Role of Porphyromonas gingivalis Gingipains in Caspase-1 Activation and Phagocytosis of Tannerella forsythia

학위논문 (박사)-- 서울대학교 대학원 : 치의학대학원 치의과학과 분자미생물치의학전공, 2016. 2. 최봉규.Objectives In the pathogenesis of periodontitis, Porphyromonas gingivalis plays a role as a keystone pathogen that dysregulates host immune responses and results in dysbiosis in oral microbial communities. Arg-gingipains (RgpA and RgpB) and Lys-gingipain (Kgp) are essential to the virulence of P. gingivalis. Therefore, gingipains are often considered as therapeutic targets. The aim of this study was to elucidate the roles of gingipains in caspase-1 activation in P. gingivalis-infected macrophages and to investigate the roles of gingipains in the modulation by P. gingivalis of phagocytosis of Tannerella forsythia by macrophages. Methods Macrophages differentiated from THP-1 cells with phorbol-12-myristate-13-acetate (PMA) and peripheral blood mononuclear cells (PBMC)-derived macrophages were infected with P. gingivalis or its gingipain mutants for 6 hours to determine whether P. gingivalis activates caspase-1 and whether gingipain mutation affects the caspase-1 activation. Caspase-1, interleukin (IL)-1β, and lactate dehydrogenase (LDH) in the culture supernatants were analyzed with immunoblot, ELISA, and LDH cytotoxicity assay. To examine the role of gingipain protease activity, macrophages were infected with P. gingivalis in the presence or absence of leupeptin, a cysteine protease inhibitor, or P. gingivalis preincubated with gingipain-specific inhibitors, KYT-1 and KYT-36. Degradation of proteins that were released from cells upon caspase-1 activation was analyzed after incubation of P. gingivalis in the culture supernatants of macrophages that had been stimulated with heat-killed P. gingivalis. Intracellular caspase-1 activity and ATP release were measured after infection with P. gingivalis and its gingipain mutants, or P. gingivalis preincubated with KYT-1 or KYT-36. To assess the effect of processing of surface proteins by gingipains on caspase-1 activation, cells were infected with the gingipain-null mutant that had been cultured overnight with gingipain-containing culture supernatants of P. gingivalis. To determine whether P. gingivalis coinfection has an effect on T. forsythia phagocytosis, PMA-differentiated THP-1 cells and PBMC-derived macrophages were infected with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled T. forsythia in the presence or absence of P. gingivalis or its gingipain mutants for 1 hour. Phagocytosis of T. forsythia was analyzed by flow cytometry and confocal microscopy. Coaggregation between the two bacterial species was assessed, and the effect of amino acids that inhibit the coaggregation on T. forsythia phagocytosis was examined. The role of gingipain protease activity was determined using KYT-1 and KYT-36. Intracellular persistence/survival of T. forsythia was analyzed by flow cytometry, confocal microscopy, and 16S rRNA-based viability assay after antibiotic protection. Results Infection with P. gingivalis at low multiplicity of infections (MOIs), but not at high MOIs, resulted in low levels of IL-1β and LDH without detectable active caspase-1 in the culture supernatants. The proteins released from caspase-1-activated cells were rapidly degraded by gingipains. However, P. gingivalis expressing gingipains induced higher intracellular caspase-1 activity in the infected cells than the gingipain-null mutant. The increased intracellular caspase-1 activity was associated with ATP release from the infected cells. In addition, growing the gingipain-null mutant in the culture supernatants containing gingipains enhanced caspase-1 activation by the mutant. In contrast, inhibition of the protease activity of Kgp or Rgp increased the caspase-1-activating potential of wild-type P. gingivalis, indicating an inhibitory effect of the protease activities of Kgp and Rgp. Phagocytosis of T. forsythia was significantly enhanced by coinfection with P. gingivalis in an MOI- and gingipain-dependent manner. Mutation of either Kgp or Rgp in the coinfecting P. gingivalis resulted in attenuated enhancement of T. forsythia phagocytosis. Inhibition of coaggregation between the two bacterial species reduced phagocytosis of T. forsythia in the mixed infection, and the coaggregation was dependent on gingipains. Inhibition of gingipain protease activities in coinfecting P. gingivalis abated the coaggregation and the enhancement of