P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

P. gingivalis é um dos principais patógenos da doença periodontal, é encontrado em biofilmes orais com S. gordonii e P. intermedia e em células endoteliais da artéria coronária in vivo. P. gingivalis necessita de ferro em seu metabolismo e pode usar certas proteínas do hospedeiro como fontes deste íon em ambientes limitantes. Assim, este estudo investigou o papel dos genes PGN0741/PG0637 (receptor dependente de TonB) e PGN0531/PG1380 (fvW) de P. gingivalis na formação de biofilme em diferentes concentrações de ferro, em biofilmes mistos com S. gordonii e P. intermedia, e na adesão e invasão de células endoteliais da artéria coronária. Os resultados mostraram divergências no papel dos genes TonB e fvW na formação dos monobiofilmes e mistos e em diferentes concentrações de ferro, demonstrando uma relação cepa-dependente. Na adesão, fvW se mostrou importante para ambas cepas, mas na persistência apenas para P. gingivalis W83. Este trabalho enfatiza, assim, a necessidade do uso de mais de uma cepa de P. gingivalis no estudo do papel de genes em ensaios experimentais. / P. gingivalis is one of the major pathogens of periodontal diseases. It is found in oral biofilms associated with S. gordonii and P. intermedia, and inside of coronary artery endothelial cells in vivo. P. gingivalis requires iron for growth and can exploit iron-carrying proteins of the host as sources in limiting environments. Thus, this work aimed to study the role of genes PGN0741/PG0637 (TonB-dependent receptor) and PGN0531/PG1380 (fvW) of P. gingivalis in biofilm formation under different iron concentrations, in mixed biofilms with S. gordonii and P. intermedia, and in the adhesion and invasion of coronary artery endothelial cells. Our data showed discordance for the role of TonB and fvW in homo- and heterotypic biofilm formation and in different iron concentrations. The relevance of both genes was strain-dependent. Gene fvW was relevant for adhesion to endothelial cells, but only for strain W83 during persistence. Therefore, our study emphasizes the importance of using different strains for a better understanding of the role of genes in experimental assays.

Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.

Surface proteins on microorganisms that build up the oral biofilm are key players in the formation of the biofilm. Antigen I/II proteins are surface adhesins found on virtually all oral streptococci and share a conserved multi-domain architecture. These adhesins bind surface components on other bacteria and on host cells. Thus, they are crucial for the development of the biofilm.     The objective of this thesis work is the structural characterization of the large multi-domain Antigen I/II protein SspB from the primary colonizing commensal bacterium Streptococcus gordonii. The crystal structure of the variable domain of SspB was determined to 2.3 Å resolution. The domain comprises a β-supersandwich and a putative binding cleft stabilized by a calcium ion. Despite high similarity in the overall structure, the cleft within SspB is significantly smaller than the cleft within the homologous protein from Streptococcus mutans, indicating that different substrates may bind in the clefts. A screen for carbohydrate binding resulted in no hits for interaction with the SspB variable domain suggesting that the cleft may not be suitable for binding sugars. This thesis also presents the high resolution 1.5 Å structure of a truncated C-terminal domain of SspB, the first of an Antigen I/II C-domain. The structure contains two structurally related domains, each containing one calcium ion and one intramolecular isopeptide bond. The SspB protein shares the feature of intramoleular isopeptide bonds with other surface proteins from Gram positive bacteria, such as pili from Streptococcus pyogenes and Corynebacterium diphtheriae. Intramolecular isopeptide bonds are suggested to be a common feature for retaining stability in a harsh environment. The SspB adherence region, shown to be the recognition motif for Porphyromonas gingivalis attachment to S. gordonii, protrudes from the core protein as a handle available for recognition. In conclusion, this thesis work has provided new knowledge about the SspB protein and increased the understanding of the common structure of AgI/II proteins.

Streptococcus gordonii, a commensal oral bacterium, is considered a good candidate to function as a live oral vaccine vector. The introduction of vaccine antigen genes into S. gordonii relies on the use of antibiotic resistance genes as selectable markers, which is undesirable. In this study, we used auxotrophic complementation (deletion of an essential gene from the chromosome and insertion into a plasmid) as a means to create an antibiotic marker-free gene delivery system in S. gordonii. S. gordonii ?thyA was created and complemented by an antibiotic marker-free expression plasmid containing the intact thyA gene, pDL276/thyAdelkan. Transformation of pDL276/thyAdelkan into the mutant gave an unexpected 100-fold increase in transformation efficiency as compared to pDL276. The transformants arose from both single and double crossing over. The increase in transformation efficiency suggests that a highly efficient antibiotic marker-free system to deliver genes to the chromosome has been created using thyA complementation.

Streptococcus gordonii is a primary coloniser of the tooth surface where it efficiently ferments carbohydrates at pH levels above 6.0. By not being able to maintain the pH of dental plaque to a level required for enamel dissolution, the dominance of S. gordonii in dental plaque is considered a sign of a healthy oral cavity. However, upon entering the bloodstream and encountering a rise in pH, S. gordonii may become pathogenic, being one of the major causative organisms associated with infective endocarditis. Proteome analyses of S. gordonii grown at steady state in a chemostat allowed the phenotypic changes associated with alterations in pH levels characteristic of these two environments to be determined. As an initial starting point to this study, a two-dimensional electrophoresis (2- DE) reference map of S. gordonii grown at pH 7.0 was produced. Although only 50% of the S gordonii genome was available in an annotated form during the course of this study, the closely related Streptococcus pneumoniae genome (with which S. gordonii shares 97.24% DNA sequence homology) had been completed in 2001. The use of both of these databases allowed many of the S. gordonii proteins to be identified by mass spectrometry. Four hundred and seventy six protein spots, corresponding to 250 different proteins, or 12.5% of the S. gordonii proteome, were identified, giving rise to the first comprehensive proteome reference map of this oral bacterium. Of the 250 different proteins, 196 were of cellular origin while 68 were identified from the extracellular milieu. Only 14 proteins were common to both compartments. Of particular interest among the 54 uniquely identified extracellular proteins was a homologue of a peptidoglycan hydrolase that has been associated with virulence in S. pneumoniae. Among the other proteins identified were ones involved in transport and binding, energy metabolism, translation, transformation, stress response and virulence. Twelve cell envelope proteins were identified as well as 25 others that were predicted to have a membrane association based on the presence of at least one transmembrane domain. The study also confirmed the existence of 38 proteins previously designated as �hypothetical� or with no known function. Mass spectral data for over 1000 protein spots were accumulated and archived for future analysis when sequencing of the S. gordonii genome is finally completed. Following the mapping of the proteome of S. gordonii, alterations in protein spots associated with growth of the bacterium at pH intervals of 0.5 units in the pH range 5.5 - 7.5 were determined. Only 16 protein spots were shown to be significantly altered in their level of expression despite the range of pH studied. Among the differentially expressed proteins was a manganese-dependent inorganic pyrophosphatase (PpaC), which regulates expression of adhesins required for coaggregation. The expression of PpaC was highest at pH 6.5 - 7.0, the pH of a healthy oral cavity, indicating that PpaC may play an important part in dental plaque formation. Another differentially expressed protein was the heat-inducible transcription repressor (HrcA). Alterations in HrcA were consistent with its role as a negative repressor in regulating heat-shock proteins at low pH, even though no changes in the level of heat-shock proteins were observed as the pH declined. This result gave rise to the hypothesis that the possible reason cariogenic bacteria, such as Streptococcus mutans, can out compete S. gordonii at low pH might simply be due to their ability to manipulate their proteome in a complex manner for survival and persistence at low pH, unlike S. gordonii. This may imply some prevailing level of genetic regulation that is missing in S. gordonii.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Indiana University-Purdue University Indianapolis (IUPUI) / The impact of tobacco smoking on human health is well documented. The influence of smoking on tooth loss and cardiovascular diseases was investigated in the current study via both epidemiology and in vitro studies. From analyzing the 2006 Behavioral Risk Factor Surveillance System (2006 BRFSS) database, we confirmed that smoking was significantly associated with the number of teeth lost in a dose-dependent manner and smoking cessation reduced the risk when compared to those subjects continuing to smoke. In addition, the virulence factors related to caries were compared between Streptococcus mutans and Streptococcus gordonii in response to cigarette smoking condensate (CSC) treatment. We observed that S. gordonii was more susceptible to CSC treatment than S. mutans. CSC significantly enhanced S. mutans sucrose-dependent and independent adherence. Western blot assays revealed that several bacterial surface proteins including glucosyltransferase (GTF), glucan-binding proteins and antigen I/II, were significantly upregulated for the treated S. mutans. These findings suggested that the oral environment with CSC may favor a cariogenic dominant composition, which may increase the risk for smokers to develop caries. We also found that smoking and oral health status modified each other and synergistically increased the risk of CVD and this joint effect was more pronounced among the youngest age group using the 2006 BRFSS database. To further understand the joint effect, we conducted an in vitro study to investigate bacterial attachment to fibronectin and endothelial cells in response to smoking condensate treatment. Our study clearly demonstrated CSC significantly enhanced S. mutans attachment to both soluble and immobilized fibronectin as well as endothelial cells. Furthermore, our data suggested that bacteria possessed several adhesins that bound to host tissues and endothelial cells also had multiple receptors for bacterial attachment. Among these adhesins, antigen I/II seemed essential for bacterial attachment to endothelial cells without CSC. The knowledge of bacterial attachment to host tissues in the presence of CSC may help in developing different preventive or therapeutic strategies against attachment and colonization of the host by S. mutans.