Escherichia coli patogênicas extraintestinais (ExPEC) causam colibacilose aviária, infecções do trato urinário e meningite neonatal em humanos. A cepa ExPEC MT78 é virulenta in vitro e in vivo, e possui a habilidade de invadir células eucarióticas. Para melhor entender o fenótipo invasivo dessa cepa, foi criada uma biblioteca de mutantes aleatórios pela técnica de mutagênese marcada com assinatura, e os mutantes foram selecionados negativamente em ensaio de invasão a fibroblastos aviários. Mutantes atenuados apresentaram mutação em genes do operon da fímbria do tipo 1 e nos genes de metabolismo de açúcares treA e bglB. Foram feitos mutantes específicos para o gene que codifica a enzima trealase periplasmática (TreA) e para o operon de metabolismo de β-glicosídeos (bgl). O mutante MT78ΔtreA apresentou, frente à cepa selvagem, diminuição na capacidade de adesão e invasão a fibroblastos aviários, na expressão da fímbria do tipo 1 e na capacidade de colonizar a bexiga de camundongos em modelo de infecção urinária. O mutante MT78Δbgl também apresentou, frente à cepa selvagem, diminuição na capacidade de adesão e invasão a fibroblastos aviários e na capacidade de colonizar a bexiga de camundongos em modelo de infecção urinária, mas não mostrou alteração na expressão da fímbria do tipo 1, medida por aglutinação de leveduras. Os resultados deste trabalho mostraram que a trealase periplasmática afeta a expressão da fímbria do tipo 1 e a virulência in vivo da cepa ExPEC MT78, enquanto o operon do metabolismo de β-glicosídeos afeta a virulência in vivo da cepa ExPEC MT78 por um mecanismo ainda não-elucidado. / Extraintestinal pathogenic E. coli (ExPEC) cause colibacillosis in birds, urinary tract infections and neonatal meningitis in humans. The ExPEC strain MT78 is virulent in vitro and in vivo, and is able to invade eukaryotic cells. In order to better understand the invasive phenotype of this strain, a library of random mutants was made using the signature-tagged mutagenesis approach. The mutants were negatively selected in invasion assays of avian fibroblasts. Attenuated mutants presented mutation in the type 1 fimbria operon and in the genes of sugar metabolism treA and bglB. Specific mutants were created for the periplasmic trehalase (TreA) gene and for the β-glycosides metabolism operon (bgl). The MT78ΔtreA mutant displayed, in comparison with the wild type strain, a reduction on the capacity of adhesion and invasion to avian fibroblastos, on type 1 fimbriae expression and on the capacity to colonize the bladder in a murine model of urinary tract infection. The MT78Δbgl mutant, compared to the wild type strain, also displayed a reduction on the capacity of adhesion and invasion to avian fibroblastos and on the capacity to colonize the bladder in a murine model of urinary tract infection, but did not show any difference on type 1 fimbriae expression as detected by yeast agglutination. Taken together, our results show that the periplasmic trehalase affects type 1 expression and in vivo virulence of the ExPEC strain MT78, and the operon for β-glycosides metabolism affects in vivo virulence by an as yet unidentified mechanism.