T. forsythia phagocytosis. However, direct effect of protease activities of gingipains on T. forsythia seemed to be minimal. Although most of the phagocytosed T. forsythia were cleared in infected cells, more T. forsythia remained in cells coinfected with gingipain-expressing P. gingivalis than in cells coinfected with the gingipain-null mutant or infected only with T. forsythia at 24 and 48 hours post-infection. Conclusion Considering that gingipains are major promising therapeutic targets, it is of great importance to fully characterize the roles of gingipains in the pathogenicity of P. gingivalis. This study demonstrated that gingipains play contradictory roles in caspase-1 activation by P. gingivalis monoinfection, and that gingipains are essential in the augmentation of T. forsythia phagocytosis in the mixed infection. This study provides clues to the role of gingipains in the mechanism by which P. gingivalis dysregulates host immune responses.I. Introduction 1 1. Periodontitis 1 1.1. Periodontitis: definition, prevalence, and outcomes 1 1.2. Pathogenesis of periodontitis 2 2. Porphyromonas gingivalis 3 2.1. General characteristics of P. gingivalis 3 2.2. Virulence factors of P. gingivalis 4 2.3. Gingipains 6 2.3.1. Characteristics of gingipains 6 2.3.2. Functions in pathogenesis of periodontitis 8 3. Tannerella forsythia 8 3.1. General characteristics of T. forsythia 8 3.2. Virulence factors of T. forsythia 9 4. Mixed infection with P. gingivalis and T. forsythia 10 4.1. Interspecies bacterial interaction 10 4.2. Synergistic pathogenicity of P. gingivalis and T. forsythia 10 5. Macrophages and phagocytosis 11 5.1. Role of macrophages in periodontitis 11 5.2. Phagocytosis in macrophages 13 6. Inflammasome 15 6.1. Inflammasome and caspase-1 activation 15 6.2. Consequences of caspase-1 activation 19 6.3. Inflammasome activation by P. gingivalis 20 7. Aims of this study 21 II. Materials and Methods 22 1. Bacterial strains and growth conditions 22 2. Treatment of P. gingivalis gingipain-null mutant with membrane vesicle-depleted culture supernatants 23 3. Cell cultures 23 4. Infection for caspase-1 activation 24 5. Immunoblotting 25 6. ELISA 26 7. Real-time reverse-transcription PCR 26 8. Measurement of cell death 27 8.1. LDH cytotoxicity assay 27 8.2. Propidium iodide uptake 28 9. Protein degradation assay 28 10. Intracellular caspase-1 activity assay 28 11. Determination of extracellular ATP concentrations 29 12. Phagocytosis of T. forsythia 29 13. Intracellular persistence 31 14. Confocal microscopy 31 15. Coaggregation assay 32 16. RNA-based viability assay of intracellular T. forsythia 33 17. Statistical analysis 34 III. Results 35 1. Role of gingipains in caspase-1 activation 35 1.1 Low IL-1β and LDH levels without active caspase-1 in the culture supernatants at low MOIs are abrogated by high MOIs of P. gingivalis infection 35 1.2. Low levels of IL-1β result from the potent proteolytic activity of P. gingivalis 40 1.3. Rgps and Kgp of P. gingivalis wipe out caspase-1-dependent proteinaceous responses 47 1.4. Gingipains differentially enhance caspase-1 activation by promoting ATP release 52 1.5. Gingipains indirectly facilitate caspase-1 activation in infected cells, possibly by processing the surface proteins of P. gingivalis 57 1.6. Protease activity of gingipains impairs extracellular ATP release and caspase-1 activation 60 2. Role of gingipains in augmentation of T. forsythia phagocytosis 62 2.1. Phagocytosis of T. forsythia is facilitated by coinfection with P. gingivalis 62 2.2. P. gingivalis gingipains are essential for the enhancement of phagocytosis of T. forsythia 64 2.3. T. forsythia augments phagocytosis of P. gingivalis 67 2.4. Coaggregation of T. forsythia with P. gingivalis that expresses gingipains contributes to the augmentation of phagocytosis of T. forsythia 69 2.5. Protease activities of P. gingivalis gingipains are partially responsible for the facilitation of phagocytosis of T. forsythia 73 2.6. Direct effect of soluble gingipains on T. forsythia is minimal 75 2.7. More T. forsythia remain viable in the infected cells in P. gingivalis coinfection 77 2.8. More T. forsythia-infected cells remain viable in the presence of P. gingivalis 84 IV. Discussion 86 1. Role of gingipains in caspase-1 activation 86 2. Role of gingipains in augmentation of T. forsythia phagocytosis 92 3. Pathological and clinical implication 96 V. Conclusion 98 VI. References 99 List of Publications 117 Abstract 118Docto

