Lors d’infections, l'identification rapide des micro-organismes est cruciale pour améliorer la prise en charge du patient et mieux contrôler l'usage des antibiotiques. L’électrochimie présente plusieurs avantages pour les tests rapides : elle permet des analyses in situ, faciles et peu chères dans la plupart des liquides. Son utilisation pour l’identification bactérienne est récente et provient de la découverte de molécules donnant de forts signaux redox dans le surnageant de bactéries du genre Pseudomonas.Cette thèse s’intéresse à l’analyse de surnageants de la bactérie Pseudomonas aeruginosa, 4e cause de maladies nosocomiales en Europe. Tout d’abord, l’intérêt de l’analyse électrochimique de surnageants de culture dans une visée d’identification a été évalué. Pour cela, après l’étude de 4 potentiels biomarqueurs de la présence de cette bactérie en solutions modèles, l’analyse électrochimiques de surnageant de plusieurs souches P. aeruginosa a été effectuée. Les résultats obtenus sont prometteurs. Ils mettent en évidence une signature électrochimique complexe et souche-dépendante du surnageant.La suite de la thèse s’est intéressée à l’amplification de la détection électrochimique grâce à l’utilisation du polymère conducteur PEDOT:PSS. Il a été choisi pour ses bonnes propriétés électrochimiques, sa biocompatibilité et sa facilité de mise en forme. Il a tout d’abord été utilisé sous forme de films minces pour confirmer son pouvoir d’amplification. Une électrode 3D a ensuite été fabriquée par lyophilisation. L’utilisation de ce type d’électrode permet d’amplifier encore la détection en augmentant la surface d’échange mais aussi en confinant les bactéries dans l'électrode. / During infections, microorganisms fast identification is critical to improve patient treatment and to better manage antibiotics use. Electrochemistry exhibits several advantages for rapid diagnostic: it enables easy, cheap and in situ analysis in most liquids. Its use for bacterial identification is recent and comes from the discovery of molecules giving strong redox signals in the bacterial supernatant of the Pseudomonas genus.This thesis focuses on the supernatants analysis of the bacterium Pseudomonas aeruginosa. This bacteria is the fourth cause of nosocomial infections in Europe. First, the interest of supernatants electrochemical analysis for identification was evaluated. For this, after the study of four redox biomarkers of this bacterium in model solutions, supernatant electrochemical analysis of several strains of P. aeruginosa was performed. The results are promising. They highlight a complex strain-dependant electrochemical signature of the supernatant.Following, we focused in the amplification of the electrochemical detection through the use of the conductive polymer PEDOT: PSS. This polymer was chosen for its good electrochemical properties, its biocompatibility and its easy shaping. It was first used as a thin films to confirm its amplification power through biomarker adsorption. Then, a 3D electrode was made by freeze drying. The use of this type of electrode can further amplify the detection by increasing the exchange surface as well as confining the bacteria in the electrode.

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Au siècle dernier, les maladies infectieuses avaient pu être endiguées dans les pays développés grâce au développement des antibiotiques. Cependant, l’apparition de bactéries résistantes aux traitements antimicrobiens remet ce problème de santé publique au premier plan. De plus, on observe l’apparition de poly-résistances de plus en plus fréquentes, notamment en milieu hospitalier, et les organisations de santé ont donc, depuis le début des années 2000, appelé à utiliser les antibiotiques avec parcimonie afin de stopper la propagation des résistances. Aujourd’hui, un effort doit être fait pour le développement de nouvelles stratégies thérapeutiques.Une des voies envisagées consiste à cibler le système de communication appelé quorum sensing que les bactéries utilisent pour réguler de façon concertée l’expression de leurs gènes afin de s’adapter aux pressions environnementales. Le quorum sensing est basé sur l’échange de molécules signal sécrétées appelées phéromones qui sont détectées par des récepteurs transmembranaires ou cytoplasmiques appelés quorum senseurs.Chez les pathogènes à Gram positif, des phéromones peptidiques re-internalisées par les bactéries viennent réguler l’activité des quorum senseurs appelés RNPP, pour les premiers membres identifiés : Rap, NprR, PlcR et PrgX. Ces systèmes de quorum sensing régulent le changement de comportement de la population bactérienne. Ils sont impliqués dans des processus cellulaires importants tels que la sporulation, la formation de biofilm, la compétence et la virulence.Mon projet a porté sur l’analyse structure-fonction de deux des membres de la famille : NprR, du groupe Bacillus cereus, et ComR, un nouveau membre récemment identifié chez les Streptocoques. En combinant la cristallographie aux rayons X avec des approches biochimiques et génétiques, nous avons pu élucider le mécanisme moléculaire qui régule l’activité de ces deux systèmes de quorum sensing.NprR est un régulateur bifonctionnel qui, chez Bacillus thuringiensis, régule la survie de la bactérie jusqu’à la sporulation dans le cadavre des insectes infectés par cet entomopathogène. Alors que NprR agit comme régulateur transcriptionnel en présence de son peptide signal NprX, nous avons montré qu’en son absence il fixe la phosphotransférase Spo0F et agit comme inhibiteur de la sporulation.ComR régule quant à lui l’état de compétence qui permet aux streptocoques de prélever une molécule d’ADN libre dans leur environnement. Nous avons mis en évidence un mécanisme de régulation par le peptide ComS différent de celui des autres régulateurs transcriptionnels de la famille RNPP. Enfin, la spécificité du peptide pour son régulateur a également été caractérisée.Enfin, la comparaison de ces résultats avec les données structurales des autres RNPP nous a permis de mettre en évidence un mode de fixation du peptide conservé mais un mécanisme d’activation propre à chaque membre de la famille. Cette étude pose donc les bases structurales nécessaires à la mise au point rationnel d’inhibiteurs de RNPP pouvant répondre au problème de résistance aux antibiotiques dans la lutte contre la recrudescence des maladies infectieuses. / In the last century, infectious diseases had been successfully stopped in developed countries through the development of antibiotics. However, the emergence of bacteria resistant to the antimicrobial treatments brings this public health issue back at the forefront of concerns. In addition, increasing appearance of poly resistances is observed since the early 2000s, particularly in hospitals, and healthcare organizations have therefore called to a reduction of the use of antibiotics in order to stop the spread of resistances. Today, an effort should be made to the development of new therapeutic strategies.One of the approaches consists in targeting the communication system called quorum sensing used by the bacteria to regulate the expression of their genes in a concerted manner in order to adapt to their environment. Quorum sensing is based on the exchange of secreted signaling molecules called pheromones, which are detected by transmembrane or cytoplasmic receptors called quorum sensors.In pathogenic Gram positive bacteria, peptidic pheromones re-internalized by bacteria regulate the activity of quorum sensors called RNPP for the first identified members: Rap, NprR, PlcR and PrgX. These quorum sensing systems regulate behavior changes in the bacterial population. They are involved in important cellular processes such as sporulation, biofilm formation, competence and virulence.My project focused on the structure-function analysis of two members of the RNPP family: NprR, from the Bacillus cereus group, and ComR, a new member recently identified in Streptococci. By combining X-ray crystallography with biochemical and genetic approaches, we were able to elucidate the molecular mechanism that regulates the activity of these two quorum sensing systems.NprR is a bifunctional regulator which, in Bacillus thuringiensis, regulates the survival of the bacteria until sporulation in the cadaver of insects infected by this entomopathogene. While NprR acts as a transcriptional regulator in the presence of its peptide signal NprX, we showed that in its absence, NprR binds to the phosphotransferase Spo0F and acts as inhibitor of sporulation.ComR regulates the competence state that allows Streptococci to uptake free DNA molecules in their environment. We showed that the regulatory mechanism of its cognate peptide ComS is different from other transcriptional regulators of the RNPP family. The specificity of the peptide for its regulator was also characterized.Finally, a comparison of these results with the structural data of other RNPP allowed us to demonstrate a conserved peptide binding mode but an activation mechanism specific to each family member. This study thus brings the structural basis for the future rational design of RNPP inhibitors which may help respond to the antibiotic resistance problem in the fight against the resurgence of infectious diseases.

Yes / Here, we present the draft genome sequence of Aeromonas caviae strain L12, which shows quorum-sensing activity. The availability of this genome sequence is important to the research of the quorum-sensing regulatory system in this isolate. / High Impact Research Grants from the University of Malaya (A000001-50001; UM-MOHE HIR Grant UM C/625/1/HIR/MOHE/CHAN/14/1, H-50001-A000027)

The success of biocontrol bacteria is often restrained due to their low persistence in the rhizosphere and fluctuations in expression of antagonistic compounds. In the first part of this thesis the ability of the biocontrol agents (BCAs) Pseudomonas brassicacearum DF41 and Pseudomonas chlororaphis PA23 to resist grazing by the bacterivorous nematode Caenorhabditis elegans was investigated. We found that both BCAs are capable of killing the nematodes through exposure to toxic metabolites. We discovered that in addition to HCN, pyrrolnitrin (PRN) is a potent nematicide produced by PA23. Unique for a pseudomonad, DF41 was also found to kill the nematodes by forming biofilms on the nematode anterior, causing starvation. Biofilm formation was dependent upon the Gac two-component system and NaCl concentration of the media. Co-culturing these BCAs in the presence of nematodes increased expression of a number of genes associated with biocontrol. We observed elevated exoproduct formation, consistent with our gene expression analysis. The nematicidal activity exhibited by DF41 and PA23 towards C. elegans bodes well for their persistence in the environment. In the second part of this thesis the role of hydrogen cyanide (HCN) and the anaerobic regulator ANR in PA23 biocontrol was explored. An hcn mutant was created and in vitro antifungal (AF) assays revealed the involvement of HCN in Sclerotinia sclerotiorum suppression. Addition of glycine promoted both AF activity and HCN production. In addition, HCN was found to be positively regulated by quorum sensing (QS). Besides a phz box, an anr box was identified in the hcnA promoter, suggesting a role for ANR in regulating hcnA. An anr mutant was generated and phenotypic characterization revealed that ANR is a key regulator governing PA23 secondary metabolite production. Through gene expression analysis, ANR was shown to positively regulate phzI/phzR and PhzR negatively regulate anr. Furthermore, expressing anr in trans partially complemented the QS-deficient phenotype with respect to several biocontrol genes and exoproducts. Overall, the global regulator ANR is vital for PA23-mediated biocontrol and a significant overlap exists between the QS and ANR regulons. / October 2016

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

The protozoan parasites Trypanosoma brucei brucei, Trypanosoma congolense and Trypanosoma vivax cause Animal African Trypanosomiasis, a disease responsible for costly livestock pathology and economic losses in Africa. Each of these African trypanosomes are vector-borne and transmitted by the blood-feeding tsetse fly. Additional blood-feeding vectors can spread T. vivax, extending its range into South America. T. b. brucei infection of the mammalian host progresses in waves, with periodic clearance of antigenic variants. Accumulation of slender parasites in the blood is accompanied by accumulation of the density-sensing factor, SIF (stumpy induction factor). SIF drives differentiation from the proliferative slender form to the growth arrested stumpy form at the peak of parasitaemia. This differentiation step aids host survival, and the stumpy form is pre-adapted for continuation of development in the tsetse fly, ensuring transmissibility. Despite facing challenges comparable to T. b. brucei during their life cycle, T. congolense and T. vivax are not found to have morphologically distinguishable slender and stumpy forms. The growth control mechanisms used by these important veterinary pathogens have been investigated in this thesis. Particular focus has been placed on the conservation of quorum sensing pathways within the African Trypanosomes. The potential for cross-species communication has implications for co-infections. T. congolense was found to undergo growth arrest at peak parasitaemia, and transcriptomic changes occurring between ascending and peak parasitaemia were identified and comparisons made to T. b. brucei slender and stumpy transcriptomes. In an examination of the conservation of the SIF-responsive pathway, expression of a T. congolense orthologue was found to rescue stumpy formation in an otherwise SIF-resistant null mutant for the corresponding T. b. brucei gene. The capacity for cross-talk between density-sensing signals in different trypanosome species was tested using conditioned medium from T. congolense bloodstream form cultures. This could activate the expression of a stumpy specific reporter protein in T. b. brucei. A cell line deficient in a SIF-responsive gene showed resistance to the conditioned medium with a delay in reporter expression. These results highlight the unanticipated capacity for different trypanosome species to exhibit intra and inter specific cell-cell communication in the mammalian bloodstream, with possible consequences for their virulence, transmission and evolution.

