L'inflammasome est complexe protéique intracellulaire de l'immunité innée permettant la reconnaissance de pathogènes intracellulaires. NLRC4, un Nod-like récepteur permettant l'activation de l'inflammasome est impliqué dans la reconnaissance du flagelle ainsi que du système de sécrétion de type 3 (SST3) de Pseudomonas aeruginosa, une bactérie majoritairement extracellulaire. Nous avons donc déterminer l'impact de la reconnaissance de P. aeruginosa par le NLRC4-inflammasome in vivo dans un modèle murin de pneumonie aiguë. De façon surprenante, l'activation du NLRC4-inflammasome par le SST3 de P. aeruginosa contribue à diminuer la survie de l'hôte en diminuant la clairance bactérienne pulmonaire et en augmentant la lésion pulmonaire induite. En effet, la perte de l'activation de l'inflammasome chez les souris NLRC4/- permet d'une part, une réponse précoce méfiée par l'IL-17A. Cette réponse dépendant de l'IL-17A conduit à une expression majeure de peptides antimicrobiens par l'épithélium pulmonaire et diminue la lésion pulmonaire en diminuant le recrutement des cellules immunitaires inflammatoires. L'administration d'IL-18 recombinante murine ou l'inhibition de cette voie par un anticorps anti-IL-17A inhibe cette réponse IL-17A dépendante. Ces résultats mettent en évidence un nouveau rôle du SST3 de P. aeruginosa, qui en plus de son effet cytotoxique et de la translocation d'exotoxines, permet d'activer l'inflammasome pour échapper à la réponse immunitaire innée de l'hôte en inhibant une voie IL-17 dépendante. / The inflammasome is thought to function as a cytosolic surveillance system against intracellular pathogens. However, we report that Pseudomonas aeruginosa, an extracellular pathogen responsible for acute lung infection, relies upon inflammasome activation to persist and worsen disease. Specifically, we show that loss of either NLRC4 expression or type-3 secretion system (T3SS)-driven activation of NLRC4, surprisingly, resulted in a robust Th-17-like immune response that enhanced bacterial clearance and attenuated virulence independently of exotoxins. This Th-17-like response correlated with marked upregulation of antimicrobial peptides and was suppressed by either neutralization of IL-17A or exogenous IL-18 administration in vivo. Together, these results unveil an adaptation mechanism through which the problem pathogen manages to evade Th17-mediated immunity and invade the lung, providing a potential mechanism for infectious complications of anti-IL17 therapy in inflammatory diseases. The T3SS exploitation of NLRC4-coupled inflammasome response may therefore represent a novel gene-for-gene model of pathogen evolution alongside host immunity.

La résistance aux antibiotiques est un problème majeur en santé publique qui mène à développer de nouvelles stratégies thérapeutiques, notamment en ciblant des facteurs de virulence bactériens. MgtC est un facteur de virulence impliqué dans la survie intra-macrophagique chez plusieurs pathogènes intracellulaires. La protéine MgtC est également présente chez le pathogène extracellulaire Pseudomonas aeruginosa. De plus, un peptide MgtR a été identifié comme étant un antagoniste naturel potentiel de MgtC. Pour étudier le rôle de MgtC dans la virulence des mycobactéries, j'ai analysé un mutant mgtC de Mycobacterium marinum dans les modèles d'infection d'embryons de danio et de macrophages en culture. Cela a permis de mettre en évidence un nouveau rôle de MgtC au niveau de l'étape de phagocytose qui est augmentée avec le mutant mgtC. Chez P. aeruginosa, nous avons montré qu'un mutant mgtC est atténué dans l'embryon de danio et présente une sensibilité accrue à l'action bactéricide du macrophage. Par ailleurs, j'ai montré que les gènes mgtC de M. marinum et de P. aeruginosa sont régulés par le magnésium. De plus, l'expression de mgtC de P. aeruginosa est fortement induite dans les macrophages. Enfin, concernant les propriétés antagonistes de MgtR, des souches de Mycobacterium bovis BCG ou de P. aeruginosa exprimants mgtR semblent se comporter de la même manière que les mutants mgtC, suggérant des propriétés anti-virulence prometteuses pour MgtR et confirmant le choix de MgtC en tant que cible dans le cadre d'une stratégie anti-virulence. / Antibiotic resistance is a major problem in public health, which leads to develop new therapeutics strategies, including strategies targeting