Bacteriophage-associated endoglycosidases selectively depolymerize capsular polysaccharides of Klebsiella pneumoniae belonging to the clonal groups 258 (clade II) and 307

Klebsiella pneumoniae is a major opportunistic pathogen responsible for severe hospital-acquired infections, frequently sustained by multidrug-resistant high-risk clones. The capsular polysaccharide (CPS) represents a key virulence factor and a primary target of bacteriophage-associated polysaccharide depolymerases. In this study, we investigated the activity and substrate specificity of two phage-associated endoglycosidases from the lytic bacteriophages ΦBO1E and vB_KpS_GP-1 (ΦGP1), infecting K. pneumoniae strains belonging to clonal group 258 clade II and clonal group 307, respectively. Purified CPS from strains KPB-1 and KP-395 were subjected to enzymatic depolymerization using intact phage particles. Enzymatic activity was qualitatively monitored by reducing sugar assays, while degradation products were isolated by size exclusion chromatography and characterized by electrospray ionization mass spectrometry and one- and two-dimensional NMR spectroscopy. The ΦBO1E-associated endoglycosidase selectively cleaves the α-L-Rhap-(1→ 3)-β-D-Galp linkage within the KPB-1 CPS, generating oligosaccharides corresponding to one and two repeating units. Similarly, the ΦGP1-associated enzyme depolymerizes the KP-395 CPS by specifically hydrolyzing the β-D-Galp-(1 → 4)-α-D-Galp linkage, yielding oligosaccharides corresponding to one and two repeating units. In both cases, the enzymes display strict specificity toward a single glycosidic bond within the CPS repeating unit. These findings provide detailed structural insights into the mode of action of phage-associated endoglycosidases targeting clinically relevant K. pneumoniae CPS types. The high substrate specificity and efficient CPS depolymerization support the potential of these enzymes as enzybiotics or anti-virulence agents for the development of alternative strategies to fight multidrug-resistant K. pneumoniae infections.