Dissertations / Theses on the topic 'Streptococcus gordonii'

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.

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.

Streptococcus gordonii is an early bacterial coloniser of the oral cavity and influences development of plaque biofilms. Interactions of Candida albicans, an opportunistic fungal pathogen, with S. gordonii are hypothesised to promote fungal carriage and persistence. Evidence suggests that signalling molecules produced by streptococci, including competence stimulating peptide (eSP) and autoinducer 2 (AI -2L affect C. albicans growth. However, less is understood about the molecular basis of these interactions.

Streptococcus gordonii is an oral commensal bacterium, and an early coloniser of the acquired salivary pellicle that coats tooth surfaces. As such, it is a key organism in the establishment of dental plaque biofilms. The amino acid L-arginine has been previously shown to play a role in biofilm formation in other oral species, and depletion of L-arginine has a significant impact upon S. gordonii growth and gene regulation. Three L-arginine-dependent transcription regulators have been identified in S. gordonii, but it is currently not clear how these co-ordinate to sense and respond to changes in the exogenous L-arginine concentration. Therefore, the major aims of this work were to (i) further elucidate the impacts of L-arginine on S. gordonii growth and biofilm formation, (ii) investigate the roles of three putative arginine-dependent regulators in modulating arginine-responsive gene regulation, and (iii) assess the effects of L-arginine-dependent gene regulators on S. gordonii biofilm formation. Initial growth experiments revealed that high concentrations (≥500 mM) of L-arginine retard S. gordonii planktonic growth in a chemically defined medium, resulting in lower growth yields than intermediate (0.5 mM) L-arginine. However, 500 mM L-arginine was not toxic to S. gordonii cells incubated in natural human saliva. S. gordonii has previously been shown to be conditionally auxotrophic for L-arginine, since it can biosynthesise L-arginine under strictly anaerobic conditions or during the gradual depletion of extracellular L-arginine, but it cannot grow following a rapid shift to medium lacking L-arginine. A similar lack of growth was also found following rapid depletion of L-histidine and the branched-chain amino acids. S. gordonii is predicted to encode all genes required for L-histidine and branched chain amino acids, and it is possible that this organism is conditionally auxotrophic for multiple amino acids. Rapid depletion of L-arginine was shown previously to result in a change in expression of >20% of the S. gordonii genome. By comparing expression levels of some of the most strongly arginine-regulated genes when cells were challenged with depletion of L-histidine or branched chain amino acids, it was shown that some of the genes (for example, argC, SGO_1686, asp5) were specifically regulated by arginine depletion, whereas others (bfbF, SGO_1699) were similarly regulated following depletion of all amino acids. Therefore, it appears that depletion of L-arginine results in both an arginine-specific response and a more generalised stress response, presumably associated with growth arrest in this medium. iv Investigation of the roles of three arginine-dependent regulators (ArcR, ArgR and AhrC) by gene expression microarrays identified a number of genes that were arginine-responsive and were differentially-regulated in the wild-type compared with the isogenic mutants ΔarcR, ΔargR or ΔahrC. There was extensive overlap between the genes regulated by the ArgR and AhrC regulators, suggesting that these regulators perform similar and interdependent roles in S. gordonii. Regulatory responses following arcR disruption were distinct from those seen in the argR and ahrC mutants. In addition to three loci that have previously been described, one particular gene, SGO_0846, encoding a hypothetical protein, was highly up-regulated in response to arcR deletion. This thesis is the first holistic study of the three arginine-dependent regulators in S. gordonii, and shows that each one plays a key role in arginine-dependent gene regulation. Finally, previous unpublished work from our group had demonstrated that S. gordonii ΔarcR displayed a defective biofilm phenotype, whereas the deletion strains of the other two regulators showed no such phenotype. To determine whether up-regulation of SGO_0846 is responsible for the biofilm attenuation in S. gordonii ΔarcR, a deletion mutant of SGO_0846 was constructed in both the wild-type and ΔarcR background. Disruption of the SGO_0846 gene showed no significant differences in biofilm formation levels in comparison to the wild-type background, and showed no effect on the biofilm defective phenotype of the ΔarcR mutant. This suggests that changes in expression of SGO_0846 are not responsible for the biofilm defects seen in the ΔarcR knockout, and that the ArcR regulator is affecting biofilm formation via another mechanism. In conclusion, this thesis provides evidence that arginine has a clear impact on gene expression and biofilm formation in S. gordonii, and furthermore, that the ArcR regulator is critical for optimal biofilm formation. It is possible that in the future, this could be used as a target for controlling S. gordonii biofilm formation, and subsequent dental plaque development.

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.<br>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.

The commensal Streptococcus gordonii expresses numerous surface adhesins with which it interacts with other microorganisms, host cells and salivary proteins to initiate dental plaque formation. However, this Gram-positive bacterium can also spread to non-oral sites such as the heart valves and cause infective endocarditis. One of its surface adhesins, Sgo0707, is a large protein composed of a non-repetitive N-terminal region followed by several C-terminal repeat domains and a cell wall sorting motif. Here we present the crystal structure of the Sgo0707 N-terminal domains, refined to 2.1 Å resolution. The model consists of two domains, N1 and N2. The largest domain, N1, comprises a putative binding cleft with a single cysteine located in its centre and exhibits an unexpected structural similarity to the variable domains of the streptococcal Antigen I/II adhesins. The N2-domain has an IgG-like fold commonly found among Gram-positive surface adhesins. Binding studies performed on S. gordonii wild-type and a Sgo0707 deficient mutant show that the Sgo0707 adhesin is involved in binding to type-1 collagen and to oral keratinocytes.

In the present thesis, the correlation between the physicochemical and antigenic properties of different recombinant Streptococcus gordonii vaccine vectors was studied. Evaluation of vaccine efficacy, antigenicity and immugenicity is a crucial step in developing vaccines, thus investigating a simple method to analyze vaccine efficacy besides other methods could be a major part of developing bacterial vaccine vectors. To approach this, isoelectric point measurements and zeta-potential titration as well as antigenicity and immunogenicity of S.gordonii vaccine vectors (with fbpA, RPS, gtfg genes mutations expressing H1 antigen) were used. These data showed that strains with more positive surface charge had higher heterologous antigen recognition and lower antibody responses in the serum of immunized mice. This correlation between surface charge and antigenicity and immunogenicity revealed the importance of using simple methods such as zeta potential titration and isoelectric point measurements to predict engineered vaccine vectors antigenicity and possible efficacy. In the second part of the thesis the immune recall in the days following Streptococcus pneumoniae lung infection by transcriptomic analysis was studied. S. pneumoniae is the most common bacterial cause of community-acquired pneumonia. Host-pathogen interaction is poorly understood, and factors that drive a more severe phenotype are unknown. One way to study host response to pathogen is using the stimulation of immune system cells with live or killed bacteria. We combined transcriptomic and cytokine level analysis on stimulated mouse splenocytes revealing the presence of a recall immune response involving both innate and adaptive immunity, stronger from the fourth day after infection. This model could analyze immune responses involved in pneumococcal infection as well as vaccine and experimental therapies efficacy in future studies. Finally, the development of a S. pneumoniae mouse model of pneumonia by intra-tracheal infection was set up. The nasopharynx of humans is the only natural reservoir for the pneumococci. To mimic human pneumonia, mice models are widely used. Bacteria can be administered to mice intranasally, intratracheally or as aerosols. Pneumococcal pneumonia was induced in mice by intra-tracheal inoculation with different doses of S. pneumoniae TIGR4. Data showed high colonization of bacteria in lung, liver and spleen starting 24 hours post-infection. Pneumonia mortalities were observed in all mice infected by 108 within 24 hours of infection. Further analysis should be done to investigate the host-pathogen interaction as well as vaccine and experimental therapies efficacy by using this model.

Trabajo de Investigación Requisito para optar al Título de Cirujano Dentista<br>La caries dental es un proceso localizado de origen multifactorial que se inicia luego de ocurrida la erupción dentaria, determinando el reblandecimiento del tejido duro del diente y que puede evolucionar hasta la formación de una cavidad. La cavidad oral contiene muchas bacterias, de las cuales son las acidogénicas las que están involucradas en el proceso carioso, a la par que también existen bacterias alcalogénicas contrarrestando esta acidogenicidad. El propósito de este trabajo es determinar la correlación entre bacterias alcalogénicas y salud oral presentes en niños de 6 y 7 años de edad. Material y métodos: Se tomaron muestras a 110 niños de 6 y 7 años de edad de saliva y placa bacteriana del sector norte de la RM, recolectando muestras de placa y muestras de saliva. Se realizó examen dentario determinando COPD/ceod. Se procedió a realizar extracción de ADN de cada muestra y determinar la cantidad de Streptococcus sanguinis, Streptococcus mutans y Streptococcus gordonii mediante qPCR. Los datos se analizaron y correlacionaron según la experiencia de caries y el número de copias por mililitros de cada bacteria analizada. Resultados: La abundancia de S. sanguinis en placa y saliva basándose en la mediana es mayor para el grupo “libres de caries” (CF) que para “caries activa” (CA), sin embargo, los resultados no son significativos. S. gordonii muestra mayor abundancia en el grupo CA que en CF siendo significativo solo para muestras de placa. En el caso de S. mutans, el recuento fue mayor en el grupo CA que en el CF para placa y saliva siendo ambos resultados significativos. Conclusiones: Las bacterias S. sanguinis y S. gordonii no son exclusivas de sujetos libres de caries, estando presentes en todos los grupos evaluados con diferente actividad cariogénica. Existe una tendencia de mayor abundancia de S. sanguinis en placa y saliva en niños CF. Por otro lado, S. gordonii no tiene una asociación positiva con sujetos libres de caries. Hay presencia de S. mutans en todos los grupos, pero su mayor recuento reside en sujetos CA, existiendo una asociación positiva significativa. Por último, la herramienta qPCR puede ser útil al contribuir a nuestro conocimiento sobre la composición de la biopelícula dental.<br>Adscrito a Proyecto FONIS SA 13/20 205

The ability to prime the immune system against an antigen, and to rapidly recall this response upon antigen reencounter is a fundamental characteristic of the adaptive immune response. The association of an antigen recognized by the immune system in a certain tissue, with the same antigen encountered at a later timepoint in a different tissue, is of primary importance to obtain a systemic and effective immune response and is the foundation behind the utilization of vaccines. The study of vaccine delivery platforms that may activate the immune system in such a manner is therefore of primary importance in the effort to design novel vaccine formulation and prime-boost strategies. The aim of the present work was to study the in vivo priming effect induced by a recombinant Streptococcus gordonii vaccine vector expressing a heterologous antigen and delivered to the vaginal tract, a unique mucosal tissue. To study the priming effect induced by the recombinant Streptococcus gordonii, we employed a prime-boost strategy and compared the cellular an humoral immune response towards the soluble antigen between recombinant- and WT-immunized mice. Using this model, we show that vaginal immunization with the recombinant Streptococcus gordonii elicited systemic production of antigen-specific antibodies, shifted the IgG subclasses profile, led to an increase in plasma cells in the lymph nodes, a higher number of antigen-specific antibody-secreting cells in the spleen and modulated the cytokine expression profile of splenocytes. The longevity of the priming effect induced by the recombinant vector was also analyzed by comparing three and six months boosting schedules. We found that the priming is boostable with a similar efficacy for at least six months (Chapter 3). These data demonstrate that vaginal priming with the recombinant S. gordonii vector results in a systemic activation of both cellular and humoral immune compartments, and that this priming effect is long-lived without significant immune waning. In this study, we also assessed the transcriptomic response of splenocytes from S. gordonii-immunized mice towards the antigen. We observed differences in immune pathways between recombinant- and WT-immunized mice, and also between the three- and six-months boosted groups (Chapter 4). In addition, we observed a gene signature correlated with antigen-specific IgG titers. These findings suggest that the immune system’s encounter with the antigen on the surface of the recombinant S. gordonii in the vaginal tract results in a differential immune activation in in response to the antigen. This work contributes to the knowledge on the capability of recombinant live vaccine vectors delivered mucosally to prime and modulate the immune response, and has important implications in the design of innovative vaccination strategies.

Streptomycin resistance in both gram-positive and gram-negative bacteria is usually caused by a single mutation in the rpsL gene. The rpsL gene encodes the S12 protein of the ribosomal complex. The rpsL genes of various bacteria have consensus regions in their sequences. Primers were designed from these consensus pockets and a fragment of the rpsL gene was sequenced from S. gordonii using PCR based methodologies. Using the Multiplex Restriction Sequence PCR(mRS PCR), which used the known primer at one end and a restriction site primer on the other, a gene walk was conducted. In streptomycin resistant strains of S. gordonii, namely GP204, SP204 and SP635, the AAA coding for Lys56 was mutated to ACA, coding for Thr56. The lysine to threonine transition, causing resistance to streptomycin was identical to that expected from the literature. The streptomycin resistance gene of S. pyogenes was mapped using similar techniques. Streptomycin resistant strains S43 ATCC, 543/192/4 and S43/192/30R were studied. In streptomycin resistant S43 ATCC and S43/192/30R strains, the lysine 56 changed to isoleucine and threonine respectively. Surprisingly, the 192/4 had two mutations, in each of the two hotspots in the rpsL gene where mutations due to streptomycin resistance occur. It had the amino acid 56, lysine, mutated to arginine and lysine 101 changed to asparagine. To check if this mutation was stable in the host animal, S43/192/4 P8 (S43/192/4 passaged eight times in mice) was sequenced and the sequence was identical to the streptomycin resistant 192/4. Hence, the lys101 mutation was stable and unlike the ancillary mutations in E.coli and S. typhimurium, which are compensated by new mutations. The pathogenesis of S. pyogenes depends in part on the ability of the pathogen to adhere to the epithelial cells of the throat and the quantity of M protein. Pathogenesis studies done on mice revealed the avirulence of S43/192/4smR strain. To elucidate the reason for this avirulence, the adherence properties and the production of M protein of the two strains S43/192/4smR and S43/192/30R were tested. Qualitative immunoblot analysis of the M protein of 192/4 and 30R revealed no significant difference. Competition ELISA was conducted to quantitate the M protein, and this also did not show any significant difference in the M protein levels. The adherence of 30R and 192/4 was measured on human pharyngeal epithelial cell line. The adherence properties of S43/192/4 SmR, was no different from other strains in this experiment. Electron microscopy, using immunogold to highlight the M protein on the cell surface showed no differences.<br>Graduation date: 2000

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.

碩士<br>國立臺灣大學<br>醫事技術學研究所<br>87<br>The oral or viridans group streptococci form a significant part of the normal flora of the human oral cavity and are associated with several disease conditions including dental caries, infective endocarditis and septicemia as well as purulent infections. Currently the viridans group streptococci taxonomically could be divided into five major clusters which included at least 20 species. These are S. mutans group, S. mitis group, S. milleri group, S. salivarius group and S. bovis group. No single system of classification suffices for the identification of this heterogeneous of organisms. Instead, classification depends on a combination of features including pattern of hemolysis observed on blood agar plates, antigenic composition, growth characteristics, biochemical reactions, and more recently, genetic analysis. The groEL gene, which encodes 60-kDa heat shock protein (GroEL), is ubiquitous and highly conserved among bacteria. It has been recently reported by using groEL gene as an alternative target for species-specific identification of staphylococci or mycobacteria. In this study, the S. gordonii groESL operon containing groES (282 bp) and groEL (1623 bp) was cloned and sequenced. The GroEL (groEL) of S. gordonii had 91% (81%) homology to S. pneumoniae and 60% (62%) to E. coli while GroES (groES) had 73% (71%) homology to S. pneumoniae and 39% (45%) to E. coli. Like other Gram-positive bacteria, a putative transcriptional promoter upstream of groES that was comprised of -35 and -10 hexamers franked downstream by the conserved Gram-positive heat shock gene regulatory sequence, CIRCE. A large inverted repeat that may function as a rho-independent transcriptional terminator was located downstream of groEL gene. In addition, the gene encoded ATP-binding cassette (ABC) transporter protein located upstream of groES and another gene located downstream of groEL were found. Degenerate PCR primers derived from conserved regions of the groESL operon of S. gordonii were used to amplify groESL of other species of viridans group streptococci. Sequences were determined for entire groESL of S. oralis and partial groEL fragment of the other species. The GroEL (groEL) homology of S. orals with S. gordonii, S. pneumoniae and E. coli were 93% (82%), 98% (91%) and 60% (62%), respectively. Comparing the sequence of GroEL and groEL among viridans group streptococci in this study, there were 75% and 85% homology among them. For application of species identification, the dot blot hybridization, direct sequencing and PCR-RFLP were used. Our results demonstrate that groEL-based identification method has the potential to be an alternative method for identification of the viridans group streptococci. Further work is required to test more strains of bacteria for designing a simple performing method with both high specificity and sensitivity.

碩士<br>國立臺灣大學<br>微生物學研究所<br>89<br>Streptococcus gordonii is a species of the viridans group streptococci, and is the major pathogen of dental caries. Superoxide dismutase, SOD, is an antioxidant enzyme, which scavenges O2-to protect cells from damage. In this study, we characterized the SOD of S. gordonii as an MnSOD by H2O2 and diethyldithiocarbamate inhibition test. Furthermore, we also found that the SOD has activity as a cambialistic enzyme, which can accept either iron or manganese as the cofactor to present enzyme activity. The activity of manganese —containing SOD was higher than that iron-containing SOD. The SOD activity of S. gordonii was variable in the growth phase of the organism, and was found three to four folds increasing from the exponential phase to the stationary phase. The sodA gene of S. gordonii was cloned in this study. The gene contains 603 bp nucleotides. The nucleotide sequence of sodA has 81.51﹪identity to that of S. pneumoniae, and 76.4﹪to that of S. agalactiae. The amino acid sequence of this gene has 89.05﹪identity to that of S. pneumoniae, and 78.6﹪to that of S. mutans. It also has 90.54﹪similarity to that of S. pneumoniae, and 80.59﹪to that of S. mutans. The coding region of gene was recombined with native expression vector pDESTTM15 and was transformed into E. coli BL21-SI strain. After induction, the transformant expressed about 23 kD protein, which showed SOD activity by native PAGE gel analysis.

碩士<br>長庚大學<br>生物醫學研究所<br>99<br>Streptococcus gordonii CH1 is one of the early colonizers of the dental plaque. The two-component system (TCS) is the major signal transduction system in bacteria. Since pH alteration is a frequent event occurring in the oral cavity, this study aims to identify TCSs that are potentially involved in the pH signaling in S. gordonii. To monitor the effects of mutations in TCS in pH signaling, the promoterless chloramphenicol acetyltransferase gene (cat) was fused with the pH-sensitive urease promoter (pureI) of Streptococcus salivarius and integrated into the gtfG of S. gordonii (SL17). Mutants of each TCS in SL17 background were generated by allelic exchange, and the expression of the pureI in cells grown at acidic pH was examined. The result revealed that TCS08- and TCS13-mutant strains exhibited aberrant cat expression comparing to the wild-type strain at pH 5.5. A lower survival rate at pH 3 was also detected in TCS08, but not in TCS13 mutant strain, comparing to the wild-type strain. The growth rate of TCS08 mutant was faster than the wild-type strain in the presence of paraquat, whereas TCS13 mutant grew more slowly than the wild-type strain. To identify the potential targets regulated by the two systems, the proteomes of these two strains were analyzed. 8 and 4 protein spots were indentified from TCS08 and TCS13 mutants, respectively, that are differentially expressed from the wild-type strain. Most of the identified proteins are involved in the metabolism, suggesting that these two systems participate in stress responses through modulating metabolic activities.

Introdução: A saúde oral é parte da saúde geral, bem-estar e qualidade de vida. Deste modo, o conhecimento da prevalência da cárie dentária na população infantil leva-nos a adotar, cada vez mais, um conjunto de medidas de prevenção, permitindo o controlo, não só dos fatores etiológicos da doença, mas também a diminuição da sua incidência. O conhecimento da microflora oral e a exploração do potencial alcalinizante de algumas bactérias orais, são uma estratégia promissora no que diz respeito à prevenção da cárie dentária. Objetivos: Este estudo visa avaliar, os comportamentos e o estado de saúde oral das crianças participantes. Estudar a associação entre o estado de saúde oral das crianças e a presença de Streptococcus mutans; Streptococcus gordonii e Streptococcus salivarius. Métodos: Foi realizado um estudo epidemiológico observacional transversal com 189 crianças dos 6 aos 10 anos de idade. Foi aplicado um questionário aos pais/encarregados de educação sobre os comportamentos de higiene oral das crianças participantes no estudo. Realizou-se uma observação intraoral que nos permitiu determinar o índice de cpo-d e o CPO-D. Por fim, realizou-se a recolha e análise molecular de biofilme oral das crianças participantes. Resultados: Das crianças participantes 70 (36,8%) apresentam cpo-d de 0, com cpo-d entre 1-3 temos 84 (44,2%), e superior a 3 temos 35 (18,4%). Relativamente ao índice de CPO-D de 0 temos 136 (71,6%) crianças, entre 1-3 temos 47 (24,7%) e superior a 3 temos 7 (3,7%). Este estudo permitiu ainda demonstrar, que crianças que apresentam o dente da recolha cariado revelam a presença de Streptococcus mutans em 80% das vezes. Por sua vez, das crianças que não apresentavam o dente da recolha cariado, foram encontrados Streptococcus gordonii e Streptococcus salivarius numa percentagem de 85,7% para ambas as bactérias. Conclusões: Este estudo estabelece uma relação entre ausência de cárie e a presença de Streptococcus gordonii e Streptococcus salivarius.<br>Introduction: Oral health is part of overall health, wellbeing and quality of life. Thus, knowledge of the prevalence of dental caries in children leads us to adopt increasingly a set of preventive measures allowing control not only of the etiological factors of the disease, but also to decrease its incidence. Knowledge of oral microflora and exploitation of alkalizing potential of some oral bacteria are a promising strategy with regard to the prevention of dental caries. Objectives: This study aims to evaluate the behavior and oral health status of children participating by studying the association between oral health status of children and the presence of Streptococcus mutans; Streptococcus gordonii and Streptococcus salivarius. Methods: An observational cross-sectional epidemiological study was conducted with 189 children 6 to 10 years old. A questionnaire was given to parents / guardians regarding oral hygiene behaviors of participating children. We conducted an intraoral observation to determine the rate of dmtf/DMTF. Finally, the collection and molecular analysis of oral biofilm was done. Results: From the 70 participating children (36.8%) have dmtf 0, with dmtf between 1-3 have 84 (44.2%), and 35 have more than 3 (18.4%). Relating to DMFT index of 0 we have 136 (71.6%), children aged 1-3 have 47 (24.7%) and greater than 3 have 6 (3.7%). This study also demonstrated that children who have the decayed teeth are colonized by Streptococcus mutans 80% of the time. In turn, 85.7% of the children who did not have decayed teeth, were colonized by Streptococcus gordonii and Streptococcus salivarius. Conclusions: This study establishes a relationship between the absence of caries and colonization by Streptococcus gordonii and Streptococcus salivarius.

Indiana University-Purdue University Indianapolis (IUPUI)<br>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.