bacterial virulence factors. MgtC is a virulence factor involved in intramacrophage survival in several intracellular pathogens. MgtC protein is also present in extracellular pathogen Pseudomonas aeruginosa. Moreover, a peptide MgtR has been identified as a potential and natural antagonist of MgtC.To study the role of MgtC in mycobacterial virulence, I have analysed a Mycobacterium marinum mgtC mutant in zebrafish embryo and macrophage infection models. This approach allowed us to uncover a new role of MgtC in phagocytosis, which is increased with mgtC mutant. In P. aeruginosa, we have shown that a mgtC mutant is attenuated in zebrafish embryos. In ex vivo experiments, mgtC mutant is more sensitive to macrophage killing. In parallel, I have shown that M. marinum and P. aeruginosa mgtC genes are regulated by magnesium. In addition, expression of P. aeruginosa mgtC is highly induced in macrophages.Finally, regarding MgtR antagonistic properties, Mycobacterium bovis BCG or P. aeruginosa strains expressing mgtR appear to mimic the behaviour of mgtC mutants, suggesting promising anti-virulence properties for MgtR and supporting the choice of MgtC as a suitable target of an anti-virulence strategy.

Les eaux minérales naturelles se distinguent de l’eau potable par leur contenu en sels minéraux et en éléments traces. Leur utilisation à des fins thérapeutiques s’effectue sous contrôle médical dans des établissements thermaux. La gestion des réseaux de distribution ainsi que la qualité microbiologique de l’eau font l’objet de réglementations. Malgré la mise en place de procédures spécifiques, les établissements thermaux sont régulièrement confrontés à des contaminations microbiologiques majoritairement dues à P. aeruginosa. Ce pathogène opportuniste possède d’importantes capacités d’adaptation, de résistance et de persistance dans l’environnement, notamment sous forme de biofilms. L’objectif de ces travaux de thèse est d’apporter des éléments de compréhension sur le comportement de P. aeruginosa dans les réseaux de distribution d’eau minérale naturelle et de valider l’efficacité de produits de traitement. L’influence de la composition minérale de 3 eaux sur les capacités à former du biofilm de 9 souches de P. aeruginosa d’origines diverses a pu être démontrée. Il apparait que les quantités de biofilms produites au bout de 24 h sont moins importantes pour la minéralisation forte. La modulation de la production de facteurs de virulence en fonction de la qualité de l’eau est corrélée à l’action spécifique de certains ions et à la biodisponibilité du fer. En conditions de minéralisation forte il a été montré que les matériaux organiques favorisent la formation de biofilm de P. aeruginosa tandis que les matériaux métalliques ont tendance à la défavoriser. L’efficacité d’une séquence de traitement de postes de soins a pu être validée sur un modèle de biofilm âgé de 24 h et sur tous les matériaux. / Natural mineral waters are distinguished from drinking water bytheir content in minerals and trace elements. Their use for therapeuticpurposes under medical control is performed in spas. Management ofdistribution networks and the microbiological quality of water are subjectto regulations. Despite the establishment of specific procedures, spas areregularly confronted with microbiological contamination mainly due to P.aeruginosa. This opportunistic pathogen has substantial adaptive capacity,resistance and persistence in the environment, under biofilm.The aim of this thesis work is to bring understanding on the P.aeruginosa behaviour in the natural mineral water distribution networksand validate the effectiveness of treatment products.The influence of the mineral composition of 3 waters on biofilmformation capacity of 9 P. aeruginosa strains of various origins could bedemonstrated. It appears that the amount of biofilm produced after 24 hare less important for the strong mineralization. Modulation of theproduction of virulence factors depending on the water quality iscorrelated to the specific action of certain ions and iron bioavailability. Inhigh mineralization conditions it has been shown that organic materialspromote biofilm formation of P. aeruginosa while metallic materials tendto disadvantage it. The efficiency of a treatment sequence of a patient pointof use has been validated on the 24 h biofilm model and on all materials.

Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.

A proteobactéria Pseudomonas aeruginosa é um patógeno oportunista em humanos, sendo associado a queimaduras e infecções pulmonares crônicas em pacientes com fibrose cística. Essas infecções são difíceis de erradicar devido à resistência intrínseca de P. aeruginosa a antibióticos e à formação de biofilmes. Essa bactéria é altamente capaz de adaptar ao ambiente, tem um metabolismo versátil e pode direcionar a expressão de genes por vários fatores sigma alternativos. Estes são subunidades para transcrição de conjuntos específicos de genes em bactérias e interagem com o cerne da RNA polimerase, levando ao reconhecimento do promotor e início da transcrição. Os fatores sigma alternativos permitem que bactérias redirecionem a sua expressão genética. Um grupo de fatores sigma alternativos é o grupo dos fatores sigma de função extracitoplasmática (ECF) que são envolvidos principalmente em funções do envelope celular. Esse trabalho teve como objetivo caracterizar dois fatores sigma ECF de função desconhecida, PA14_21550 e PA14_46810. A linhagem mutante Δ21550 foi analisada quanto a sua sobrevivência a diferentes estresses, observando-se que é mais resistente ao choque de 45°C que a linhagem selvagem. Esse fator sigma não é essencial para crescimento da bactéria em meio LB e meio mínimo M63 acrescido de glicose ou succinato. Além disso, observou-se que a superexpressão desse fator sigma aumenta a expressão da proteína hipotética PA14_30100, usando-se uma abordagem proteômica. O mutante de transposon para o fator sigma PA14_46810 apresenta melhor crescimento que a bactéria selvagem em meio M63 acrescido de glicose. Essa linhagem mostrou mesmo fenótipo para biofilme e formação de exopolissacarídeo que a bactéria selvagem. Ademais, foi realizada análise de transcritoma por RNA-Seq com a superexpressão do fator sigma PA14_46810 na linhagem selvagem. Na linhagem de superexpressão Observou-se que ocorre indução de genes envolvidos com a desnitrificação, transporte de moléculas e metabolismo de uma maneira geral, em relação à linhagem controle. Por outro lado, o excesso de PA14_46810 reprime principalmente genes envolvidos com a tradução de proteínas e síntese de espermidina. Este trabalho, portanto, trouxe novas informações sobre as funções de diferentes fatores sigma ECF de P. aeruginosa, contribuindo assim para um maior entendimento da fisiologia desta bactéria e sua adaptação a diferentes condições. / The proteobacterium Pseudomonas aeruginosa is an opportunistic pathogen in humans, and it is associated to chronic pulmonary infections in patients with cystic fibrosis and burn wounds. These infections are difficult to eradicate due to P. aeruginosa intrinsic resistance to antibiotics and formation of biofilms, which allow the bacteria to adhere to biotic and abiotic surfaces. This bacterium is highly adaptaptable to the environment has a versatile metabolism and can direct the expression of genes by several alternative sigma factors. The sigma factors bind to the RNA polymerase core, providing recognition to promoter and transcription initiation. Therefore, the alternative sigma factors can redirect bacterial genetic expression by recognizing specific promoters. One subfamily of alternative sigma factors is the extracytoplasmic function (ECF) sigma factors, involved mostly in cell envelope functions. The aim of this work was characterize two ECF sigma factors with unknown function in P. aeruginosa, PA14_21550 and PA14_46810. The strain Δ21550 was analyzed for its survival in different stress conditions and it is more resistant in heat shock conditions at 45°C than the wild type strain. It was also observed that PA14_21550 sigma factor is not essential for bacterial growth in LB and M63 minimal medium added with glucose or succinate as the carbon source. Furthermore, overexpression of this sigma factor increases the expression of hypothetical protein PA14_30100, as verified by a proteomic approach. A strain insertionally inactivated in the PA14_46810 gene has better growth than the wild type strain in M63 added with glucose and the same phenotype regarding to biofilm formation and exopolysaccharide production as the wild type strain. Moreover, transcriptome analysis was carried out by RNA-Seq with overexpression of the PA14_46810 sigma factor in the wild type strain. Induction of genes involved in denitrification, transport of molecules and energetic metabolism in relation to the control strain was observed. On the other hand, excess of PA14_46810 represses genes involved in protein translation and spermidine synthesis. This work, therefore, brought new information about the functions of two ECF sigma of P. aeruginosa, thus contributing to a greater understanding of the physiology of this bacterium and its adaptation to different conditions.

A infecção crônica das vias aéreas por Pseudomonas aeruginosa (PA) é a principal causa de morbidade e mortalidade em pacientes com fibrose cística (FC), devido à contínua degradação do tecido pulmonar, que leva ao declínio da função pulmonar, gerada pela infecção e pelo processo inflamatório. O objetivo do presente estudo foi analisar características genéticas de PA que levam à sua adaptação às vias aéreas destes pacientes com infecção pulmonar crônica, atendidos no Centro de Referência em FC do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP e relacionar com resultados de tipagem molecular, resistência a antibióticos, cronicidade e dados clínicos dos pacientes em acompanhamento clínico no período de julho/2011 a abril/2014. As características genéticas dos isolados investigados englobam pesquisa de 18 genes de virulência e genes do sistema quorum sensing (genes lasR e rhlR), associação entre mutações e conversão para fenótipo mucoide (operon algTmucABD) e caracterização de linhagens hipermutantes (genes mutS e mutL). A identificação de P. aeruginosa foi realizada por PCR e MALDI-TOF, que mostraram alta concordância. Foram considerados os dados clínicos dos pacientes: índice de massa corpórea, escore de Shwachman, medidas de capacidade vital forçada e volume expiratório forçado no primeiro segundo. A porcentagem de pacientes com infecção pulmonar crônica por PA observada foi similar aos dados disponíveis na literatura, entretanto, a alta incidência em pacientes jovens foi preocupante. O perfil de macrorrestrição do DNA genômico por PFGE se mostrou útil para definição de colonização crônica/intermitente em associação com critérios clínicos e, juntamente com a detecção de mutações nos genes mucA e mucD confirmaram transmissão interpacientes. Foi observada alta ocorrência dos genes de fatores de virulência pesquisados para grande maioria dos isolados de pacientes crônicos. A resistência aos antibióticos pesquisados dos isolados de P. aeruginosa foi baixa e está de acordo com a literatura nacional e internacional e com a antibioticoterapia adotada no hospital. Não foi observada resistência aos carbapenêmicos e às fluoroquinolonas devido à presença de genes de resistência plasmideais. As mutações no gene mucA foram o principal mecanismo de conversão para o fenótipo mucoide e o fenótipo revertente não-mucoide ocorreu principalmente por mutações no gene algT. Foram detectadas novas mutações nos genes mutS e mutL que também suportam a ideia que hipermutação em PA está associada com mutações do sistema mismatch de reparo do DNA. O sistema quorum sensing dos isolados estudados está parcialmente prejudicado devido às várias mutações no gene lasR, mas todos conservam o gene rhlR intacto, que sustenta alguma atividade quorum sensing envolvida na produção de fatores de virulência importantes. Pacientes com infecção pulmonar crônica por PA com isolamento de outros bacilos gram-negativos não-fermentadores apresentaram maior alteração da função pulmonar quando comparados com pacientes com infecção pulmonar crônica por PA com ou sem isolamento de Staphylococcus aureus. As alterações presentes no operon algTmucABD, quorum sensing e hipermutabilidade contribuem para a cronicidade dos pacientes com FC em relação à infecção por P. aeruginosa. / The chronic airway infection by P. aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients, due to continuous degradation of the pulmonary tissue. This leads to decline in lung function, which is generated by the related infection and inflammation. The aim of this study was to analyze genetic characteristics associated with the adaptation of PA to the airways of patients with chronic pulmonary infection attended at the CF Reference Center from the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP; and to correlate these findings with the results of molecular typing, antibiotic resistance, chronicity and clinical data of patients in clinical follow-up from July/2011 to April/2014. The genetic characteristics of isolates investigated includes the research of 18 virulence genes and the quorum sensing system genes (lasR and rhlR genes), association between mutations and conversion to the mucoid phenotype (algTmucABD operon), and characterization of hypermutable strains (mutS and mutL genes). Identification of PA was performed by PCR and MALDI-TOF, which showed a high correlation. The