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.

En raison de l'efficacité pour délivrer des antigènes directement dans le cytoplasme des CAPs in vivo, les vecteurs bactériens atténués et basés sur les propriétés du système de sécrétion de type 3 (SST3) attirent de plus en plus l'attention grâce à leur potentiel dans le développement des vaccins contre le cancer. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le SST3. Dans nos travaux précédents, le potentiel de souches atténuées de P. aeruginosa dans le domaine de la vaccination anti-tumorale a été démontré. Dans ce travail, nous avons optimisé des vecteurs vaccinaux basés sur le SST3 de P. aeruginosa pour des applications cliniques. Dans un premier temps, la performance de ces vecteurs bactériens a été améliorée en utilisant différents modèles de tumeurs murines. Ceci par : 1) l'ajout d'un épitope spécifique des lymphocytes CD4+ Th aux vecteurs; 2) l'application d'un modèle d'expression bi-antigénique aux vecteurs; 3) la construction de vecteurs induisant une réponse humorale. Dans un deuxième temps, la performance thérapeutique du vecteur bactérien a été optimisée par la modulation de la fréquence des injections et l'intervalle qui les sépare. Cette performance a été confirmée dans des modèles différents de tumeurs murines. Dans un troisième temps, un candidat qui pourrait être appliqué en clinique a été généré par l'adaptation d'un mutant (CHA-OAL) de P. aeruginosa totalement avirulent dans un milieu chimiquement défini. La très faible infectiosité de cette souche a été surmontée par en vaccinant à des emplacements multiples. Par la suite, le potentiel du vecteur bactérien dans l'immunothérapie humaine a été également évalué- dans un premiers temps-dans un modèle de souris humanisées (HHD). Enfin, nous avons observé qu'une immunité anti-vecteur pré-existante n'a pas d'effet sur l'efficacité de la vaccination par le vecteur bactérien. L'ensemble de nos résultats a mis en évidence le potentiel de nos vecteurs vivants et atténués de P. aeruginosa pour des applications dans des essais cliniques pertinents. / Due to the endowed effective ability to deliver antigen to cytoplasm of APCs in vivo, T3SS based attenuated bacterial vectors attracted more and more attention for their potential interest in cancer vaccine development. Pseudomonas aeruginosa est un pathogène opportuniste responsable d'infections graves chez les personnes immunodéprimées, les grands brûlés et les patients atteints de la mucoviscidose. Cette pathogénicité repose sur de nombreux facteurs de virulence dont le système de sécrétion de type III (SSTT). In our previous work, the potential of attenuated P. aeruginosa strains as the carriers for anti-tumor vaccination purpose has been reported. In this work, we would like to strengthen P. aeruginosa T3SS based vaccine vectors and direct the development of these bacterial vectors toward clinical applications. First, the performance of these bacterial vectors has been improved in different murine cancer models by: 1) adding one CD4+ Th epitope to vectors; 2) applying bi-antigen expression pattern to vectors; 3) constructing potential humoral response inducing vectors. Second, the therapeutic performance of bacterial vector has been optimized by modulating injection frequency and interval and then be confirmed in murine tumor models. Third, one clinically applicable candidate has been generated by adapting one totally avirulent P. aeruginosa mutant (CHA-OAL) in a chemically defined medium and the poor infectivity of this new strain has been overcome by vaccinations at multiple loci. Fourth, the potential of bacterial vector for human immunotherapy has been further evaluated in one first level humanized mice (HHD) model. Finally, we observed that the pre-existing anti-vector immunity didn't impair the vaccination efficiency of bacteria vector. Taken together, our results highlight the potentials of our live attenuated P. aeruginosa vectors for applications in relevant clinical trials.

Pseudomonas aeruginosa, une bactérie à Gram négatif responsable d'infections nosocomiales, possède de nombreux facteurs de virulence lui permettant d'infecter ses hôtes. En particulier, le Système de Sécrétion de Type III (SST3) lui permet d'injecter des effecteurs directement dans le cytoplasme de la cellule cible eucaryote. Durant cette thèse, deux protéines du SST3 de P. aeruginosa ont été étudiées : l'ATPase PscN et la protéine ExsB. Plusieurs approches ont été utilisées afin d'étudier l'ATPase PscN, indispensable à l'activité du SST3. Des mutations ponctuelles réalisées dans PscN conduisent à des souches de P. aeruginosa non cytotoxiques, et cet effet est dominant négatif. Une autre approche a permis l'obtention de fractions partiellement purifiées de l'ATPase PscN active, sous forme de grands complexes visualisés en microscopie électronique. Ces fractions contiennent également d'autres protéines du SST3, qui pourraient être des partenaires de PscN. La protéine ExsB a été caractérisée pour la première fois. Après avoir vérifié son expression chez P. aeruginosa, son association à la membrane externe de la bactérie a été démontrée. Son rôle a ensuite été étudié par une analyse du phénotype d'une souche de P. aeruginosa dépourvue du gène exsB. Nous n'avons pas identifié d'activité de ExsB dans la régulation du SST3. Après avoir constaté l'implication de ExsB dans la virulence de la bactérie dans des modèles d'infections aiguës chez les animaux, son rôle dans l'activité du SST3 a été établi. Nous avons enfin pu montrer que ExsB a une activité de pilotine, car elle participe à l'assemblage de la sécrétine, le composant de la membrane externe du SST3. / Pseudomonas aeruginosa, a Gram negative bacterium responsible for nosocomial infections, exhibits numerous virulence factors to infect its hosts. In particular, the Type III Secretion System (T3SS) allows the injection of effectors directly into the host cell cytoplasm. This work focuses on the study of two proteins from the T3SS of P. aeruginosa: the ATPase PscN and the ExsB protein. Several approaches were used to study the ATPase PscN, an enzyme essential for T3SS activity. Site-directed mutations, made on PscN, lead to non cytotoxic strains, and this effect is dominant negative. Another approach allowed the partial purification of active PscN, visualized as large complexes by electron microscopy. These partially purified samples also contain other T3SS proteins, which could interact with PscN. The ExsB protein was characterized for the first time. After checking its expression in P. aeruginosa, its association with the outer membrane was shown. The phenotypic analysis of a strain lacking exsB gene gave insights into the role of this protein. We did not identified any function of ExsB in the T3SS regulation. After showing the involvement of ExsB in the bacterial virulence during acute animal infections, ExsB role in T3SS activity was established. Finally, we showed that ExsB has a pilotin activity as it participates in the assembly of the secretin, the outer membrane component of T3SS.

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.

Xanthomonas oryzae pv. oryzae (Xoo) est une bactérie phytopathogène responsable de la bactériose vasculaire du riz, maladie pouvant engendrer de fortes pertes de rendement à travers le monde. La course à l'armement entre la bactérie et sa plante hôte correspond d'une part à la mise en place de la virulence par le microorganisme et d'autre part en la résistance du végétal face à l'agression. Comprendre les mécanismes par lesquels Xoo accompli son cycle infectieux est d'une importance cruciale pour le développement futur de nouvelle méthode de luttes. Plusieurs approches complémentaires ont été mises en uvre afin de caractériser des éléments associés au pouvoir pathogène de Xoo.Dans un premier temps nous avons effectué une analyse protéomique comparative. Cette approche a permis l'identification chez une souche Africaine de Xoo d'un jeu de protéines induites par HrpX et susceptibles de jouer un rôle dans la virulence. Dans un second temps, l'implication de deux peptides dans la virulence Xoo a été étudiée. Le premier de ces peptides, supposé être le facteur d'avirulenceAvrXa21, a fait l'objet d'une caractérisation fonctionnelle et phylogénique. Le second peptide est synthétisé par un cluster NRPS, similaire à l'un de ceux présent chez Xanthomonas albilineans. Afin d'élucider l'importance de la molécule synthétisée par cette voie pour Xoo, une étude préliminaire impliquant la mutation d'un élément régulateur des NRPS a été effectuée. En dernier lieu, des informations nouvelles ont été apportées sur la topologie de la protéine membranaire HrcR qui est une composante essentielle du système de sécrétion de type III chez la plupart des bactéries appartenant au genre Xanthomonas. / Xanthomonas oryzae pv. oryzae (Xoo) is the agent of bacterial leaf blight BLB in rice, a disease which causes considerable yield losses throughout the world. In the arms race underlying the interactions between the microorganism and the host, the presence of virulence factors in the former parallels that of resistance factors in the latter. Understanding the mechanisms of Xoo's infectious cycle is of paramount importance for the elaboration of new fighting strategies to combat BLB. To achieve this, several complementary approaches to characterize components of Xoo's pathogenicity have been employed.First, we performed comparative proteomics that allowed us to identify novel HrpX-induced candidate pathogenicity factors of an African Xoo strain. Second, the involvement of two peptides in Xoo's pathogenicity has been investigated. One was speculated to be the avirulence factor AvrXa21 and has been characterized both functionally and phylogenetically. The other one was found to be synthesized by a Non-Ribosomal Peptide Synthetase (NRPS), reminescent to NRPS genes found in Xanthomonas albilineans. In order to determine the role of NRPS-mediated synthesis in Xoo virulence, we studied a strain carrying a mutated regulatory gene of the NRPS pathway. Finally, we provide new information on the topology of the HrcR membrane protein which is a conserved component of the type III secretion system of most Xanthomonas.

