Gingipaine sind Cysteinproteinasen des wohl in Ätiologie und Pathogenese der chronischen Erwachsenenparodontitis bedeutsamsten bakteriellen Erregers und zugleich die wichtigsten Virulenzfaktoren der Spezies Porphyromonas gingivalis. Die für diese extrazellulären Produkte kodierenden Gene sind rgpA, rgpB und kgp. Deren Produkte sind entsprechend HRgpA, RgpB und Kgp. HRgpA und RgpB verursachen eine Steigerung der Gefäßpermeabilität durch die Aktivierung des Kallikrein/Kinin-Systems und aktivieren die Blutgerinnung, welche potentiell mit der Synthese der Sulkusflüssigkeit und dem Fortschreiten der Entzündung bis hin zum Verlust des alveolären Knochens assoziiert sind. Offenbar wird dieses durch die Aktivierung von Matrixmetalloproteinasen begünstigt. Kgp ist von den dreien die potenteste fibrinogen-und fibrinabbauende Proteinase und bei der Blutungsneigung der erkrankten Stellen involviert. HRgpA aktiviert besonders Blutgerinnungsfaktoren. Gingipaine stören das Komplementsystem und manipulieren den Zytokinhaushalt der Entzündungskaskaden. Die Gingipaine unterstützen die Kolonisierung von P. gingivalis durch die Bindung zu anderen Bakterien des subgingivalen Biofilms und der Bindung zu epithelialen Zellen. Sie vermögen an Laminin, Fibrinogen, Fibronektin, Hämoglobin und an manchen Typen von Kollagen zu binden. Alle können den Rezeptor CD14 auf Makrophagen abbauen und so die Leukozytenaktivierung hemmen. Sie regulieren die Infektionsintensität, den Bakterienhaushalt, die Aminosäurenaufnahme aus Wirtsproteinen und die Fimbrienreifung. Die Genetik, die Chemie und die virulenzverursachenden Eigenschaften der Gingipaine stehen seit Mitte der 90iger Jahre des letzten Jahrhunderts im Blickpunkt des wissenschaftlichen Interesses. Aufgrund ihrer Schlüsselrolle bei der Pathogenese der Parodontitis und der mikrobiellen Infektion sind die Gingipaine Ziele für die mögliche Entwicklung von Hemmstoffen respektive für Immunisierungsstrategien gegen die chronische Parodontitis.:Erklärungen I Genehmigungsvermerk II Inhaltsverzeichnis III 1. Einleitung und Fragestellung 4 1.1 Parodontitis 4 1.1.1 Epidemiologie 4 1.1.2 Klassifikation 4 1.1.3 Ätiologie und Pathogenese 5 1.1.4 Mikrobiologie 7 1.1.5 Biofilm 8 1.2 Porphyromonas gingivalis 10 1.3 Verwandtschaftsverhältnisse von P. gingivalis 12 1.4 Mit P. gingivalis assoziierte Proteinasen 15 1.5 Fragestellung 16 2. Material und Methoden 17 3. Ergebnisse und Diskussion 18 3.1 Einführung – Gingipaine als Enzyme von P. gingivalis 18 3.1.1 Definition der Gingipaine 18 3.1.2 RgpA- und RgpB-Gen-Gingipaine 19 3.1.3 Kgp-Gen-Gingipain 20 3.2 Bedeutung der Gingipaine für die Pathogenität von P. gingivalis 21 3.2.1 Gingipaine und fimbrienvermittelte Adhäsion, intrazelluläre Invasion und parazellulärer Pfad von P. gingivalis 21 3.2.2 Effekte auf die Integrität des Bindegewebes 24 3.2.3 Aktivierung des Kallikrein-Kinin-Systems 25 3.2.4 Aktivierende Einflüsse auf das Gerinnungssystem 27 3.2.5 Hemmende Einflüsse auf das Fibrinogen-Fibrin-System 28 3.2.6 Beeinträchtigung der Immunabwehr des Wirts 28 3.3 Gingipaine im biochemischen Regulationsmechanismus von P. gingivalis 35 3.3.1 Die Rolle des proteolytischen Systems von P. gingivalis beim Nährstofferwerb 35 3.3.2 Gingipainreifung und die Steuerung des intrazellulären Haushalts von P. gingivalis 39 3.3.3 Regulation der Proteinaseexpression von P. gingivalis 40 3.3.4 Gingipaine als bakterielle Hämagglutinine, Adhäsine und hämoglobinbindende Proteine 42 3.3.5 Proteinasegenmutationen bei P. gingivalis und ihre enzymatische Aktivität 42 3.4 Gingipaine und systemische Effekte 44 3.5 Synergismen mit anderen Spezies und quorum sensing 45 3.6 Hemmstoffe der Gingipaine 46 3.7 Immunisierung gegen Gingipaine 48 4. Zusammenfassung / Summary 52 5. Literaturverzeichnis 53 Anhang 87 Abbildungsverzeichnis 87 Tabellenverzeichnis 87 Danksagung 88 Lebenslauf 89 / Gingipains are cysteine proteases of the probably most important bacterial pathogen of the adult periodontitis in the field of etiology as well as pathology. At the time, they are the most important virulence factors of the species Porphyromonas gingivalis. The encoding genes of this extracellular products are rgpA, rgpB and kgp. Their products are corresponding HRgpA, RgpB and Kgp. HRgpA and RgpB induce vascular permeability enhancement through activation of the kallikrein/kinin system and activate the blood coagulation, processes potentially associated with gingival crevicular fluid synthesis and progression of inflammation which ultimately can lead to alveolar bone loss. Obviously, this will be favoured by matrix metalloproteinases. Kgp is the most potent fibrinogen/fibrin degrading enzyme of the three gingipains involved in the bleeding tendency at the diseased sites. HRgpA especially activates coagulation factors. Gingipains disturb the complement system and manipulate