Characterization of the Fiber Protein C-Terminal Domain from Klebsiella pneumoniae Phage KlebP_144 and Evaluation of Its Anti-Capsular Activity

Klebsiella pneumoniae, a member of the ESKAPEE group of priority pathogens, has become one of the most challenging bacterial pathogens in modern clinical practice, largely due to its multidrug resistance and the immune-evasive effect of its capsular polysaccharide (CPS). Phage-encoded depolymerases, which selectively degrade the capsular polysaccharide, have emerged as promising antimicrobial agents capable of restoring bacterial susceptibility to both immune clearance and phage infection. The fragment corresponding to the C-terminal region of a putative depolymerase of bacteriophage KlebP_144, namely DepKP144ΔC, was cloned, expressed in E. coli, and purified using immobilized metal affinity chromatography. DepKP144ΔC displays an enzymatic activity against capsular polysaccharides of 100% K1 capsular-type strains and 85% K2 capsular-type strains, including classical and hypervirulent isolates. It was demonstrated that this protein is capable of inhibiting K. pneumoniae biofilm formation, but it is unable to disrupt mature biofilms. In vivo experiments using a murine K. pneumoniae infection model further confirmed its therapeutic potential: treatment with DepKP144ΔC improved survival rate in mice infected with K2-type K. pneumoniae, indicating significant attenuation of bacterial virulence. Therefore, these results demonstrate the potential role of the C-terminal domain of the bacteriophage KP144 tail-fiber protein in phage entry and show that its carbohydrate-recognition motifs possess enzymatic activity against the Klebsiella capsular polysaccharides.