Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-16T21:26:49Z No. of bitstreams: 1 Elsio Augusto Wunder Júnior Patogênese da leptospirose...pdf: 2609622 bytes, checksum: aa34c959355bc105a6e849e74258cf78 (MD5) / Made available in DSpace on 2012-07-16T21:26:49Z (GMT). No. of bitstreams: 1 Elsio Augusto Wunder Júnior Patogênese da leptospirose...pdf: 2609622 bytes, checksum: aa34c959355bc105a6e849e74258cf78 (MD5) Previous issue date: 2010 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A leptospirose é uma zoonose de importância global e um importante problema de saúde pública principalmente em países em desenvolvimento. É causada por bactérias do gênero Leptospira, uma espiroqueta móvel e de alta morbidade capaz de se disseminar nos tecidos e causar doença crônica em animais hospedeiros. Uma barreira importante para o controle e prevenção da doença tem sido o pouco conhecimento da patogênese do agente, em parte pela falta de ferramentas disponíveis e eficazes de manipulação genética. Um dos objetivos desse estudo foi caracterizar duas cepas mutantes de Leptospira interrogans. A interrupção do gene lipl32, que codifica para a proteína LipL32, a mais abundante proteína no gênero Leptospira e expressa somente na superfície das leptospiras patogênicas, foi realizada através da inserção do transposon Himar1 no sorovar Manilae. A cepa mutante não apresentou nenhuma diferença de crescimento ou de aderência em componentes da matriz celular, comparada com a cepa parental. O mutante foi capaz de produzir doença aguda no modelo animal de hamster e causar colonização crônica no modelo animal de rato, mostrando que LipL32 não possui um papel nestes modelos de infecção. A interrupção do gene ligB foi realizada com a utilização, pela primeira vez em leptospiras patogênicas, da técnica de recombinação homóloga por mutagênese dirigida, onde o gene que codifica para a proteína LigB teve uma parte substituída por um cassete de resistência de espectinomicina (Spcr). Essa proteína, identificada como um possível fator de virulência, reconhecida pelo soro de pacientes infectados e importante para a aderência em componentes da matriz celular, mostrou não ser importante para a infecção aguda ou crônica, quando testada frente aos modelos animais, além de não ser necessária para a aderência em cultura de células. Outro objetivo desse trabalho foi estudar a cinética de disseminação da Leptospira interrogans no modelo animal de hamster, utilizando uma dose alta (108 leptospiras) e baixa (250 leptospiras) de inóculo, além de diferentes rotas de infecção. Nossos resultados demonstraram que leptospiras se disseminam rapidamente em todos os tecidos 01 hora após a infecção com uma alta dose de inóculo e que possivelmente a carga do agente nos tecidos é mais importante para a patogênese do que a sua habilidade para a disseminação. Também demonstramos que a motilidade não é essencial para disseminação, mas pode ser essencial para a carga nos tecidos e letalidade. / Leptospirosis is a worldwide zoonosis and a major public health problem especially important in developing countries. Caused by bacteria of Leptospira genus, a motile lifethreatening spirochete which is able to disseminate to tissues and causes chronic carriage in animal hosts. A significant barrier to the control and prevention of leptospirosis has been the limited understanding of its pathogenesis, due in part to the lack of tools available for the genetic manipulation of this pathogen. One of the purposes of this study was to characterize two mutant strains of Leptospira interrogans. Interruption of lipL32 gene, encoding LipL32, the most abundant protein of pathogenic leptospires and its major outermembrane lipoprotein, was achieved in serovar Manilae using transposon mutagenesis with Himar1. The mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. The mutant was able to cause acute severe disease manifestations in the hamster model and chronic colonization in the rat model, showing that LipL32 doesn’t play a role in neither of those models of infection. Interruption of ligB gene was achieved using the homologous recombination by target mutagenesis for the first time in pathogenic leptospires and a spectinomycin resistance (Spcr) gene replaced a portion of the ligB coding sequence. This Lig protein, previously identified as a putative virulence factor, recognized by the sera of infected patients and important for the adherence in extracellular matrix components, showed not to be important for acute or chronic infection when tested with animal models and it’s not required to mediate bacterial adherence to cultured cells. Another purpose of this study was to determine and analyze the kinetics of dissemination of Leptospira interrogans in the hamster model, using a high (108 leptospires) and low (250 leptospires) dose of inoculum and different routes of infection. Our results demonstrated that leptospires can rapidly disseminate through all tissues after 1 hour post-challenge with a high inoculum dose and that perhaps the load of the agent in target tissues is more important for pathogenesis than its ability for dissemination. We also demonstrate that motility is not essential for dissemination but can be essential for tissue load and lethality.

The work in this thesis is concerned with the study of bacterial adaptation on short and long timescales. In the first section, consisting of three chapters, I describe a recently developed high-throughput technology for probing gene function, transposon-insertion sequencing, and its application to the study of functional differences between two important human pathogens, Salmonella enterica subspecies enterica serovars Typhi and Typhimurium. In a first study, I use transposon-insertion sequencing to probe differences in gene requirements during growth on rich laboratory media, revealing differences in serovar requirements for genes involved in iron-utilization and cell-surface structure biogenesis, as well as in requirements for non-coding RNA. In a second study I more directly probe the genomic features responsible for differences in serovar pathogenicity by analyzing transposon-insertion sequencing data produced following a two hour infection of human macrophage, revealing large differences in the selective pressures felt by these two closely related serovars in the same environment. The second section, consisting of two chapters, uses statistical models of sequence variation, i.e. covariance models, to examine the evolution of intrinsic termination across the bacterial kingdom. A first collaborative study provides background and motivation in the form of a method for identifying Rho-independent terminators using covariance models built from deep alignments of experimentally-verified terminators from Escherichia coli and Bacillus subtilis. In the course of the development of this method I discovered a novel putative intrinsic terminator in Mycobacterium tuberculosis. In the final chapter, I extend this approach to de novo discovery of intrinsic termination motifs across the bacterial phylogeny. I present evidence for lineage-specific variations in canonical Rho-independent terminator composition, as well as discover seven non-canonical putative termination motifs. Using a collection of publicly available RNA-seq datasets, I provide evidence for the function of some of these elements as bona fide transcriptional attenuators.

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Bacillus amyloliquenaciense FZB42 ist ein bekanntes Pflanzenwachstum-stimulierendes Rhizobakterium. Es produziert neben einer Vielzahl an Sekundärmetaboliten mit antibakterieller und antifungaler Wirkung, auch das Pflanzenhormon IAA. Obwohl viele dieser Mechanismen diskutiert werden, ist wenig darüber bekannt, auf welche Weise die Bakterien das Pflanzenwachstum fördern. In dieser Arbeit wurde eine Transposonmutagenese mithilfe des ‘mariner-transposons’ durchgeführt, und so eine Transposonbibliothek erstellt. Diese wurde dann auf geeignete Phänotypen untersucht, um die Gene zu finden, welche bestimmte Phänotypen verursachen. So konnten drei Mutanten erzeugt werden, die auf Grund der gestörten Biofilmbildung und der Fähigkeit zu schwärmen die Pflanzenwurzeln nicht mehr kolonialisieren konnten. Eine solche degU-Mutante, welche in der Biofilmbildung und ‚Swarming’ defizitär war und zwei Mutanten (yusV und pabB), die eine Beeinträchtigung in der Biofilmbildung aufwiesen, konnten durch Komplementation und Retransformation bestätigt werden. Mithilfe des Lemna-Biosystems und anderer Analysen mit A. thaliana konnten drei Gene bei B. amyloliqufaciens FZB42 gefunden werden, die wichtig für die Förderung des Pflanzenwachstums sind. Koloniesierungsexperimente der Wurzeln von A. thaliana mit diesen Mutanten zeigten deutlich verändertes Wachstum, verglichen mit dem Wildtypstamm. Ein weiteres Ziel dieser Arbeit war es neue Antibiotika in Mutanten, die in ihren nicht-ribosomalen Synthesen blockiert sind, zu finden. So konnten durch die Untersuchungen der Transposonbibliothek der Mutanten zwei neue Antibiotika entdeckt werden. Genauere Analysen dieser Antibiotika bestätigten, dass es sich um ein neues Bacteriocin (Amylocyclicin A) und ein neues Thiazol/Oxazole-modifiziertes Microcin (Plantazolicin) handelt. Die abschließenden Arbeiten beschäftigten sich dann mit Untersuchungen von Genen, welche für die Produktion von Substanzen gegen Nematoden verantwortlich sind. Hierbei konnten vier Mutanten gefunden werden, die durch eine Transposoninsertion eine schlechtere. / Bacillus amyloliqufaciens FZB42 has been known as PGPR which has an impressive effect to improve plant growth. It produces not only vast array of secondary metabolites with antibacterial and antifungal activities, but also produces the plant hormone IAA. Although many mechanisms have been elucidated, our knowledge about basic molecular mechanisms responsible for its beneficial action is far from complete. In this study, transposon mutagenesis based on mariner tranposon was applied to generate tranposon library which then was screened to identify the genes involved in plant growth-promoting activity. Three mutants that were impaired in their ability to colonize plant surface due to defects in biofilm formation and swarming motility were found. One mutant (degU mutant) showed defect in biofilm formation and swarming motility, as well, two mutants (yusV mutant and pabB mutant) impaired in biofilm formation were confirmed by complementation and retransformation. Screening by the Lemna biosystem and further assays with A. thaliana revealed three genes responsible for reduction in plant growth promoting activity of B. amyloliqufaciens FZB42. Colonization studies of these mutants in A. thaliana roots revealed patterns different to the wild type. A further issue pursued in this study was to discover new antibiotics using a mutant which has been blocked in its nonribosomally pathway. Screening of tranposon librabries from this mutant led to the finding of two novel ribosomally synthesized antibiotics. Further characterization revealed that these new antibiotics belonged to a novel bacteriocin (Amylocyclicin A) and a novel thiazole/oxazole-modified microcin (Plantazolicin). Last work in this study was looking for genes responsible for nematocidal production. Four mutants which showed reduction in nematocidal activity due to transposon insertion were found.

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Pour étudier la capacité cellulaire à s’adapter aux changements de signalisation dépendante des RTKs, nous utilisons un modèle de souris où l’expression du RTK Met sauvage peut être accrue dans un tissu spécifique. La plupart des tissus se protègent contre cette expression anormale des RTK. Mais certains types cellulaires sont sensibles aux altérations des RTKs, c’est le cas du mésenchyme du membre pendant l’embryogenèse. En effet, l’expression de certains gènes du mésenchyme est modifiée et celui-ci n’est plus accessible aux myoblastes qui le colonisent, conduisant à des déficits des muscles du membre. Chez l’adulte une augmentation de l’expression de Met dans le foie (Alb-R26Met) perturbe l’homéostasie tissulaire, conduisant à la tumorigenèse. Pour identifier des gènes qui coopèrent avec les RTKs pendant l’initiation de la tumorigenèse, nous avons combiné les souris Alb-R26Met avec le système de mutagenèse Sleeping Beauty (SB) transposon. 285 gènes putatifs liés au cancer ont été identifiés. Certains sont des proto-oncogènes ou suppresseurs de tumeurs déjà connus, validant le système. D’autres gènes n’avaient, jusqu’à présent, jamais été associés à ce processus. 9 candidats ont été fonctionnellement validés. Pour identifier des signaux assurant le maintien de la tumeur, nous avons analysé le phosphokinome, testé l’efficacité de composés et identifié de nouvelles combinaisons de drogues qui agissent en synergie pour tuer les cellules cancéreuses dérivées de Alb-R26Met. En conclusion, ces travaux montrent qu’une approche génétique non-biaisée combinée à une approche génomique permet d’identifier de nouveaux mécanismes pertinents pour la biologie du cancer. / We explore the cell competence to deal with slight changes in RTK inputs during embryogenesis and tissue homeostasis using a mouse model in which wild-type RTK Met levels can be moderately enhanced in a tissue specific manner. Most tissues buffer enhanced RTK levels thus avoiding perturbation of developmental programs and tissue homeostasis. Nevertheless, certain cell types are vulnerable to RTK levels. During embryogenesis, the limb mesenchyme is sensitive to alterations of the spatial distribution of RTKs, as illustrated by gene expression changes and by loss of accessibility to incoming myoblasts, which lead to limb muscle defects. At adulthood, liver enhanced Met levels (Alb-R26Met) perturbs tissue homeostasis, leading to tumorigenesis. To uncover new genes that cooperate with RTKs during tumour initiation, we combined Alb-R26Met mice with the Sleeping Beauty (SB) transposon mutagenesis system. 285 putative cancer-related genes have been identified. Some correspond to known proto-oncogenes or tumour suppressors, thus validating the overall strategy we employed for cancer gene discovery. Others have not been previously linked to cancer. 9 new tumour suppressors have been functionally validated, demonstrating the validity of our screen strategy. To identify signals involved in tumour maintenance, we employed a phosphokinome-guided drug screen and identified new synergistic drugs deleterious for cancer cells modelled by the Alb-R26Met genetic setting. The overall strategy and outcomes strengthen the value of combining unbiased genetic and genomic approaches to identify new mechanisms relevant for cancer biology and new therapeutic interventions.