Les carbapénèmes, β-lactamines possédant le spectre d’activité le plus large, sont souvent la dernière option thérapeutique des infections sévères dues à des germes multi-résistants. Les entérobactéries résistantes aux carbapénèmes, bien que rares en France, sont épidémiques voir même endémiques dans de nombreux pays. Cette résistance est principalement due à la production d’enzymes, les carbapénèmases, comme les enzymes de type KPC (Klebsiella pneumoniae Carbapenemase) dont il existe plusieurs variants. Les souches produisant ces enzymes ont rapidement disséminé dans de nombreuses régions du monde. Les objectifs de ce travail étaient de comprendre les mécanismes moléculaires de la multi-résistance aux antibiotiques des souches productrices de KPC et de déterminer lesfacteurs génétiques responsables de leur diffusion. Nous avons montré que le gène blaKPC est associé à un transposon de type Tn3, le transposon Tn4401, dont il existe trois isoformes.Nous avons aussi montré que Tn4401 est un transposon actif, capable de mobiliser le gèneblaKPC, et qui participe également à l’expression de ce gène par apport de séquencespromotrices. Puis, nous avons étudié une collection de souches de Klebsiella pneumoniae et de Pseudomonas aeruginosa exprimant le gène blaKPC. Nous avons ainsi montré que plusieurs clones de K. pneumoniae diffusent actuellement dans différentes régions du monde, avec unclone majoritaire, le clone ST258. Ces clones se caractérisent par des plasmides différents etpar la présence constante de Tn4401. Nous avons montré que plusieurs clones de P.aeruginosa disséminent dans les hôpitaux de Colombie et sont associés à des structuresgénétiques variables encadrant le gène blaKPC. Enfin, nous avons évalué une nouvelle méthode de détection des souches productrices de BLSE et de carbapénèmases, basée sur une puce à ADN. Cet outil s’est révélé rapide, sensible et spécifique pour tous les gènes recherchés. / Carbapenems are β-lactams with the broadest spectrum of activity and are often the last therapeutic option for treating severe infections due to multi-resistant organisms.Carbapenem-resistant Enterobacteriaceae remain rare in France, but are endemic in someareas. Carbapenem-resistance is mainly due to the production of carbapenemases, such as KPC (Klebsiella pneumoniae Carbapenemase). Several variants of KPC enzymes have beenidentified and KPC-producers are increasingly isolated worldwide. The aim of this study wasto determine the molecular mechanisms involved in multi-resistance of KPC-producers and tocharacterize the genetic elements involved in blaKPC gene mobilization and diffusion. Wehave described a new Tn3-based transposon, Tn4401, and characterized three isoforms. We have demonstrated that Tn4401 is an active transposon, capable of mobilizing blaKPC, and isinvolved in blaKPC gene expression. We have analysed several Klebsiella pneumoniae andPseudomonas aeruginosa isolates harboring the blaKPC-2 gene. We have assessed the spread ofseveral clones of K. pneumoniae isolates, including a major clone, ST258. These clones arecharacterized by different plasmids but an identical genetic structure, Tn4401. We havedemonstrated that several clones of P . aeruginosa are disseminating in Colombia, differingby MLST type, genetic support of blaKPC-2 and Tn4401-like structures. Finally, we have evaluated a new commercial system, based on microarray and dedicated to the identification of ESBL- and carbapenemase-producers. We found this method fast, sensitive and specific.