Salmonella enterica sérovar Typhi est une bactérie pathogène humain-spécifique et l’agent étiologique de la fièvre typhoïde. Parmi ses facteurs de virulence, il y a 14 systèmes d’adhésion putatifs nommés fimbriae qui ont été identifiés dans le génome de S. Typhi. Les fimbriae sont regroupés en opérons qui codent pour des structures protéiques extracellulaires, pour une machinerie de sécrétion et d’assemblage et parfois pour des régulateurs. Ceux-ci sont peu exprimés en conditions de laboratoire et peu étudiés chez S. Typhi. Parmi les 14 fimbriae de S. Typhi, 12 appartiennent à la classe des chaperon-placier, c’est-à-dire qu’ils possèdent un chaperon et un placier qui leur sont dédiés pour la formation de la structure fimbriaire. Je crois que ces fimbriae sont importants pour la pathogenèse de S. Typhi. Le but de ce projet est l’analyse fonctionnelle des fimbriae de type chaperon-placier chez S. Typhi. Pour ce faire, j’ai voulu établir une caractérisation générale des 12 fimbriae de type chaperon-placier, puis j’ai concentré l’étude sur la régulation de 2 de ces fimbriae, c’est-à-dire Fim et Std. La caractérisation générale des fimbriae de type chaperon-placier consistait à déterminer l’expression des promoteurs fimbriaires lors de la croissance en différentes conditions de culture mimant l’infection, à déterminer la présence et la morphologie des fimbriae à la surface de la bactérie et à évaluer l’effet des fimbriae sur la pathogenèse de S. Typhi (formation de biofilm, interactions avec les cellules de l’hôte et motilité bactérienne). L’expression maximale des fimbriae a été obtenue principalement en milieu minimal. J’ai observé pour la première fois 6 des 12 fimbriae par microscopie électronique à transmission. Chaque fimbria présentait des effets sur au moins une étape testée sur la pathogénèse. La régulation de std et fim a été étudiée en déterminant le rôle de régulateurs globaux et par criblage d’une banque de mutants par insertion de transposon. Principalement, j’ai découvert que le promoteur std était activé par Crp, responsable de la répression catabolique, tandis que fim voit son expression modulée par la chaîne de transport d’électrons (Ndh) et des perturbations de l’enveloppe (OmpR). Finalement, nos résultats démontrent que les fimbriae de type chaperon-placier sont importants pour la 6 pathogenèse de S. Typhi et que deux de ceux-ci sont régulés par des signaux environnementaux importants rencontrés par la bactérie lors de l’infection. / Salmonella enterica serovar Typhi is a human-specific pathogenic bacteria and the etiologic agent of typhoid fever. Among its virulence factors, there are 14 putative adhesion systems named fimbriae identified in the S. Typhi genome. Each fimbria is clustered in an operon that encodes for extracellular proteinaceous structures, for the secretion and assembly machinery and sometime for regulators. Fimbrial genes are poorly expressed under laboratory conditions, with few studied in S. Typhi. Among the 14 fimbriae, 12 belong to the chaperone-usher class, where each one encodes a dedicated chaperone and usher that form the fimbrial structure. I propose that fimbriae are important for S. Typhi pathogenesis. The aim of this project is the functional analysis of all the chaperone-usher fimbriae of S. Typhi. My goals were to establish a general characterization of the 12 chaperone-usher fimbriae, and to study specifically the regulation of 2 fimbriae, Fim and Std. The general characterization of chaperone-usher fimbriae includes the determination of the expression of fimbrial promoters in different growth conditions mimicking infection, the observation of the presence and morphology of fimbriae at the bacterial surface, and the evaluation of the role of fimbriae on S. Typhi pathogenesis (biofilm formation, host-cells interactions and motility). Fimbrial expression was generally higher when cells were grown in minimal medium. I was able to observe for the first time the presence of 6 out of 12 fimbriae by transmission electron microscopy. Regarding the role of fimbriae in pathogenesis, each fimbria was involved in at least one step. Regulation of std and fim was studied by evaluating the implication of several general regulators and by screening a transposon-based library. Overall, I discovered that the std promoter was activated by Crp, responsible of catabolic repression, and that fim was modulated by the activity of the electron transport chain and by envelope perturbations. Finally, my results demonstrated that the chaperone-usher fimbriae are important for S. Typhi pathogenesis and two of them are regulated by important environmental signals encountered during bacterial infection.