patients\' clinical data considered were: body mass index, Shwachman score, forced vital capacity measures and forced expiratory volume in one second. The percentage of patients with chronic PA infection observed was similar to the data available in the literature; however, a worrying high incidence in young patients was noticed. The macrorestriction profile of genomic DNA by PFGE proved to be useful to define chronic/intermittent colonization in association with clinical criteria and it confirmed interpatient transmission, in combination with the detection of mutations in the mucA and mucD genes. High occurrence of virulence genes was detected for the vast majority of isolates from chronic CF patients. Antibiotic resistance for PA isolates was low and is in accordance with national and international literature and antibiotic therapy adopted in the hospital. There was no resistance to carbapenems and fluoroquinolones by the presence of plasmid mediated resistance genes. Mutations in the mucA gene were the main mechanism to conversion to mucoidy, and the non-mucoid revertants occurred mainly by mutations in the algT gene. New mutations in mutS and mutL genes were detected, which support the idea that hypermutation in PA is associated with mutations in the DNA mismatch repair system. The quorum sensing system of the isolates is partially damaged due to several mutations in the lasR gene, but all isolates maintain an intact rhlR gene, which holds some quorum sensing activity with production of important virulence factors. Patients with chronic PA infection with isolation of other non-fermenting gram-negative rods had greater change in lung function compared with patients with chronic PA infection with or without isolation of Staphylococcus aureus. The changes presented in the algTmucABD operon, quorum sensing and hypermutability contribute to the chronicity of CF patients in relation to infection by P. aeruginosa.

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las. / Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent.

Pseudomonas Aeruginosa est un pathogène opportuniste qui adapte son comportement aux molécules présentes dans son milieu. Il augmente en virulence lorsqu’il détecte des peptides natriurétiques dans son environnement. L’isatine est une molécule qui bloque cet effet. Pour mieux comprendre sur quel récepteur l’isatine agit, la conception d’un biocapteur a été menée. Un biocapteur est un outil alliant un bioélément, réagissant spécifiquement avec une cible biologique, et un support physique permettant la transduction du signal pour le rendre mesurable. Les travaux de thèse suivants décrivent la préparation d’un transducteur polymérique, le copolymère d’oléfines cycliques (COC), par greffage de sels de diazonium en surface. La modification de l’isatine par couplage pallado-catalysé compose la partie principale des travaux de synthèse organique effectués. Les méthodes de couplages peptidiques en surface et techniques d’accroche des isatines modifiées sur les surfaces des transducteurs (or ou COC) sont également décrites dans ce manuscrit. Pour finir, l’évaluation des effets biologiques des isatines modifiées et des biocapteurs conçus a été effectuée sur Pseudomonas Aeruginosa et sur d’autres bactéries. / Pseudomonas Aeruginosa is an opportunistic pathogen which can adapt its behavior to the present molecules in its surrounding. It increases in virulence when it detects natriuretics peptides in its environment. Isatin is a molecule which can block this effect. To determine on which receptor isatin acts on, the conception of a biosensor have been conducted. A biosensor is a tool combining a bioelement, reacting specifically with a biological target, and a physical support allowing the transduction of the signal to make it measurable. The following thesis describes the preparation of a polymeric transducer, the cyclic olefin copolymer (COC), by diazonium salts surface grafting. Isatin modification by palladium-catalysed coupling reaction represents the major part of the organic synthesis carried out. Surface peptide coupling methods and techniques to link the modified isatins to the surfaces of transducers (gold or COC) are also described in this dissertation. To finish, biological effects of the modified isatins and designed biosensors have been evaluated on Pseudomonas Aeruginosa and others bacteria.

Les bactériophages sont des virus qui injectent leur génome dans une bactérie après fixation à des récepteurs sur la surface de celle-ci, puis effectuent un cycle de multiplication de leur ADN, la synthèse des protéines de structure, l’encapsidation du génome viral et la lyse de la bactérie. Les phages virulents réalisent uniquement des cycles lytiques alors que les phages tempérés peuvent également intégrer leur génome dans celui de la bactérie, donnant ainsi naissance à une bactérie dite lysogène. Les phages peuvent parfois être maintenus dans la bactérie sans effectuer un cycle lytique ni s’intégrer, dans un état encore peu compris, connu sous le nom de pseudolysogénie. Pseudomonas aeruginosa est une espèce bactérienne présente dans l’environnement et associée à de nombreux hôtes, végétaux et animaux. Elle est responsable de graves infections nosocomiales et on observe de plus en plus souvent des souches multirésistantes aux antibiotiques, ayant une grande capacité à former des biofilms, et en conséquence très difficiles à éradiquer. Il faut donc absolument trouver des approches thérapeutiques nouvelles telle que la phagothérapie. De nombreuses données cliniques obtenues dans les pays de l’est de l’Europe et en Russie attestent de l’efficacité et de l’innocuité de la phagothérapie, mais il reste des incertitudes en particulier concernant la nature et la fréquence des résistances naturelles. Notre projet vise à évaluer le potentiel thérapeutique des phages et à mieux comprendre la dynamique de leur interaction avec leur hôte. Nous avons étudié les mécanismes de résistance mis en place par la souche de P. aeruginosa, PAO1, à quatre bactériophages virulents appartenant à des genres différents: deux podovirus, Ab05 (ФKMV-like) et Ab09 (LIT1-like), et deux myovirus, Ab27 (PB1-like) et Ab17 (KPP10-like), tous isolés par notre laboratoire. Des infections simples ou multiples de PAO1 ont été réalisées, et une collection de variants résistants aux phages a été isolée et étudiée. La fréquence des bactéries résistantes était de 10⁻⁵ pour l'infection par un phage seul et 10⁻⁶ pour les infections par des combinaisons de deux ou quatre phages. Le phénotype et la mobilité des variants résistants étaient fréquemment affectés.Le génome de 27 variants a été entièrement séquencé par la technologie Illumina, et la comparaison avec le génome de la souche PAO1 a permis l'identification de mutations ponctuelles ou de petites indels. Quatre variants supplémentaires ont été caractérisés par une approche «gène candidat». Des mutations affectant 14 gènes différents et 1 région régulatrice ont été observées. Les gènes mutés codent pour des protéines impliquées dans la biosynthèse des pili de type IV (T4P) et des lipopolysacharides (LPS), très fréquemment utilisés comme récepteurs par les phages. Des mutations de la synthèse des alginates ont été également observées. La moitié des variants possède des mutations de variation de phase qui se sont révélées être instables. Par contre, les gènes impliqués dans la biosynthèse du T4P montrent des délétions stables. Nous avons aussi observé que la pseudolysogénie est une conséquence fréquente de l'infection par ces phages virulents et que la sélection de mutants (très souvent des mutants doubles) est favorisée par la production continue de phages par les pseudolysogènes. La présence d'ADN de phage libre a été observée en liaison avec l'exclusion de surinfection. Pour conclure, si les phages sélectionnent des bactéries résistantes possédant des altérations dans les gènes impliqués dans la biogenèse ou la régulation des déterminants de la virulence, celle-ci sera probablement modifiée, d'une manière bénéfique ou préjudiciable, ce qui reste à étudier. L'utilisation du cocktail par rapport à l’infection simple, ne réduit pas de manière significative la fréquence de la résistance aux phages et en outre, nous montrons que la pseudolysogénie est un acteur majeur de la sélection de mutations. / Bacteriophages are obligate parasites of bacteria that can be defined as virulent or temperate according to their lifestyle: virulent phages perform a lytic cycle by injecting their genome in the bacterial cell and immediately multiply. Temperate phages, instead, can either perform a lytic, or a lysogenic cycle by integrating their genome into the bacterial chromosome and persisting in a dormant state until the lytic cycle is resumed. The viral genome can also be maintained in the bacterial cell in an episomal form for an undetermined period of time in a stage known as pseudolysogeny. P. aeruginosa, a bacterium commonly found in the environment and in association with many hosts including plants and animals, is responsible for severe nosocomial infections. A proportion of clinical strains are multidrug-resistant, possessing a high ability to form biofilms which are very difficult to eradicate with conventional treatments. It is therefore essential to find new therapeutic approaches, such as phage therapy. Numerous clinical data obtained in Eastern Europe and Russia attest the effectiveness and safety of phage therapy. However, there remain uncertainties related to their therapeutic use and particularly the high frequency of natural resistance. Our project aimed to better understand the dynamic of phage/bacteria interactions by studying the resistance mechanisms acting in the reference strain P. aeruginosa PAO1, against virulent phages. Infections were performed by combining phages belonging to four different genera: Ab05, a ФKMV-like podovirus, Ab09, a LIT1-like podovirus, Ab27, a PB1-like myovirus and Ab17, a KPP10-like myovirus, all isolated in our laboratory. Single or multiple infections of P. aeruginosa PAO1 were performed, and a collection of phage-resistant variants was isolated and analysed. The frequency of phage-resistant variants selection was 10⁻⁵ for single phage infection, and 10⁻⁶ for infections with cocktails of two or four phages. The phenotype and mobility of the variants was often affected, as compared to the parental strain. The genome of 27 variants was entirely sequenced by Illumina technology in order to identify mutations responsible for the resistance. Other variants were analysed by a candidate gene approach. We identified point mutations or small indels: in total, 27 independent mutations affected 14 genes and 1 regulatory region. The affected genes encode proteins involved in biosynthesis of type IV pili (T4P) and lipopolysaccharide (LPS), frequently used as receptors by the phages. Other mutations were observed in genes necessary for alginate production. Of interest, we found that half of the variants with mutations in genes involved in LPS biosynthesis possessed unstable phase variation mutations, responsible for translation frameshift. In contrast, genes involved in pilus type IV biogenesis were mainly subjected to deletions. Surprisingly, the presence of free phage DNA was found in association with exclusion of superinfection in half of the variants and no chromosomal mutation could be found in three of them. Thus, we showed that pseudolysogeny is a frequent outcome of infection by virulent phages of P. aeruginosa. Moreover, double mutants were selected at high frequency and this could presumably due to evolutionary pressure exerted by re-activation of lytic cycle in some cells of the pseudolysogen population. In conclusion, if phage predation selects for variants with alterations in genes involved in biogenesis or regulation of virulence determinants such as LPS or alginate, the resulting phage-resistant variants could potentially exhibit altered levels of virulence in a beneficial or detrimental way. The use of cocktail does not lower significantly the frequency of phage-resistance and in addition we show that pseudolysogeny is a major actor in the selection of mutations.

