Poster Presentation The 48th Lorne Conference on Protein Structure and Function 2023

Developing phage depolymerase enzymes to disarm Klebsiella pneumoniae (#353)

Daniel E Williams 1 2 , Slawomir Michniewski 3 4 , Chathura D Suraweera 1 2 , Stephen Harrop 5 , Trevor J Lithgow 1 2 , Eleanor Jameson 3 6 , Simon R Corrie 2 7 , Rhys A Dunstan 1 2 , Sheena McGowan 1 2
  1. Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC, Australia
  2. Centre to Impact AMR, Monash University, Clayton, VIC, Australia
  3. School of Life Sciences, University of Warwick, Coventry, United Kingdom
  4. University of Leicester, Leicester , United Kingdom
  5. Australian Synchrotron, Clayton, VIC, Australia
  6. School of Natural Sciences, Bangor University, Bangor, United Kingdom
  7. Department of Chemical and Biological Engineering, Monash University, Clayton, VIC, Australia

In Australia, hospital acquired infections (HAIs) result in 2 million extra days of hospitalisation each year (1). HAIs with multi-drug resistant (MDR) Klebsiella pneumoniae (Kp) present a serious risk for patients due to limited effective treatment options (2). Despite rigorous cleaning protocols, the most common cause of HAIs are from bacteria already present in the hospital environment (3). Novel strategies are needed, and one approach involves examining bacteriophages, which produce a multiplicity of enzymes that have anti-bacterial and anti-virulence properties. One such enzyme family known as depolymerases, can degrade both the protective sticky capsule that covers the Kp cell as well as bacterial biofilms, rendering the bacteria avirulent. Depolymerase activity could thus be exploited to provide a solution to HAIs by disarming pathogenic bacteria before they reach the patient as part of a disinfectant regimen. However, presently depolymerases are poorly characterised in the literature and are highly variable in sequence, enzymatic mechanism, specificity, and structure.

We have mined our extensive Kp-targeting phage collection and identified putative depolymerases. In this study, we are building a knowledge base of the depolymerases' biological activity, mechanism of action and structure using crystallographic and cryo-EM approaches. To date, we have purified seven recombinant depolymerase enzymes and have shown them to be active against multiple Kp strains (including hypervirulent strains). These enzymes additionally demonstrate remarkable shelf-life and stability in harsh chemical and thermal environments. The data gained is transformative to the knowledgebase of these underappreciated enzymes, and, given these highly advantageous biochemical properties, we expect these enzymes to serve as an excellent platform to prevent Kp contamination via a disinfectant strategy as part of surface decontamination formulations or as an anti-fouling coating on hospital surfaces. These future bio-agents can be engineered in order to generate candidates with superior properties to confront the emergence of MDR Kp.

  1. [1] Australian Safety and Quality Goals for Healthcare: Goal 1.2: Healthcare-Associated Infection Action Guide 2012 20 May 2020:[12p.]. Available from: https://www.safetyandquality.gov.au/ sites/default/files/migrated/1.2-Healthcare-Associated-Infection.pdf
  2. [2] Limbago BM, Rasheed JK, Anderson KF, Zhu W, Kitchel B, Watz N, et al. IMP- producing carbapenem-resistant Klebsiella pneumoniae in the United States. Journal of Clinical Microbiology. 2011;49(12):4239-45.
  3. [3] Hughes R. Patient Safety and Quality: An Evidence-Based Handbook for Nurses. 2008