A citricultura é de grande importância para as atividades agrícolas brasileiras, uma vez que o Brasil é o principal produtor e exportador de suco de laranja. O cancro cítrico, causado pela bactéria Xanthomonas axonopodis pv. citri (Xac) é um grave problema nesse setor, causando um elevado prejuízo na produção de frutos e seus derivados. O fator sigma é a subunidade da RNA polimerase que tem a função de direcionar o núcleo da RNA polimerase a uma classe específica de sequências promotoras. Como a maioria das bactérias sintetiza diversos fatores sigma, essa característica proporciona à bactéria a oportunidade de manutenção basal da sua expressão gênica, assim como, a regulação em resposta a alterações ambientais e a sinais durante o desenvolvimento bacteriano. O genoma de Xac codifica para 14 fatores sigma. Nesse presente trabalho, detectamos interações dos fatores σECF (Xac2814. Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) e seus fatores anti-σ cognatos (Xac2815. Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). Além disso, observamos interações entre o fator σFliA (Xac1933) e o anti-σFlgM (Xac1989), seu fator anti-σ cognato. A caracterização das cepas nocautes para alguns fatores σ apontaram o envolvimento do fator σ54Xac1969 no mecanismo de formação de flagelo, a contribuição do fator σECFXac1682 na resposta ao choque térmico e a participação do fator σECFXac2191 no crescimento bacteriano em condições de carência de ferro. / Citriculture is an important sector of the economy of the State of São Paulo. Citrus canker, caused by Xanthomonas axonopodis pv. citri (Xac), is a devastating disease responsible for large agribusiness losses every year. several sigma factors. The sigma factor is the subunit of RNA polymerase that serves to direct the RNA polymerase core to a specific class of promoter sequences. Most bacteria code for more than one sigma factor, which provides the cell with the means by which to maintain basal gene expression while at the same time modulate the expression of specific genes in response in environmental changes and signals during bacterial growth. The Xac genome codes for 14 sigma factors which are the objects of study in this thesis. We demonstrate that many of the sigma factors of the σECF family (Xac2814, Xac3989, Xac0922, Xac1319, Xac1380, Xac1682, Xac4129 e Xac2191) interact with cognate anti- factors (Xac2815, Xac3988, Xac0921, Xac1320, Xac1379, Xac1681, Xac4130 e Xac2192). These sigma-anti-sigma pairs are all coded by neighboring genes. Interactions between the sigma factor σFliA (Xac1933) and anti-σFlgM (Xac1989) were also observed. Xac strains with gene knockouts for several sigma factors were produced. The characterization some these knockout strains point to the involvement of σ54Xac1969 in the biosynthesis of flagella, participation of σECFXac1682 in the ability to survive heat shock and involvement of σECFXac2191 in the response to iron deficiency.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012

No description available.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citossol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. Estudos anteriores realizados em nosso laboratório relataram a potente atividade pró-inflamatória de ExoU, responsável por um intenso recrutamento de neutrófilos para o sítio de infecção. No presente trabalho, o efeito de ExoU na modulação da ativação do fator transcricional NF-κB e na regulação da expressão e da secreção da quimiocina para neutrófilos IL-8 foi avaliado em culturas de células epiteliais respiratórias e endoteliais humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU) ou com a mutante deletada no gene exoU, PA103κexoU. Análises por RT-PCR semi-quantitativo mostraram que a infecção pela cepa produtora de ExoU levou ao aumento dos níveis de mRNA de IL-8, enquanto ensaios de alteração da mobilidade eletroforética (EMSA), supershift e com gene repórter mostraram que ExoU induziu a translocação nuclear do heterodímero transativador p65/p50 de NF-κB e a ativação da transcrição de genes dependente deste fator transcricional. Adicionalmente, o tratamento das culturas celulares com um inibidor de NF-κB antes da infecção bacteriana reduziu significativamente os níveis de mRNA de IL-8 e da secreção desta quimiocina. Em conjunto, estes resultados mostram que ExoU ativa NF-κB e, consequentemente, estimula a expressão e