Orientador: Reginaldo Bruno Gonçalves / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-05T08:24:42Z (GMT). No. of bitstreams: 1 Napimoga_MarceloHenrique_D.pdf: 980827 bytes, checksum: d6e89bf1b359918d8c502f0a7367d2c1 (MD5) Previous issue date: 2005 / Resumo: Porphyromonas gingivalis (Pg) sintetiza diversas classes de fosfolipídeos. Entretanto, pouco se sabe sobre os efeitos biológicos que estes lipídeos exercem sobre a reabsorção óssea. Células precursoras de osteoclastos de camundongos, incluindo RAW264.7 e células da medula óssea, foram estimuladas com um lipídeo recém caracterizado isolado de Pg (F24), LPS de Pg, RANKL e um isômero estrutural isolado de mamíferos (DMPE), seguida de coloração das células por fosfatase ácida (TRAP) para detecção de osteoclastos. A formação de lacunas de reabsorção em discos de dentina foi realizados para identificar a presença de osteoclastos maduros. Proteínas de células precursoras RAW264.7 com afinidade a F24 foram identificadas como sendo nucleolin através de espectometria de massa, e confirmada sua presença através de ensaio em microscopia confocal. Utilizando Western-blot e RNAi, a sinalização intracelular desencadeada pela F24 durante a osteoclastogênese foi analisada. A F24 mas não o LPS ou DMPE induziu números significantemente maiores de células multinucleadas TRAP+ quando comparado ao RANKL, assim como a formação de lacunas de desmineralização em discos de dentina, o qual não foi inibida pelo repressor do RANKL, a OPG. LPS de Pg, mas não a F24, induziu a produção de TNF-a, IL-1ß e IL-6 pelas células RAW264.7. Microscopia confocal mostrou que a F24 está co-localizada com este receptor na superfície de células RAW264.7. A F24 induziu a fosforilação de maior intensidade do p38 e JNK comparado ao RANKL. Este novo fosfolipídeo isolado de P. gingivalis, distinto do LPS, mostrou-se capaz de induzir osteoclastogênese utilizando nucleolin como a molécula receptora através de uma via independente do RANK / Abstract: Porphyromonas gingivalis (Pg) synthesizes several classes of complex phospholipids in addition to LPS. However, little is known about the biological effects of these phospholipids on bone resorption. Mouse osteoclast precursors cells including, RAW264.7 and bone marrow cells, were stimulated with phospholipids isolated from Pg (F24), Pg LPS, RANKL and a mammalian structural isomer (DMPE). Tartrate-resistant acid phosphatase (TRAP) staining and a dentine-pit formation assay were used for the identification of activated mature osteoclasts. F24¿s counter-ligand expressed on RAW264.7 cells were identified by affinity purification and mass-spectrometry based proteomics, and counter-examined by a confocal microscopy. Using Western-blot, signaling pathways triggered by F24 during osteoclastogenesis were examined with RNAi technology. F24, but not LPS or DMPE, induced significantly higher number of TRAP+ multinuclear cells from osteoclast precursors than RANKL, along with dentine-pit formation, which was not inhibited by RANKL decoy receptor OPG. Pg LPS, but not F24, induced TNF-a, IL-1ß and IL-6 by RAW264.7 cells. Proteomics analyses identified nucleolin as a ligand for F24. Confocal microscopy revealed the co-localization of F24 and its ligand on the surface of RAW264.7 cells. F24 induced stronger phosphorylation of p38 MAP kinase than RANKL. A novel P. gingivalis phospholipid that is distinct from LPS represents a new class of RANK-independent osteoclast differentiation factor / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental

Biofilme subgengival, conteúdo vaginal, âmnio e parênquima placentários foram obtidos de 93 puérperas. Os periodontopatógenos (BPPG) P.g., A.a, F.n e T.f foram identificadas por PCR, corioamnionite e vilosite por histopatologia e analisados estatisticamente. Roturas de membranas (22,2%), cesáreas (65,6%), pretermo (36,6%), baixo peso (34,4%), morte perinatal (5,4%), CAM (34,4%) e vilosite (5,5%) foram observados. Periodontite agressiva (62,4%) e cáries (83,9%). Periodontite ou BPPG em qualquer sitio não se associou com maior freqüência ou com maior risco de corioamnionite ou de desfecho reprodutivo ruim. No biofilme observou-se Aa em 2 (2,1%), Fn em 16 (17,20%), Pg em 30 (32,30%) e Tf em 29 (31.2%); na vagina, Aa em 3 (3,2%), Fn em 2 (2,1%), Pg em 16 (17,2%) e Tf em 3 (3,2%); no âmnio, Fn em 4 (4,2%) e Pg em 9 (9,7%); na placenta, Fn em 1 (1,07%), Pg em 4 (4,3%) e Tf em 1 (1,1%). P.g e F.n foram observados simultaneamente: 6/30 casos de Pg na boca estavam na vagina, 3 no âmnio e 1 na placenta; dos 16 Fn na boca, 1 foi encontrado na placenta. Esta taxa de disseminação sugere que as BPPG na vagina, âmnio ou placenta não se originaram na boca das puérperas. / Subgingival biofilm and buccal samples, vaginal contents, amnion and placental parenchyma were obtained from 93 pregnant. The periodontopathogens (PPG) Pg, Aa, Fn, and Tf were identified by PCR, the diagnosis of chorioamnionitis (CAM) and villitis by histopathology methods and its relationships with adverse perinatal outcome (APO) statically analyzed. Rupture of membranes (22.2%), cesarean (65.6%), preterm (36.6%), low fetal weight (34.4%), perinatal death (5.4%), CAM (34.4%), and villitis (5.5%) were observed. Aggressive periodontitis (62,4%), and 83.9% had caries. Periodontitis or PPG (Aa, Fn, Pg, and Tf) in any site not associated with greater frequency or at greater risk of CAM or APO. In the subgingival biofilm Aa was observed in 2 (2.1%), Fn in 16 (17.20%), Pg in 30 (32.30%) and Tf in 29 (31.2%); in the vagina, 3 in Aa (3.2%), Fn 2 (2.1), Pg 16 (17.2%) and Tf 3 (3.2%); amnion, Fn in 4 (4.2%) and Pg 9 (9.7%); in the placenta, Fn 1 (1.07%), Pg 4 (4.3%) and Tf 1 (1.1%). Only Fn and Pg were observed simultaneously: 30 mothers with Pg in the mouth, 6 were detected in the vagina, 3 in amnion and 1 in the placenta; Fn 16 with the mouth, 1 was found in the placenta. This low rate of spread suggests that most of periodontopathogens present in vagina, amnion or placenta did not belong to the pregnant mouth.

Prevotella intermedia e Prevotella nigrescens são espécies comumente associadas Porphyromonas gingivalis. os objetivos foram verificar a co-agregação entre cepas de P. intermedia, P. nigrescens e P. gingivalis; quantificar a biomassa, avaliar a proporção dos microrganismos nos biofilmes mistos; verificar a interferência do co-cultivo pela técnica de dois compartimentos, avaliar os biofilmes em ensaios de Hibridização In Situ (FISH) e verificar o papel dos genes PINA0102 e PIN0398 de P. intermedia no biofilme. Assim, 9 isolados clínicos de P. intermedia e 5 de P. nigrescens, as cepas padrão P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277, os mutantes Pi17D0398 e Pi17D0102 foram avaliados em variados consórcios. Os resultados mostraram que as interações investigadas são cepa específicas. / Prevotella intermedia and Prevotella nigrescens are commonly associated with periodontal diseases. The goals of this study were to determine the co-aggregation of strains of P. intermedia, P. nigrescens and P. gingivalis; to quantify the biomass of these associations, to evaluate the ratio of these microorganisms in heterotypic biofilms, to verify the modulation of biofilms in co-culture using a trans-well system; to evaluate the structure of the biofilms by Fluorescent Hybridization In Situ (FISH) and to determine the role of genes PINA0102 and PIN0398 of P. intermedia in the modulation of its biofilm. Therefore, 9 clinical isolates of P. intermedia, 5 of P. nigrescens, type strains P. intermedia 17, P. intermedia 25611, P. nigrescens 33563, P. gingivalis W83, P. gingivalis 33277 and mutant strains Pi17D0398 and Pi17D0102 were grown in consortia of two strains. Our data demonstrate that the associations of P. intermedia, P. nigrescens and P. gingivalis are strain-specific.

