I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

La fibrose kystique (FK) est la maladie génétique mortelle la plus répandue chez les jeunes Canadiens. La colonisation des poumons par une bactérie opportuniste, Pseudomonas aeruginosa, est la principale cause de morbidité et de mortalité chez les patients FK. La maladie est causée par des mutations du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator) qui agit comme canal à ions chlorures. Ces modifications entraînent notamment une surexpression d'oligosaccharides fucosylés à la surface de l'épithélium pulmonaire. Le processus d'adhésion de la bactérie à la surface des cellules de l'épithélium pulmonaire est causé par la présence de deux lectines à la surface de la bactérie. Nous nous intéressons principalement à l'une d'entre elles, une lectine calcium dépendante qui reconnaît particulièrement le L-fucose (PA-IlL). Des études cristallographiques menées sur PA-IlL, en complexe avec divers ligands naturels tel que le Lewis a, ont permis d'identifier plusieurs éléments essentiels à l'obtention d'une forte interaction ligand-lectine. Basé sur ces études, le projet de recherche a consisté en la synthèse de différents analogues d'un disaccharide composé d'une unité fucose et glucosamine du type L-Fuc-a(1→4)-D-GIcNAcβ intimement impliqué dans le site de liaison de la protéine. Différents glycoclusters du disaccharide ont été synthétisés en utilisant la 'Click Chemistry'. De plus, quelques disaccharides modifiés en position C-2 et C-6 ont aussi été synthétisés. Les disaccharides ainsi que les glycoclusters ont été testés sur la PA-IlL en utilisant un test d'inhibition compétitive (ELLA). Les dérivés disaccharidiques ont montrés une constante de dissociation (Kd = 310 nM) dans le même ordre de grandeur que celle du meilleur ligand naturel Lewis a (Kd = 210 nM) connu jusqu'à maintenant pour la PA-IlL. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Pseudomonas aeruginosa, Fibrose kystique, Lectine PA-IlL, Lewis a, Glycosylation, «click chemistry», Glycoclusters.

Differential gene expression of P. aeruginosa in a rhizosphere biofilm on poplar tree roots was examined in order to identify new virulence factors from this human pathogen. Changes in gene expression for poplar trees contacted with P. aeruginosa was examined as well to identify the response of poplar roots to P. aeruginosa infection. This is the first study of the whole-transcriptome analysis of P. aeruginosa on a plant tree root. The 20 most highly-induced genes of P. aeruginosa were examined for their role in biofilm formation, rhizosphere colonization, barley germination, and poplar tree killing assays. Seven previously uncharacterized virulence genes (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150, and PA4295) were identified. The role of PA2663, a hypothetical protein discovered in the microarrays of P. aeruginosa while killing poplar trees, was examined in further detail. Expression of PA2663 protein increases biofilm formation in P. aeruginosa PAO1 drastically. By complementing the PA2663 mutation in trans and by studying with DNA microarrays and RT-PCR the PA2663 mutant vs. the wild-type strain, PA2663 was confirmed to be related to biofilm formation and was found that it is the first protein to control the psl operon in P. aeruginosa PAO1. Furthermore, PA2663 protein increases pyoverdine synthesis and quorum sensing (QS)- regulated phenotypes. A biofilm formation-related hypothetical protein, PA0939, was identified in this study. The effects of indole and 7-hydroxyindole on P. aeruginosa virulence factors were also examined for the first time. Indole and 7HI repressed expression of mexGHI-opmD multidrug efflux pump genes and genes involved in synthesis of QS-regulated virulence factors (pyocyanin, rhamnolipid, PQS, and pyoverdine production). In addition, the effects of an anti-cancer uracil analog, 5-fluorouracil (5-FU) on P. aeruginosa virulence factors and E. coli K-12 biofilm formation were examined. 5-FU repressed biofilm formation, abolished quorum-sensing phenotypes, and reduced virulence in P. aeruginosa. DNA microarray and biofilm studies with 5-FU in E. coli revealed that 5-FU controls biofilm formation through the AriR protein in E. coli K-12 strain. The effects of lsrR and lsrK mutations on E. coli biofilm formation were also examined by flow cell experiments.

Reproduction de : Thèse d'exercice : Médecine. Pneumologie : Nantes : 2008. / Bibliogr.

Zugl.: Berlin, Techn. Univ., Diss., 2009

Thesis (PhD)--Montana State University--Bozeman, 2009. / Typescript. Chairperson, Graduate Committee: Michael Franklin. Includes bibliographical references.

Thesis (M.S.)--University of North Carolina at Wilmington, 2003. / Includes bibliographical references (leaves : 85-91).

Thesis (M. Sc.)--University of Hong Kong, 1970. / Mimeographed.

Transcription and translation are the two most important central mechanisms to control gene regulation in living organism. Although these two mechanisms have been studied intensively for last several decades, it is still not clear how all the information encoded on genomic DNA is converted to mRNA and proteins, the molecular functional components that change the characteristics of cells, depending on their needs. Here, I investigated the gene regulation of opportunistic bacterium Pseudomonas aeruginosa, using recently developed high-throughput techniques, microarray for transcriptomics and LC-MS/MS for proteomics. By analyzing transcriptome of 17 strains isolated over time from three individuals with cystic fibrosis, I identified 24 genes showing significant expression changes consistently across all strains, as evidence of parallel evolution of common traits that were beneficial in establishing chronic infection. Also, by analyzing proteome and transcriptome of two reference Pseudomonas aeruginosa strains, PAO1 and PA14, under growth condition mimicking in vivo nutrition environment in cystic fibrosis patients, I revealed that protein abundances are less correlated than mRNA abundance between them, and many proteins known as virulence factors showed different abundances only in protein level, demonstrating that post-transcriptional regulation is important to understand pathogenesis of Pseudomonas aeruginosa. To boost sensitivity both in identification and quantification in shotgun proteomics, I also created a novel integrative database search algorithm, and released freely available software package termed in MSblender. These results would be valuable information for the research community to understand the regulatory mechanism of gene expression both in transcription and translation, especially related to infectious diseases caused by pathogenic bacteria. Also, I present an integrative analysis method would be generally beneficial to extract more information from conventionally used shotgun proteomics experiments. / text

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy