Certains microorganismes sont capables de communiquer en utilisant des molécules et d’utiliser ce système pour réguler des comportements en fonction de la densité cellulaire. Ce système de communication, appelé quorum sensing (QS), régule des comportements bactériens, tels que la virulence et la formation de biofilm. Les molécules signal les plus étudiées sont les acyle-homosérine lactones (AHLs). Les enzymes qui sont capables de dégrader ces molécules peuvent couper la communication bactérienne, et se comportent ainsi comme des inhibiteurs de virulence et de biofilm. Ce phénomène, appelé quorum quenching (QQ), est une approche prometteuse pour le contrôle bactérien sans les tuer, ainsi que pour le développement de nouvelles thérapies contre les bactéries résistantes aux antibiotiques. Les travaux menés durant ma thèse ont permis d’isoler et de caractériser biochimiquement, enzymatiquement et structuralement de nouvelles lactonases provenant d’organismes thermophiles, capables d’inhiber le QS. J’ai résolu les structures de trois lactonases et en complexe avec différents types de lactones. Ces données ont révélées l’extrême polyvalence des sites actifs de ces enzymes, et ont permis d’identifier les résidus potentiellement impliqués dans la spécificité de substrat de ces enzymes. Ces résultats serviront de bases aux futurs projets d’ingénierie visant à changer la spécificité de ces enzymes. Enfin, mes travaux de caractérisation sur ces lactonases très stables ont permis de les utiliser hors du laboratoire et de démontrer l’importance de la signalisation bactérienne dans des processus biologiques complexes tels que la formation de biofilm et la bio-corrosion. / Numerous microorganisms are able to communicate using molecules and use this signaling system to coordinate behaviors in a cell-density-dependent manner. This communication system, dubbed quorum sensing (QS), regulates bacterial behaviors such as biofilm formation. The most popular system utilizes acyl homoserine lactones (AHLs) as signals. Enzymes that can degrade these signaling molecules can effectively disrupt bacterial signaling, and thereby behave as potent biofilm and virulence inhibitors. Therefore, the inhibition of QS, termed quorum quenching (QQ) by these enzymes is a promising approach to control microbes without killing them and develop therapies on multidrugs resistant strains. During this thesis, I have isolated and characterized biochemically, enzymatically and structurally novel lactonases from thermophilic sources. I have determined the structures of three lactonases in complex with different types of lactones. This enabled me to elucidate their catalytic mechanisms, as well as the unique binding modes of structurally different lactones. These data revealed the extreme catalytic versatility of the active sites of these enzymes, and allowed for the determination of residues possibly involved in substrate specificity. These data, in combination with structural data obtained on improved lactonase mutants, will serve as a foundation to guide future engineering studies aiming at altering lactonases’ specificity. Lastly, isolation and characterization work on these thermostable lactonases allowed to demonstrate the importance of bacterial signaling in complex biological processes, in the field, including biofilm formation and biocorrosion.

Des bactéries utilisent un système de communication, quorum sensing (QS), qui leur permet de synchroniser leur comportement proportionnellement à la densité de population. Des bactéries pathogènes utilisent le QS pour coordonner leur virulence et la formation de biofilm. Bloquer le QS, quorum quenching (QQ), constitue donc une piste pour étendre l'arsenal thérapeutique antibactérien. Durant cette thèse, SsoPox, une lactonase hyperstable hydrolysant les acyl homosérine lactones (AHL) impliquées dans le QS de bactéries à Gram négative pathogènes comme P. aeruginosa, a été étudiée. Son utilisation dans des pansements contre les infections à P. aeruginosa a été évaluée. La résistance aux contraintes industrielles liées aux procédés de fabrication des pansements a été évaluée. SsoPox s'est montrée résistante à la chaleur, aux solvants organiques, au stockage à température ambiante et à la stérilisation. L’efficacité de l’enzyme a été montrée sur 2 souches modèles et 51 isolats cliniques de P. aeruginosa. SsoPox a été plus efficace que 2 inhibiteurs du QS. L’efficacité de l’enzyme est aussi conservée après immobilisation. Une étude phénotypique et moléculaire du QQ enzymatique de P. aeruginosa a été réalisée entre SsoPox et une autre lactonase GcL ayant un spectre d'action distinct sur les AHL. In vitro, il a été mis en évidence des impacts différents entre les 2 enzymes sur les facteurs de virulence et le biofilm. In vivo, il a été montré que seul SsoPox réduit la virulence de P. aeruginosa. L’expression des gènes du QS et le protéome ont confirmé des différences entre GcL et SsoPox. Ainsi SsoPox semble constituer un bon candidat pour le développement de pansements innovants. / Bacteria use a communication system, or quorum sensing (QS), to synchronize group behaviors according to population density. Some pathogens use QS to coordinate virulence and biofilm formation. Interfering with QS, or quorum quenching, represents a target to extent antibacterial drug resources. Along this PhD project, SsoPox, a hyperstable lactonase, hydrolyzing acyl homoserine lactones (AHL) involved in the QS of Gram-negative pathogen bacteria like Pseudomonas aeruginosa was studied. SsoPox was studied and characterized for its use in wound dressing against P. aeruginosa infection. The enzyme resistance to industrial constraints encountered during wound dressings manufacturing processes was estimated. SsoPox demonstrated a high tolerance to heat, organic solvents, ambient temperature storage and to sterilization processes. The enzyme efficiency was shown on 2 model strains and 51 clinical isolates of P. aeruginosa. SsoPox was also more efficient than 2 well characterized QS inhibitors. The enzyme kept its efficiency even when immobilized. A last part was dedicated to phenotypical and molecular study of enzymatic QQ of P. aeruginosa. SsoPox and another lactonase GcL, having distinct AHL specificities, were compared. In vitro experiments highlighted different impacts between the enzymes on virulence factors and biofilm. An in vivo study showed that SsoPox, unlike GcL, was able to reduce P. aeruginosa virulence. QS gene expression and proteome study confirmed distinct impacts between each enzyme treatment s. Thus, SsoPox seems to be a prime candidate for development of innovative medical devices.

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Nitric oxide (•NO) is a molecule with diverse biological effects involved in both signaling and defense mechanisms in mammalian systems. The production of •NO is catalyzed from L-Arginine by a family of enzymes known as nitric oxide synthases (NOS). All mammalian isoforms contain an active site oxygenase domain and an electron-donating reductase domain joined by a calmodulin binding region. Recently, prokaryotic homologues to the oxygenase domain of mammalian NOS enzymes have been identified. Although several bacterial NOS (bNOS) enzymes have been characterized, their function is still unknown. Possible roles for this enzyme could include: (i) intercellular signaling to coordinate cellular/infectious activity, (ii) regulation via •NO-mediated posttranslational modifications, or (iii) nitration of compounds in different biosynthetic pathways. Efforts, contained in this thesis, to determine a probable role for this enzyme are two-fold: (i) via a search for possible interacting protein partners, and (ii) via a proteomic analysis of the effects of a knockout of the bNOS gene. Bacterial-NOS knockouts in Bacillus subtilis and Bacillus cereus have been created. 2D-Differential in gel electrophoresis (DIGE) has been used to analyze the proteomic effects of the expression of this gene in B. subtilis. Thus far 16 proteins which exhibited significant changes in expression, with a p value ≤ 0.05 and fold change ≥│2│, have been isolated and identified by peptide mass fingerprinting (PMF) in order to shed light on the relevance of this bacterial NOS homologue. The proteins which have been identified thus far are involved in cellular metabolism, amino acid metabolism and nitrogen metabolism. The identification of further proteins is required for a broader view of the impact of the expression of this gene on the proteome.

Nitric oxide (•NO) is a molecule with diverse biological effects involved in both signaling and defense mechanisms in mammalian systems. The production of •NO is catalyzed from L-Arginine by a family of enzymes known as nitric oxide synthases (NOS). All mammalian isoforms contain an active site oxygenase domain and an electron-donating reductase domain joined by a calmodulin binding region. Recently, prokaryotic homologues to the oxygenase domain of mammalian NOS enzymes have been identified. Although several bacterial NOS (bNOS) enzymes have been characterized, their function is still unknown. Possible roles for this enzyme could include: (i) intercellular signaling to coordinate cellular/infectious activity, (ii) regulation via •NO-mediated posttranslational modifications, or (iii) nitration of compounds in different biosynthetic pathways. Efforts, contained in this thesis, to determine a probable role for this enzyme are two-fold: (i) via a search for possible interacting protein partners, and (ii) via a proteomic analysis of the effects of a knockout of the bNOS gene. Bacterial-NOS knockouts in Bacillus subtilis and Bacillus cereus have been created. 2D-Differential in gel electrophoresis (DIGE) has been used to analyze the proteomic effects of the expression of this gene in B. subtilis. Thus far 16 proteins which exhibited significant changes in expression, with a p value ≤ 0.05 and fold change ≥│2│, have been isolated and identified by peptide mass fingerprinting (PMF) in order to shed light on the relevance of this bacterial NOS homologue. The proteins which have been identified thus far are involved in cellular metabolism, amino acid metabolism and nitrogen metabolism. The identification of further proteins is required for a broader view of the impact of the expression of this gene on the proteome.