The main objective of the study was to understand the mode of interaction of protamine (Ptm), a cationic antibacterial peptide from fish milt on the Gram negative bacterial envelope. The present study was designed to resolve the question of Ptm translocation across the seemingly impermeable Gram negative cell envelope. The Gram negative pathogen Pseudomonas aeruginosa was studied as an example of a microorganism that is Ptm-sensitive but doesn’t lyse even at bactericidal concentrations. Acquired resistance to Ptm was induced in P. aeruginosa by continuous sub-culturing in nutrient rich media containing increasing concentrations of Ptm. Alterations in bacterial surface charge, LPS composition, cell morphology and Ptm localisation on acquiring resistance were also examined. Expression of outer membrane proteins significantly decreased as P. aeruginosa acquired resistance to Ptm. OprF, the major porin in P. aeruginosa was found to be stably expressed in control, revertant (Ptm-Rev) and resistant (Ptm-Res) groups. No change in expression of efflux proteins was observed as a result of induced Ptm resistance, indicating that efflux is not among the Ptm resistance mechanisms at least in P. aeruginosa. OprM, which is part of the major efflux system (MexAB-OprM) in P. aeruginosa, was found to be down-regulated in Ptm-resistant P. aeruginosa. Another outer membrane protein down-regulated in Ptm-resistant P. aeruginosa was found to be petidyl-prolyl cis trans isomerase (PPIase) which plays a major role in proper folding and maturation of channel proteins in the outer membrane. Among the sarcosinate soluble proteins, DNA dependent RNA polymerase ? and ?’ subunits were found to be down-regulated in Ptm-resistant group indicating lower transcription levels in them. Lipopolysaccharide (LPS) from the three groups of P. aeruginosa under study was isolated and separated by SDS-PAGE. LPS composition of Ptm-Res P. aeruginosa was found to be significantly different from that of the control and Ptm-Rev but was found to be similar with that of LPS from O-antigenic mutant (A+B-, which possessed only A band structures). Comparison of the zetapotential of control, Ptm-Rev and Ptm-Res P. aeruginosa, proved that electrostatic shielding was coincidental in acquired resistance to Ptm in P. aeruginosa. The MIC of the parent strain of P. aeruginosa (A+B+) and the O-antigenic mutants (A+B-, A-B+ and A-B-) were found to be the same which may be indicating that alterations in O-antigenic components alone cannot contribute to Ptm resistance. Effects of Ptm treatment on morphologies of E. coli, S. typhimurium and P. aeruginosa whole cells and spheroplasts were also studied using transmission immuno-electron microscopy. Condensation of cytoplasmic contents was observed when whole cells and spheroplasts were treated with Ptm. Also, Ptm-treated cells and spheroplasts were stained with colloidal gold-labelled antibodies against Ptm to determine distribution within the target cells. It was quite evident that Ptm internalised in whole cells and spheroplasts without lysis and was found to be concentrated in the cytoplasm. Morphological changes observed in Ptm-Rev P. aeruginosa when exposed to Ptm were comparable with that of the control. Condensation of cytoplasmic contents was not observed in Ptm-Res P. aeruginosa when challenged with Ptm. Most of the Ptm was localized at or near the outer membrane of Ptm-treated Ptm-Res P. aeruginosa, indicating decreased outer membrane permeability. Results obtained from these experiments confirm that the resistance to Ptm observed in P. aeruginosa is at the very least, coincidental with the pleiotropic mutations involving change in outer surface including change in LPS composition, loss of porins and or alterations of porin size in OprF.

