Oral Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Guided missile strikes: Targeting mechanisms by novel bacteriophages. (#49)

Rhys A Dunstan 1 , Rebecca Bamert 1 , Francesca Short 2 , Murray White 1 , Derek Pickard 2 3 , David Goulding 2 , Matthew Belousoff 4 , Richard Strugnell 5 , Gordon Dougan 2 3 , Trevor Lithgow 1
  1. Infection & Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton 3800, VIC, Australia
  2. Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire CB10 1SA, United Kingdom
  3. Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge, UK
  4. Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia
  5. Department of Microbiology and Immunology, The University of Melbourne, Melbourne, Vic, Australia

Bacteriophages, or phages, are the most varied and abundant biological entities on the planet and differ substantially in the bacterial hosts they infect. In order to infect its host a phage must first recognize a receptor on the surface of the bacterial cell. Tailed phages use a broad range of receptor binding proteins, such as tail fibres and tail spikes, to target their cognate receptor. These receptors may include lipopolysaccharide (LPS), capsular polysaccharide (CPS), outer membrane proteins (OMPs) such as porins or other more elaborate proteinaceous structures such as fimbriae and flagella. We have isolated several novel phages that target Klebsiella pneumoniae, a pathogen that has become a major healthcare burden, particularly in the hospital setting. The genomes of these phages have been sequenced and their structural morphology characterized by electron microscopy (EM). Transposon Directed Insertion Sequencing (TraDIS) was used to identify bacterial mutants that were resistant to each individual phage detailing the host genes required for phage infection. These genes included the porin ompK36 and several other genes involved in the biosynthesis or secretion of CPS. We have also identified novel capsule hydrolases produced by these phages which act to degrade the bacterial capsule. Using in vitro assays, we have characterized the host range, activity and stability of one of these enzymes and have determined its structure using single particle Cryo-EM. This work provides a greater understanding of the targeting mechanisms used by Klebsiella-targeting phages and provides a framework for their potential applications as therapeutics or use in the biotechnology industry.