CRISPR-Cas9 pour l'édition de génomes viraux et l'étude des gènes du phage virulent p2
|Abstract:||Phages are viruses that specifically infect and kill bacteria. They can be found in every ecosystem, including milk products. Despite decades of research, virulent phages infecting Lactococcus lactis strains used for milk fermentation still threatens the production process and cheese quality. Phage p2 is a model for the study of virulent lactococcal phages, but almost half of its genes encode proteins of unknown functions. The study of virulent phages is a challenge in itself because the modification of their genome is limited to the short infection cycle within a bacterial host. The first objective of this thesis was to adapt a CRISPR-Cas9-based genetic tool to inactivate genes of interest in the genome of phage p2. The CRISPR-Cas9 technology is derived from a natural antiviral system that allows some prokaryotes to defend themselves against invasive nucleic acids. The bacterial host of phage p2, L. lactis MG1363, is naturally deprived of this system. The second objective was to study the viral and bacterial proteomes during phage infection, making use of high throughput mass spectrometry-based proteomics. Lastly, the third objective was to study the roles of inactivated genes on phage replication and bacterial growth, including the impact on their proteomes. Amongst other, with an integrative approach combining genomic, phenotypic and proteomic analysis, I compared the mutant phage p2Δ47, lacking a functional orf47 gene, to the wild-type phage p2. These analyses allowed me to hypothesize about protein ORF47 function. Phages are ubiquitous, abundant and can replicate quickly. Despite their importance and over a century of research, many aspects of phage biology are still poorly understood. By designing a tool for the modification of virulent phages and by optimizing protocols for proteomic analysis, I developed a robust pipeline to investigate uncharacterized phage proteins and to study phage-host interactions.|
|Document Type:||Thèse de doctorat|
|Open Access Date:||2 October 2019|
|Collection:||Thèses et mémoires|
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