Leveraging CRISPR-Cas to Examine the Interplay Between Legionella pneumophila and Mobile Genetic Elements

Legionella pneumophila is a Gram-negative, intracellular bacterium and an accidental human pathogen. Much of the research to date has focused on L. pneumophila's ability to cause disease, and while this is important for developing treatments, studying the factors that influence its environmental persistence is critical to mitigating future outbreaks of disease. To this end, I focused on leveraging the CRISPR-Cas adaptive immune system to examine the impact that mobile genetic elements could have on L. pneumophila. I provided bioinformatic and experimental evidence to support the hypothesis that plasmid-based CRISPR-Cas systems are likely circulated by horizontal gene transfer, then leveraged by the bacterium to strengthen and restore its defences against environmental threats. Next, I expanded on our lab's previous work to generate a comprehensive, bioinformatic catalogue of the mobile genetic elements that L. pneumophila encounters in its environment. Using an expanded catalogue of CRISPR spacer sequences and a custom bioinformatics workflow, I showed that one or more lytic gokushoviruses and the prophage-like Legionella mobile element-1 (LME-1) are frequently encountered by L. pneumophila, providing the first evidence that the bacterium is challenged by lytic phages in the environment. In light of my findings, I re-visited the hypothesis that LME-1 could form phage-like particles under one or more experimental conditions, as opposed to being a phage remnant. I used transmission electron microscopy to show that under axenic growth conditions in rich media, LME-1 could form podophage-like particles, and that these particles were abolished in LME-1 mutants. I also showed that the LME-1 genome was protected from DNase treatment in extracellular fractions, and that a Xer recombinase, XerL, was implicated in LME-1 excision. Finally, I designed and validated a new assay to assess if the LME-1 phage-like particle is required for transfer between Legionella strains. Taken together, the data presented in my graduate research thesis highlight the role that mobile genetic elements could have on L. pneumophila's environmental persistence, and provide avenues for future research to mitigate outbreaks of Legionnaires' disease.

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