Structural characterisation of pbunavirus pv141: a lytic pseudomonas-infecting bacteriophage

Thesis

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative bacterium that causes opportunistic infections in humans that can be severe and multidrug resistant. Increasing antibiotic resistance has led P. aeruginosa to become a pathogen of high significance, for which novel treatments are urgently needed. As antibiotic resistance has increased, interest in bacteriophages (phages), viruses that infect bacteria, has also increased. Despite this increased interest, the majority of sequenced phages are not well characterised and fundamental structural knowledge is lacking. The PB1-like myovirus pv141 (14-1) is a lytic phage, able to infect and subsequently kill P. aeruginosa through cell lysis. Despite 14-1 being used in initial clinical trials to treat P. aeruginosa infection, 14-1 has not been structurally characterised. In this thesis, I explore structurally, for the first time through electron cryomicroscopy, the fundamental major structural components of 14-1, including the genome filled and unfilled capsid head, both at an overall resolution of 3.0 Å, and the repeating lower tail structure in an extended state at an overall resolution 3.5 Å. The capsid head is formed of the major capsid protein gp23, forming the classical HK97-fold, embellished with b-tulip fold gp22 decoration proteins. The repeating tail structure is formed of the major tail tube protein gp30, forming a b-sandwich fold, in addition to the multi-domain sheath protein gp29. The structural components of the phage virion that I have solved to date provide structural insight into overall phage morphology and insight into how the phage functions to infect and kill P. aeruginosa. I also present electron cryotomography of 14-1 infection of P. aeruginosa, which shows 14-1 in several states of the lytic infection cycle. This work is complemented by preliminary neutralisation assays and single particle analysis of samples of 14-1 with lipopolysaccharide (LPS), which is the receptor for 14-1 and a major component of the Gram-negative outer membrane. In this thesis, I present work that increases our structural understanding of the 14- 1 virion which could aid in the development of novel treatments in the future. Furthermore, the work I present investigating the 14-1 lytic infection cycle will develop the basis for future studies investigating 14-1 interaction with the P. aeruginosa outer membrane, providing mechanistic and structural understanding of phage-host interactions and advancing phage research towards in situ structural biology.

Authors: Letham, SC

• DOI: 10.5287/ora-qzjgxwrdj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: cryo-ET; Pbunavirus; pseudomonas; bacteriophage; cryo-EM
• Subjects: Structural Biology; Microbiology