the cytokine network of the inflammation cascades. Gingipains support colonizing of P. gingivalis due to their connection to other bacteria of the subgingival biofilm and to epithelial cells. They are able to bind to laminin, fibrinogen, fibronectin, hemoglobin and some types of collagen. All of them are able to degrade macrophage CD14 receptor, thus preventing activation of the leukocytes. They regulate the intensity of infection and the bacterial housekeeping, including amino acid uptake from host proteins and fimbriae maturation. Genetics, chemistry and the virulence inducing properties of gingipains are the focus of scientific attention since the middle of the nineties of the last century. Due to their key role in pathogenesis of periodontitis and microbial infection the gingipains are targets for the possible development of inhibitory substances, respectively for immunization strategies against chronic periodontitis.:Erklärungen I Genehmigungsvermerk II Inhaltsverzeichnis III 1. Einleitung und Fragestellung 4 1.1 Parodontitis 4 1.1.1 Epidemiologie 4 1.1.2 Klassifikation 4 1.1.3 Ätiologie und Pathogenese 5 1.1.4 Mikrobiologie 7 1.1.5 Biofilm 8 1.2 Porphyromonas gingivalis 10 1.3 Verwandtschaftsverhältnisse von P. gingivalis 12 1.4 Mit P. gingivalis assoziierte Proteinasen 15 1.5 Fragestellung 16 2. Material und Methoden 17 3. Ergebnisse und Diskussion 18 3.1 Einführung – Gingipaine als Enzyme von P. gingivalis 18 3.1.1 Definition der Gingipaine 18 3.1.2 RgpA- und RgpB-Gen-Gingipaine 19 3.1.3 Kgp-Gen-Gingipain 20 3.2 Bedeutung der Gingipaine für die Pathogenität von P. gingivalis 21 3.2.1 Gingipaine und fimbrienvermittelte Adhäsion, intrazelluläre Invasion und parazellulärer Pfad von P. gingivalis 21 3.2.2 Effekte auf die Integrität des Bindegewebes 24 3.2.3 Aktivierung des Kallikrein-Kinin-Systems 25 3.2.4 Aktivierende Einflüsse auf das Gerinnungssystem 27 3.2.5 Hemmende Einflüsse auf das Fibrinogen-Fibrin-System 28 3.2.6 Beeinträchtigung der Immunabwehr des Wirts 28 3.3 Gingipaine im biochemischen Regulationsmechanismus von P. gingivalis 35 3.3.1 Die Rolle des proteolytischen Systems von P. gingivalis beim Nährstofferwerb 35 3.3.2 Gingipainreifung und die Steuerung des intrazellulären Haushalts von P. gingivalis 39 3.3.3 Regulation der Proteinaseexpression von P. gingivalis 40 3.3.4 Gingipaine als bakterielle Hämagglutinine, Adhäsine und hämoglobinbindende Proteine 42 3.3.5 Proteinasegenmutationen bei P. gingivalis und ihre enzymatische Aktivität 42 3.4 Gingipaine und systemische Effekte 44 3.5 Synergismen mit anderen Spezies und quorum sensing 45 3.6 Hemmstoffe der Gingipaine 46 3.7 Immunisierung gegen Gingipaine 48 4. Zusammenfassung / Summary 52 5. Literaturverzeichnis 53 Anhang 87 Abbildungsverzeichnis 87 Tabellenverzeichnis 87 Danksagung 88 Lebenslauf 89

Objectifs: Le but de cette étude clinique était de comparer un groupe d’adultes ayant un parodonte sain avec un groupe d’adultes atteints de parodontite chronique en terme de risque carieux et mesures cliniques et microbiologiques de la carie. Méthodes: Quatre-vingt-seize individus ont été divisés en deux groupes en fonction de leur état de santé parodontal et ont été appariés pour l'âge, le sexe et l'origine ethnique. Trente-huit sujets étaient atteints de parodontite chronique définie comme ayant au moins quatre dents avec ≥ 1 site avec une profondeur de sondage ≥ 4 mm et une perte d'attache clinique ≥ 2 mm, et 58 sujets présentaient un parodonte sain. Par la suite, les groupes ont été subdivisés en deux groupes en fonction de leur statut carieux : les participants ayant au moins une lésion carieuse non traitée sur une surface dentaire et ceux n’ayant pas de lésion carieuse non traitée. Les données ont été recueillies par le biais d’un questionnaire, un examen clinique et des échantillons de plaque supra- et sous-gingivale. L’évaluation de la charge buccale de Streptococcus mutans et de six agents pathogènes parodontaux a été réalisée par la technique d'amplification de la réaction en chaine de la polymérase (PCR). Les données ont été analysées à l'aide d’analyses statistiques descriptives et bivariées. Résultats: Les individus atteints de parodontite chronique étaient 3,5 fois plus susceptibles d'avoir des caries que les individus en bonne santé (OR 3,5 ; IC: 1,5 - 8,3 ; P = 0,006). Les sujets à la fois atteints de parodontite chronique et de caries dentaires ont eu un niveau d’éducation significativement plus faible que les sujets ayant un parodonte sain et sans caries dentaires (OR 6,0 ; IC: 1,7 à 21,7 ; P = 0,04). La proportion de sujets ayant une charge buccale élevée de Porphyromonas gingivalis (P. g.) et Treponema denticola (T. d.) était significativement plus élevée chez les patients atteints de parodontite chronique et de carie que chez les patients sains présentant des caries (P. g.: OR 8,6 ; IC: 