs stimulated various factors involved in inflammation and osteoclastogenesis, such as IL-1β, IL-6, IL-8, TNF-α, COX-2, PGE2, and MMP-9, in host cells, including monocytes and fibroblasts.
T. denticola Td92 could activate caspase-1, pro-IL-1β, and IL-1β secretion, which resulted from ATP release and K+ efflux. Td92-induced caspase-1 activation was markedly decreased by the knockdown of NLRP3 or ASC, while the knockdown of NLRC4 did not affect Td92-induced caspase-1 activation. The rapid uptake of Td92 by a protein transfection reagent into the cells decreased Td92-induced caspase-1 activation, and blocking Td92 internalization by treatment with cytochalasin D did not affect caspase-1 activation or proIL-1β expression. Integrin α5β1 played a critical role in NLRP3 inflammasome activation because integrin α5β1 is a receptor for T. denticola Td92. Although Td92 was able to bind integrin α5β1 and fibronectin, Td92-induced NLRP3 inflammasome activation was mediated via direct binding to integrin α5β1, independent of fibronectin binding. The integrin α5β1 priming by Td92 led to the induction of pro-IL-1β expression, NLRP3 expression, and caspase-1 activation, which were mediated via NF-κB activation.

Td92 directly interacts with integrin α5β1 to activate the NLRP3 inflammasome and upregulate proIL-1β synthesis, which could lead to uncontrolled IL-1β secretion. Therefore, integrin α5β1 could be regarded as a principal cell membrane receptor for both NLRP3 inflammasome activation and IL-1β transcription by Td92. Understanding of the interaction between Td92 and integrin α5β1 may contribute to the development of a new therapeutic target for periodontitis treatment.
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