Abstract
Bacteriophages (phages) are bacterial infecting viruses that are co-evolving alongside their hosts. Their ability to infect specific host bacteria makes them a promising therapeutic tool against antibiotic-resistant infections in both clinical and agricultural settings (phage therapy). Phages are extraordinarily diverse and infect a wide range of bacterial hosts, yet many of their genes remain annotated as hypothetical, leaving their roles in host–phage interactions unresolved. One area of growing interest is the Chimalliviridae jumbo phage family, with genomes ≥200 kb, they avoid host DNA targeting defence systems by forming a proteinaceous nucleus-like structure around genome during infection. Their large genomes encode numerous genes, many of which remain functionally uncharacterised. This thesis investigates host–phage interactions in such systems, focusing on identifying essential host factors and phage genes required for successful infection.
Past assumptions of jumbo phage meant that their large genomes were assumed to allow them to be more independent of their host compared to smaller phage. Using transposon mutagenesis and deep screening, and CRISPRi interference, we identified over 90 host genes involved in the infection of the nucleus-forming jumbo phage PCH45. These results demonstrated that, although jumbo phage utilise many host factors, they are still highly vulnerable to receptor mutations, which can lead to cross-resistance to other phage.
The ability to mutate phage genomes is essential to be able to assign unknown gene functions and deliver genetic cargo to enhance phage functionality. However, many existing technologies require in-depth understand of the phage genome prior to engineering them. As no genome wide unbiased insertional mutational approach previously existed, we designed phage Tn-seq a random transposon mutagenesis and deep sequencing method designed to probe gene essentiality in phage and enable the rapid delivery of genetic cargo into phage without prior genomic knowledge. Using CRISPR-anti-CRISPR (Acr) based selection, we assigned gene essentiality to the nucleus-forming jumbo phage PCH and demonstrated that the system could be readily adapted to deliver alternative cargo into diverse phage. Overall, this study expands our ability to manipulate and understand bacterium–phage interactions, providing new insight into host factors essential for jumbo phage infection and establishing a platform for investigating phage genomes.