Salmonella enterica sérovar Typhi (S. Typhi) est l’agent responsable de la fièvre typhoïde et cause environ 200 000 morts et 27 millions de cas annuellement. C’est un pathogène entérique dont le réservoir est restreint à l’Homme. Les raisons de cette restriction d’hôte sont méconnues et pourraient dépendre de l’expression de facteurs d’adhésion à des étapes importantes au cours de la pathogenèse. L’annotation bioinformatique du génome de S. Typhi identifie 12 fimbriae de type chaperon-placier (FCP), un curli ainsi qu’un pilus de type IV. L’objectif de ce projet de recherche est d’étudier ces systèmes d’adhésion peu caractérisés. D’abord, le niveau d’expression de ces gènes a été évalué dans différentes conditions de culture in vitro en utilisant une approche de gènes rapporteurs. L’expression des 14 systèmes d’adhésion a été détectée. Nos résultats indiquent qu’une carence en fer favorise l’expression des opérons bcf et csg. Indépendamment du fer, l’expression de bcf, csg, pil, sef, sta, stc, stg et sth est influencée par la richesse nutritive du milieu. L’incubation en milieu LB liquide favorise l’expression de la plupart des systèmes d’adhésion par rapport à un milieu LB liquide sans agitation ou un milieu LB solide. En somme, l’expression des systèmes d’adhésion de S. Typhi a été observée et est influencée par des conditions environnementales. Dans un second volet, nous avons tent de surexprimer les différents systèmes d’adhésion chez une souche d’E. coli ou de S. Typhi afimbriaire. Avec cette approche, nous avons été en mesure de démontrer que l’opéron tcf encode pour un fimbria fonctionnel que l’on a pu observer en microscopie électronique. L’expression de tcf chez une souche afimbriaire d’E. coli et S. Typhi a également diminué leur capacité d’adhésion à des cellules épithéliales intestinales humaines lors d’essais in vitro. Nos observations démontrent que l’expression des systèmes d’adhésion retrouvés chez S. Typhi est influencée par les conditions enviroi9onnementales. Au moins un de ces systèmes est fonctionnel. Ceci suggère une contribution des systèmes d’adhésion retrouvés chez S. Typhi lors de l’interaction de ce pathogène avec l’humain. / Salmonella enterica serovar Typhi (S. Typhi) is the etiological agent of typhoid fever which causes more than 200 000 deaths and 27 million cases worldwide, mostly in south Asia. This pathogen can only cause significant symptoms in humans, which are the only recognized animal reservoir. This host restriction is not clearly understood and could depend on the expression of adhesion systems during critical pathogenesis steps. Bioinformatic studies on S. Typhi predict 12 chaperone-usher fimbriae, one curli and one type IV secretion system. The aim of the project was to study those poorly described adhesion systems using two different methodologies. First, transcription levels were evaluated in different in vitro growth conditions using both gfp and β-galactosidase reporter genes. The expression of the 14 adhesion systems was detected, even if some of them were poorly expressed. The expression of bcf and csg was higher during iron-deficiency. Also, the availability of nutrients had an impact on bcf, csg, pil, sef, sta, stc, stg and sth expression, independently of the presence of iron. Most of the adhesion systems showed higher expression levels in liquid LB media with aeration compared to the same media without aeration or supplemented with agar. Secondly, several S. Typhi adhesion systems were cloned into an inducible expression plasmid introduced in both an afimbriated E. coli K-12 strain (ORN172) and an afimbriated S. Typhi strain (ISP1820). This approach enabled us to directly observe the presence of tcf by electron microscopy. Furthermore, the expression of tcf was correlated with a reduction of the capacity of bacteria to adhere to INT-407 human intestinal epithelial cells in an in vitro assay. In summary, this work demonstrates that the putative adhesion systems found in S. Typhi can indeed be expressed and this expression can be regulated by environmental signals. Furthermore, tcf encodes for a functional fimbria which has never before been observed. Taken together, our results suggest a significant contribution of the putative adhesion systems during normal pathogenesis.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de β-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a γ-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.

Pseudomonas aeruginosa é uma gamaproteobactéria ubíqua capaz de infectar indivíduos imunocomprometidos e causar infecções hospitalares. Entre duas repetições diretas situadas na ilha de patogenicidade PAPI-1 da linhagem PA14, encontram-se dois grupos de genes transcritos em direções opostas. O primeiro, composto de pvrS, pvrR, rcsC e rcsB, codifica proteínas de sistemas de dois componentes e está relacionado com virulência, enquanto o segundo compreende cinco genes (cupD1-D5) e codifica uma fímbria do tipo chaperoneusher, com alta similaridade com o grupo cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Fímbrias da mesma família são importantes na patogenicidade de outras bactérias. Com o objetivo de estudar a relação entre esses dois grupos de genes, procurou-se caracterizar sua organização por ensaios de RT-PCR, que possibilitaram observar a disposição dos genes dos sistemas de dois componentes em dois operons distintos (pvrRS e rcsCB) e, pelo menos, cupD1-cupD2 como uma unidade transcricional, com indícios apontando para a organização de cupD1-D5 em um único operon. Os inícios de transcrição e as regiões promotoras de cada operon foram caracterizados por experimentos de RACE 5 e extensão de oligonucleotídeo marcado em busca de seqüências relevantes para a ativação da expressão desses genes. Visando investigar a regulação da expressão da fímbria CupD pelos sistemas de dois componentes codificados pelos genes adjacentes, foram realizados ensaios de qRT-PCR, os quais mostraram uma menor expressão de cupD na linhagem mutante para rcsB. RcsB apresenta um domínio de ligação a DNA e, apesar da falta de sucesso em ensaios de ligação dessa proteína à região promotora de cupD, os dados obtidos por qRT-PCR 1 indicam fortemente que essa proteína funciona como um ativador de transcrição dos genes da fímbria. Corroborando esses achados, somente quando se utilizou RNA extraído de P. aeruginosa PA14 superexpressando RcsB foi possível visualizar no gel a banda referente ao início de transcrição de cupD1-D2 no ensaio de extensão de oligonucleotídeo. Ao contrário do efeito positivo de RcsB observado na transcrição de cupD1, cupD2 e cupD5, a histidina quinase RcsC atua negativamente na expressão dos genes da fímbria, sugerindo que, nesse caso, sua atividade predominante sobre RcsB seja a de fosfatase. PvrS e PvrR parecem atuar de forma indireta e positiva sobre cupD. Como um segundo objetivo do trabalho, ensaios de virulência de P. aeruginosa no hospedeiro-modelo Dictyostelium discoideum foram otimizados, com o estabelecimento de uma técnica para se testar alguns genes estudados no laboratório que podem ser relevantes para a virulência dessa bactéria. Resultados desses ensaios confirmaram a atenuação de mutantes para uma suposta metiltransferase, já observada em modelos de planta, camundongo e drosófila. Em conjunto, os resultados desse trabalho tornam-se informações valiosas para serem usadas na pesquisa de controle de infecções por P. aeruginosa, que depende de fímbrias para colonizar com sucesso superfícies abióticas que servem de veículo de disseminação desse agente infeccioso e para que as bactérias possam persistir no organismo do hospedeiro. / Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria able to infect immunocompromised individuals and to cause nosocomial infections. Between two direct repeats in the PAPI-1 pathogenicity island present in strain PA14, there are two gene clusters transcribed in opposite directions. The first, composed of pvrS, pvrR, rcsC and rcsB, encodes two-component systems proteins and is implicated in virulence, whereas the second comprises five genes (cupD1-D5) encoding a chaperone-usher fimbria with high similarity to cupA, a gene cluster involved in biofilm formation in other strains of P. aeruginosa. Fimbriae belonging to the same family of Cup are related to pathogenicity of other bacteria. In order to study the relationship between these two clusters, the organization of the genes in operons was characterized using RT-PCR, which results lead to the conclusion that the twocomponent systems genes are arranged into two different operons (pvrSR and rcsCB) and that cupD1-D2 share the same promoter, with some evidences that the operons extends from cupD1 to cupD5. The transcription start sites and the promoter regions of each operon were characterized with RACE 5 and primer extension assays to look for sequences that could be relevant to the activation of expression of these genes. Quantitative RT-PCR assays were carried out to investigate whether the expression of CupD fimbriae is regulated by the twocomponent systems encoded by the adjacent genes and the results showed a lower expression of cupD in the rcsB mutant strain than in the wild-type. RcsB bears a DNA-binding domain and, although our assays of DNA-protein interactions have failed, data obtained by qRT-PCR 1 strongly indicate that this protein functions as a transcription activator of fimbrial genes. These findings were corroborated by the primer extension assay, in which the band corresponding to the transcriptional start of cupD1-D2 was visible only when the reaction was performed with the RNA extracted of P. aeruginosa overexpressing RcsB. Unlike the effect observed for RcsB in cupD1, cupD2 and cupD5 transcription, the histidine quinase RcsC acts negatively in the fimbrial genes expression, suggesting that it might function predominantly as a RcsB phosphatase. PvrS and PvrR seem to regulate cupD positively and indirectly. As a second aim of this work, virulence assays of P. aeruginosa in the model host Dictyostelium discoideum were optimized, and a technique for testing genes studied in the laboratory that could be important for Pseudomonas virulence was established. These assays confirmed the attenuation-in-virulence of strains mutant in a putative methyltransferase gene, as observed before in plant, mouse and drosophila models. The results obtained in this work may contribute to P. aeruginosa infection control research, since this bacterium depends on fimbriae to successfully colonize abiotic surfaces that act as a dissemination vehicle, and to allow the bacteria to persist into the host organism.

Pseudomonas aeruginosa é uma gamaproteobactéria ubíqua capaz de infectar indivíduos imunocomprometidos e causar infecções hospitalares. Entre duas repetições diretas situadas na ilha de patogenicidade PAPI-1 da linhagem PA14, encontram-se dois grupos de genes transcritos em direções opostas. O primeiro, composto de pvrS, pvrR, rcsC e rcsB, codifica proteínas de sistemas de dois componentes e está relacionado com virulência, enquanto o segundo compreende cinco genes (cupD1-D5) e codifica uma fímbria do tipo chaperoneusher, com alta similaridade com o grupo cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Fímbrias da mesma família são importantes na patogenicidade de outras bactérias. Com o objetivo de estudar a relação entre esses dois grupos de genes, procurou-se caracterizar sua organização por ensaios de RT-PCR, que possibilitaram observar a disposição dos genes dos sistemas de dois componentes em dois operons distintos (pvrRS e rcsCB) e, pelo menos, cupD1-cupD2 como uma unidade transcricional, com indícios apontando para a organização de cupD1-D5 em um único operon. Os inícios de transcrição e as regiões promotoras de cada operon foram caracterizados por experimentos de RACE 5 e extensão de oligonucleotídeo marcado em busca de seqüências relevantes para a ativação da expressão desses genes. Visando investigar a regulação da expressão da fímbria CupD pelos sistemas de dois componentes codificados pelos genes adjacentes, foram realizados ensaios de qRT-PCR, os quais mostraram uma menor expressão de cupD na linhagem mutante para rcsB. RcsB apresenta um domínio de ligação a DNA e, apesar da falta de sucesso em ensaios de ligação dessa proteína à região promotora de cupD, os dados obtidos por qRT-PCR 1 indicam fortemente que essa proteína funciona como um ativador de transcrição dos genes da fímbria. Corroborando esses achados, somente quando se utilizou RNA extraído de P. aeruginosa PA14 superexpressando RcsB foi possível visualizar no gel a banda referente ao início de transcrição de cupD1-D2 no ensaio de extensão de oligonucleotídeo. Ao contrário do efeito positivo de RcsB observado na transcrição de cupD1, cupD2 e cupD5, a histidina quinase RcsC atua negativamente na expressão dos genes da fímbria, sugerindo que, nesse caso, sua atividade predominante sobre RcsB seja a de fosfatase. PvrS e PvrR parecem atuar de forma indireta e positiva sobre cupD. Como um segundo objetivo do trabalho, ensaios de virulência de P. aeruginosa no hospedeiro-modelo Dictyostelium discoideum foram otimizados, com o estabelecimento de uma técnica para se testar alguns genes estudados no laboratório que podem ser relevantes para a virulência dessa bactéria. Resultados desses ensaios confirmaram a atenuação de mutantes para uma suposta metiltransferase, já observada em modelos de planta, camundongo e drosófila. Em conjunto, os resultados desse trabalho tornam-se informações valiosas para serem usadas na pesquisa de controle de infecções por P. aeruginosa, que depende de fímbrias para colonizar com sucesso superfícies abióticas que servem de veículo de disseminação desse agente infeccioso e para que as bactérias possam persistir no organismo do hospedeiro. / Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria able to infect immunocompromised individuals and to cause nosocomial infections. Between two direct repeats in the PAPI-1 pathogenicity island present in strain PA14, there are two gene clusters transcribed in opposite directions. The first, composed of pvrS, pvrR, rcsC and rcsB, encodes two-component systems proteins and is implicated in virulence, whereas the second comprises five genes (cupD1-D5) encoding a chaperone-usher fimbria with high similarity to cupA, a gene cluster involved in biofilm formation in other strains of P. aeruginosa. Fimbriae belonging to the same family of Cup are related to pathogenicity of other bacteria. In order to study the relationship between these two clusters, the organization of the genes in operons was characterized using RT-PCR, which results lead to the conclusion that the twocomponent systems genes are arranged into two different operons (pvrSR and rcsCB) and that cupD1-D2 share the same promoter, with some evidences that the operons extends from cupD1 to cupD5. The transcription start sites and the promoter regions of each operon were characterized with RACE 5 and primer extension assays to look for sequences that could be relevant to the activation of expression of these genes. Quantitative RT-PCR assays were carried out to investigate whether the expression of CupD fimbriae is regulated by the twocomponent systems encoded by the adjacent genes and the results showed a lower expression of cupD