2,4 - 30,3 ; P = 0,004 et T. d.: OR 10,0 ; CI: 2,6 - 38.1 ; P = 0,003). Conclusions: Les résultats de cette étude suggèrent que, chez les sujets adultes atteints de la parodontite chronique, la fréquence des caries est plus élevée que chez les sujets ayant un parodonte sain. De plus, le faible niveau d'éducation influence négativement le statut parodontal des individus. / Aim: The aim of this clinical study was to compare adults with a healthy periodontium and those with chronic periodontitis, in terms of caries’ risk and caries’ clinical and microbiological measures. Methods: Ninety-six healthy adults were divided into chronic periodontitis (n= 38) and healthy periodontium (n=58) based on their periodontal status, and matched for age, gender, and ethnic background. Chronic periodontitis was defined as having at least four teeth with ≥1 site with a pocket depth ≥4 mm and clinical attachment loss ≥2 mm. Each group were subsequently subdivided in 2 groups according to their caries status: participants having at least one untreated decayed surface and those with no untreated caries. Data were collected by means of self-administrated questionnaire, clinical examination, and supra- and subgingival plaque sampling. Assessments of oral levels of Streptococcus mutans and six periodontal pathogens were conducted by PCR amplification techniques. Data were analyzed using descriptive and bivariate statistical tests. Results: Individuals with chronic periodontitis were 3.5 times more likely to have caries than healthy individuals (OR 3.5; CI: 1.5 – 8.3; P = 0.006). Subjects with both chronic periodontitis and dental caries had a significantly lower level of education than periodontally healthy subjects without dental caries (OR 6.0; CI: 1.7 – 21.7; P = 0.04). A significant higher proportion of subjects with high oral levels of Porphyromonas gingivalis (P. g.) and Treponema denticola (T. d.) was found among subjects with chronic periodontitis and untreated caries compared to periodontally healthy subjects with untreated caries (P. g.: OR 8.6; CI: 2.4 – 30.3; P = 0.004 and T. d.: OR 10.0; CI: 2.6 – 38.1; P = 0.003). Conclusion: The results from this study suggest that, adults with chronic periodontitis are more prone to caries disease than those adults with a healthy periodontium. Furthermore, low educational level could have a negative impact on the periodontal status of individuals.

Microorganismos da cavidade oral têm sido admitidos como causadores de doenças sistêmicas com reconhecido mecanismo de disseminação via corrente sangüínea. Diferentes fatores, incluindo a presença da doença periodontal, têm influência no risco de bacteremia oral, podendo ocasionar endocardite infecciosa por Streptococcus viridans. Sendo assim, a manutenção da saúde bucal adquire elevado grau de importância em gestantes portadoras de doença valvar reumática, em que o risco de endocardite infecciosa é eminente. A escassez científica fez deste tema o objetivo deste estudo: investigar a condição clínica periodontal de gestantes portadoras de cardiopatia valvar, identificando agentes periodontopatógenos nas amostras coletadas de saliva, sulco/bolsa periodontal, Para tanto, foram estudadas 52 gestantes cardiopatas (GC) e 70 gestantes não-cardiopatas (GNC). A condição periodontal foi avaliada empregando-se profundidade clínica de sondagem (PCS), nível clínico de inserção (NCI), linha esmalte cemento/margem gengival (LEC/MG), índice de sangramento (IS) e índice de placa bacteriana (IP). As seguintes médias foram obtidas para os parâmetros periodontais avaliados: PCS: 1.52 (GC) e 1.45 (GNC); NCI: 1.13 (GC) e 1.02 (GNC); LEC/MG: 0.41 (GC) e 0.40 (GNC); IS: 7.34 (GC) e 6.27 (GNC) e IP: 12.19 (GC) e 13.48 (GNC). Não houve diferença entre os grupos para o NCI (p= 0,612). A presença da Porphyromonas gingivalis na saliva foi maior (p= 0,007) no GNC, porém não houve diferença nas amostras de sulco/bolsa periodontal. / Microorganisms of the oral cavity are known to cause systemic diseases, spread through sanguine current. Different factors, including the presence of periodontal disease, influencing the risk of oral bacteremia could cause infectious endocarditis for Streptococcus viridans. Nevertheless, the maintenance of the oral health is extremely important in pregnant women with rheumatic valvar disease, in which the risk of infectious endocarditis is eminent. The aim of this study was to investigate the clinical periodontal condition of pregnant women with valvar disease and to identify the presence of Porphyromonas gingivalis in saliva and subgingival samples. For these purposes, we studied 52 pregnant with valvar disease (GC) and 70 healthy pregnant women (GNC). The following periodontal parameters were evaluated: probing depth (PCS), clinical attachment level (NCI), gingival margin location (LEC/MG), bleeding on probing (IS) and plaque index (IP). The following mean periodontal parameters were obtained: PCS: 1.52 (GC) e 1.45 (GNC); NCI: 1.13 (GC) e 1.02 (GNC); LEC/MG: 0.41 (GC) e 0.40 (GNC); IS: 7.34 (GC) e 6.27 (GNC) e IP: 12.19 (GC) e 13.48 (GNC). There was no statistical difference for NCI among the groups. There was no difference between periodontal clinical conditions in pregnant women with valvar disease and healthy pregnant women. The presence of the Porphyromonas gingivalis in saliva samples of healthy pregnant women is statistically higher than in pregnant woman with valvar disease; however, there was no difference in periodontal samples