Made available in DSpace on 2014-06-12T15:51:44Z (GMT). No. of bitstreams: 2 arquivo4521_1.pdf: 3119722 bytes, checksum: 150947b593a878f27c87a2a02052057f (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / A interferência da bactéria P. aeruginosa sobre o grupo coliforme é conhecida nas análises de água e sua enumeração tem sido questionada durante os últimos anos por ser considerada apenas um patógeno oportunista não habitante do trato intestinal de animais de sangue quente. No entanto, a grande versatilidade metabólica, daria vantagens à bactéria P. aeruginosa sobre outros microrganismos presentes na água. Neste trabalho, foi investigada a ação antagonista de 16 linhagens da bactéria P.aeruginosa, isoladas de diferentes amostras de águas de consumo humano e águas residuárias, contra bactérias do grupo coliforme. A existência de antagonismo foi observada em todas as linhagens no teste em meio sólido. As três linhagens mais agressivas foram submetidas ao teste em meio líquido. O contato se deu entre a bactéria P. aeruginosa com 72h de vantagem de crescimento, contra bactérias do grupo coliforme com inóculo inicial cerca de 102UFC/mL em caldo Műeller-Hinton em uma concentração de 10% da composição original. Entre 24h e 96h após o contato, o NMP/100mL das duas populações foi estimado utilizando a técnica dos tubos múltiplos em série de cinco. O fenômeno de antagonismo foi observado quando os inóculos iniciais partiram de 102UFC/mL e evidenciado quando a piocianina estava presente. E. aerogenes se mostrou mais sensível que E. coli. Este trabalho reascende uma discussão sobre a inclusão da bactéria P. aeruginosa como uma ferramenta adicional nas análises de água de consumo

Made available in DSpace on 2014-06-12T18:06:32Z (GMT). No. of bitstreams: 2 arquivo6313_1.pdf: 1481990 bytes, checksum: 994600fb8e4272b6f803e8477c45a54d (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2005 / Pseudomonas aeruginosa é uma bacteria Gram-negativa ubíqua e oportunista. Na rotina hospitalar, os marcadores fenotípicos nem sempre revelam a diversidade das bactérias distribuídas nos diversos setores, assim, aplicamos três métodos moleculares baseados na amplificação por PCR do locus de rDNA e tDNA para caracterizar a diversidade genética de linhagens de P. aeruginosa isoladas em um hospital público em Recife-PE, Brasil. O rDNA-PCR detectou 15% de variabilidade genética, contra 23% do tDNA-PCR e 23% do Duplex-PCR. O setor com maior diversidade genética foi a Unidade de Tratamento Intensivo do hospital, o qual apresentou quatro genótipos bacterianos diferentes. A ocorrência de linhagens de P. aeruginosa pertencentes ao mesmo genótipo e mesmo perfil de resistência a múltiplas drogas (MDR), em diferentes setores do hospital, sugere que há infecção cruzada entre pacientes. Os dados apresentados pelo rDNA-PCR, tDNA-PCR e Duplex-PCR, em associação ao perfil de susceptibilidade antimicrobiana provêem valiosas informações epidemiológicas para o controle de infecções hospitalares causadas por P. aeruginosa

La voie de signalisation impliquant l’interleukine (IL)-22 a un rôle majeur dans le maintien des fonctions de barrière des surfaces exposées du corps humain. Elle est indispensable pour la promotion de l'immunité antimicrobienne épithéliale, de l'inflammation et de la réparation tissulaire. Des situations pathologiques impliquant une altération de cette voie de signalisation ont déjà été décrites, mais rarement au niveau pulmonaire. Ce travail étudie cette voie de signalisation dans trois pathologies respiratoires. Lors de la broncho-pneumopathie chronique obstructive, un excès de sécrétion de protéases à sérine secondaire au recrutement de neutrophiles est responsable d’une protéolyse du récepteur à l’IL-22. Cette protéolyse inhibe les mécanismes de défense épithéliale dépendante de l’IL-22. Lors d’infection pulmonaire à P. aeruginosa, un facteur de virulence sécrété par cette bactérie dégrade l’IL-22 et inhibe la sécrétion épithéliale de peptides antimicrobiens. Enfin, une surexpression du récepteur à l’IL-22 a été observée dans le cancer du poumon non à petites cellules. Cette surexpression est associée à une surmortalité. / The (interleukin) IL-22/IL-22 receptor (R) pathway is critical in the maintenance of barrier function at exposed surface of the body. This pathway is also essential to promote innate mucosal immunity, inflammation and tissue homeostasis. Dysregulation of IL-22/IL-22R pathway has been described in human diseases, but has been barely studied in respiratory pathologies. This work reveals three lung diseases with altered IL-22/IL-22R pathway. During chronic obstructive pulmonary disease, the proteolytic action of neutrophil-derived enzymes cleave the IL-22R and inhibit IL-22-mediated epithelial cell response. Next, we demonstrated that P. aeruginosa used its own proteolytic system to escape from host defenses through the proteolysis of IL-22 leading to negative regulation of antimicrobial peptides. Finally, we observed that higher IL-22R expression is correlated with squamous cell lung carcinoma and is associated with increase mortality.

