Orientador: Adriano Sakai Okamoto / Resumo: Uma das principias doenças transmitida por alimento é a salmonelose, causada pela bactéria Salmonella spp., ela está presente em vários aviários, e com o aumento da produção avícola, se disseminou nos aviários, tornando-se um problema pertinente e de grande importância na saúde pública e animal. Com isso medidas de controle foram instituídas, entre elas o uso de antimicrobiano, causando diversas discussões, principalmente em relação a resistência bacteriana á antimicrobianos utilizados na medicina humana, onde medidas alternativas estão sendo desenvolvidas para evitar o uso dos antimicrobianos. Dessa forma o objetivo do projeto foi avaliar a capacidade dos Lactobacillus spp., isolados na inibição da Salmonella Heidelberg (SH), pelo mecanismo de quorum sensing in vitro e in vivo. Foram colhidos de suabes cloacal de frangos de corte que passaram por provas fenotípica e molecular. Esses isolados foram submetidos ao teste de inibição em placa para avaliar a capacidade de inibição da SH. Em seguida foram produzidos os filtrados, a partir dos Lactobacillus sp. (ATCC), separadamente, com contato com a SH. Esses isolados foram cultivados com os filtrados, separadamente e em seguida submetidos novamente ao teste de inibição em placa. Dessa maneira, foi possível medir e comparar os halos de inibição, para a verificação da ocorrência da comunicação bacteriana entre os Lactobacillus sp. (ATCC) e os isolados. Foi realizada a análise estatística selecionando um Lactobacillus spp. isolado d... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: One of the main food-borne diseases is salmonellosis, caused by the bacterium Salmonella spp., It is present in several aviaries, and with the increase of poultry production, it has spread in aviaries, becoming a relevant problem and of great importance in health public and animal. With this, control measures were instituted, including the use of antimicrobial, causing several discussions, mainly regarding bacterial resistance to antimicrobials used in human medicine, where alternative measures are being developed to avoid the use of antimicrobials. Thus, the objective of the project was to evaluate the ability of Lactobacillus spp., isolated in the inhibition of Salmonella Heidelberg (SH), by in vitro and in vivo quorum sensing mechanism. They were harvested from cloacal swabs of chickens that underwent phenotypic and molecular tests. These isolates were subjected to plaque inhibition test to assess the inhibitory capacity of SH. Filtrates were then produced from Lactobacillus sp. (ATCC), separately, with SH. These isolates were cultured with the filtrates separately and then replate inhibited. In this way, it was possible to measure and compare inhibition halos to verify the occurrence of bacterial communication between Lactobacillus sp. (ATCC) and the isolates. Statistical analysis was performed by selecting a Lactobacillus spp. isolated from chickens and a filtrate which obtained better results in the in vitro test. The in vivo test was divided into four treatments, Lacto... (Complete abstract click electronic access below) / Mestre

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Les bactéries utilisent la communication intercellulaire pour se coordonner afin de réguler des processus bactériens majeurs comme la virulence, la sporulation, la compétence ou la formation de biofilms en fonction de la densité cellulaire. Ce processus repose sur la sécrétion de petites molécules signal appelées phéromones. Chez les bactéries à gram positif, ces phéromones sont de petits peptides qui peuvent être, soit reconnus au niveau de la membrane externe par des systèmes à deux composants dits systèmes indirects, soit être re-internalisés afin de se fixer directement sur un régulateur transcriptionnel cytoplasmique. Dans la recherche de nouveaux agents antimicrobiens, le potentiel thérapeutique d’inhibiteurs ciblant les systèmes de communication intercellulaire est bien étudié chez les bactéries à gram négatif qui utilisent des homosérine lactones comme phéromones. Quelques études s’intéressent aux systèmes indirects de bactéries pathogènes à gram positif mais le potentiel des systèmes directs, plus récemment identifiés, n’a pas encore été explorés.Les récepteurs cytoplasmiques directement régulés par des peptides signal re-internalisés forment la famille des RNPP. Ces récepteurs sont caractérisés par un domaine de fixation du peptide composé d’une répétition de motifs en hélice  appelés TetratricoPeptide Repeats (TPR). La plupart de ces récepteurs sont des régulateurs transcriptionnels contenant un domaine N-terminal de fixation de l’ADN de type Hélice-Tour-Hélice (HTH). Les études précédentes ont montré que, malgré une structure conservée, les modes de régulation des différents membres de la famille RNPP suivent des mécanismes moléculaires distincts. L’un de ces systèmes directs le mieux caractérisé est le système ComRS qui régule la compétence chez les streptocoques. La compétence permet aux bactéries d’internaliser des fragments d’ADN exogènes pour l’acquisition de nouveaux phénotypes tels que la résistance aux antibiotiques ou la virulence. Dans le groupe salivarius, il a été montré que les récepteurs ComR de deux espèces très proches, S. thermophilus et S. vestibularis, ne sont pas capables de coordonner leur état de compétence par échange de leurs peptides ComS respectifs.L'objectif de ma thèse a été d'étudier les déterminants structuraux de la spécificité du système ComRS. J’ai produit, purifié et cristallisé le récepteur ComR de S. vestibularis afin d’en déterminer la structure cristalline, seul et en présence de son peptide signal ComS. La comparaison de ces structures avec celles précédemment résolues chez S. thermophilus, conjointement à une étude fonctionnelle par mutagénèse dirigée réalisée chez nos collaborateurs (P. Hols, UCLouvain, Belgique), a permis d’aller plus loin dans la compréhension du mécanisme de régulation de ComR mais aussi d’identifier les résidus responsables de la spécificité du système ComRS. En parallèle, j’ai également initié la caractérisation structurale de deux paralogues de ComR chez S. salivarius, ScuR et SarF, qui ne sont pas activés par ComS et ne reconnaissent pas les mêmes cibles ADN que ComR malgré des séquences très conservées. Une analyse par SEC-MALS et SAXS m’a permis de montrer que ScuR semble suivre un mécanisme similaire à celui de ComR alors que SarF se comporte différemment en solution. J’ai proposé un modèle par homologie pour ScuR et cristallisé SarF. La résolution de sa structure est en cours. Cette étude permet donc de mieux comprendre la régulation de la compétence chez les streptocoques et ouvre la voie à d’éventuelles applications biotechnologiques ou biomédicales. / Bacteria use intercellular communication to coordinate and regulate major bacterial processes such as virulence, sporulation, competence or biofilm formation, as a function of cell density. This process relies on the secretion of small signal molecules called pheromones. In gram-positive bacteria, these pheromones are small peptides that can be either recognized at the outer membrane by two-component systems called indirect systems, or can be re-internalized to directly interact with a cytoplasmic transcriptional regulator. In the search for new antimicrobial agents, the therapeutic potential of inhibitors targeting intercellular communication systems is well studied in gram-negative bacteria that use homoserine lactones as pheromones. Some studies focus on the indirect systems of gram-positive pathogenic bacteria, but the potential of the more recently identified direct systems has not yet been explored.The cytoplasmic receptors directly regulated by re-internalized signal peptides form the RNPP family. These receptors are characterized by a peptide binding domain consisting of repeats of  helical motifs called TetratricoPeptide Repeats (TPR). Most of these receptors are transcriptional regulators containing an N-terminal DNA binding domain of the helix-turn-helix (HTH) type. Previous studies have shown that, despite a conserved structure, the modes of regulation of the different members of the RNPP family follow distinct molecular mechanisms. One of the best characterized direct systems is the ComRS system that regulates competence in streptococci. Competence allows bacteria to internalize exogenous DNA fragments for the acquisition of new phenotypes such as antibiotic resistance or virulence. In the salivarius group, it has been shown that the ComR receptors of two closely related species, S. thermophilus and S. vestibularis, are not able to coordinate their state of competence by exchange of their respective ComS peptides.The aim of my thesis was to study the structural determinants of the specificity of the ComRS system. I produced, purified and crystallized the ComR receptor from S. vestibularis in order to determine its crystal structure, alone and in the presence of its ComS signal peptide. The comparison of these structures with those previously solved with ComR from S. thermophilus, together with a functional study by directed mutagenesis performed by our collaborators (P. Hols, UCLouvain, Belgium), allowed us to go further in the understanding of the regulatory mechanism of ComR but also to identify residues responsible for the specificity of the ComRS system. In parallel, I also initiated the structural characterization of two ComR paralogs from S. salivarius, ScuR and SarF, which are not activated by ComS and do not recognize the same DNA targets as ComR despite highly conserved sequences. SEC-MALS and SAXS analyses allowed me to show that ScuR seems to follow a mechanism similar to that of ComR whereas SarF behaves differently in solution. I proposed a homology model for ScuR and crystallized SarF. The resolution of its structure is in progress. This study therefore provides a better understanding of the regulation of competence in streptococci and opens the way to potential biotechnological or biomedical applications.