Dentro do contexto do novo paradigma da doença periodontal, alguns estudos têm sugerido que a doença periodontal poderia influenciar o desenvolvimento de doenças sistêmicas, incluindo os acidentes vasculares cerebrais. O objetivo deste estudo foi avaliar as condições periodontais de pacientes com acidente vascular cerebral (AVC), comparativamente à amostra populacional sem AVC, bem como identificar e quantificar o nível de bactérias periodontopatogênicas presentes nas áreas de bolsa periodontal com a finalidade de investigar a correlação da doença periodontal com acidente vascular cerebral. Para tanto, foram selecionados 80 pacientes de ambos os sexos, com idade entre 30-80 anos. O grupo experimental foi constituído por 20 pacientes internados em ambiente hospitalar devido à ocorrência de AVC. O grupo controle foi constituído de 60 pacientes provenientes da amostra populacional da cidade de Bauru que não apresentavam sinais e sintomas clínicos ou história familiar de AVC. Um questionário de saúde investigando as possíveis causas do AVC e outras condições sistêmicas foi aplicado a todos os pacientes. Os dois grupos foram avaliados periodontalmente quanto às medidas de profundidade de sondagem, nível de inserção, sangramento à sondagem e índice de placa. Para identificar e quantificar as bactérias periodontopatogênicas, Porphyromonas gingivalis e Aggregatibacter (Actinobacillus) actinomycetemcomitans, em ambos os grupos, foi coletada amostra de placa dentomicrobiana subgengival dos dois sítios com maior profundidade de sondagem de todos os pacientes, por meio da introdução de tira de papel esterilizada (PerioPaper) no sulco gengival. A análise qualitativa e quantitativa dessas bactérias foi realizada por meio de PCR convencional e em tempo real. Os dados obtidos foram analisados por meio de análise de variância (ANOVA) complementado pelo método de Tukey, teste de correlação de Spearman, teste \"t\" de Student, Mann-Whitney, Qui-Quadrado e \"Odds Ratio\" para avaliar a correlação entre os diferentes parâmetros clínicos periodontais com o AVC e os resultados obtidos pelo PCR convencional e PCR em tempo-real, com nível de confiança de 95%. Os resultados obtidos mostraram maior prevalência da doença periodontal no grupo teste (95%) do que no controle (28,3%). O nível de inserção à sondagem, índice de placa e índice gengival estavam significativamente aumentados nos pacientes com AVC (p<0,001). Entretanto, as medidas de profundidade de sondagem não mostraram diferenças significantes entre os grupos (p=0,051), embora estivessem aumentadas para o grupo teste. A presença e quantidade da P.gingivalis foi estatisticamente maior no grupo teste do que no controle (p<0,05). Não foi encontrado A.actinomycetemcomitans em nenhum dos grupos estudados. Do total de pacientes com AVC, 65% desenvolveram AVC-I e 35% AVC-H. Pacientes com AVC-H abrigavam maiores níveis de P.gingivalis do que pacientes com AVC-I. Entretanto, nesse grupo, houve correlação positiva entre bolsas mais profundas e contagem de bactérias (p<0,05), o que não foi observado para AVC-H. A análise de risco por \"odds ratio\" identificou que pacientes com doença periodontal apresentam risco elevado de desenvolvimento de AVC (OR=48,06, IC=95%). Esses achados indicam que a doença periodontal é mais prevalente e severa em pacientes com AVC-I ou AVC-H, com grande quantidade de bactérias, especialmente P. gingivalis, presente em bolsas periodontais mais profundas, sugerindo que a doença periodontal poderia atuar como fator de risco ao desenvolvimento de acidentes vasculares cerebrais. / Inside of the context of the new paradigm of the periodontal disease, some studies have suggested that the periodontal disease could influence the development of systemics diseases, including vascular cerebral accident. The aim of this study was to evaluate the periodontal conditions of patients with vascular cerebral accidents (VCAs), comparatively to the population sample without VCA, as well as identifying and quantifying the level of periodontopathic bacteria presents in the areas of periodontal pocket in order to investigate the correlation of the periodontal disease with vascular cerebral accident. For this