Quality characteristics of alcoholic beverage, vinegar and rice cake using pleurotus ostreatus, flammulina velutipes and grifola frondosa

학위논문(석사) --서울대학교 대학원 :식품영양학과, 2009.2.Maste

나노컴포지트에서 acidulated phosphate fluoride 적용에 따른 Streptococcus mutans 부착량 변화

Thesis(master`s)--서울대학교 대학원 :치의학과 소아치과학전공,2005.Maste

Effects of collagenase and esterase on dentin bonding

학위논문(박사)--서울대학교 대학원 :치의학과 소아치과학 전공,2007.Docto

Changes in adhesion of Streptococcus mutans to nanocomposite resins after acidulated phosphate fluoride gel applicaion

복합레진 표면에 대한 APF gel 도포는 표면을 거칠게 하고 세균 부착을 증가시킨다. 본 연구는 Filtek Z250(FZ), Filtek Supreme Universal(FS)과, 실험적으로 나노충전재를 각각 0%, 3%, 6% 포함시켜 만든 복합레진(E0, E3, E6)으로 제작한 레진 시편을, APF gel을 적용한 군과 적용하지 않은 군으로 나누어 Streptococcus mutans의 부착량과 표면 조도를 측정하고 비교, 평가하여 다음과 같은 결과를 얻었다. 1. APF gel을 적용하지 않은 레진 시편에 대한 S. mutans 부착량은 FS에서 가장 적었고, FZ, E3, E6에 대한 부착량보다는 유의하게 낮았다(p0.05). Topical application of APF gel can increase the surface roughness of resin composites and the roughened surfaces may allow increased bacterial accumulation and surface staining. Resin specimens of two proprietary resin composites, Filtek Z250(FZ) and Filtek Supreme Universal(FS), and experimental resin composites containing 0%, 3%, 6% nanofillers(E0, E3, E6) were fabricated and divided into two groups of the same number; APF treatment group and no treatment group. The amount of S. mutans adhered to specimens and the mean surface roughness(Ra) were measured. The results were as follows; 1. In no treatment group, the amount of S. mutans adhered to FS was the smallest. It was significantly different from those of FZ, E3, E6(p0.05). 2. For all resin composites used, the amount of S. mutans adhesion in APF treatment group was significantly greater than that in no treatment group(p0.05).본 연구는 보건복지부 보건의료기술연구개발사업의 지원에 의하여 이루어진 것임 (03-PJ1-PG1-CH09-0001)

매복된 하악 견치의 치험례

Impaction of mandibular canine is not common, and transmigration of mandibular canine is rare. Treatment of impacted canine can be removal of physical obstacle and periodic observation, surgical exposure of impacted tooth and orthodontic traction, autotransplantation, surgical extraction. Management of impacted canine depends on existence of physical obstacle, position and direction of impacted tooth, space available for canine eruption, stage of root development. Of the two case in this report, one case involved impaction of lower canine with odontoma and dentigerous cyst that is treated by surgical exposure and orthodontic traction. The other case involved transmigration of lower canine with supernumerary teeth. It was thought difficult to treat only by orthodontic treatment, so the impacted canine was transplanted to its normal position and orthodontic treatment was conducted

나노 충전제 함량에 따른 복합레진의 표면 미세경도 차이

The objective of this study was to evaluate the effects of nanofiller content on the microhardness and polymerization of experimental microhybrid composites. The nanofiller contorts in the experimental composites were varied (0%, 1%, 2%, 3%), while the total filler content remained constant as 76%wt. We obtained the following results: 1. The microhardness of the top surface for the 2% 3% nanofilled microhybrid composites were significantly higher than those for the 0%, 1% nanofilled composites (p0.05). 3. As the nanofiller level increased, the difference between microhardness of top and bottom surfaces significantly decreased (p0.05)

Collagenase와 esterase가 상아질 접착강도와 nanoleakage에 미치는 영향

The purpose of this study was to evaluate the effects of collagenase and esterase on dentin bond strength and nanoleakage. Resin composites were bonded to occlusal dentin of premolars with Single Bond 2(SB) and Clearfil SE Bond(SE). After the microtensile specimens were prepared and stored in PBS for 24 hours(I) or, PBS(II), collagenase(III), esterase(IV) solution for 4 weeks, the specimens were stained with silver nitrate solution. Microtensile bond strength() and silver penetration area were measured and, the results were as follows: 1. For group II, III, and IV, the bond strengths of SB were lower than those of SB(p0.05). 2. Silver penetration areas of SB were higher than those of SE for all storage groups(p0.05). 3. SE I, II, and III showed inverse relationship between the bond strengths and the silver penetration areas(p<0.05)

상아질 접착에서 collagenase와 esterase가 미세인장결합강도에 미치는 영향

The purpose of this study was to evaluate the effect of collagenase and esterase on the microtensile bond in dentin bonding. After resin composites were bonded to occlusal dentin. specimens were formed and stored in PBS, collagenase, or esterase solution After 4-week storage, was determined and, the results were as follows : 1. values of Single Bond 2 were lower than those of Clearfil SE Bond for all storage medium (p0.05). 3. In Clearfil SE Bond group, esterase solution lowered bond strength more than PBS(p>0.05). Collagenase solution lowered bond strength more than esterase solution(p>0.05) and PBS(p<0.05)