Au moment de la division cellulaire, l’ADN contenu dans les chromosomes doit être transmis de la cellule mère à chacune des cellules filles. Pour cela l’ADN est d’abord copié (réplication de l’ADN) puis séparé (ségrégation des chromosomes) dans chacune des cellules filles. Chez les eucaryotes, cette séparation se fait au moment de la mitose c’est à dire après que les chromosomes soient complètement répliqués. Chez les bactéries qui en général possède un unique chromosome circulaire, cette ségrégation des chromosome se fait au fur et à mesure de la réplication et deux grands types d’acteurs sont souvent impliqués dans ce processus : Les condensines bactériennes (de type SMC) et les systèmes de partition (ParABS).Les systèmes de partitions sont composés de deux protéines ParA et ParB et de séquences spécifiquement reconnues par ParB nommées parS. Ces séquences sont en nombres variables selon les espèces et sont souvent localisées proche de l’origine de réplication (oriC) sur le chromosome. Pendant ma thèse, je me suis interessée à l’importance de ces séquences et à l’importance de leur positionnement sur le chromosome chez Pseudomonas aeruginosa. J’ai pu montrer qu’un seul site parS suffit pour une ségrégation correcte des chromosomes s’il est situé dans une région s’étendant de -200 à + 450 kb autour d’oriC. Les limites de cette région appelée « zone de compétence » serait liées à la distance oriC-parS et son asymétrie serait due à la présence de l’opéron ribosomique rrnD à 220 kb à gauche d’oriC. En plus de donner une meilleur compréhension de la ségrégation chez P. aeruginosa, cette partie du projet à permis de mettre en evidence un lien entre oriC et sites parS qui pourrait expliquer leurs localisation proches sur le chromosome.Je me suis également interéssée au complexe SMC-ScpAB et à son rôle dans la ségrégtion du chromosome chez P. aeruginosa. J’ai montré que si SMC n’a pas un rôle majeur dans la ségrégation des chromosomes, il est important pour le positionnement du chromosome dans la cellule. J’ai aussi mis en evidence un lien entre SMC et le système ParABS en étudiant l’effet du déplacement d’un site parS sur l’organisation et la ségrégation des chromosomes. / When a mother cell is dividing, DNA inside chromosomes needs to be transmitted to daughter cells. For that, the DNA is copied (replication) and the two copies are separated in each daughter cell (segregation). In eukaryotes this separation occurs during mitosis after complete replication of DNA. In a bacterium, which in general has a unique and circular chromosome, segregation occurs concomitantly with replication and two mains actors are often involved in this segregation: SMC complexes and partition systems (ParABS).A partition system contains 3 elements: two proteins ParA and ParB and DNA sequences named parS. These sequences are highly conserved and are found in variable numbers depending on bacteria. Their chromosomal localization is almost always close to oriC. During my thesis I worked on the importance of these sequences and on the importance of their chromosomal localization for the functioning of ParABS in Pseudomonas aeruginosa.I have shown that one parS site is enough for correct chromosome segregation if it is located between -200 and +450 kb around oriC. Limits of this region called “zone de competence” are probably linked with the distance between oriC-parS. The asymmetry of the “competence zone” is probably due to the presence of a ribosomal operon (rrnD) at -220kb. This part of the project allow us to understand better chromosome segregation in Pseudomonas aeruginosa but also to highlight a fonctionnal link between oriC and parS which can explain why this sequences are located close from each other on the chromosome in lots of bacteria.I also studied the SMC-ScpAB complex and its role in chromosome segregation. For that I analyzed the conformation and segregation of a ∆smc mutant. I highlighted a link between SMC-ScpAB and ParABS system by characterization of mutants with parS displaced on the chromosome.