study, it had been selected 80 patients of both genders, with age between 30-80 years. The experimental group consisted of 20 hospitalized patients presenting VCA. The control group was consisted of 60 patients proceeding from the population sample of Bauru who did not present clinical signs and symptoms or family history of VCA. A health questionnaire investigating the possible causes of the VCA and others systemics conditions was applied to all patients. The both groups were periodontally evaluated according to probing depth measures; bleed on probing and plaque index. In other to identify and quantify the periodontopathic bacteria Porphyromonas gingivalis and Aggregatibacter (Actinobacillus) actinomycetemcomitans, in both groups, a sample of subgengival plaque was collected from the two deepest sites of all patients by the introduction of sterilized paper strip (PerioPaper). The qualitative and quantitative analysis of these bacteria was performed by conventional PCR and Real Time PCR. Data were analyzed by variance analysis test (ANOVA) complemented by the Tukey test, Pearson correlation test, Student \"t\" test, Mann-Whitney test, Qui-Square test and Odds Ratio to evaluate the different correlations between the different periodontal clinical parameters VCA, and the results obtained from the Real Time PCR, with a 95% confidence level. The analysis of the results showed significantly bigger prevalence of the periodontal disease in the test group (95%) than the control group (28,3%). The level of insertion, plaque index and bleeding on probing were significantly increased in the patients with VCA (p<0,001). However, the probing depth measures had not shown significant differences between the groups (p=0,051), even so were increased for the test group. The presence and amount of the P.gingivalis were statistically bigger in the test group than the control group (p<0,05). A.actinomycetemcomitans was not found in any of the studied groups. Considering all patients with VCA, 65% developed VCA-I and 35% VCA-H. Patients with VCA-H showed greater levels of P.gingivalis than patient with VCA-I. However, in this group, it had positive correlation between deeper pockets and number of bacteria (p<0,05), what it was not observed for VCA-H. The analysis of risk for \"odds ratio\" identified that patient with periodontal disease present high risk of VCA development (OR=48,06, IC=95%). These findings show that the periodontal disease is more prevalent and severe in patients with VCA-I or VCA-H, with great amount of bacteria, especially P. gingivalis, present in deeper periodontal pockets, suggesting that the periodontal disease could play as risk factor on the development of vascular cerebral accident.

Porphyromonas gingivalis é um dos principais mircrorganismos associados à periodontite. O objetivo do presente estudo foi testar a hipótese de que a colonização por diferentes genótipos fimA de P.gingivalis resultaria em diferenças na resposta ao tratamento periodontal. Foram analisados 20 pacientes com periodontite crônica, fumantes, portadores de P.gingivalis, divididos em 2 grupos: 1 grupo recebeu tratamento periodontal mecânico (RAR) e o outro recebeu, além da RAR, antibioticoterapia (amoxicilina e metronidazol). O efeito do tratamento foi avaliado com relação a parâmetros clínicos, prevalência e níveis subgengivais de P. gingivalis e dos genótipos fimA II e fimA IV em estudo quantitativo por PCR em tempo real, antes e 180 dias após o tratamento. Os dados sugerem que RAR associado a antibiotioticoterapia sistêmica é mais eficiente na redução dos níveis de P.gingivalis do que apenas RAR. Não foram detectadas diferenças na resposta ao tratamento periodontal quanto à presença dos genótipos fimA II ou fimA IV em relação aos demais genótipos de P.gingivalis. / Porphyromonas gingivalis is one of the main mircrorganisms associate to periodontitis. The present study proposed to test the hypothesis that the colonization by different P.gingivalis genotypes fimA would lead to different results on periodontal treatment. Twenty chronic periodontitis patients, smokers, bearers of P. gingivalis was analyzed and divided in 2 groups: one group received mechanic periodontal treatment (SRP) and the other group received, besides SRP, antibiotic therapy (amoxicillin and metronidazole). The effect of treatment was appraised under clinical parameters, prevalence and subgingival levels of P. gingivalis and genotypes fimA II and fimA IV in quantitative study by Real time PCR, before and after 180 days of the treatment. Data suggest that SRP associated with systemic antibiotic administration is more efficient in reducing P. gingivalis levels than SRP only. No difference on periodontal treatment results was detected about the presence of fimA II or fimA IV genotypes related to other P.gingivalis genotypes.

