Effect of Structural Trimming on the Therapeutic Efficacy of K2 Capsule Depolymerase’s Klebsiella pneumoniae Phage B1

Background/Objectives: The treatment of multidrug-resistant and hypervirulent Klebsiella pneumoniae is one of today’s biggest healthcare challenges. The depletion of therapeutic options necessities the application of new methods. One promising approach is to use phage-derived enzymes that target the capsule and make the causative more visible to the immune system. As proteins, these enzymes must be optimised by reducing their size and antigenicity to make them suitable for repeated use. Methods: In this study, the recently isolated and cloned K2B1orf61 K2 capsule-specific depolymerase was analysed structurally, and certain amino acid residues were deleted by cloning. The following amino acid residues were removed from the 80th (D2_N80), 115th (D3_N115), and 200th (D4_N200) N-terminal, and 250 (D5_C250) and 20 (D6_C20) from the C-terminal positions of the wild-type molecule (D1_wt). The resulting derivative molecules were compared with in vitro and in vivo tests. Results: In the presence of wild-type depolymerase, human serum was able to eliminate the target bacterium and save the lives of mice challenged with the bacterium in an originally lethal intraperitoneal model. However, the D5_C250 derivative lost all activity, meaning that the bacteria with the K2 capsule in the serum survived and all the mice died. Derivatives D2_N80, D3_N115, and D6_C20 exhibited prolonged activity in the serum killing assay, effectively eliminating bacteria within 5 h. Similar activity differences were revealed in the intraperitoneal experiment: D5_C250 had no rescue effect; however, D1_wt, D2_N80, D3_N115, D4_200, and D6_20 resulted in survival rates of 100%, 60%, 80%, 100%, and 20%, respectively. Conclusions: Our results demonstrate that molecular trimming is a promising procedure for developing ideal therapeutic depolymerases.