The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.
Our study focuses on 'nucleus-forming' jumbo phages, which can avoid bacterial immune systems by shielding their DNA during infection. To be used to treat disease, we wanted to know what bacterial genes could mutate and stop nucleus-forming phage infection. We did this by combining transposon sequencing (Tn-seq) and CRISPR interference (CRISPRi) to interrupt gene function, followed by phage challenge, to see which genes were important during infection. We found the production and regulation of the main receptor for phage attachment was vital for infection. Many other biological processes were involved in infection and were also used by non-jumbo phages. We show that nucleus-forming phages remain vulnerable to bacterial mutations, including receptor loss, which should be evaluated prior to therapeutic use.
© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.