Les maladies parodontales sont causées par une infection bactérienne touchant les tissus entourant les dents, appelés parodonte. L’inflammation aggravée du parodonte peut conduire au déchaussement ou à la chute des dents. Porphyromonas gingivalis est une bactérie anaérobique qui sécrète des toxines appelées gingipaïnes, considérées comme le facteur de virulence majeur. Le système de sécrétion de type IX (T9SS) a été récemment mis en évidence exclusivement chez les membres de la famille des bacteroidetes. Chez P. gingivalis, ce système et directement relié à la sécrétion des gingipaïnes est donc sa pathogénicité. Des études ont montré que plus d’une quinzaine de protéines sont impliquées dans l’assemblage, la fonction et la régulation de ce système de sécrétion. Parmi ces protéines, PorK, PorL, PorM, PorN forment un complexe membranaire au cœur de la machinerie de sécrétion, enchâssé dans les deux membranes bactériennes. L’objectif de ce travail de thèse a été de mettre en place une méthodologie d’extraction et de solubilisation du complexe membranaire PorK-L-M-N afin d’étudier sa structure moléculaire par des méthodes de biochimie intégrative. Trois sous-complexes ont été étudiés successivement. Le complexe de membrane externe PorK-N, le complexe de membrane externe étendu PorK-N-M et le complexe de membrane interne PorL-M. Les résultats obtenus montrent que le complexe de membrane externe PorK-N présente une structure en forme d’anneau de 50nm de diamètre et le complexe de membrane interne PorL-M possède une structure étendue avec une base sphérique de 25nm. Ces résultats valident une méthodologie qui pourra par la suite être utilisée pour d'autres études du T9SS. / Periodontal diseases are caused by a bacterial infection affecting the tissues surrounding the teeth, called periodontal. The aggravated inflammation of the periodontium may lead to loosening or falling of the teeth. Porphyromonas gingivalis is an anaerobic bacterium able to secrete toxins called gingipains, considered as the major virulence factor. First called PorSS, the type IX secretion system (T9SS) was recently found exclusively in members of bacteroidetes. In P. gingivalis this system is directly related to the secretion of gingipains and is therefore its pathogenicity. Studies have shown that more than fifteen proteins are involved in the assembly, function and regulation of this secretory system. Among these proteins PorK, PorL, PorM, PorN form a membrane core complex, the central part of the secretory machinery embedded in the two bacterial membranes. The objective of this thesis was to set up a methodology of extraction and solubilization of the PorK-L-M-N membrane complex in order to study its molecular structure by integrative biochemistry methods. Three sub-complexes have been studied successively. PorK-N the outer membrane complex, PorK-N-M extended outer membrane complex and PorL-M the inner membrane complex. The results show that the PorK-N outer membrane complex has a ring-shaped structure of 50nm in diameter, confirming published results, and the PorL-M inner membrane complex has an extended structure of 25nm with a spherical base. These results validate the established methodology that can subsequently be used to continue the structural study of T9SS.

Dentro do contexto do novo paradigma da doença periodontal, alguns estudos têm sugerido que a doença periodontal poderia influenciar o desenvolvimento de doenças sistêmicas, incluindo os acidentes vasculares cerebrais. O objetivo deste estudo foi avaliar as condições periodontais de pacientes com acidente vascular cerebral (AVC), comparativamente à amostra populacional sem AVC, bem como identificar e quantificar o nível de bactérias periodontopatogênicas presentes nas áreas de bolsa periodontal com a finalidade de investigar a correlação da doença periodontal com acidente vascular cerebral. Para tanto, foram selecionados 80 pacientes de ambos os sexos, com idade entre 30-80 anos. O grupo experimental foi constituído por 20 pacientes internados em ambiente hospitalar devido à ocorrência de AVC. O grupo controle foi constituído de 60 pacientes provenientes da amostra populacional da cidade de Bauru que não apresentavam sinais e sintomas clínicos ou história familiar de AVC. Um questionário de saúde investigando as possíveis causas do AVC e outras condições sistêmicas foi aplicado a todos os pacientes. Os dois grupos foram avaliados periodontalmente quanto às medidas de profundidade de sondagem, nível de inserção, sangramento à sondagem e índice de placa. Para identificar e quantificar as bactérias periodontopatogênicas, Porphyromonas gingivalis e Aggregatibacter (Actinobacillus) actinomycetemcomitans, em ambos os grupos, foi coletada amostra de placa dentomicrobiana subgengival dos dois sítios com maior profundidade de sondagem de todos os pacientes, por meio da introdução de tira de papel esterilizada (PerioPaper) no sulco gengival. A análise qualitativa e quantitativa dessas bactérias foi realizada por meio de PCR convencional e em tempo real. Os dados obtidos foram analisados por meio de análise