Les intégrons sont des éléments génétiques d'ADN mobiles tout comme les transposons et les plasmides R. Ils sont grandement impliqués dans la dissémination par transfert horizontal de gènes de résistance qui se retrouvent sous forme de cassette. Une cassette est constituée d'un gène associé à un site de recombinaison spécifique nommé attC reconnu par une intégrase. L'intégrase est l'enzyme capable d'exciser des gènes sous forme de cassettes et de les intégrer dans son intégron au niveau du site attl. Il existe deux types d'intégrons : les intégrons de résistance et les intégrons chromosomiques. L'intégron chromosomique de Treponema denticola est le premier à avoir été identifié chez un spirochète et est le premier exemple où le gène codant pour l'intégrase est dans le même sens que les cassettes. De plus, cet intégron semble plus près des intégrons de résistance que les autres intégrons chromosomiques. Des expériences in vivo chez E. coli nous permettent de constater que des interactions entre l'intégron chromosomique de T. denticola et les intégrons de résistance sont possibles et viennent appuyer notre hypothèse que les intégrons chromosomiques sont à l'origine des intégrons de résistance.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

The major outer sheath protein (Msp) is the most prominent surface antigen of the periodontal pathogen Treponema denticola. It mediates adhesion to extracellular matrix and dysregulation of cytoskeletal homeostasis of host cells. Disassembly of actin filaments and the coincident subcortical de novo synthesis of actin filaments in fibroblasts upon exposure to Msp were investigated with a barbed-end fluorescent labeling method. The functional impact of actin cytoskeleton disorganization was determined with a scratch wound migration assay in fibroblast monolayers and a videomicroscopy migration assay in neutrophils. Msp pretreatment had a significant inhibitory effect on the migration of the fibroblasts across a collagen substratum and inhibited the neutrophil chemotactic migration towards a chemoattractant. In a study originally aimed to find the biologically active domains of Msp that may perturb actin, short peptides were selected from the deduced and predicted surface exposed regions of Msp and investigated for their role in actin dynamics and cell motility. A novel BSA-conjugated peptide (P34BSA) was found serendipitously to induce stress fiber formation and stability in fibroblasts. This activity was found to be mediated by Rho activation and cofilin phosphorylation, which are important tandem signaling pathways in the regulation of a variety of actin-dependent cellular functions. P34BSA was internalized by the cells. Yet, a mechanistic study using low-temperature treatments and depletion of cholesterol with methyl-β-cyclodextrin (MβCD) revealed that P34BSA most likely induces actin stress fiber formation extracellularly through a Rho-dependent signaling pathway. P34BSA induced Rho activation via binding of guanosine nucleotide exchange factor p114RhoGEF to RhoA, one of many exchange factors that have been shown to play a role in activating Rho signaling. Pretreatment with P34BSA partially protected the fibroblasts against the actin-disrupting effects of cytochalasin D and latrunculin B, and the cells maintained most of their actin filaments. P34BSA treatment caused retardation of fibroblast migration on a collagen substratum. It also inhibited the chemotactic movement of neutrophils towards the chemoattractant fMLP. P34 may represent a novel amino acid sequence of a bacterial virulence protein that, when conjugated to BSA, can be used as a chemical reagent to investigate RhoA signaling pathways in host cells.

Zugl.: Berlin, Univ., Diss., 2008

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 111-134).

Periodontitis is a polymicrobially-induced, chronic inflammatory disease of the tissues that support and surround the tooth. While greater than 500 organisms are found in dental plaque, Treponema denticola is one of only a few species associated with disease. It has been hypothesized that oral bacteria disrupt host homeostasis through manipulation of the innate immune system. In this study, we examine the impact of binding and subsequent cleavage of factor H, a complement regulator, by T. denticola in the evasion and subversion of complement. The molecular interaction between the sole FH-binding protein, FhbB, and FH was detailed by x-ray crystallography, site-directed mutagenesis, and inhibition analyses. Negatively-charged amino acids of FhbB formed salt-bridges with positively-charged residues of FH within the complement control protein domains (CCP) 6 and 7. In support of its critical role in disease, FhbB was universal among T. denticola isolates, although, different strains produce highly divergent FhbB proteins. Despite extensive sequence variation, predominantly within the FH-binding determinant, all FhbB proteins have similar structure and interact with FH using nearly identical molecular mechanisms. In addition to differences in FhbB, many isolates of T. denticola displayed significant variation in the activity of the chymotrypsin-like protease, dentilisin. Neither FhbB type produced nor dentilisin activity influenced serum resistance, as all strains tested were highly serum resistant. While sequence diversity of FhbB did not influence the interaction with FH or serum resistance, FhbB proteins elicited type-specific antibodies that blocked FH binding. Collectively, these analyses indicate that FH binding is an essential complement evasion mechanism of T. denticola and characterizes the uniquely complex interaction between T. denticola and complement. The data presented here provide novel insight into the pathogenesis of disease and begins to explore a hypothetical molecular mechanism by which this key periopathogen disrupts the host innate immune system, leading to periodontitis.

Periodontitis is a chronic inflammatory disease that affects over 116 million adults in the United States. A shift in the normal microflora occurs as periodontal disease develops resulting in a larger number of Gram-negative anaerobes and spirochetes. An increase in the oral spirochete, Treponema denticola, is highly correlated with periodontal disease progression and severity. The ability of this periopathogen to thrive in the subgingival crevice is dependent on complement evasion mechanisms. Earlier analyses demonstrated that the primary mechanism of T. denticola serum resistance is binding of the human complement regulatory protein, Factor H (FH), to the factor H-binding protein (FhbB). FH serves as cofactor in the Factor-I mediated cleavage of C3b and accelerates the decay of the C3 convertase complex, leading to downregulation of C3b production. Several pathogens bind FH, and a number of these bacterial binding proteins have been shown to bind plasminogen. Plasminogen is a plasma glycoprotein that circulates as a zymogen. Its active form, plasmin, degrades components of the extracellular matrix and cleaves complement proteins C3b and C5 inhibiting complement pathway progression. Through molecular and biochemical analyses, this study demonstrates that FhbB simultaneously binds plasminogen and FH to residues located on its positively and negatively charged surfaces, respectively, and that the two ligands do not compete for binding. This study also shows that the surface-bound plasminogen is available for proteolytic cleavage into the active serine protease plasmin. The activated plasmin could break down components of the periodontal tissue leading to increased nutrient availability and creation of a larger anaerobic environment where the bacteria can flourish, thereby promoting periodontal disease.

Selenoproteins are unique proteins in which selenocysteine is inserted into the polypeptide chain by highly specialized translational machinery. They exist within all three kingdoms of life. The functions of these proteins in biology are still being defined. In particular, the importance of selenoproteins in pathogenic microorganisms has received little attention. We first established that a nosocomial pathogen, Clostridium difficile, utilizes a selenoenzyme dependent pathway for energy metabolism. Following this initial characterization, we demonstrate that this pathway is linked to production of toxins by this organism. Finally, we show that interruption of selenium metabolism is a viable pathway for development of antimicrobials against this, and other selenoprotein dependent pathogens. We investigated whether Stickland reactions (paired amino acid fermentation) might be at the heart of C. difficile's bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases; glycine and D-proline reductase. These selenoenzymes were expressed upon addition of the corresponding Stickland acceptor (glycine, proline or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys containing) proteins. D-proline reductase utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. The enzyme was found to be independent of divalent cations, and zinc was a potent inhibitor. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of D-proline reductase may differ significantly from similar enzymes from non-pathogenic species. C. difficile pathogenesis is due to the production of toxins, A and B, members of the large clostridial cytotoxin family. Previous studies have shown that toxin production by this organism is influenced by the composition of the growth medium. We examined the impact of Stickland acceptor amino acids (Stickland acceptors; glycine, proline and hydroxyproline) on growth kinetics and yield, protein synthesis, toxin production and gene expression. Although addition of Stickland acceptors moderately increases growth yield and total protein synthesis, there does not appear to be a clear impact on entry into stationary phase. Glycine dramatically increases the amount of toxin released into the growth medium. Conversely, the addition of hydroxyproline suppresses toxin production. We examine possible mechanisms of regulation and demonstrate that CodY, a regulator of toxin gene transcription does not appear to mediate this effect. Given the importance of selenium dependent Stickland reactions to C. difficile growth and toxin production we aimed to examine the efficacy of blocking such pathways as a means of antimicrobial development. Selenide is the only known substrate for selenophosphate synthetase, the first enzyme involved in the specific incorporation of selenium into selenoproteins. We have identified a stable complex formed upon reaction of auranofin (a gold containing drug) with selenide in vitro. Auranofin potently inhibits the growth of C. difficile but does not similarly affect other clostridia that do not utilize selenoproteins to obtain energy. Moreover, auranofin inhibits the incorporation of radioisotope selenium (75Se) in selenoproteins in both E. coli, the prokaryotic model for selenoprotein synthesis, and C. difficile without impacting total protein synthesis. Auranofin blocks the uptake of selenium and results in the accumulation of the auranofin-selenide adduct in the culture medium. Addition of selenium in the form of selenite or L-selenocysteine to the growth media significantly reduces the inhibitory action of auranofin on the growth of C. difficile. Based on these results, we propose that formation of this complex and the subsequent deficiency in available selenium for selenoprotein synthesis is the mechanism by which auranofin inhibits C. difficile growth. The antimicrobial potential of blocking selenium metabolism is further demonstrated in the dental pathogen Treponema denticola. We show that auranofin blocks the growth this organism which also participates in Stickland fermentation. In addition, we provide evidence that the antimicrobial action of stannous salts against T. denticola is also mediated through inhibition of the metabolism of selenium. These studies clearly show that, at least in a subset of microbes that use selenium for the synthesis of selenoproteins, the need for this metalloid can be a useful target for future antimicrobial development.

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.

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.