Les métaux biologiques jouent un rôle clé en tant que cofacteurs, contribuant à la structuration des macromolécules et catalysant les réactions biochimiques dans les cellules. Ils sont nécessaires pour une croissance bactérienne optimale mais deviennent toxiques lorsqu'ils sont présents en excès. Par conséquent, l'homéostasie de ces métaux doit être finement régulée, et tout déséquilibre dans leur concentration pourrait affecter la viabilité cellulaire. Lors de cette thèse nous avons investigué les mécanismes moléculaires impliqués dans l'homéostasie du Fe chez Pseudomonas aeruginosa, en étudiant les deux principaux sidérophores produits par cette bactérie, pyoverdine (PVD) et pyochéline (PCH). Avec notre approche, nous avons identifié des nouveaux mécanismes de régulation des voies d’acquisition du fer par PVD et PCH. Nous avons également étudié comment d'autres métaux biologiques peuvent interférer avec ces voies, et le Co a montré une forte propension à pirater et à polluer la voie PCH. / Biological metals (Fe, Zn, Co, Ni, Mn, Cu) play a key role by acting as co-factors, contributing to macromolecule structuration, and catalyzing biochemical reactions into the cells. They are required for optimal bacterial growth but also become toxic when present in excess. Consequently, the homeostasis of these metals has to be finely regulated, and any disequilibrium in their concentration into bacteria could affect cell viability. We have further investigated the molecular mechanisms implicated in Fe homeostasis in Pseudomonas aeruginosa involving the two major siderophores produced by this bacterium, pyoverdine (PVD) and pyochelin (PCH). With our approach we identified new regulation mechanisms of both PVD and PCH pathways. In parallel, we have also investigated how other biological metals than Fe can interfere with these iron uptake pathways. Our data showed a strong propensity of Co to pirate and pollute the PCH iron uptake pathway.

A Fibrose Cística (FC) é uma doença letal, de caráter autossômico recessivo, que acomete populações de diferentes etnias. A doença caracteriza-se pelo comprometimento sistêmico das glândulas exócrinas e, na maioria dos pacientes, a doença pulmonar acaba tornando-se a patologia predominante. A infecção por P. aeruginosa é a principal causa de mortalidade dos pacientes com FC. O Sistema de Secreção Tipo III da bactéria é expresso na fase aguda da doença e é responsável por injetar proteínas citotóxicas no interior da célula eucariótica. Há um grande interesse em se investigar a resposta de anticorpos anti P. aeruginosa em pacientes com FC a fim de diagnosticar a colonização e ou infecção pulmonar antes da cultura, permitindo a antibioticoterapia preventiva, a fim de se evitar a infecção pulmonar crônica. Nesta tese, investigamos a resposta de anticorpos (IgG+IgM+IgA) contra as proteínas do SSTT de P. aeruginosa, através do Western-Blot. Participaram do estudo 51 pacientes com FC, de 1.1 a 16.8 anos acompanhados no Departamento de Pneumologia do Instituto Fernandes Figueira - FioCruz, durante um período aproximado de 2 anos. De cada paciente foram coletadas de 1 a 4 amostras de sangue, com intervalo médio de 6 meses entre as coletas. O grupo controle negativo consistiu de 28 indivíduos não fibrocísticos, de 2 a 17 anos, atendidos no Hospital Universitário Pedro Ernesto - HUPE UERJ. As proteínas do SSTT foram extraídas das cepas PAO1 e PAOΔExsA (regulador da expressão do SSTT) de P. aeruginosa. Controles positivos e negativos foram utilizados em todas as reações. Para a identificação das proteínas do SSTT na reação utilizou-se antisoro de camundongos imunizados com a proteína recombinante PcrV. Doze (75%) dos 16 pacientes fibrocísticos considerados não infectados por P. aeruginosa tiveram a primeira sorologia positiva para PopB e 15 (93,75%) para ExoS/ExoT, indicando a colonização ou infecção por P. aeruginosa. Aproximadamente 25% e 35,7% dos soros do grupo controle mostraram reatividade fraca com PopB ou ExoS/ExoT, respectivamente. O tempo decorrido entre a primeira sorologia positiva e o primeiro isolamento de P. aeruginosa nestes pacientes variou de 18 a 30 meses. Concluindo, é possível fazer o diagnóstico sorológico da infecção pulmonar por P. aeruginosa antes do isolamento da bactéria pela cultura. / Cystic Fibrosis (CF) is a lethal disease of autosomal recessive character, which affects people of different ethnicities. The disease is characterized by the involvement of systemic exocrine glands and in most patients, the lung disease becomes the predominant pathology. The infection with P. aeruginosa is the leading cause of mortality in patients with CF. The