Throughout existence, fungi and bacteria have long shared ecological niches and thus engage in numerous interactions to mutually enhance survival or antagonistically gain competitive advantages. Of importance to human health are those interactions that involve bacteria with the opportunistic fungi, Candida albicans. An important virulence factor of C. albicans is the ability to control morphology, which allows the transition between yeast, pseudohyphal, and hyphal phenotypes. Morphological control in C. albicans is governed by quorum sensing and the secreted autoregulatory molecule farnesol. Quorum sensing allows individual cells to sense the environment and respond as a group. Bacteria also use quorum sensing to communicate and control virulence. Despite their abundance in nature, very little is known about the interactions of C. albicans with bacteria on a genetic and molecular level. The objective of our research is to identify the genetic elements involved in C. albicans-bacterial interactions and characterize the genes that may participate in these relationships. To accomplish this, we screened a C. albicans mutant library for the ability to filament in the presence of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, where 3 typically, these three bacterial species inhibit C. albicans filamentation. We identified 836 C. albicans mutants that displayed a filamentous phenotype in the presence of bacteria. Collectively, 295 of these mutants filamented in the presence of all 3 bacterial species. Candidates were subsequently sequenced to identify the location of the mutation and the affected genetic element. CDR4, a putative ABC transporter, and ALS6, a putative adhesion, were further characterized for their specific involvement in Candida-bacterial interactions. Using a filamentation assay, cdr4 and als6 deletion strains demonstrated a decreased response to the inhibitory effects of farnesol, as well as bacterial molecules known to inhibit the production of hyphal-filaments. Additionally, the ability of cdr4 and als6 deletion strains to attach and form biofilms was significantly enhanced even in the presence of farnesol and bacterial inhibitors. The results of this study contribute to the body of knowledge involving polymicrobial interactions and these findings may lead to new antifungal targets for therapeutic interventions.

The objectives of this work are: 1) to agarose-stabilize fragile biofilms for quantitative structure analysis; 2) to understand the influences of LuxS on biofilm formation; 3) to improve teacher quality by preparing Georgia’s middle school science teachers to integrate inquiry-based, hands-on research modules in the classroom. Quantitative digital image analysis demonstrated the effectiveness of the agarose stabilization technique for generating reproducible measurements of three dimensional biofilm structure. The described method will also benefit researchers who transport their flow cell-cultivated biofilms to a core facility for imaging. AI-2-dependent and independent effects of LuxS on biofilm-related phenotypes were revealed, suggesting that LuxS is a versatile enzyme, possessing multiple functions in E. coli ecology that could assist E. coli in adapting to diverse conditions. Overall, the work presented in this dissertation supported the concept that quorum sensing, biofilm formation, and cell adhesion are largely related. Additionally, through this project, teachers enhanced content knowledge and confidence levels, mastered innovative teaching strategies and integrated inquiry-based, inter-disciplinary, hands-on activities in the classroom. As a result, student learning was enhanced, and teachers are better equipped to give Georgia’s students a solid foundation in the sciences.

This dissertation describes the development and application of an approach for creating multiphoton crosslinked protein microchambers to characterize bacterial group behaviors in small populations (~10¹ - 10⁵ cells). Porous protein cavities of desired size and geometry are made with sub-micrometer three-dimensional (3D) resolution using a dynamic mask-based multiphoton lithography (MPL) technique previously developed in the Shear Group. One aspect of this dissertation focuses on basic characterizations of properties of these materials key to their utility in studying entrapped bacteria. Studies are presented on the mass transport across microcavity walls (important for growth and signaling), and the temperature- and light-induced volume response (used to open/close microchamber apertures for cell entry/exit). Fabrication parameters are optimized to trap and manipulate small populations under in vitro conditions that are relevant to in vivo environments. The ability to culture bacteria at physiologic growth rates within protein microstructures has provided a unique platform to study the group behaviors of quorum sensing (QS) and antibiotic resistance in biologically relevant population sizes, a platform I have exploited to study group behaviors in the opportunistic pathogen, Pseudomonas aeruginosa. This work presents the first experimental evidence supporting the efficiency sensing QS model by showing that QS-dependent gene expression is affected by both the population size and density, as well the external flow rate in the surrounding environment. The onset of antibiotic resistance is observed in as few as ~150 P. aeruginosa cells, and is shown to increase with cell density. Lastly, the development of a gelatin-based MPL approach that is demonstrated in situ to create confined populations of non-motile cells, free-floating 3D cultures, nested colonies, and spatially patterned polymicrobial communities of P. aeruginosa and Staphylococcus aureus. / text