in the rcsB mutant strain than in the wild-type. RcsB bears a DNA-binding domain and, although our assays of DNA-protein interactions have failed, data obtained by qRT-PCR 1 strongly indicate that this protein functions as a transcription activator of fimbrial genes. These findings were corroborated by the primer extension assay, in which the band corresponding to the transcriptional start of cupD1-D2 was visible only when the reaction was performed with the RNA extracted of P. aeruginosa overexpressing RcsB. Unlike the effect observed for RcsB in cupD1, cupD2 and cupD5 transcription, the histidine quinase RcsC acts negatively in the fimbrial genes expression, suggesting that it might function predominantly as a RcsB phosphatase. PvrS and PvrR seem to regulate cupD positively and indirectly. As a second aim of this work, virulence assays of P. aeruginosa in the model host Dictyostelium discoideum were optimized, and a technique for testing genes studied in the laboratory that could be important for Pseudomonas virulence was established. These assays confirmed the attenuation-in-virulence of strains mutant in a putative methyltransferase gene, as observed before in plant, mouse and drosophila models. The results obtained in this work may contribute to P. aeruginosa infection control research, since this bacterium depends on fimbriae to successfully colonize abiotic surfaces that act as a dissemination vehicle, and to allow the bacteria to persist into the host organism.

Bacteria have the ability to sense different environmental signals. When an environmental stress is detected, bacteria rapidly adjust their gene expression profile to be able to survive and thrive. The transduction of such environmental signals often requires the coordinated involvement of several factors that constitute complex regulatory networks. Hence, depending on the combination of signals, a unique gene expression profile required to adapt to the specific stress conditions is generated. Proteins are the best-known regulatory factors. However, non-proteinaceous molecules are also important in signal-responsive control of bacterial gene expression. Alarmones are low molecular weight non-proteinaceous regulatory factors which can characteristically be rapidly turned-over to mediate instant changes in gene expression. One such alarmone is the modified nucleotide ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of this alarmone are expected to rapidly increase in response to any environmental stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that likewise directly targets RNA polymerase, has been suggested to be required for both the positive and negative regulation mediated by ppGpp in Escherichia coli. This thesis describes dissection of the role of ppGpp and DksA on transcriptional regulation, primarily using the fim genetic determinant that encodes for the type 1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial adhesion/invasion of bladder cells, as well as in biofilm formation. We found that ppGpp regulates phase variation by increasing the sub-population of cells that express the fimbriae. The effect of ppGpp was ultimately traced to its role in transcription of the fimB gene that encodes a recombinase involved in the phase variation process (paper 1). In contrast, we unexpectedly found that lack of DksA causes an increase, rather than a decrease, in transcription from the fimB P2 promoter in vivo. However, in vitro transcription studies demonstrated that ppGpp and DksA, both independently and co-dependently, stimulate transcription from the fimB P2 promoter. These seemingly contradictory results from the in vivo and in vitro transcriptional studies were shown to be, at least in part, a consequence of the increased association of Gre-factors with RNA polymerase that can occur in the absence of DksA in vivo (paper 2). The results outlined above have implications for the role of ppGpp and/or DksA in global gene expression. Using gene expression profile (microarray analysis) during the transition from logarithmic to stationary phase of E. coli, we found that while most of the genes regulated by ppGpp and DksA are regulated in the same direction by the two factors, many were not. In addition to the fim genes, genes involved in flagella functioning, taxis responses, and a few genes encoding different transport systems are also differentially regulated in ppGpp- and DksA-deficient strains in vivo. Our results clearly indicate that the effect of deficiencies in ppGpp and DksA is far more complex than phenotypic similarity of the corresponding mutants anticipated by the proposed concerted action of ppGpp and DksA on gene expression (paper 2 & 3).