Type III Secretion System (TTSS) of bacteria is expressed during acute disease and injects cytotoxic proteins inside the host cell. There is a great interesting in investigate the antibody response to P. aeruginosa in CF patients in order to diagnose a pulmonary infection or colonization before the culture. Then, preventive antibiotic treatment can be initiated before the installation of chronic lung infection. We investigated the antibody response (IgG + IgA + IgM) against TTSS proteins of P. aeruginosa by Western-blot. The study included 51 patients with CF, from 1.1 to 16.8 years attending the Pediatric Pulmonology Unit of Fernandes Figueira Institute (IFF) FIOCRUZ, Rio de Janeiro, for a period of approximately 2 years. Most patients had or 4 blood samples collected for antibody analyses. Samples were obtained with a mean interval of 6 months. The negative control group consisted of 28 non-CF individuals, from 2 to 17 years, attended at Pedro Ernesto University Hospital - HUPE - UERJ. The TTSS proteins were extracted from strains PAO1 and PAOΔExsA (regulator of TTSS proteins expression) of P. aeruginosa. Positive and negative controls were used in all reactions. For the identification of TTSS proteins in the reaction we used antisera from mice immunized with the recombinant protein PcrV. Twelve (75%) of 16 CF patients considered not infected by P. aeruginosa had their first serology positive for "PopB" and 15 (93.75%) for "ExoS/ExoT. These results indicated that these patients were colonized or infected by P. aeruginosa. About 25% e 35,7% of negative control sera showed a weak reactivity with PopB or ExoS, respectively. The time between the first positive serology and the first isolation of P. aeruginosa in these patients ranged from 18 to 30 months. In conclusion, it is possible to make a serological diagnosis of pulmonary infection by P. aeruginosa before the isolation of the bacterium by culture.

A Fibrose Cística (FC) é uma doença letal, de caráter autossômico recessivo, que acomete populações de diferentes etnias. A doença caracteriza-se pelo comprometimento sistêmico das glândulas exócrinas e, na maioria dos pacientes, a doença pulmonar acaba tornando-se a patologia predominante. A infecção por P. aeruginosa é a principal causa de mortalidade dos pacientes com FC. O Sistema de Secreção Tipo III da bactéria é expresso na fase aguda da doença e é responsável por injetar proteínas citotóxicas no interior da célula eucariótica. Há um grande interesse em se investigar a resposta de anticorpos anti P. aeruginosa em pacientes com FC a fim de diagnosticar a colonização e ou infecção pulmonar antes da cultura, permitindo a antibioticoterapia preventiva, a fim de se evitar a infecção pulmonar crônica. Nesta tese, investigamos a resposta de anticorpos (IgG+IgM+IgA) contra as proteínas do SSTT de P. aeruginosa, através do Western-Blot. Participaram do estudo 51 pacientes com FC, de 1.1 a 16.8 anos acompanhados no Departamento de Pneumologia do Instituto Fernandes Figueira - FioCruz, durante um período aproximado de 2 anos. De cada paciente foram coletadas de 1 a 4 amostras de sangue, com intervalo médio de 6 meses entre as coletas. O grupo controle negativo consistiu de 28 indivíduos não fibrocísticos, de 2 a 17 anos, atendidos no Hospital Universitário Pedro Ernesto - HUPE UERJ. As proteínas do SSTT foram extraídas das cepas PAO1 e PAOΔExsA (regulador da expressão do SSTT) de P. aeruginosa. Controles positivos e negativos foram utilizados em todas as reações. Para a identificação das proteínas do SSTT na reação utilizou-se antisoro de camundongos imunizados com a proteína recombinante PcrV. Doze (75%) dos 16 pacientes fibrocísticos considerados não infectados por P. aeruginosa tiveram a primeira sorologia positiva para PopB e 15 (93,75%) para ExoS/ExoT, indicando a colonização ou infecção por P. aeruginosa. Aproximadamente 25% e 35,7% dos soros do grupo controle mostraram reatividade fraca com PopB ou ExoS/ExoT, respectivamente. O tempo decorrido entre a primeira sorologia positiva e o primeiro isolamento de P. aeruginosa nestes pacientes variou de 18 a 30 meses. Concluindo, é possível fazer o diagnóstico sorológico da infecção pulmonar por P. aeruginosa antes do isolamento da bactéria pela cultura. / Cystic Fibrosis (CF) is a lethal disease of autosomal recessive character, which affects people of different ethnicities. The disease is characterized by the involvement of systemic exocrine glands and in most patients, the lung disease becomes the predominant pathology. The infection with P. aeruginosa is the leading cause of mortality in patients with CF. The Type III Secretion System (TTSS) of bacteria is expressed during acute disease and injects cytotoxic proteins inside the host cell. There is a great interesting in investigate the antibody response to P. aeruginosa in CF patients in order to diagnose a pulmonary infection or colonization before the culture. Then, preventive antibiotic treatment can be initiated before the installation of chronic lung infection. We investigated the antibody response (IgG + IgA + IgM) against TTSS proteins of P. aeruginosa by Western-blot. The study included 51 patients with CF, from 1.1 to 16.8 years attending the Pediatric Pulmonology Unit of Fernandes Figueira Institute (IFF) FIOCRUZ, Rio de Janeiro, for a period of approximately 2 years. Most patients had or 4 blood samples collected for antibody analyses. Samples were obtained with a mean interval of 6 months. The negative control group consisted of 28 non-CF individuals, from 2 to 17 years, attended at Pedro Ernesto University Hospital - HUPE - UERJ. The TTSS proteins were extracted from strains PAO1 and PAOΔExsA (regulator of TTSS proteins expression) of P. aeruginosa. Positive and negative controls were used in all reactions. For the identification of TTSS proteins in the reaction we used antisera from mice immunized with the recombinant protein PcrV. Twelve (75%) of 16 CF patients considered not infected by P. aeruginosa had their first serology positive for "PopB" and 15 (93.75%) for "ExoS/ExoT. These results indicated that these patients were colonized or infected by P. aeruginosa. About 25% e 35,7% of negative control sera showed a weak reactivity with PopB or ExoS, respectively. The time between the first positive serology and the first isolation of P. aeruginosa in these patients ranged from 18 to 30 months. In conclusion, it is possible to make a serological diagnosis of pulmonary infection by P. aeruginosa before the isolation of the bacterium by culture.

Durant ma thèse, nous avons identifier PlpA comme une cytotoxine conservée dans des isolats cliniques d'origines diverses, contrairement à son homologue, le facteur de virulence ExoU de Pseudomonas aeruginosa. Un rôle dans la cytotoxicité envers des cellules phagocytaires de l'immunité innée a été attribué à PlpA, et celui-ci dépend de l'intégrité de la dyade catalytique Ser/Asp, caractéristique des protéines de la famille des patatines. Un interactome réalisé in vivo dans des cellules hôtes nous a permis d'identifier des transporteurs de la mitochondrie comme partenaires de PlpA. L'interaction de PlpA avec ses partenaires mitochondriaux, aurait de manière inattendue un effet anti-apoptotique sur les macrophages, mais conduit cependant, à la mort de ceux-ci vraisemblablement par un phénomène de nécrose induite.PlpD a précédemment été caractérisée par Salacha et ses collaborateurs comme étant l'archétype du Système de Sécrétion de Type Vd (2010). Bien que le mécanisme précis de sécrétion de cette protéine reste à ce jour mal connu, nos travaux ont permis de lui attribuer un rôle dans la compétition bactérienne, conférant ainsi un avantage compétitif aux souches qui la possède. D'ailleurs, l'analyse phylogénétique de PlpD (Salacha et al., 2010 ; Heinz & Lithgow 2014) révèle la conservation de cette protéine au sein de nombreuses espèces vivants dans un environnement hostile, suggérant ainsi la nécessité de cette protéine dans l'implantation et la conservation de niches écologiques, que se soit dans l'environnement ou au cours d'infections polymicrobiennes chez un organisme hôte. / During my PhD, in the PAO1 strain of Pseudomonas aeruginosa, we identified PlpA as a cytotoxin conserved in clinical isolates of various origins, contrary to its virulence factor ExoU homologues. A cytotoxic role of PlpA has been highlighted against phagocytic cells, and showed to depend on the integrity of its Ser/Asp catalytic dyad. An in vivo interactome allowed us to identify mitochondrial transporters as partners of PlpA. Interestingly, PlpA interaction with these partners has an anti-apoptotic effect on macrophages but ultimely allows macrophages death probably by a necroptosis phenomenon. PlpD was previously described by Salacha and collaborators as the SST5d archetype (Salacha et al., 2010). While its exact secretion mechanism remains poorly understood, our work allowed showing that it played a role in bacterial competition. PlpD phylogenetic analysis (Salacha et al., 2010 ; Heinz & Lithgow 2014) revealed its conservation in many species living in hostile environments, suggesting its necessity in the implantation and conservation of ecological niches in the environment or during polymicrobial infections into host organism.

