In Australia, hospital acquired infections (HAIs) result in 2 million extra days of hospitalisation each year (1). HAIs with multi-drug resistant 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 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 with little structural information available.
We have mined our extensive Kp-targeting phage collection and identified putative depolymerases based on genomic data. To date, we have purified four recombinant depolymerase enzymes and have shown them to be active against multiple Kp strains (including hypervirulent strains), whilst also demonstrating their enzymatic mechanism. These enzymes additionally exhibit remarkable shelf-life (>2 years at 37 °C), as well as exceptional stability in harsh thermal and chemical environments - including when formulated with currently approved TGA-listed cleaning agents. Moreover, we have generated high-resolution cryo-EM structures of three of these depolymerases, at up to 1.86 Å. The data gained in this study 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 either as part of surface decontamination formulations or as an anti-fouling coating on hospital surfaces.