Alexandre Augusto ZaiaOrientador: / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-22T16:52:01Z (GMT). No. of bitstreams: 1 Suzuki_ClaudiaLealSampaio_M.pdf: 1692911 bytes, checksum: 11c9ab9ff10af76a78f2989485f0ed25 (MD5) Previous issue date: 2013 / Resumo: O objetivo do estudo foi avaliar se as bactérias Streptococcus mutans, Enterococcus faecalis e Porphyromonas gingivalis alteram a secreção de colágeno tipo ? por fibroblastos 3T3 e odontoblastos MDPC-23 em cultura celular; e, havendo alteração, se é dependente do contato célula/bactéria ou apenas dos subprodutos bacterianos. Fibroblastos 3T3 e odontoblastos MDPC-23 foram cultivados e incubados com as bactérias Streptococcus mutans, Enterococcus faecalis e Porphyromonas gingivalis (MOI 1:1) nos períodos de 2, 4 e 8 horas. As bactérias ficaram em contato direto com as células, e em contato indireto através do uso de inserts de 0.4?m. A produção de colágeno tipo I secretado pelas células foi quantificada pelo Ensaio de Imunoabsorbância Ligado à Enzima (Enzyme-Linked Immunosorbent Assay, ELISA), e sua organização estrutural (birrefringência) foi visualizada em microscopia de polarização após coloração com picrosirus. Assim, pode-se concluir que a infecção com S. mutans, E. faecalis e P. gingivalis estimularam um aumento nos níveis de colágeno tipo I nos períodos de 2 e 4 horas em cultura de odontoblastos MDCP-23, e um aumento progressivo nos níveis de colágeno tipo I entre 2 e 8 horas em cultura de fibroblastos 3T3. Também se observou que o estímulo para o aumento na produção de colágeno se deve a ação dos subprodutos liberados pelas bactérias e não pelo contato célula/bactéria / Abstract: The aim of the study was to evaluate whether the bacteria Streptococcus mutans, Enterococcus faecalis and Porphyromonas gingivalis alter the secretion of type I collagen by 3T3 fibroblasts and MDPC-23 odontoblast-like cells in cell culture, and, with change, if it is dependent on the contact cell/bacteria or only bacterial by-products. 3T3 fibroblasts and MDPC-23 odontoblast-like cells were cultured and incubated with the bacteria S. mutans, E. faecalis and P. gingivalis (MOI 1:1) for the periods of 2, 4 and 8 hours. The bacteria were in direct contact with the cells, and indirect contact through the use of inserts 0.4 ?m. The production of type I collagen secreted by cells was quantified by Enzyme-Linked Immunosorbent Assay (ELISA), and its structural organization (birefringence) was visualized in polarized light microscopy after staining with picrosirus. Thus, it can be concluded that infection with S. mutans, E. faecalis and P. gingivalis stimulated an increase in the levels of type I collagen in periods 2 and 4 hours in cultured MDPC-23 odontoblast-like cells, and a progressive increase in the levels of type I collagen between 2 and 8 hours in cultured 3T3 fibroblasts. It was noted that the stimulus for the increase in collagen production is due to the action of the by-products released by bacteria and not by contact cell/bacteria / Mestrado / Endodontia / Mestra em Clínica Odontológica

A Bacteroides gingivalis adhesin mediating attachment to red blood cells and buccal epithelial cells was isolated, cloned and characterized. The isolation procedure involved gentle stirring of the cells followed by ammonium sulphate precipitation, ion-exchange and gel chromatography. The native molecule had a Mr in excess of 10⁶ kDa and was made up of subunits with an Mr of 43 kDa. Antisera raised to the adhesin and its subunits reacted with antigens on the surface of B. gingivalis cells. No reaction with fimbriae was seen. The IgG fractions from these antisera inhibited the adherence of B. gingivalis to host tissue. Proteolytic enzymes destroyed binding capability of whole cells and of the purified adhesin but the molecular weight of the haemagglutinin was not altered. A genomic library of B. gingivalis DNA was created in E. coli JM83. 