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Tableau d'honneur de la Faculté des études supérieures et postdoctorales, 2017-2018 / Pseudomonas aeruginosa est une bactérie pathogène opportuniste qui cause des infections pulmonaires chroniques chez les gens atteints de fibrose kystique (FK). Certaines souches, adaptées au microenvironnement des poumons FK, se distinguent, entre autres, par une résistance accrue aux antibiotiques et une formation abondante de biofilm. L'étude d'isolats provenant de patients FK est nécessaire afin de comprendre les déterminants génétiques expliquant cette adaptation et trouver de nouveaux moyens de lutter contre cette bactérie. Une librairie de mutants de LESB58, souche épidémique chez les patients FK, a été créée en 2009. Dans l’étude présentée ici, un criblage successif dans trois hôtes différents a été réalisé sur ces mutants, permettant d'identifier un mutant particulièrement peu virulent : le mutant STM PALES_11731. Ce mutant, contrairement à la souche sauvage, s'est avéré incapable de former des biofilm-like structures (BLS), une forme d'agrégation bactérienne ressemblant à du biofilm, mais flottant dans le milieu de culture. Afin d’évaluer ce phénomène chez différentes souches, la formation de biofilm adhéré et de BLS chez LESB58 et trois autres souches (la référence PAO1 et deux souches environnementales, PPF-1 et Urg-7) a été comparée à l'aide d'une nouvelle approche de quantification par analyse d'images. L’effet de cations divalents sur la formation de ces structures a également été exploré. Une diversité dans les phénotypes de formation de biofilms et de BLS et a été observée. Un manque de corrélation entre la formation de biofilms adhérés et de BLS a également été noté, suggérant que ces phénomènes pourraient ne pas être directement reliés et soulevant plusieurs questions sur les mécanismes de formation des BLS. Les résultats de ce projet de maîtrise indiquent que les BLS pourraient jouer un rôle dans la virulence de P. aeruginosa chez les patients FK et soulèvent l’importance de déterminer les mécanismes moléculaires ou physiques responsables de leur formation. / Pseudomonas aeruginosa is an opportunistic bacterial pathogen known to cause chronic lung infections in cystic fibrosis (CF) patients. Some strains, adapted to the CF lung microenvironment, show distinguishing phenotypes such as an increased resistance to antibiotic treatments and an enhanced biofilm formation. The study of P. aeruginosa isolates from CF patients is necessary to understand the genetic determinants explaining this adaptation and to allow the development of new ways to fight this bacterium. A library of LESB58 mutants has been created in 2009. LESB58 is an epidemic strain among CF patients. In the study presented here, a sequential screening in three different hosts has been performed, leading to the identification of a mutant with a strong virulence defect: the STM PALES_11731 mutant. This mutant, contrary to the wild type, was unable to form biofilm-like structures (BLSs), a type of bacterial aggregation resembling biofilm, but floating in the culture medium. To assess this phenomenon among P. aeruginosa strains, the formation of adhered biofilm and BLSs in LESB58 and three other strains (the reference strain PAO1 and two environmental strains, PPF-1 and Urg-7) was compared using a novel image analysis quantification approach. The impact of the addition of divalent cations on the formation of those structures was also assessed. The results obtained demonstrate some diversity of biofilm and BLS formation in this bacterial species. They also reveal a lack of correlation between the BLS and adhered biofilm formation response to the ion treatment, suggesting that these two phenomena might not be directly related and raising questions about the mechanisms of BLS formation. The results of this project indicate that BLSs could play a role in the virulence of P. aeruginosa in CF patients and highlight the importance, in a future study, of studying the molecular and physical mechanisms responsible for their formation.

The bacterium Burkholderia strain 2.2 N is a soil isolate and a member of a group of non-obligative predator bacteria that can prey on other microorganisms or grow saprophytically. The bacterium has anti-bacterial, anti-fungal, anti-yeast and anti-protozoan activities. Burkholderia strain 2.2 N culture shows hydrolysis on Milk Casein Agar, indicating the bacterium also produces a protease. Azocoll hydrolysis was used to detect and measure protease activity. Protease activity was two-fold higher at pH of 7.5 than pH 9.0 and 25-fold at pH 4.0. Cultures grown in media containing 1.0 % yeast extract (YE), tryptic soy, tryptone or beef extract had protease activity, whereas activity was absent in cultures grown in media containing peptone, soytone, casitone, or tryptone as sole protein source. Addition of 1.0 % sucrose or glucose to 1.0 % YE medium increased protease activity 1.8-fold and 1.4-fold, respectively. Protease activity was 2-fold higher in cultures grown in media containing 1.0 % YE and 10 mM MgCl₂ or FeCl₂, than in 1.0 % YE medium lacking metals or containing 10 mM MnCl2 or CaCl2. The 1.0 % YE medium containing either ZnCl₂ or CuCl₂ lacked protease activity (< 5.0 %). In cultures grown in 1.0 % YE at 30°C with rotation at 120 rpm, protease activity was higher in stationary phase (0.38 units /mg protein) than in exponential phase (0.04 units/mg protein). The Burkholderia strain 2.2 N protease is evidently exported from cells because 86 % of the total proteolytic activity of cells was found in the cell-free culture medium. The cell free filtered culture supernatant medium assayed at 4°C had protease activity, however at a three-fold lower specific activity compared to the same supernatant assayed at 3°C. Protease activity was lower in filtered culture supernatants stored at 4°C, room temperature, and 30°C. Protease activity in samples stored at 4°C was only 40 % (24 hours) and 15 % (48 hours) of activity at time zero. Protease activity in samples stored at room temperature was only 45 % (24 hours) and 35 % (48 hours) of activity at time zero. Protease activity in samples stored at 30°C was only 78 % (24 hours) and 9 % (48 hours) of activity at time zero. Purification of the protease from filtered culture supernatant medium by ammonium sulfate precipitation, increased the protease activity 20-fold. An eluted protein fraction from DEAE-Sepharose column chromatography had 50-fold higher protease activity. Protease activity was inhibited by 10 mM 1-10-phenathroline, EDTA and EGTA, all metalloprotease inhibitors. Purified protease activity inactivated with 10 mM 1-10-phenanthroline or 10 mM EGTA was regained through the addition of Ca2+ or Mg2+. Protease activity was reduced by exposure to dithiothreitol (29 % with 1 mM and 84 % with 10 mM), a disulfide bond inhibitor. Protease activity was not inhibited by leupeptin or phenylmethylsulphonyl flouride. Casein polyacrylamide zymography revealed a band of hydrolysis at approximately 60,000 Da. SDS-PAGE resolved a doublet band present at 60,000 Da present in both the filtered culture supernatant sample and the ammonium sulfate / DEAE-Shepharose column chromatography purified protease sample. Burkholderia strain 2.2 N protease is a metalloprotease with a broad temperature range of activity. It has a molecular weight of approximately 60,000 Da. / Master of Science

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 150-153).