Bacteria produce and respond to external stimuli using molecules termed autoinducers. Poultry meat contains inhibitors which interfere with AI-2 signaling. The primary objective of this work was to understand the effects of AI-2 on the virulence and growth of Salmonella Typhimurium, and if the introduction of AI-2 inhibiting compounds would influence these effects. Using DNA microarray analysis, expression of 1136 virulence-related genes in a Salmonella Typhimurium wild type and a luxS mutant strain, PJ002 (unable to produce AI-2), was monitored after exposure to treatments containing in vitro synthesized AI-2 (AI-2) and poultry meat (PM) inhibitors. Responding gene expression was unique in the presence of AI-2, with 23 genes differentially expressed at least 1.5-fold (p < 0.05). The combined AI-2 + PM treatment resulted in 22 genes being differentially expressed. Identification of inhibitory compounds was attempted using GC analysis on a hexane solvent extract obtained from a PM wash. From this analysis, chemical standards of linoleic, oleic, palmitic, and stearic acid were tested for inhibition using V. harveyi BB170. Combined fatty acids (FA) demonstrated inhibition against AI-2 at 60 % while 10-fold and 100-fold concentrations had inhibition of 84 % and 70 %, respectively. Growth of PJoo2, was studied using M-9 minimal medium with FA of varying concentrations, supplemented with either AI-2, or 1X phosphate buffered saline (PBS). Comparative analysis was done calculating the growth constants based on OD 600 values for each treatment. No significant difference in the combined FA + AI-2 treatments was observed against the AI-2 treatment. A significant increase in the growth rate constants of the AI-2 treatments was observed, however, compared to the PBS control (P = 0.01). Bacterial invasiveness, using a murine macrophage cell line, RAW 264.7, was also studied. AI-2 decreased cell invasiveness (P = 0.02), while the addition of combined FA improved invasiveness to normal levels. The results of these studies indicate that AI-2 does have an effect on the growth and virulence of Salmonella, but this is not uniformly modulated by the introduction of fatty acids, that inhibit AI-2 activity, suggesting that inhibition may be based on species specific transport systems.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

Orientadores: Reginaldo Bruno Gonçalves, Renata de Oliveira Mattos-Graner / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-07T15:16:39Z (GMT). No. of bitstreams: 1 Klein_MarliseInez_D.pdf: 1313647 bytes, checksum: 7404c2e04298de29087b5abb74a318d4 (MD5) Previous issue date: 2006 / Resumo: Streptococcus mutans são os principais patógenos da cárie dentária, um importante problema de saúde pública no país. Para um melhor entendimento da biologia desta espécie bacteriana, o objetivo desta tese de doutorado foi avaliar características genéticas e fisiológicas relacionadas à virulência de isolados clínicos de S. mutans. Neste microrganismo, o sistema de indução da competência, "quorum-sensíng", é dependente da densidade celular e está envolvido na capacidade de crescimento em biofilme. Foram pesquisados, os genes de sistema "quorum-sensíng" e os genes envolvidos no processo de transformação da espécie S. mutans. Foi demonstrada a presença de todos os genes avaliados em S. mutans, bem como a presença de polimorfismo em alguns genes pesquisados, dados ainda inéditos na literatura. Posteriormente, genótipos clínicos de S. mutans foram submetidos a uma análise genética e fisiológica em que foram avaliadas a tolerância e adaptação ao pH ácido, a capacidade de formação de biofilme e curvas de crescimento sob diferentes condições (pHs e diferentes fontes de carbono). Os dados de expressão gênica confirmaram os resultados obtidos nas análises fisiológicas. As cepas clínicas apresentaram um comportamento heterogêneo frente aos mesmos desafios ambientais, o que pode favorecer a sobrevivência das mesmas na cavidade bucal, um ambiente com diversas condições de estresse. Os dados obtidos, comparados aos existentes na literatura, sugerem que além da atividade A TPase, os sistemas de transporte de açúcares fosfotransferase fosfoenolpiruvato:açúcar dependente (pep-PTS) também estariam envolvidos na tolerância de S. mutans ao estresse ácido. Em conjunto, as observações decorrentes das análises efetuadas, nesta tese, podem contribuir para a compreensão dos processos biológicos de S. mutans. / Abstract: Streptococcus mutans is considered the primary etiological agent of human dental caries, an important public health problem in Brazil. The purpose of this thesis was to evaluate genetic and physiologic properties related with virulence in clinical isolates of Streptococcus mutans. The quorum-sensing system, that induces and regulates genetic competence, depends on cellular density and also may play a role in biofilm growth and structure in S. mutans species. A screening of genes of the quorum-sensing system and genes involved with genetic transformation was performed. The analysis revealed that ali genes are widespread within the S. mutans species, and some genes presented polymorphisms. These data are new in the scientific literature. Furthermore, clinical genotypes of S. mutans were subjected to genetic and physiological analysis, including tolerance and acid adaptation to low pH, ability to form stable biofilm and growth kinetic under different conditions (pH and carbon source). The profile of gene expression confirms the data found in the physiologic assays. The studied strains shown heterogenic behavior at the same environmental challenge, that could favor these strains survive in the oral cavity, an environmental with several stress conditions. The data obtained here, and supported by scientific literature, suggest that besides ATPase activity' the sugar:phosphotransferase systems (PTS) could help to mount an adaptive acid tolerance response in S. mutans. Taken together all data obtained in this thesis may help to understand S. mutans biological processes. / Doutorado / Microbiologia e Imunologia / Doutor em Biologia Buco-Dental