La maladie de Crohn (MC) est une affection inflammatoire chronique du tube digestif dont l'étiologie est multifactorielle. Les lésions iléales des patients atteints de MC sont anormalement colonisées par des souches pathogènes de Escherichia coli appartenant au pathovar AIEC pour " Adherent-Invasive E. coli ". Ces souches sont capables d'adhérer et d'envahir les cellules épithéliales intestinales, et ont la capacité de survivre et de se multiplier fortement en macrophages en induisant une synthèse intense de TNF-α. L'objectif de ce travail s'inscrit dans la compréhension des mécanismes permettant aux bactéries AIEC de coloniser la muqueuse intestinale et d'induire les stades précoces de la pathologie. Une précédente étude menée au laboratoire avait permis de mettre en évidence l'importance de l'activation de la voie de régulation dépendante du facteur bactérien sigma alternatif RpoE (ou σE) dans le processus d'adhésion et d'invasion des cellules épithéliales intestinales par la souche AIEC de référence LF82 via l'expression des pili de type 1 et des flagelles. En continuité de ces travaux, nous montrons que l'activation de la voie de signalisation dépendante du facteur σE est également primordiale pour la capacité des souches AIEC à former des biofilms, et une analyse bioinformatique ayant pour but d'identifier les gènes régulés par σE a montré que l'opéron waaWVL, impliqué dans la biosynthèse du lipopolysaccharide, est primordial pour la formation de biofilm par les souches AIEC. De plus, nous avons mis en évidence que les long polar fimbriae (LPF) sont impliqués dans le ciblage de l'épithélium associé aux plaques de Peyer par les bactéries AIEC, et ceci en leur permettant de cibler spécifiquement les cellules M. L'inactivation du gène Nod2, gène de susceptibilité à la MC, conduit à une augmentation du nombre de plaques de Peyer ainsi que des cellules M à leur surface, indiquant que les bactéries AIEC pourraient tirer avantage d'une susceptibilité génétique pour cibler les plaques de Peyer.

