Les PLLs sont une famille d’enzyme dont l’activité catalytique est double. L’activité lactonase permet entre autre de détruire les AHLs, molécules médiatrices de la communication chez les bactéries Gram négatives, empêchant ainsi la synchronisation à l’échelle de la population de la sécrétion de facteurs de virulence ou la formation de biofilm. Elles sont non seulement capables d’hydrolyser les molécules possédant un noyau lactone, mais aussi les phosphotriésters par promiscuité de substrat. L’activité phosphotriestérase permet de dégrader les composés organophosphorés (OPs) hautement toxiques, que ce soit les insecticides (Paraoxon) ou les agents neurotoxiques de guerre (Sarin, VX). Les travaux effectués ont consisté à évaluer l’efficacité de la PLL SsoPox, provenant de l’archée extrêmophile Sulfolobus solfataricus, pour empêcher la mise en place de la virulence et du biofilm chez Pseudomonas aeruginosa PAO1. Cette évaluation a été effectuée in vitro et in vivo via un modèle d’infection pulmonaire chez le rat. D’autre part, une étude in vitro a été réalisée sur 73 souches cliniques de P. aeruginosa isolés sur des patients atteints de pied diabétique, en évaluant la quantité de pyocyanine et l’activité protéolytique. Enfin, une phase prospective pour identifier de nouvelles PLLs a permis la caractérisation enzymatique et structurale de 2 nouvelles enzymes, SacPox et VmoLac, contribuant ainsi à affiner la connaissance sur cette famille et leur potentiel d’amélioration par ingénierie protéique. Au final, les PLLs offrent un intérêt biotechnologique majeur et peuvent mener à une valorisation concrète pour la santé humaine mais également pour la bioremédiation des OPs. / Phosphotriesterase-Like Lactonases (PLLs) are a family of enzyme displaying a dual catalytic activity. Lactonase activity allows for, among others, the destruction of Acyl Homoserine Lactones (AHLs). These molecules mediate the communication in Gram negative bacteria, allowing them to synchronize group behavior like virulence factor secretion or biofilm formation. Beside their ability to hydrolyze molecules with a lactones moiety, PLL also show substrate promiscuity for hydrolyzing highly toxic organophosphate compounds (OPs), such as pesticides (i.e. Paraoxon) or Chemical Warfare Nerve Agents (CWNAs) (e.g. Sarin & VX). The work described here consisted in evaluating the efficacy of the PLL SsoPox, originating from the extremophile Archaea Sulfolobus solfataricus, to prevent virulence and biofilm formation on the model strain Pseudomonas aeruginosa PAO1. This evaluation was performed both in vitro and in vivo, by using a rat pulmonary infection model. A study has been performed on a strain collection of 73 clinical strains of P. aeruginosa isolated from diabetic foot, to assess the enzyme effects on pyocyanin secretion and protease activity. Finally, a prospective phase to identify new PLLs allowed for the enzymatic and structural characterization of two new enzymes, SacPox and VmoLac, thus contributing to refine the knowledge about this enzyme family and their engineering potential. In conclusion, PLLs are of prime biotechnological interest and could lead to developments in human health, but also for OPs bioremediation with a non aggressive decontamination solution.

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

Biomining is a maturing technology that uses the activity of sulfur and iron oxidising microorganisms to liberate valuable metals from ores but suffers from slow reaction kinetics. Increasing the reaction kinetics of a biomining process could produce significant bottom line improvements for mining companies worldwide and encourage the use of biomining as a green mining technology. Quorum sensing molecules have been shown to successfully modulate the behaviours of biomining bacteria in manners that may be able to improve bioreactor retention times. This study tests the potential for two different quorum sensing treatments to improve the nickel leaching ability of a biomining bacterial consortium. A novel method of delivering quorum sensing treatments to bacterial cultures is described while doubt is cast on established methods. Laboratory scale bioreactors were constructed and the leaching of nickel into solution was followed via ICP-AES to quantify improvements in bioleaching ability. Similar bioreactors were used to exhibit the inhibitory effect that a commonly used organic solvent can have on the leaching ability of bioleaching consortia. Ultimately a qualitative improvement in the bioleaching of nickel is produced using a mixture of tetradecanoyl-acylhomoserine lactone (C14-AHL) and its two derivatives, but the use of C14-AHL alone did not improve bioleaching kinetics. Use of small volumes of the solvent DMSO produced large inhibitory effects on the leaching of nickel by bioleaching consortia.

Le contrôle du biofouling sur des surfaces inertes immergées ou en atmosphère humide est une nécessité dans le secteur marin, tant pour des raisons économiques qu’environnementales. La formation de biofilm microbien, étape préalable à la formation du biofouling, est souvent intrinsèquement liée chez les bactéries au système de communication “Quorum Sensing” (QS). Chez certaines bactéries Gram négatif, le QS est basé sur la perception de petites molécules diffusibles appelées N- Acyl Homosérine Lactones (AHLs). L’une des stratégies antifouling en voie de développement de nos jours repose sur l’inhibition du QS bactérien. L’objectif de cette thèse est d’utiliser certaines bactéries marines afin d’identifier des molécules anti-QS capables de perturber la formation de biofilm. Ce travail a donc porté sur la mise en évidence de molécules AHLs impliquées dans le QS chez certaines bactéries marines isolées de la rade de Toulon, l’étude de la modulation de certains phénotypes dont la formation du biofilm, par ces molécules et, la mise en place d’un test préliminaire d’inhibition du QS. Parmi les trois bactéries isolées de la rade de Toulon (TC8, TC14 et TC15) du genre Pseudoalteromonas, connues pour produire de nombreuses molécules actives, et testées pour leur capacité à sécréter des AHLs, seule Pseudoalteromonas sp. TC15 a produit la C12-HSL. P. ulvae TC14, capable de produire un biofilm conséquent et de la violacéine, ne produit aucune AHL. Afin d’évaluer la possibilité d’utiliser une bactérie marine comme outil de criblage anti-QS, interférant avec les AHLs et les conséquences sur son biofilm, des AHLs exogènes ont été testées sur la production de violacéine, la formation de biofilm et la mobilité de TC14. Certaines AHLs ont montré qu’elles pouvaient réguler la production de violacéine et la formation de biofilm chez TC14, suggérant l’existence d’un récepteur AHLs fonctionnel. Des tests préliminaires d’inhibition du QS ont été effectués avec des molécules commerciales et des analogues synthétiques. La 3-oxo-C6-HSL commerciale, ainsi que l’esculétine et la p- benzoquinone, connues pour interférer avec le QS bactérien, ont été capables d’inhiber la production de violacéine ainsi que la formation de biofilm de TC14 à des concentrations n’affectant pas sa croissance. Cette étude suggère donc que P. ulvae TC14 pourrait être utilisée comme un outil de recherche de molécules anti-QS en conditions proches de celles trouvées dans l’environnement marin, et ce dans le but d’être ultérieurement testées sur la formation de biofilm. L’objectif à plus long terme reste de trouver un moyen de limiter la formation du biofilm en utilisant des molécules non toxiques pour l’environnement. / The biofouling control on immersed inert surfaces or in moist atmosphere is a necessity in the marine sector for both economic and environmental reasons. Microbial biofilm formation, the initial step of biofouling development, is intrinsically linked to the communication system “Quorum sensing” (QS). In some Gram negative bacteria, QS is based on the perception of small diffusible signaling molecules called Acyl Homoserine Lactones (AHLs). The inhibition of bacterial QS is part of the different antifouling strategies currently developed. This present work focused on the detection of AHLs molecules involved in this communication system in bacteria isolated from Toulon harbor and the study of modulation of some phenotypes, including biofilm formation, by these molecules as well as the development of a preliminary anti-QS assay. Three marine bacteria isolated from Toulon harbor (TC8, TC14 and TC15), belonging to the Pseudoalteromonas genus, known to synthesize many active molecules, have been tested for their ability to produce AHLs. Only Pseudoalteromonas sp. TC15 produced the C12-HSL. P. ulvae TC14 a violacein-producing and biofilm-forming bacteria, did not secrete any AHLs. Few marine bacteria are used as an anti-QS screening tool, especially by interfering with AHLs with the goal of studying the consequences on biofilm formation. In order to evaluate the possibility to use TC14 with this purpose, exogenous AHLs were tested on the violacein production, the biofilm formation and the motility of TC14. Some AHLs were able to regulate violacein production and biofilm formation suggesting the presence of a functional AHLs receptor in TC14. Preliminary QS inhibition assays were performed with commercial molecules and synthetic analogues. The commercial 3-oxo-C6-HSL as well as esculetin and p-benzoquinone, known to interfere with bacterial QS, were able to inhibit QS and biofilm formation at a non-toxic concentration. Overall, this study suggests that the marine strain P. ulvae TC14 may be used as a tool for the detection of anti-QS molecules in conditions closed to the marine environment. These molecules may subsequently be tested on the biofilm formation of marine bacteria. The long term objective is to find a way to limit biofilm formation, using non-toxic molecules for the environment.

Orientador: Douglas Roberto Monteiro / Coorientador: Alberto Carlos Botazzo Delbem / Banca: Farli Aparecida Carrilho Boer / Banca: Debora de Barros Barbosa / Resumo: O tirosol é uma molécula de quorum-sensing (QS) que participa do controle da morfogênese em Candida albicans. Contudo, seu efeito como agente antimicrobiano sobre biofilmes de Streptococcus mutans permanece desconhecido. Assim, o objetivo deste estudo foi avaliar o efeito do tirosol em diferentes concentrações sobre a produção de ácido e formação de biofilmes por S. mutans ATCC 25175, bem como quantificar biofilmes pré-formados desta espécie desenvolvidos sobre espécimes de hidroxiapatita (HA) e tratados com tirosol. A concentração inibitória mínima (CIM) de tirosol sobre células no estado planctônico foi determinada de acordo com o método da microdiluição. Na sequência, biofilmes de S. mutans foram formados durante 48 horas sobre espécimes de HA na presença de diferentes concentrações de tirosol (11,25, 22,5, 50, 100 e 200 mmol l-1), usando saliva artificial como meio de cultura. Após, a produção de ácido foi avaliada através da mensuração do pH do meio, enquanto a formação de biofilmes foi determinada através da quantificação da biomassa total (BT), atividade metabólica (AM) e número de unidades formadoras de colônias (UFCs). Ainda, biofilmes pré-formados (24 horas) de S. mutans foram tratados com tirosol a 100 e 200 mmol l-1 por 1 minuto, duas vezes ao dia, durante três dias, totalizando biofilmes de 96 horas. Em seguida, a atividade antimicrobiana foi avaliada por meio da quantificação da BT, AM, número de UFCs e composição da matriz extracelular (proteínas e carboidratos). Gluconato de clorexidina (490 μmol l-1) foi usado como controle positivo. Os dados foram analisados por ANOVA a um critério, seguido pelos testes de Tukey e Holm-Sidak (α = 0,05). Microscopia eletrônica de varredura (MEV) foi utilizada na análise da estrutura dos biofilmes. A CIM de tirosol foi 90 mmol l-1. Tirosol em concentrações... / Abstract: Tyrosol is a quorum-sensing molecule (QS) that participates in the control of Candida albicans morphogenesis. However, its effect as an antimicrobial agent on Streptococcus mutans biofilms remains unknown. Thus, the aim of this study was to evaluate the effect of tyrosol at different concentrations on the acid production and biofilm formation by S. mutans ATCC 25175, as well as to quantify pre-formed biofilms of this species developed on hydroxyapatite (HA) specimens and treated with tyrosol. Minimum inhibitory concentration (MIC) of tyrosol against planktonic cells was determined in accordance with the microdilution method. Subsequently, S. mutans biofilms were formed during 48 hours on HA specimens in the presence of different concentrations of tyrosol (11.25, 22.5, 50, 100 and 200 mmol l-1), using artificial saliva as culture medium. Next, the acid production was assessed by pH determination of the medium, while the biofilm formation was determined through quantification of total biomass (TB), metabolic activity (MA) and number of colony-forming units (CFUs). Further, S. mutans pre-formed biofilms (24 h) were treated with tyrosol at 100 and 200 mmol l-1 twice a day for 1 min, during 3 days, totaling 96-h biofilms. Then, the antimicrobial activity was evaluated through quantification of TB, MA, number of CFUs and composition of biofilms' extracellular matrix (proteins and carbohydrates). Chlorhexidine gluconate (490 μmol l-1) was used as positive control. Data were analyzed by one-way ANOVA, followed by Tukey's and Holm-Sidak's tests (α = 0.05). Scanning electron microscopy (SEM) observations were performed in order to analyze biofilms' structure. MIC of tyrosol was 90 mmol l-1. Tyrosol at sub-inhibitory concentrations (11.25 and 22.5 mmol l-1) was not able to significantly reduce acid production by S. mutans biofilms. However, biofilms... / Mestre

Le Quorum sensing (QS) est un système de communication bactérienne capable de réguler divers processus cellulaires qui dépendent de la densité de la population microbienne. Chez les bactéries à Gram négatif, cela se produit par production de molécules de signalisation auto-inductrices (AI), les acyl homosérine lactones (AHL). La libération d´AHL à l'extérieur de la cellule est détectée par la population bactérienne provoquant en réponse la régulation de l'expression de certains gènes (régulon QS).Notre laboratoire a étudié et identifié un système QS fonctionnel dans la souche Acidithiobacillus ferrooxidans ATCC 23270T. En outre, nous avons montré que des analogues synthétiques d´AHL modulent l´adhésion d´At. ferrooxidansT sur un substrat minéral, tels des coupons de soufre.Dans ce projet de recherche, nous nous proposons d'identifier les gènes qui sont régulés par le QS chez At. ferrooxidansT, en particulier ceux impliqués dans la biogénèse du biofilm. Notre hypothèse est que des analogues synthétiques d’AHL induisent le système QS. Ainsi, nous nous proposons de moduler l´adhésion sur substrat minéral grâce à l'utilisation de ces molécules. L'utilisation de ces AHL permettra de caractériser le régulon QS dans cette souche bactérienne.L'identification d'analogues synthétiques d´AHL qui favorisent l'adhésion à des coupons de soufre nous a permis d'étudier le transcriptome d´At. ferrooxidansT dans des conditions où le régulon QS est stimulé. Puces à ADN d´At. ferrooxidansT avec/sans ces analogues synthétique d´AHLs nous a permis de caractériser le régulon QS et les gènes impliqués dans la biogénèse du biofilm dans les conditions utilisées. / Quorum sensing (QS) is a bacterial communication system capable of controlling several cellular processes dependent on the density of the microbial population. In Gram-negative bacteria, it occurs mainly through the production by bacteria of small diffusible signaling molecules, termed autoinducers (AI), of the acyl homoserine lactones type (AHLs). The release of AHLs outside the cell is detected by the bacterial population generating the regulation of the expression of several genes (regulon QS).Our laboratory has studied and identified a functional QS system in the Acidithiobacillus ferrooxidans ATCC 23270T type strain. Besides, by using synthetic analogs of AHLs, we have shown that AHL-type QS molecule analogs modulate adhesion of At. ferrooxidansT to minerals, such as sulfur coupons. In this research, we propose to identify the genes that are regulated by QS in At. ferrooxidansT, particularly those that are associated with biofilm formation. For this, we propose to modulate the adhesion of At. ferrooxidansT to mineral substrate through the use of a synthetic AHL analog. Our working hypothesis postulates that AHLs molecules induce the QS system, and that their use will allow the characterization of the QS regulon of this bacterial strain by transcriptomic analysis.The identification of synthetic AHLs improving adherence of At. ferrooxidansT on sulfur coupons allowed us to study the transcriptome of this organism in conditions in which QS regulon is stimulated. DNA microarrays of At. ferrooxidansT with/without one of these AHLs synthetic analogues allowed us to identify the QS regulon and to determine genes involved in biofilm formation.

Swimming bacteria are ubiquitous in aqueous environments ranging from oceans to fluidic environments within a living host. Furthermore, engineered bacteria are being increasingly utilized for a host of applications including environmental bioremediation, biosensing, and for the treatment of diseases. Often driven by chemotaxis (i.e. biased migration in response to gradients of chemical effectors) and quorum sensing (i.e. number density dependent regulation of gene expression), bacterial population dynamics and emergent behavior play a key role in regulating their own life and their impact on their immediate environment. Computational models that accurately and robustly describe bacterial population behavior and response to environmental stimuli are crucial to both understanding the dynamics of microbial communities and efficiently utilizing engineered microbes in practice. Many existing computational frameworks are finely-detailed at the cellular level, leading to extended computational time requirements, or are strictly population scale models, which do not permit population heterogeneities or spatiotemporal variability in the environment. To bridge this gap, we have created and experimentally validated a scalable, computationally-efficient, agent-based model of bacterial chemotaxis and quorum sensing (QS) which robustly simulates the stochastic behavior of each cell across a wide range of bacterial populations, ranging from a few to several hundred cells. We quantitatively and accurately capture emergent behavior in both isogenic QS populations and the altered QS response in a mixed QS and quorum quenching (QQ) microbial community. Finally, we show that the model can be used to predictively design synthetic genetic components towards programmed microbial behavior. / Master of Science / Bacteria are an integral part of life and possess a host of characteristics that make them a powerful tool with which to confront many modern day problems. Using genetic engineering and the burgeoning field of synthetic biology, these single-celled organisms can be manipulated to perform many useful tasks such as detecting oil spills or other environmental pollutants, producing pharmaceuticals such as insulin, and even invading and killing cancer cells. Accurate computational simulations of microbial behavior will aid in the efficient design of such synthetic bacteria-based systems and reduce dependency on the current time-intensive “guess and check” paradigm. Towards this goal, we have built a comprehensive computer simulation of bacterial swimming behavior, response to chemo-effector concentration gradients called chemotaxis, a form of microbial communication called quorum sensing (QS), and a form of communication disruption called quorum quenching (QQ). Not only do we demonstrate an unprecedented level of accuracy in predicting experimental results, but we also couple the simulation with synthetic biology to precisely tune bacteria QS behavior, neither of which have previously been reported in literature. The overarching outcome of this thesis is a tool that could be used to improve the design process of useful bacteria-based systems in diverse areas of biotechnology.

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