Porphyromonas gingivalis, a gram-negative anaerobic bacterium, is implicated as a major etiological agent in the initiation and progression of severe forms of periodontal disease. It has been reported that gingivitis and periodontal disease affect roughly 50.3% and 35% of the adult population, respectively. Therefore, approximately over 85% of the adult population may suffer from some form of gingival disease. Porphyromonas gingivalis, an established periodontopathogen, requires hemin for growth. Although multiple hemin uptake systems appear to be present in this organism, their specific role in hemin uptake and virulence remains unknown. Pigmentation is thought to result from the accumulation of iron protoporphyrin IX (FePPIX) derived from erythrocyte hemoglobin. It has been observed that mutations abolishing activity of the Lys-X specific cysteine protease, Kgp, resulted in loss of black pigmentation of P. gingivalis W83; they were less virulent than their wild-type counterparts. Thus, we have observed that Kgp degradation of fibrinogen deregulates the clotting cascade, thereby minimizing the availability of free erythrocytes. Additionally, Kgp binds erythrocytes and degrades them, releasing hemoglobin. The interference with mechanisms involved in the accumulation of black pigmentation may be significant in controlling the pathogenic potential of P. gingivalis. These results suggest that Lys-gingipain protease is a principal protein involved in acquisition of hemin from hemoglobin as well as a major factor in transport, by affecting the accumulation of FePPIX on the bacterial cell surface. Microarray analysis indicates a change in the expression of key enzymes and proteins required for hemin uptake, iron storage, electron transport and oxidative stress. Therefore, interference with mechanisms involved in accumulation of black pigmentation may be significant in controlling the pathogenic potential of P. gingivalis.

Pesquisas recentes investigando o receptor ativado por protease do tipo 2 (PAR-2) sugerem uma associação entre este receptor e a inflamação periodontal. Além disso, é sabido que a gingipaína, protease bacteriana secretada por um importante periodontopatógeno, Porphyromonas gingivalis (Pg), tem a capacidade de ativar o PAR-2. Ademais, um estudo anterior do grupo, verificou que quanto mais profunda a bolsa periodontal, maior era a expressão do receptor PAR-2. No entanto, não é sabido se a expressão de PAR-2 é proporcional a severidade de doença periodontal e a quantidade de gingipaína expressa na bolsa periodontal. Desta forma, o presente estudo, verificou no fluido gengival a correlação entre a expressão gênica de PAR-2 (Real Time-PCR) com os parâmetros clínicos periodontais, e a expressão gênica da protease gingipaína em pacientes com periodontite crônica severa e moderada, antes e após o tratamento periodontal não-cirúrgico. A expressão de PAR-2 e da protease gingipaína foi estatisticamente maior nos pacientes do grupo periodontite crônica severa (PS) em comparação com os pacientes do grupo periodontite crônica modera (PM) e controle (C). Além disso, o tratamento periodontal levou à redução significativa (p<0.05) da expressão de PAR-2 nos pacientes com periodontite crônica moderada. Em conclusão, dentro dos limites do presente estudo, nós demonstramos que a severidade da doença periodontal e a expressão de gingipaína influenciaram a expressão de PAR-2 no fluido gengival de pacientes com periodontite crônica. / Recent studies investigating the protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. In addition, it is known that gingipain, a bacterial protease secreted by an important periodontopathogen, Porphyromonas gingivalis (Pg), has the ability to activate PAR- 2. Furthermore, a previous study from our group found that the deeper the periodontal pocket, the higher the expression of the PAR-2 receptor. However, it is not known whether the expression of PAR-2 is associated to the severity of periodontal disease and the amount of gingipain expressed in the periodontal pocket. Thus, the present study verified, in the gingival fluid, the correlation between the PAR-2 gene expression (Real Time-PCR) with the clinical periodontal parameters, and the gene expression of the gingipain protease in patients with moderate and severe chronic periodontitis before and after non-surgical periodontal treatment. PAR-2 expression and gingipain protease were statistically more expressed in patients of the severe chronic periodontitis group (PS) compared with those in the moderate chronic periodontitis group (PM) and control group (C). Furthermore, periodontal treatment led to a significant reduction (p <0.05) in the expression of PAR-2 in patients with moderate chronic periodontitis. In conclusion, within the limits of the present study, we demonstrated that the severity of periodontal disease and the expression of gingipain influenced the PAR-2 expression in the gingival fluid of patients with chronic periodontitis.