Pseudomonas aeruginosa é uma proteobactéria do grupo gama, que pode se comportar como um patógeno oportunista. A linhagem PA14 apresenta duas ilhas de patogenicidade. A maior delas, PAPI-1, contém dois grupos de genes envolvidos com virulência, transcritos de maneira oposta e que estão entre duas seqüências repetidas diretas. O primeiro grupo compreende quatro genes dispostos em dois operons, que codificam para proteínas de sistemas de dois componentes (PvrS, PvrR, RcsC e RcsB). PvrS e RcsC são proteínas sensoras híbridas, que apresentam domínios de histidina-quinase e de reguladores de resposta. PvrR é um regulador de resposta com um domínio EAL com atividade de fosfodiesterase de diGMP cíclico e RcsB apresenta um domínio de ligação a DNA, além de um domínio fosfoaceptor. O outro grupo é composto de cinco genes, cupD1 a cupD5, que codificam para uma fímbria do tipo chaperone-usher e que apresenta alta similaridade com cupA, envolvido na formação de biofilme em outras linhagens de P. aeruginosa. Trabalhos anteriores mostraram que pvrS, pvrR, rcsC, rcsB e cupD2 estão relacionados com a virulência de PA14. Como estes grupos de genes parecem ter sido inseridos na ilha em um único evento de recombinação, este trabalho investigou se os sistemas de dois componentes estão relacionados com a regulação da expressão de cupD. Foi observado que a expressão de cupD é maior a 28ºC do que que a 37ºC e é influenciada positivamente pelo regulador global de expressão, MvaT, uma proteína tipo H-NS. Ensaios de β-galactosidase a partir de uma fusão de transcrição mostraram que a atividade promotora de cupD é cerca de 50% menor numa linhagem com deleção em rcsB em relação à linhagem selvagem. Nenhuma diferença consistente foi observada entre as linhagens com deleções em pvrS, pvrR, rcsC e rcsB e PA14 em relação a motilidade dos tipos swarming, swimming ou twitching ou à formação de biofilme. A linhagem de P. aeruginosa PA14 superexpressando RcsB mostrou níveis exacerbados de mRNA de cupD1, sendo a atuação de RcsB específica em cupD, já que os outros grupos de genes cup presentes em PA14 não mostraram a mesma variação na expressão, conforme analisado por RT-PCR quantitativo. Essa linhagem mostrou também um aumento na formação de biofilme, sem que a motilidade fosse alterada. Ainda visando elucidar os mecanismos de regulação de cupD, linhagens que superexpressam pvrR também foram analisadas quanto a estes fenótipos. Nesse caso, a superexpressão de pvrR diminuiu a formação de biofilme, conforme esperado, aumentou a motilidade do tipo swarming, porém não alterou a expressão de cupD. Os dados do presente trabalho demonstraram que a cupD é regulado pelos genes do sistemas de dois componentes adjacentes a ele e que o ativador de transcrição RcsB está relacionado com a formação de biofilme em tubos de vidro, provavelmente via a fímbria CupD. / Pseudomonas aeruginosa is a γ-proteobacteria that can behave as an opportunistic pathogen. The strain PA14 carries two pathogenicity islands, the largest of them, PAPI-1, contains two gene clusters between two direct repeat sequences that are transcribed in opposite directions and are involved in virulence. The first group consists of four genes arranged in two operons encoding two-component system proteins (PvrS, PvrR, RcsC and RcsB). PvrS and RcsC are hybrid sensor proteins, which contain domains of histidine kinase and response regulator domains. PvrR is a response regulator with a phosphodiesterase EAL domain and RcsB presents a C- terminal HTH DNA biding domain, in addition to a phosphoaceptor domain. The other group is composed of five genes, cupD1-5, that encodes components and assembly factors of a putative fimbrial CupD, which has high similarity with CupA, involved in the biofilm formation in other P. aeruginosa strains. Earlier work showed that pvrS, pvrR, rcsC, rcsB and cupD2 are related to the virulence of PA14. As these groups of genes appear to have been inserted on the island in a single event of recombination, this study investigated whether the two-component systems are related to the regulation of cupD expression. It was observed that cupD promoter activity is higher at 28oC than at 37oC and it is positively influenced by the global regulator, MvaT, a H-NS like protein. A lacZ transcriptional fusion showed about 50% less promoter activity of cupD from a strain with deletion in rcsB as compared to PA14. No consistent differences were found among the strains with deletions in pvrS, pvrR, rcsC and rcsB and PA14 on swarming, swimming and twitching motilities or biofilmsformation. A strain overexpressing overexpression showed heigher levels of cupD1mRNA of, and the role of RcsB as an activator is specific to cupD, as the other groups of cup genes present in PA14 did not show the same variation in the expression, as analyzed by quantitative RT-PCR. This strain also showed an increase in biofilm formation. In further assays aiming to elucidate the mechanisms of regulation of cupD, a strains overexpressing pvrR was also analyzed. Overexpression of pvrR decreased the formation of biofilm, as expected, and increased swarming motility, but did not alter the expression of cupD. The data from this study demonstrated that cupD is regulated by RcsB, and that this transcriptional activator is involved in the formation of biofilm in glass tubes, probably via CupD fimbriae.