a secreção de IL-8 por células epiteliais respiratórias e células endoteliais infectadas com P. aeruginosa / ExoU, a cytotoxin produced by the opportunistic pathogen Pseudomonas aeruginosa that is translocated into host cell cytosol by the type three secretory system, has been associated with severity of acute infections. We have previously described the potent ExoU proinflammatory activity, which accounts for a market recruitment of neutrophils to infected tissues. In this present study, the effect of ExoU on the activation of the transcriptional factor NF-B and on the regulation of the expression and secretion of the chemokine IL-8 was investigated in human epithelial respiratory and endothelial cell cultures infected with the ExoU-producing PA103 P. aeruginosa or with the bacterial mutant with the deletion of the exoU gene PA103exoU. By semi-quantitative RT-PCR, ExoU was shown to significantly increase the expression of IL-8 mRNA. By electrophoretic mobility shift assay (EMSA), supershift and reporter assay ExoU was shown to induce the nuclear translocation of the NF-κB p65/p50 transactivator heterodimer as well as the NF-κB-dependent transcriptional activity. In addition, treatment both the IL-8 mRNA expression and the protein secretion. Together, our results show that ExoU activates NF-B and stimulates IL-8 expression and secretion by P. aeruginosa-infected human epithelial respiratory and endothelial cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / ExoU, uma citotoxina produzida pelo patógeno oportunista Pseudomonas aeruginosa e translocada para o citossol de células hospedeiras via sistema de secreção do tipo III, é associada à gravidade de infecções agudas. Estudos anteriores realizados em nosso laboratório relataram a potente atividade pró-inflamatória de ExoU, responsável por um intenso recrutamento de neutrófilos para o sítio de infecção. No presente trabalho, o efeito de ExoU na modulação da ativação do fator transcricional NF-κB e na regulação da expressão e da secreção da quimiocina para neutrófilos IL-8 foi avaliado em culturas de células epiteliais respiratórias e endoteliais humanas infectadas com a cepa PA103 de P. aeruginosa (produtora de ExoU) ou com a mutante deletada no gene exoU, PA103κexoU. Análises por RT-PCR semi-quantitativo mostraram que a infecção pela cepa produtora de ExoU levou ao aumento dos níveis de mRNA de IL-8, enquanto ensaios de alteração da mobilidade eletroforética (EMSA), supershift e com gene repórter mostraram que ExoU induziu a translocação nuclear do heterodímero transativador p65/p50 de NF-κB e a ativação da transcrição de genes dependente deste fator transcricional. Adicionalmente, o tratamento das culturas celulares com um inibidor de NF-κB antes da infecção bacteriana reduziu significativamente os níveis de mRNA de IL-8 e da secreção desta quimiocina. Em conjunto, estes resultados mostram que ExoU ativa NF-κB e, consequentemente, estimula a expressão e a secreção de IL-8 por células epiteliais respiratórias e células endoteliais infectadas com P. aeruginosa / ExoU, a cytotoxin produced by the opportunistic pathogen Pseudomonas aeruginosa that is translocated into host cell cytosol by the type three secretory system, has been associated with severity of acute infections. We have previously described the potent ExoU proinflammatory activity, which accounts for a market recruitment of neutrophils to infected tissues. In this present study, the effect of ExoU on the activation of the transcriptional factor NF-B and on the regulation of the expression and secretion of the chemokine IL-8 was investigated in human epithelial respiratory and endothelial cell cultures infected with the ExoU-producing PA103 P. aeruginosa or with the bacterial mutant with the deletion of the exoU gene PA103exoU. By semi-quantitative RT-PCR, ExoU was shown to significantly increase the expression of IL-8 mRNA. By electrophoretic mobility shift assay (EMSA), supershift and reporter assay ExoU was shown to induce the nuclear translocation of the NF-κB p65/p50 transactivator heterodimer as well as the NF-κB-dependent transcriptional activity. In addition, treatment both the IL-8 mRNA expression and the protein secretion. Together, our results show that ExoU activates NF-B and stimulates IL-8 expression and secretion by P. aeruginosa-infected human epithelial respiratory and endothelial cells