Pseudomonas aeruginosa est un pathogène opportuniste de l’Homme responsable de nombreuses infections des voies pulmonaires et urinaires chez des patients immunodéprimés. Largement étudié pour son implication dans la sévérité des symptômes liés à la mucoviscidose, le« bacille pyocyanique » constitue un enjeu majeur en termes de sécurité sanitaire puisqu’il représente après Escherichia coli et Staphylococcus aureus la 3ème cause d’infections nosocomiales en France (INVS, Enquête nationale de prévalence des infections nosocomiales en France, 2012). La persistance de P. aeruginosa notamment dans le cadre médical, est largement due à sa grande capacité à s’adapter et à se développer en communauté organisée au sein de biofilm. Le génome de P. aeruginosa contient de nombreux gènes codant des systèmes de régulation dont la majorité est impliquée dans des mécanismes de perception-transduction de signaux, conférant à la bactérie son fort pouvoir d’adaptation. Parmi ces systèmes, les facteurs sigma à fonction extracytoplasmique (ECF), des sous-unités transitoires de l’ARN polymérase, jouent un rôle fondamental dans la résistance et l’adaptation aux stress. SigX est un facteur sigma ECF, impliqué dans la virulence et la formation de biofilms, ainsi que dans la production des acides gras à courte chaine. Au cours de cette étude, les fonctions cellulaires de SigX ont été précisées, Nous avons montré que SigX joue un rôle important dans la composition, la fluidité et la perméabilité membranaires, et par conséquent dans le métabolisme de la cellule. L’activation de SigX en réponse à des conditions entrainant un stress de l’enveloppe, telles que la perte de la porine majoritaire OprF, la présence d’une concentration de sucrose dans le milieu de culture ou d’une concentration sub-inhibitrice de tobramycine, suggère que cet ECF, comme AlgU, pourrait appartenir à la classe des ECF de type RpoE.De manière remarquable, certaines altérations de l’enveloppe pourraient induire la formation de biofilm, un phénotype impliquant au moins partiellement SigX. Il conviendra à présent de caractériser les mécanismes moléculaires conduisant à l’activation de SigX et de préciser le rôle de ce facteur sigma dans la formation de biofilm. / Pseudomonas aeruginosa is a major opportunistic pathogen causing many infectious diseases in immunocompromised patients. Widely studied because of its involvement in lung infections of cysticfibrosis suffering patients, this bacterium is a major public health challenge. P. aeruginosa persistence is largely due to its ability to adopt a multicellular lifestyle called biofilm. P. aeruginosa genome encodes numerous genes predicted to be involved in signal transduction allowing this bacterium to adapt to many environments. Among these systems, the extracytoplasmic function sigma factors, which are transitory subunits of the RNA polymerase, are of major importance for stress resistance and adaptation. SigX is an ECF sigma factor that has been involved in virulence, biofilm formation and in short chain fatty acidsbiosynthesis. This work led to precise the cellular functions of SigX. We have shown that SigX is of major importance for membrane homeostasis, including composition, fluidity and permeability. As a consequence, SigX was shown to be involved in P. aeruginosa metabolism. SigX activity is enhanced in conditions leading to a cell wall stress, as the lack of the major outer membrane porin OprF, high concentrations of sucrose or sublethal concentration of tobramycin, suggesting that this ECF, as AlgU,is a new cell wall stress responsive sigma factor. Remarkably, some alterations could induce biofilm formation, a phenotype involving at least partially SigX. The molecular mechanisms leading to SigX activity should now be deciphered and the role of this ECF in biofilm formation should be precised.