Pesquisas recentes investigando o receptor ativado por protease do tipo 2 (PAR-2) sugerem uma associação entre este receptor e a inflamação periodontal. Além disso, é sabido que a gingipaína, protease bacteriana secretada por um importante periodontopatógeno, Porphyromonas gingivalis (Pg), tem a capacidade de ativar o PAR-2. Ademais, um estudo anterior do grupo, verificou que quanto mais profunda a bolsa periodontal, maior era a expressão do receptor PAR-2. No entanto, não é sabido se a expressão de PAR-2 é proporcional a severidade de doença periodontal e a quantidade de gingipaína expressa na bolsa periodontal. Desta forma, o presente estudo, verificou no fluido gengival a correlação entre a expressão gênica de PAR-2 (Real Time-PCR) com os parâmetros clínicos periodontais, e a expressão gênica da protease gingipaína em pacientes com periodontite crônica severa e moderada, antes e após o tratamento periodontal não-cirúrgico. A expressão de PAR-2 e da protease gingipaína foi estatisticamente maior nos pacientes do grupo periodontite crônica severa (PS) em comparação com os pacientes do grupo periodontite crônica modera (PM) e controle (C). Além disso, o tratamento periodontal levou à redução significativa (p<0.05) da expressão de PAR-2 nos pacientes com periodontite crônica moderada. Em conclusão, dentro dos limites do presente estudo, nós demonstramos que a severidade da doença periodontal e a expressão de gingipaína influenciaram a expressão de PAR-2 no fluido gengival de pacientes com periodontite crônica. / Recent studies investigating the protease-activated receptor type 2 (PAR-2) suggest an association between the receptor and periodontal inflammation. In addition, it is known that gingipain, a bacterial protease secreted by an important periodontopathogen, Porphyromonas gingivalis (Pg), has the ability to activate PAR- 2. Furthermore, a previous study from our group found that the deeper the periodontal pocket, the higher the expression of the PAR-2 receptor. However, it is not known whether the expression of PAR-2 is associated to the severity of periodontal disease and the amount of gingipain expressed in the periodontal pocket. Thus, the present study verified, in the gingival fluid, the correlation between the PAR-2 gene expression (Real Time-PCR) with the clinical periodontal parameters, and the gene expression of the gingipain protease in patients with moderate and severe chronic periodontitis before and after non-surgical periodontal treatment. PAR-2 expression and gingipain protease were statistically more expressed in patients of the severe chronic periodontitis group (PS) compared with those in the moderate chronic periodontitis group (PM) and control group (C). Furthermore, periodontal treatment led to a significant reduction (p <0.05) in the expression of PAR-2 in patients with moderate chronic periodontitis. In conclusion, within the limits of the present study, we demonstrated that the severity of periodontal disease and the expression of gingipain influenced the PAR-2 expression in the gingival fluid of patients with chronic periodontitis.

Syfte: Syftet med denna studie var att experimentellt bestämma om Parvimonas micra ökar tillväxt och gingipainaktiviteten hos olika stammar av Porphyromonas gingivalis. En ökning av gingipainaktiviteten indikerar att P. micra skulle kunna öka virulensen i P. gingivalis.Material och metod: Fem stammar av P. gingivalis 1A, 16A, 50A, 33F och W50 odlades med P. micra och jämfördes med kontrollen som endast innehöll P. gingivalis. Arginin gingipain (Rgp) och lysin gingipain (Kgp) aktivitet mättes med hjälp av Bikkam-16 respektive Bikkam-14. Bakterietillväxt mättes genom att mäta optisk densitet.Resultat: Experimenten visade en ökad tillväxt av stammarna 1A, 16A och 50A när de odlades med P. micra, 33F visade måttlig tillväxt och W50 ingen tillväxt.Rgp aktiviteten ökade i vissa P. gingivalis-stammar (1A och 16A) i närvaro P. micra men ökade inte i andra (W50, 33F och 50A)Kgp aktiviteten ökade i P. gingivalis lA i närvaro av P. micra men ingen ökning skedde i de andra stammarna (16A, W50, 33F och 50A).Slutsats: Resultaten från denna studie visade att P. micra påverkar tillväxten och gingipainaktiviteten hos vissa stammar av P. gingivalis men inte alla. / Aim: The aim of this study was to experimentally determine if Parvimonas micra increase growth and gingipain activity in different strains of Porphyromonas gingivalis. An increase in gingipain activity could indicate that P. micra induces pathogenic expression of P. gingivalis.Material and method: Five strains of P. gingivalis 1A, 16A, 50A, 33F and W50 was cultivated together with P. micra while control samples contained only P. gingivalis. Activity of arginine gingipain (Rgp) and lysine gingipain (Kgp) was measured using Bikkam-16 respectively Bikkam-14. Bacterial growth was determining by measuring the optical density.Results: The experiments show an increased growth of some strains (1A, 16A and 50A) of P. gingivalis when grown with P. micra, one showed moderate growth (33F) and one showed no growth (W50).The gingipain activity (Rgp) increased in P. gingivalis strains (1A and 16A) in the presence of P. micra but did not increase in other strains (W50, 33F and 50A)The gingipain activity (Kgp) increased in one P. gingivalis strains (1A) when mixed with P. micra and did not increase in others (16A, W50, 33F and 50A). Conclusion: The results from this study indicates that P. micra effect the growth and gingipain activity in certain strains of P. gingivalis but not all.