5500 colonies were screened by a colony immunoassay with anti-S. gingivalis serum and by a direct haemagglutinating assay. 337 clones tested positive by the immunoassay and two clones, 1-3,and 1-49 tested positive for haemagglutinating activity. Both haemagglutinating positive recombinants had inserts of 3.2 kb. One clone, 1-49 was chosen for further characterization. E. coli 1-49 expressed a protein of 43 kDa that was not present in E. coli JM 83 control as seen by SDS-PAGE and Western blot analysis. Anti-1-49 serum inhibited the haemagglutinating activity of B. gingivalis and E. coli 1-49. This serum reacted with surface molecules on B. gingivalis and E. coli 1-49 as seen by immunogold electron microscopy and immunofluorescence, and to the purified haemagglutinin by Western blot analysis. Like the haemagglutinin on B. gingivalis, the haemagglutinating activity of E. coli 1-49 was destroyed by heating and proteolytic enzymes but the apparent size of the molecule as determined by SDS-PAGE was not affected. A bacterial coaggregating adhesin from B. gingivalis was isolated and characterized. The isolation procedure involved adsorption of the solubilized adhesin on S. mitis followed by elution with glycine buffer. SDS-PAGE of the boiled adhesin revealed a protein with an Mr of 46 kDa. Proteolytic digestion destroyed all bacterial aggregating activity and hydrolysed the 46 kd protein. Antisera raised to the 46 kDa protein reacted with surface molecules on all strains of B. gingivalis tested. This antiserum inhibited the coaggregation reaction between B. gingivalis and other bacteria. Vesicles produced by B. gingivalis were found to enhance the binding of S. sanguis to serum coated hydroxy apatite (SeHA). Maximum vesicle mediated binding took place at 37°C and was destroyed by heating. The lipopolysaccharide from several black pigmented bacteroides were isolated and characterized physically, chemically and immunologically. All of the LPS were of the smooth type and contained the sugars rhamnose, glucose, galactose, glucosamine and galactosamine; no KDO or heptose were found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A busca por relações entre a infecção pelo Vírus da Imunodeficiência Humana (HIV) e a agressividade da doença periodontal (DP) tem motivado inúmeras pesquisas clínicas. Em acréscimo, a literatura atual também é divergente em relação ao exato processo de sucessão bacteriana que ocorre com a progressão da DP em pacientes HIV-positivos (HIV+). O objetivo deste trabalho foi comparar parâmetros clínicos periodontais e microbiológicos (prevalência de Porphyromonas gingivalis- Pg) em pacientes portadores do HIV. Foram selecionados setenta pacientes (35 HIV+, selecionados no Serviço Ambulatorial Especializado da cidade de Ji-Paraná/RO e 35 HIV-, selecionados da clínica de Periodontia da Faculdade São Lucas de Porto-Velho/RO). Os parâmetros clínicos periodontais avaliados foram: Índice de placa bacteriana (IP); Índice gengival (IG); Profundidade de sondagem (PS); Nível de inserção clínica (NIC) e Índice de sangramento gengival (ISG). A análise microbiológica para a determinação da prevalência de Porphyromonas gingivalis foi realizada através da PCR. Os resultados de todos os parâmetros periodontais analisados no grupo teste (HIV+) mostraram-se estatisticamente superiores quando comparados ao grupo controle (HIV-). Adicionalmente, a prevalência total de Pg também se mostrou significantemente maior no grupo teste em relação ao controle (77,14% e 52,95% para os grupos: teste e controle respectivamente). O modelo experimental realizado permitiu concluir que os indivíduos portadores do HIV apresentaram maior gravidade da doença periodontal acompanhada de um aumento na prevalência de Porphyromonas gingivalis em relação a indivíduos HIV. Estes achados sugerem que a terapia de suporte periodontal periódica pode se constituir como importante aliado na