de variância (ANOVA) complementado pelo método de Tukey, teste de correlação de Spearman, teste \"t\" de Student, Mann-Whitney, Qui-Quadrado e \"Odds Ratio\" para avaliar a correlação entre os diferentes parâmetros clínicos periodontais com o AVC e os resultados obtidos pelo PCR convencional e PCR em tempo-real, com nível de confiança de 95%. Os resultados obtidos mostraram maior prevalência da doença periodontal no grupo teste (95%) do que no controle (28,3%). O nível de inserção à sondagem, índice de placa e índice gengival estavam significativamente aumentados nos pacientes com AVC (p<0,001). Entretanto, as medidas de profundidade de sondagem não mostraram diferenças significantes entre os grupos (p=0,051), embora estivessem aumentadas para o grupo teste. A presença e quantidade da P.gingivalis foi estatisticamente maior no grupo teste do que no controle (p<0,05). Não foi encontrado A.actinomycetemcomitans em nenhum dos grupos estudados. Do total de pacientes com AVC, 65% desenvolveram AVC-I e 35% AVC-H. Pacientes com AVC-H abrigavam maiores níveis de P.gingivalis do que pacientes com AVC-I. Entretanto, nesse grupo, houve correlação positiva entre bolsas mais profundas e contagem de bactérias (p<0,05), o que não foi observado para AVC-H. A análise de risco por \"odds ratio\" identificou que pacientes com doença periodontal apresentam risco elevado de desenvolvimento de AVC (OR=48,06, IC=95%). Esses achados indicam que a doença periodontal é mais prevalente e severa em pacientes com AVC-I ou AVC-H, com grande quantidade de bactérias, especialmente P. gingivalis, presente em bolsas periodontais mais profundas, sugerindo que a doença periodontal poderia atuar como fator de risco ao desenvolvimento de acidentes vasculares cerebrais. / Inside of the context of the new paradigm of the periodontal disease, some studies have suggested that the periodontal disease could influence the development of systemics diseases, including vascular cerebral accident. The aim of this study was to evaluate the periodontal conditions of patients with vascular cerebral accidents (VCAs), comparatively to the population sample without VCA, as well as identifying and quantifying the level of periodontopathic bacteria presents in the areas of periodontal pocket in order to investigate the correlation of the periodontal disease with vascular cerebral accident. For this study, it had been selected 80 patients of both genders, with age between 30-80 years. The experimental group consisted of 20 hospitalized patients presenting VCA. The control group was consisted of 60 patients proceeding from the population sample of Bauru who did not present clinical signs and symptoms or family history of VCA. A health questionnaire investigating the possible causes of the VCA and others systemics conditions was applied to all patients. The both groups were periodontally evaluated according to probing depth measures; bleed on probing and plaque index. In other to identify and quantify the periodontopathic bacteria Porphyromonas gingivalis and Aggregatibacter (Actinobacillus) actinomycetemcomitans, in both groups, a sample of subgengival plaque was collected from the two deepest sites of all patients by the introduction of sterilized paper strip (PerioPaper). The qualitative and quantitative analysis of these bacteria was performed by conventional PCR and Real Time PCR. Data were analyzed by variance analysis test (ANOVA) complemented by the Tukey test, Pearson correlation test, Student \"t\" test, Mann-Whitney test, Qui-Square test and Odds Ratio to evaluate the different correlations between the different periodontal clinical parameters VCA, and the results obtained from the Real Time PCR, with a 95% confidence level. The analysis of the results showed significantly bigger prevalence of the periodontal disease in the test group (95%) than the control group (28,3%). The level of insertion, plaque index and bleeding on probing were significantly increased in the patients with VCA (p<0,001). However, the probing depth measures had not shown significant differences between the groups (p=0,051), even so were increased for the test group. The presence and amount of the P.gingivalis were statistically bigger in the test group than the control group (p<0,05). A.actinomycetemcomitans was not found in any of the studied groups. Considering all patients with VCA, 65% developed VCA-I and 35% VCA-H. Patients with VCA-H showed greater levels of P.gingivalis than patient with VCA-I. However, in this group, it had positive correlation between deeper pockets and number of bacteria (p<0,05), what it was not observed for VCA-H. The analysis of risk for \"odds ratio\" identified that patient with periodontal disease present high risk of VCA development (OR=48,06, IC=95%). These findings show that the periodontal disease is more prevalent and severe in patients with VCA-I or VCA-H, with great amount of bacteria, especially P. gingivalis, present in deeper periodontal pockets, suggesting that the periodontal disease could play as risk factor on the development of vascular cerebral accident.

