Génomique fonctionnelle de Pseudomonas aeruginosa et analyse moléculaire fine d'un facteur sigma-anti-sigma
|Advisor:||Lévesque, Roger C.|
|Abstract:||Pseudomonas aeruginosa is an opportunistic pathogen that can cause pulmonary infections in cystic fibrosis patients (CF). To overcome innate self defense, P. aeruginosa possesses a wide arsenal of virulence factors. These include degradation enzymes such as proteases, lipases and phospholipases and the production of three specific toxins: exotoxin A and exoenzymes S and T. Sequencing of the complete P. aeruginosa chromosome (strain PAO1) of 6.3 Mb revealed a highly regulated and complex genomic organization. In order to better understand host-pathogen molecular interactions, we developped a new signature-tagged mutagenesis (STM) approach based on PCR screening. The PCR-based STM technology lead to the identification of 214 mutants deficient in their ability to maintain a chronic pulmonary infection in the rat lung. In that pool of STM mutants, STM2895, which contains a transposon insertion in functional PA2895, was the most frequently drafted during the whole mutant library screening. Phenotypic analyses of the STM2895 strain allowed us to identify an exoprotease production defect as compared with wild type strain PAO1. The biochemical characterization of that proteolytic default using specific degradation assays combined with western blotting revealed that at least two (LasA and LasB) of the four major exoproteases from P. aeruginosa STM2895 strain are inactive. In fact, LasA and LasB elastases were shown to be present in the STM2895 culture supernatant, correctly processed but inactive due to a probable misfolding of proteins. The PA2895 gene (unknown function) encodes a protein with a predicted transmembrane domain. Basic genomic context analyses strongly suggest a cotranscription unit with the downstream gene PA2896, a putative sigma 70 factor from ECF (extracytoplasmic function) type. Microarray experiments on the STM2895 strain and an insertional mutant of the PA2896 gene were performed to establish a link between the putative PA2895-PA2896 operon and the metabolism of iron. Transcriptome analysis also demonstrated a repressive action of PA2895 on the transcription of PA2896 putative sigma factor. Finally, in vivo studies in the rat lung chronic infection model clearly showed a ten-fold decrease in survival capacity of the mutant strain when compared to the PAO1 wild-type strain.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||12 April 2018|
|Collection:||Thèses et mémoires|
All documents in CorpusUL are protected by Copyright Act of Canada.