manutenção da saúde bucal deste grupo de pacientes. / The search for relations between infection by the Human Immunodeficiency Virus (HIV) and the aggressiveness of periodontal disease (PD) has motivated many clinical research. In addition, the current literature differs in relation to the exact process of bacterian succession that occurs with the progression of PD in HIVpositive patients (HIV +). The objective of this study was to compare clinical periodontal parameters and microbiological (prevalence of Porphyromonas gingivalis-Pg) in HIV patients . Were selected seventy patients (35 HIV +, selected in the Specialized Ambulatory Service - the town Ji-Paraná/RO and 35 HIV+, selected from the Clinic of Periodontics of the St. Lukes Academy of Porto-Velho/RO). The periodontal clinical parameters were evaluated: plaque Index (PI); gingival index (GI); depth survey (DS); level of clinical Insertion(LCI) and gingival bleeding Index (GBI). The microbiological analysis to determination the prevalence of Porphyromonas gingivalis was performed by PCR. The results of all parameters examined in the test group periodontal (HIV +) showed a higher statistically when compared to the control group (HIV-). Additionally, the overall prevalence of Pg also was significantly higher in group testing in relation to the control (77.14% and 52.95% for the groups: test and control respectively). The experimental model done conducted found that those HIV people bearers had greater severity of periodontal disease accompanied by an increase in the prevalence of Porphyromonas gingivalis regarding HIV people. These findings suggest that periodontal therapy regular support can be as important ally in oral maintaining health of this group of patients.

O presente estudo teve como hipótese que a adrenalina, a noradrenalina e o cortisol, hormônios liberados em grandes quantidades durante o estresse fisiológico, poderiam ser capazes de alterar o crescimento e de aumentar a virulência de Porphyromonas gingivalis, estimulando a expressão de genes relacionados à virulência, estresse oxidativo e metabolismo do ferro, podendo agravar a condição periodontal em indivíduos com periodontite. Objetivos: Assim o presente projeto visou avaliar a interferência desses hormônios relacionados ao estresse sobre o crescimento, viabilidade, susceptibilidade antimicrobiana e virulência de Porphyromonas gingivalis. Método: Culturas de Porphyromonas gingivalis W83 foram expostas à adrenalina, noradrenalina e cortisol, utilizando três meios de cultura (TSB-HM, SAPI e SAPI-HM) e foram incubadas em estufa de anaerobiose para avaliação quanto ao crescimento e viabilidade. Essas culturas foram testadas quanto à sensibilidade ao metronidazol após exposição às catecolaminas e ao cortisol. Possíveis alterações da expressão de genes relacionados ao estresse oxidativo, metabolismo do ferro e fatores de virulência foram verificados pela técnica de qRT-PCR. Resultados: As catecolaminas e o cortisol, de forma geral, não interferiram no crescimento de P. gingivalis, independente das condições nutricionais a que ela foi exposta e dos tempos avaliados (p>0.05, ANOVA). A sensibilidade de P. gingivalis ao antimicrobiano metronidazol não se alterou na presença de adrenalina, noradrenalina ou cortisol (p>0.05, Kruskall Wallis). No entanto, a exposição bacteriana a adrenalina, noradrenalina e/ou cortisol elevaram os níveis de RNAm de genes relacionados à obtenção de ferro (hmuR); estresse oxidativo (tpx, oxyR, dps, sodB, aphC), hemólise (hem, hagA) e proteína de superfície imunodominante (ragA) (teste de modo pareado fixo de realocação ao acaso, p<0.05). Os resultados do presente estudo sugerem que as catecolaminas e o cortisol podem influenciar na expressão de fatores relacionados à virulência e ao estresse oxidativo de P. gingivalis. / This study hypothesized that adrenaline, noradrenaline and cortisol , hormones released in large quantities during the physiological stress , might be able to alter the growth and increase the virulence of Porphyromonas gingivalis by stimulating the expression of genes related to virulence , oxidative stress and iron metabolism and may aggravate periodontal status in subjects with periodontitis. Objectives : So this project aimed to evaluate the effect of these stress-related hormones on growth , viability , virulence and antimicrobial susceptibility of Porphyromonas gingivalis . Method : Porphyromonas gingivalis W83 cultures were exposed to adrenaline , noradrenaline and cortisol , using three culture media ( TSB - HM , SAPI and SAPI - HM ) and were incubated in anaerobiosis for review on the growth and viability . These cultures were tested for sensitivity to metronidazole after exposure to catecholamines and cortisol. Possible changes in the expression of genes related to oxidative stress , iron metabolism and virulence factors were verified by qRT-PCR technique . Results: The catecholamines and cortisol , in general , did not affect the growth of P. gingivalis , independent of nutritional conditions to which she was exposed and evaluated times ( p> 0.05 , ANOVA ) . The sensitivity of P. gingivalis antimicrobial metronidazole did not change in the presence of adrenaline , noradrenaline and cortisol ( p > 0:05 , Kruskal Wallis ) . However , bacterial exposure to adrenaline, noradrenaline and / or cortisol increased mRNA levels of genes related to iron acquisition ( hmuR ), oxidative stress ( tpx , oxyR , dps , sodB , aphC ) , hemolysis ( hem , hagA ) and immunodominant surface protein ( ragA ) ( test paired mode relocation fixed at random, p <0,05 ) . The results of this study suggest that catecholamines and cortisol can influence the expression of factors related to oxidative stress and virulence of P. gingivalis.

Porphyromonas gingivalis is a major etiological agent that is responsible for the cause and progression of periodontal diseases. The bacterium is exposed to various environmental conditions and oxidative stress conditions while it is in the oral cavity. So, P. gingivalis should have an efficient regulatory system in order to adjust and survive in the oral cavity. But little is known about the regulatory mechanisms that help the bacteria to survive in the oral cavity. So, it is essential to understand and characterize these regulatory mechanisms. The response and adaptation of P. gingivalis to environmental stress conditions occur at the level of transcription which involves the alternative sigma factors. Extracytoplasmic function (ECF) sigma factors are the largest group of alternative sigma factors that play a major role in bacterial response to environmental stress conditions. Here we characterize the σ-70 factor, SigH and SigG, the extracytoplasmic function sigma factors encoded in P. gingivalis genome. Our results show that the expression of SigH is upregulated when P. gingivalis is grown in the presence of oxygen. However, there is no change in the expression of SigG when grown in the presence of oxygen. Furthermore several genes involved in oxidative stress protection such as sod, trx, tpx, ftn, feOB and the hemin uptake locus, hmu, are downregulated in the mutant deficient in SigH designated as V2948. Our RNA-seq analysis of SigG showed that there is no change in the regulation of genes involved in oxidative stress protection and metal homeostasis in SigG deficient mutant designated as V3085. Our survival studies showed that both SigH and SigG are essential for P. gingivalis to grow in host cells. Collectively our studies demonstrate that SigH is a positive regulator of gene expression required for survival of the bacterium in the presence of oxygen and oxidative stress, hemin uptake and virulence. However our studies show that SigG is essential for the bacteria to grow in host cells and hence helps in the virulence of P. gingivalis.

Periodontal diseases are bacterially induced, inflammatory diseases which are responsible for loss of alveolar bone and connective tissue supporting the teeth which results in loss of teeth. Gram negative anaerobic bacteria are highly associated with these diseases. One of them is Porphyromonas gingivalis belonging to the phylum Bacteroidetes. Infection by P. gingivalis is recurrent after physical removal of the bacteria from the oral cavity and even after antibiotic treatment as development of resistance is not rare. Hence complete understanding the biology of this bacterium is of significance. This gram negative obligate anaerobe, being aerotolerant, manages to survive inside the oral cavity, where oxidative stress is ubiquitous. Genome sequence of P. gingivalis shows the presence of a transcriptional regulator OxyR which is a homologue of OxyR present in E. coli. P. gingivalis OxyR induces the expression of antioxidant defense genes like sod, ahpC-F, dps to protect the bacteria from oxidative stress. Expression of P. gingivalis OxyR regulon is not very well understood. Microarray studies carried out in our lab using P. gingivalis W83 to study gene regulation by OxyR, indicated that several genes in P. gingivalis are co-regulated by iron-and OxyR. Literature also supports that in iron deplete conditions genes involved in oxidative stress are down-regulated. These studies formed the basis of our hypothesis that OxyR might regulate the genes in P. gingivalis in an iron dependent manner. To study the mechanism of regulation by P. gingivalis OxyR and to determine whether OxyR regulation is iron dependent, two approaches were applied - in vitro characterization of binding and in vivo characterization. First step of in vitro characterization was to perform CHIP-chip assay to determine OxyR-binding sites present on the genomic DNA of P. gingivalis. As this assay was performed under completely anaerobic conditions, the target fragments to which OxyR was found to bind during this assay were not same as reported in literature. These and the fragments reported in literature were used for EMSA. EMSAs carried out using crude cell lysates and in vitro OxyR protein preparations showed expected results but the results were not reproducible. In vivo expressed and purified P. gingivalis OxyR never bound to the target fragments used. Preparation of a stable protein preparation and improvement in the parameters of EMSA is very important to further investigate the binding in vitro. The second approach is based on in vivo characterization of binding. This requires tagging the P. gingivalis OxyR at its C-terminus with fluorescent protein to observe its binding to the target DNA sequences. Fluorescently tagged OxyR, is expected to emit fluorescence from a highly localized area to produce sharp fluorescent spots when it is bound to its target sequences. Unbound OxyR is expected to emit a fluorescent signal which is spread over the entire area of the cell. This technique will help to determine the conditions under which OxyR binds to its target DNA sequences. This provides a means to confirm the results obtained from in vitro characterization instead of just extrapolating them.

Porphyromonas gingivalis, a gram-negative anaerobic, pathogenic bacterium is a major etiological agent in the initiation and progression of periodontal disease. Due to the ever-changing environment of the oral cavity, inhabitants like Porphyromonas gingivalis must possess the ability to adapt to changes in environmental conditions like pH, temperature, oxygen tension, and metal concentration. P. gingivalis should therefore have an efficient regulatory system in order to adapt and survive in the oral cavity. This response adaptation occurs at the transcriptional level, which involves alternative sigma factors. Extracytoplasmic function sigma (ECF-s) factors are the largest group of alternative sigma factors that play a role in the bacterial response to environmental stress conditions. Here we analyze the s-70 factor gene, PG0214, an extracytoplasmic function sigma factor encoded in the P. gingivalis genome, and examine its role in the bacterial response to environmental stress and virulence. Our findings indicate that the PG0214 gene is important in regulating major functional gene groups and pathways in the P. gingivalis genome. Strains deficient in the PG0214 gene were analyzed and shown to have decreased protease activity, as well as reduced survivability and invasion rates in eukaryotic host cells when compared against wild-type W83 and ATCC 33277 strains. Collectively our studies demonstrate that the PG0214 gene is a positive regulator of gene expression for the survival and virulence of P. gingivalis in the presence of oxidative- and iron-stress, although further study is needed to fully characterize the gene and determine its specific function.