Objectifs: Le but de cette étude clinique était de comparer un groupe d’adultes ayant un parodonte sain avec un groupe d’adultes atteints de parodontite chronique en terme de risque carieux et mesures cliniques et microbiologiques de la carie. Méthodes: Quatre-vingt-seize individus ont été divisés en deux groupes en fonction de leur état de santé parodontal et ont été appariés pour l'âge, le sexe et l'origine ethnique. Trente-huit sujets étaient atteints de parodontite chronique définie comme ayant au moins quatre dents avec ≥ 1 site avec une profondeur de sondage ≥ 4 mm et une perte d'attache clinique ≥ 2 mm, et 58 sujets présentaient un parodonte sain. Par la suite, les groupes ont été subdivisés en deux groupes en fonction de leur statut carieux : les participants ayant au moins une lésion carieuse non traitée sur une surface dentaire et ceux n’ayant pas de lésion carieuse non traitée. Les données ont été recueillies par le biais d’un questionnaire, un examen clinique et des échantillons de plaque supra- et sous-gingivale. L’évaluation de la charge buccale de Streptococcus mutans et de six agents pathogènes parodontaux a été réalisée par la technique d'amplification de la réaction en chaine de la polymérase (PCR). Les données ont été analysées à l'aide d’analyses statistiques descriptives et bivariées. Résultats: Les individus atteints de parodontite chronique étaient 3,5 fois plus susceptibles d'avoir des caries que les individus en bonne santé (OR 3,5 ; IC: 1,5 - 8,3 ; P = 0,006). Les sujets à la fois atteints de parodontite chronique et de caries dentaires ont eu un niveau d’éducation significativement plus faible que les sujets ayant un parodonte sain et sans caries dentaires (OR 6,0 ; IC: 1,7 à 21,7 ; P = 0,04). La proportion de sujets ayant une charge buccale élevée de Porphyromonas gingivalis (P. g.) et Treponema denticola (T. d.) était significativement plus élevée chez les patients atteints de parodontite chronique et de carie que chez les patients sains présentant des caries (P. g.: OR 8,6 ; IC: 2,4 - 30,3 ; P = 0,004 et T. d.: OR 10,0 ; CI: 2,6 - 38.1 ; P = 0,003). Conclusions: Les résultats de cette étude suggèrent que, chez les sujets adultes atteints de la parodontite chronique, la fréquence des caries est plus élevée que chez les sujets ayant un parodonte sain. De plus, le faible niveau d'éducation influence négativement le statut parodontal des individus. / Aim: The aim of this clinical study was to compare adults with a healthy periodontium and those with chronic periodontitis, in terms of caries’ risk and caries’ clinical and microbiological measures. Methods: Ninety-six healthy adults were divided into chronic periodontitis (n= 38) and healthy periodontium (n=58) based on their periodontal status, and matched for age, gender, and ethnic background. Chronic periodontitis was defined as having at least four teeth with ≥1 site with a pocket depth ≥4 mm and clinical attachment loss ≥2 mm. Each group were subsequently subdivided in 2 groups according to their caries status: participants having at least one untreated decayed surface and those with no untreated caries. Data were collected by means of self-administrated questionnaire, clinical examination, and supra- and subgingival plaque sampling. Assessments of oral levels of Streptococcus mutans and six periodontal pathogens were conducted by PCR amplification techniques. Data were analyzed using descriptive and bivariate statistical tests. Results: Individuals with chronic periodontitis were 3.5 times more likely to have caries than healthy individuals (OR 3.5; CI: 1.5 – 8.3; P = 0.006). Subjects with both chronic periodontitis and dental caries had a significantly lower level of education than periodontally healthy subjects without dental caries (OR 6.0; CI: 1.7 – 21.7; P = 0.04). A significant higher proportion of subjects with high oral levels of Porphyromonas gingivalis (P. g.) and Treponema denticola (T. d.) was found among subjects with chronic periodontitis and untreated caries compared to periodontally healthy subjects with untreated caries (P. g.: OR 8.6; CI: 2.4 – 30.3; P = 0.004 and T. d.: OR 10.0; CI: 2.6 – 38.1; P = 0.003). Conclusion: The results from this study suggest that, adults with chronic periodontitis are more prone to caries disease than those adults with a healthy periodontium. Furthermore, low educational level could have a negative impact on the periodontal status of individuals.

Porphyromonas gingivalis é um dos principais mircrorganismos associados à periodontite. O objetivo do presente estudo foi testar a hipótese de que a colonização por diferentes genótipos fimA de P.gingivalis resultaria em diferenças na resposta ao tratamento periodontal. Foram analisados 20 pacientes com periodontite crônica, fumantes, portadores de P.gingivalis, divididos em 2 grupos: 1 grupo recebeu tratamento periodontal mecânico (RAR) e o outro recebeu, além da RAR, antibioticoterapia (amoxicilina e metronidazol). O efeito do tratamento foi avaliado com relação a parâmetros clínicos, prevalência e níveis subgengivais de P. gingivalis e dos genótipos fimA II e fimA IV em estudo quantitativo por PCR em tempo real, antes e 180 dias após o tratamento. Os dados sugerem que RAR associado a antibiotioticoterapia sistêmica é mais eficiente na redução dos níveis de P.gingivalis do que apenas RAR. Não foram detectadas diferenças na resposta ao tratamento periodontal quanto à presença dos genótipos fimA II ou fimA IV em relação aos demais genótipos de P.gingivalis. / Porphyromonas gingivalis is one of the main mircrorganisms associate to periodontitis. The present study proposed to test the hypothesis that the colonization by different P.gingivalis genotypes fimA would lead to different results on periodontal treatment. Twenty chronic periodontitis patients, smokers, bearers of P. gingivalis was analyzed and divided in 2 groups: one group received mechanic periodontal treatment (SRP) and the other group received, besides SRP, antibiotic therapy (amoxicillin and metronidazole). The effect of treatment was appraised under clinical parameters, prevalence and subgingival levels of P. gingivalis and genotypes fimA II and fimA IV in quantitative study by Real time PCR, before and after 180 days of the treatment. Data suggest that SRP associated with systemic antibiotic administration is more efficient in reducing P. gingivalis levels than SRP only. No difference on periodontal treatment results was detected about the presence of fimA II or fimA IV genotypes related to other P.gingivalis genotypes.

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells.